Mouse Development: Patterning, Morphogenesis, and Organogenesis

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Mouse Development Patterning, Morphogenesis, and Organogenesis

This Page Intentionally Left Blank

ouse e/,/e 0 men Patterning, Morphogenesis, and Organogenesis

Edited by

Janet Rossant Samuel Lunenfeld Research Institute Mount Sinai Hospital Toronto, Ontario, Canada

Patrick P. L. Tam Children's Medical Research Institute University of Sydney Westmead, New South "Wales, Australia

ACAA)EMIC PRESS An Imprint of Elsevier

San Diego

San Francisco New York Boston London Sydney Tokyo

Co~erphotoqraph. An E13.5 mouse embryo stained for cartilage and nerves. Courtesy' of R Akin,.vunmi and R. Behringer..kiD Anderson Cancer Center. Houston. USA. This book is printed on acid-flee paper.(~ Copyright g 2002 by ACADEMIC PRESS All rights reser-ved. No part of this publication max be reproduced or transmitted in any form or by any' means, electronic or mechanical, including photocopy, recording, or any' information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science and Technology Rights Department in Oxford, UK. Phone: (44) 1865 843830, Fax: (44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage: http://www.elsevier.com by selecting "Customer Support" and then "Obtaining Permissions".

Academic Press An Imprint of Elsevier 525 B Street, Suite 1900, San Diego. California 92101-4495, USA http://www.academicpress.com Academic Press Harcourt Place. 32 Jamestown Road. London NW1 7BY. UK http://www.academicpress.com Library" of Congress Catalog Card Number: 2001098457 ISBN- 13: 978-0-12-597951-1 ISBN-10:0-12-597951-7 PRINTED IN C A N A D A 05 06 FR 9 8 7 6 5 4 3 2

Dedicated to the memory of our dear friend and colleague, Rosa Beddington, for all her great insights into the intricacies of the early mouse embryo.


Contents

Contributors xiii A b o u t the Editors

IV. Specification of the Polarity of the AnteriorPosterior Axis of the Fetus? 29 V. Conclusions 32 References 33

xvii

I Establishment of Body Patterns

3 Anterior Posterior Patterning of the Mouse Body A~is at Gastrulation Stew-Lan Ang and Richard R. Behringer 1. Introduction 37 11. Gastrulation 38 III. The Node: Morphogenesis, Cell Fate, and Cell Movement 38 IV. The Organizer Phenomenon: Conserved Properties of 'vertebrate Organizers 40 V. The Vertebrate Organizer is a Dynamic, Nonhomogeneous, and Renewable Cell Population at Gastrulation 40 VI. Insights into the Function of the Mouse Organizer Gained from Genetic and Embryological Studies 41 VII. Genetic Analysis of Organizer Function: Mouse Mutants Showing Defects in Organizer Function 42 VIII. Inhibitory Signals Secreted by the Organizer and Its Derivatives 44 IX. Specification of the Primitive Streak and the Organizer 44 X. Rote of the AVE in Anterior Patterning in Mouse 45 XI~ Embryological and Genetic Analysis of the Function of the AVE in Anterior Patterning 46

1 Fertilization and Activation of the Embryonic Genome Davor Soiter, Wilhelmine N. de Vries, Alexei V. Evsikov, Anne E. Peaston, Frieda H. Chen, and Barbara B. Knowles 1. Introduction 5 il. Oogenesis 6 111.Meiosis and the Beginning of Oocyte Asymmetry 7 IV. Fertilization 8 V. Transcription andlts Control 8 Vi. mRNA Utilization during Oocyte Maturation and Preirnplantation Development I0 VII. Gene Expression in the Early Mouse Embryo 11 VIII. Functional Analysis 13 References I5 2 A s y m m e t r y a n d P r e p a t t e r n in Mammalian Development R. L. Gardner I. Introduction 2I !1. Asymmetries in Early Development 23 II1. Asymmetry of the Btastocyst 27

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Contents IV. CNS Dorsal-Ventral Patterning Involves a Tug of War between Dorsal and Ventral Signaling 120 V. Summary 122 References t22

XII. A Model for AVE Function in Anterior Patterning 47 XIII. Conclusions and Future Directions 48 References 49

4

I. !/. III. IV. V. VI. VII. VIII. IX.

Left-Right A s y m m e t r y Hiroshi Harnada Introduction 55 Morphological LeR-Right Asymmetries 56 Genetic/Molecular Pathway Governing LeftRight Determination 58 Molecular Readout of the First Asymmetry 61 Role of the Midline 64 Readout of Left-Right Asymmetry in Later Development 65 Miscellaneous Mutations/ Gene Factors 67 Diversity among Vertebrates 68 Future Challenges 69 References 70

5 P a t t e r n i n g , R e g i o n a l i z a t i o n , a n d Cell D i f f e r e n t i a t i o n in t h e F o r e b r a i n Oscar Marin and John L. R. Rubenstein 1. Organization of the Forebrain 75 11. Early Patterning and Regional Specification of the Forebrain 78 111.Morphogenetic Mechanisms in the Forebrain 85 IV. Control of Neurogenesis and Cell-Type Specification in the Forebrain 87 References 97

7 Somitogenesis: Segmentation of the Paraxiai M e s o d e r m a n d t h e D e l i n e a t i o n of Tissue C o m p a r t m e n t s Achim Gossler and Patrick P. L. Tam I. Overview of Somite Development 127 11.Allocation of Progenitor Cells to the Paraxial Mesoderm 132 I11. Cells Are in Transit in the Presomitic Mesoderm 132 IV. Regionalized Genetic Activity Points to a Prepattern of Prospective Somites 133 V. Emergence of Anterior-Posterior Somite Compartments 134 VI. Role of Notch Signaling in the Establishment of Somite Borders and Anterior-Posterior Polarity 134 VII. A Molecular Clock 9 in the Paraxial Mesoderm to Control the Kinetics of Somite Formation 138 VIII. Specification of Lineage Compartments by Inductive Interactions 139 IX. Summary and Open Questions 142 References 144

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Lineage Specification and Differentiation 8

6 Establishment of Anterior-Posterior and D o r s a l - V e n t r a l P a t t e r n in t h e Early C e n t r a l N e r v o u s System Alexandra L. Joyner I. Overview of Early CNS Development and Patterning 107 11.Anterior-Posterior Patterning of the Mesencephalon and Metencephalon 110 II1. Hindbrain Anterior-Posterior Patterning Involves Segmental Units of Development 117

Extraembryonic Lineages Janet Rossant and James C. Cross I. Introduction 155 I1. Early Development of the Trophoblast and Primitive Endoderm Lineages 156 III. Cell Lineage Analysis and the Extraembryonic Lineages 156 158 IV. Setting Aside the Blastocyst Lineages V. Molecular Specification of the Blastocyst Cell Lineages 159 VI. Differentiation of the Yolk Sacs t 61 VII. Morphogenetic Events in Development of the Chorioallantoic Placenta 161

Contents VIII. Comparative Aspects of Development of Extraembryonic Membranes 162 IX. Molecular Control of Primitive Endoderm Development 164 X. Signaling Pathways in Early Trophoblast Development 166 XI. Control of Spongiotrophoblast and Giant Cell Fate 168 Xll. Trophoblast Giant Cell Development: Gene Pathways and Control of Endoreduplication t 69 Xlll. Initiating Chorioallantroic Fusion 170 XIV. Gcm I Regulates the Initiation of Chorioallantoic Branching 170 XV. Growth Factor Signaling Regulates Branching Morphogenesis of the Labyrinth 171 XVI. Placental Development and Pregnancy Complications 173 References 174

9 G e r m Cells Christopher Wylie and Robert Anderson 1. General Concepts 181 Ii. Early Appearance of Germ Cells in the Mouse 182 111.Specification of Germ Cells in the Mouse 183 IV. Migration of Germ Cells 185 V. Motility of Germ Cells 185 VI. Guidance of Germ Cell Migration 186 Vii. Adhesive Behavior of Germ Cells during Migration t 87 VIII. Survival and Proliferation of Germ Cells during Migration 188 References 189

10 D e v e l o p m e n t o f t h e V e r t e b r a t e H e m a t o p o i e t i c System Nancy Speck, Marian Peeters, and Elaine Dzierzak 1. Introduction 191 !!. Cellular Aspects of Blood Development in the Mouse Embryo 192 111.Molecular Genetic Aspects of Blood Development in the Mouse Embryo 202 IV. Current Cellular and Molecular Conceptual Frameworks for Hematopoietic Ontogeny 205

ix V. Future Directions References 206

206

1 1 Vasculogenesis and Angiogenesis Thomas N. Sato and Siobhan Loughna i. Introduction 211 11. Overview of Vascular Development 211 I11.Generation of Endothelial Cells 212 IV. Vascular Morphogenesis 220 V. Concluding Remarks 228 References 228 1 2 Stem Cells of t h e N e r v o u s System sean J. Morrison 1. Introduction 235 11. Lineage Determination of Neural Stem Cells 237 I11. Do Stem Cells Retain Broad or Narrow Neuronal Potentials? 241 IV. Regulation of Neural Stem Cell Self-Renewal 242 V. Differences between Hematopoietic Stem Cells and Neural Stem Cells 243 VI. Im ~ v o Function of Neural Stem Cells 244 VII. Surprising Potential of Neural Stem Cells 245 VIIi. Are Neural Stem Cells Involved in Disease? 246 IX. Outstanding Issues 248 References 248 13 Cellular a n d M o l e c u l a r M e c h a n i s m s R e g u l a t i n g Skeletal M u s c l e D e v e l o p m e n t Atsushi Asakura and Michael ~_ Rudnicki I. Introduction 253 11. Embryonic Origin of Skeletal Muscle 254 II!. MyoD Family of Myogenic Regulatory Factors 256 It,," Muscle-Specific Transcriptional Regulation 262 V. Inductive Mechanisms of Myogenesis 262 Vl. Specification of Muscle Fiber Types 268 VII. Muscle Regeneration 269 VIII. Conclusion 2_72 References 272

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Contents

14

Deconstructing the Molecular Biology of Cartilage and Bone Formation Benoit de Crombrugghe, Veronique Lefebvre, and Kazuhisa Nakashirna 1. Introduction 279 II. Sox Transcription Factors: Essential Roles in the Chondrocyte Differentiation Program 281 Iit. Parathyroid Hormone-Related Peptide (PTHrP) and Parathyroid Hormone (PTH)/PTHrP Receptor: Gatekeepers of the Zone of Hypertrophic Chondrocytes 284 IV. FGFs and FGF Receptor 3: Counterintuitive Inhibitors of Chondrocyte Proliferation 286 V. ihh: A Central Coordinator of Endochondral Bone Formation 287 VI. The Two Roles of the Transcription Factor Cbfal in Endochondral Bone Formation 288 VII. Other Transcription Factors Involved in Bone Formation 290 VIII. Gelatinase B and Vascular Endothelial Growth Factor: Additional Coordinators of Endochondrat Bone Formation 290 IX. Conclusion 291 References 292

III Organogenesis 15

Development of the Endoderm a n d Its Tissue D e r i v a t i v e s Brigid L. M. Hogan and Kenneth S. Zaret I. Introduction and Overview 301 I!. Endoderm Development prior to Organogenesis 302 Ill. Patterning and Differentiation of the Digestive Tract 307 IV. Development of Tissues That Bud from the Endoderm 310 V. Perspectives and Remaining Issues on Organogenesis from the Endoderm 322 References 322 1 6 Molecular Determinants of Cardiac Development and Congenital Disease Richard P. Harvey I. Introduction 332 !i. Overview of Heart Structure and Development 332

111.A Conserved Pathway for Cardiac Induction and Morphogenesis 334 IV. Cardiac Induction: The Role of Endoderm 334 V. Bone Morphogenetic Proteins as Cardiac Inducing Molecules 336 Vi. Other Factors Involved in Cardiac Induction 336 VII. A Role for Anterior Visceral Endoderm in Cardiac Induction in the Mouse? 338 VIII. The Heart Morphogenetic Field 339 IX. The Size and Shape of the Heart Field 339 X. The Timing and Stability of Cardiac Induction 340 XI. Migration of Cardiac Precursors 340 XII. Cellular Proliferation and Death in the Forming Heart 341 Xlll. Cardiac Myogenesis 341 XIV. Modulation of Myogenesis in Heart Chambers 343 XV. Regionality in the Developing Heart 343 XVI. Plasticity of Heart Regionalization : 344 XVil. The Segmental Model of Cardiac Morphogenesis 344 XVIli. An Inflow/Outflow Model of Early Heart Tube Patterning 345 XIX. A Role for Retinoic Acid Signaling in Inflow/ Outflow Patterning 346 XX. A Role for the Delta/Notch Pathway in Primary Heart Patterning 347 XXI. Cardiac Chamber Formation 347 XXil. Ventricular Specification: Knock-Out and Transgenic Phenotypes 348 XXlil. Transcriptional Circuits Acting in Chamber Formation 351 XXIV. The Cardiac Left-Right Axis 351 XXV. Developmental Pathways and Congenital Heart Disease 356 XXVI. Horizons 357 References 358

1 7 Sex D e t e r m i n a t i o n a n d D i f f e r e n t i a t i o n Amanda Swain and Robin Loveli-Badge I. Introduction 371 !i. Gonad Development 372 !11. Sex Determination 376 IV. Testis Differentiation 380 V. Cell Movement and Proliferation in the Early Gonad 382

Contents VI. VII. VIII. IX.

Ovary Differentiation 384 Sexual Development 384 Evolution and Sex Determination Conclusion 388 References 389

xi 21

386

18

D e v e l o p m e n t o f t h e E x c r e t o r y System Gregory R. Dressier 1. Introduction 395 11. Patterning of the Intermediate Mesoderm 396 111.Growth of the Nephric Duct and Ureteric Bud Diverticulum 400 IV. Inductive Interactions 404 V. Mesenchyme-to-Epithelial Conversion 407 Vi. Glomerular Development and Vascularization 412 VII. Developmental Basis of Human Renal Disease 414 VIII. Future Perspectives 416 References 416

19 C r a n i o f a c i a l D e v e l o p m e n t Michael J. Depew, Abigail S. Tucker, and Paul T. Sharpe I. Introduction 421 il. Primordial Cells of the Head 422 !il. Organ Development 433 IV. Conclusion 454 V. Appendix 1: Descriptive Dental Development 454 Vi. Appendix 2: Morphological Organization of the Murine Skull 456 VII. Appendix 3: Molecular Regulators of Craniofacial Pattern and Development 465 References 481 20 Pituitary Gland Development Sally Camper, Hoonkyo Suh, Lori Raetzman, Kristin Douglas, Lisa Cushman, Igor Nasonkin, Heather Burrows, Phil Gage, and Donna Martin 1. Pituitary Gland Anatomy and Function 499 il. Development of the Pituitary Primordia and Cell Specification 500 !11. Expansion of Committed Cell Types 510 IV. Conclusion 512 References 513

Development of the Eye Hisato Kondoh 519 1. Overview of Eye Development II. Development of the Retina 521 I11. Lens Development 528 IV. Conservation and Divergence of the Transcriptional Regulatory Systems in the Eye Development 533 References 535 2 2 D e v e l o p m e n t o f t h e M o u s e I n n e r Ear Axny E. Kiernan, Karen P. Steel, and Donna M. Fekete 1. introduction 539 II. Anatomy of the Inner Ear 540 111.Development of the Inner Ear 541 IV. Early Development of the Otic Blacode and Otocyst 542 V. Pattern Formation in the Inner Ear 546 VI. Sensory Di~erentiation 552 VII. Neurogenesis 558 VIII. The Stria Vascularis 559 IX. Future Directions 560 References 561 23 Integumentary Structures carolyn Byrne and Matthew Hardman 1. Introduction 567 11. Mature Skin 569 I1t. Non-Neural Embryonic Ectoderm 570 IV. Stratification 571 V. Dermal Development 572 VI. Epidermal Appendage Morphogenesis 574 VII. Model for Follicle Formation: The First Dermal Signal 574 VIII. Follicle Spacing 577 IX. Follicle Morphogenesis and Differentiation 578 X. Follicle Morphogenesis and Follicle Cycling 578 XI. Molecular Parallels between Skin Tumorigenesis and Skin Development 579 XII. Early Terminal Differentiation 579 XtlI. Regulation of Transit to Late Stages of Terminal Differentiation 580 XIV. Late Terminal Differentiation: Formation of Stratum Corneum and Skin Barrier 581

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xv. Periderm Disaggregation 583 XVl. Conclusions and Future Directions References 584

Contents

584

Author Index Subject Index

591 691

Contributors

Benoit de Crombrugghe (279) Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Wilhelmine N. De Vries (5) The Jackson Laboratory, Bar Harbor, Maine 04609 Michael J. Depew (421 ) Nina Ireland Laboratory of Developmental Neurobiology and Department of Oral Biology, University of California, San Francisco, San Francisco, California 94143 Kristin Douglas (499) Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 Gregory R, Dressier (395) Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109 Elaine Dzierzak ( 191 ) Department of Cell Biology and Genetics, Erasmus University, Rotterdam 3000, The Netherlands Alexi V. Evsikov (5) The Jackson Laboratory, Bar Harbor, Maine 04609 Donna M. Fekete (5391 Department of Biological Sciences, Purdue University, "West Lafayette, Indiana 47907 Phil Gage (499) Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 R. L. Gardner (21 ) Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom Achim Gossler ( 1271 Institute fur Molekularbiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany Hiroshi Hamada (55 ) Division of Molecular Biology, Institute for Molecular

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Robert Anderson ( 181 ) University of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Siew-Lan Ang (37) Institut de G6n6tique et de Biologie Mol6culaire et Cellulaire, C N R S / I N S E R M , Universit6 Louis Basteur, 67404 Illkirch cedex, C.U. de Strasbourg, France Atsushi Asakura (253) Pro~am in Molecular Genetics, Ottawa Hospital Research Insititute, Ottawa, Ontario, Canada K1H 8L6 Richard R, Behringer (37) Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Heather Burrows (499) Graduate Pro~am in Cellular and Molecular Biology, University of Michigan Medical School, Ann .Arbor, Michigan 48109 Carolyn Byrne (567) School of Biological Sciences, University of Manchester, Manchester M 13 9PT, United Kingdom Sally Camper (499) Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 Freida H. Chen (5) Max Planck Institute of Imrnunobiology, 79108 Freiburg, Germany James C. Cross ( 155) Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada T2N 4N 1 Lisa Cushman (499) Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 xiii

xiv

Contributors

and Cellular Biology. Osaka University. Osaka 565-0871, Japan M a t t h e w Hardrnan ( 567) School of Biological Sciences. University of Manchester, Manchester M 13 9PT. United Kingdom Richard P. Harvey (331 ) The Victor Chang Cardiac Research Institute. St. Vincent's Hospital. Darlinghurst 2010. Australia and Faculties of Medicine and Life Sciences. University of New South Wales. New South Wales 2052. Australia Brigid L. M. Hogan (301) Department of Cell Biology. Howard Hughes Medical Institute. Vanderbilt University Medical Center. Nashville. Tennessee 37232 Aiexandra k. Joyner ( 1071 Developmental Genetics Program. Skirball Institute of Biomolecular Medicine. New York University School of Medicine. New York. New York 10016 Amy E. Kiernan' (539) MRC Institute of Hearing Research. Nottingham NG7 2RD. United Kingdom Barbara B. Knowles (5) The Jackson Laboratory. Bar Harbor. Maine 04609 Hisato Kondoh (519) Institute for Molecular and Cellular Biology. Osaka University. Osaka 565-0871. Japan V & o n i q u e Lefebvre (279) Department of Biomedical Engineering. Lerner Research Institute. Cleveland Clinic Foundation. Cleveland. Ohio 44195 Siobhan koughna (211) The University of Texas Southwestern Medical Center at Dallas. Dallas. Texas 75390 Robin kovell-Badge (371 ) Section of Gene Function and Regulation. Chester Beatty Laboratories. London SW3 6JB. United Kingdom Oscar Marin (75 ) Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, Langley Porter Psychiatric Institute. University of California. San Francisco, San Francisco. California 94143 Donna Martin (499) Department of Pediatrics. University of Michigan Medical School. Ann Arbor. Michigan 48109 Sean J. Morrison (235) Departments of Internal Medicine and Cell and Developmental Biology. Howard Hughes Medical Institute. University of Michigan. Ann Arbor. Michigan 48109

Present address: The Jackson Laboratory. Bar Harbor. Maine 04609.

Kazuhisa Nakashirna (279) Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 igor Nasonkin (499) Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 Anne E. Peaston (5) The Jackson Laboratory. Bar Harbor. Maine 04609 Marian Peeters ( 191 ) Department of Cell Biology and Genetics. Erasmus University, Rotterdam 3000. The Netherlands kori Raetzrnan (499) Department of Human Genetics. University of Michigan Medical School. Ann Arbor, Michigan 48109 Janet Rossant ( 155) Samuel Lunenfeld Research Institute, Mount Sinai Hospital. Toronto. Ontario. Canada M5G 1X5 John k. R, Rubenstein (75) Nina Ireland Laboratory, of Developmental Neurobiology. Department of Psychiat W. Langley Porter Psychiatric Institute. University of California, San Francisco. San Francisco. California 94143 Michael A. Rudnicki (253) Program in Molecular Genetics, Ottawa Hospital Research Insititute. Ottawa. Ontario, Canada K1H 8L6 Thomas N. Sato (211 ) The University of Texas Southwestern Medical Center at Dallas. Dallas. Texas 75390 Paul Y. Sharpe (421 ) Department of Craniofacial Development, GKT School of Dentistry. Guy's Hospital, London SE1 9RT, United Kingdom Davor Solter (5) Max Planck Institute of Immunobiology, 79108 Freiburg, Germany: and The Jackson Laboratory, Bar Harbor. Maine 04609 Nancy Speck ( 191 ) Department of Biochemist~,, Dartmouth Medical School. Hanover, New Hampshire 03755 Karen P. Steel (539) MRC Institute of Hearing Research, Nottingham NG7 2RD. United Kingdom Hoonkyo Suh (499) Graduate Program in Neuroscience, University of Michigan Medical School, Ann Arbor, Michigan 48109 A m a n d a Swain (371 ) Section of Gene Function and Regulation, Chester Beatty Laboratories, London SW3 6JB, United Kingdom Patrick P. k. Tam (127) Embryology' Unit. Children's Medical Research Institute, University of Sydney, Westmead. New South Wales 2145, Australia

Contributors

Abigail S. Tucker (421 ) MRC Centre for Developmental Neurobiology, GKT School of Biomedical Sciences, Guy's Hospital, London SE 1 1UL, United Kingdom Chris Wylie ( 181 ) Division of Developmental Biology, Children's Hospital Research Foundation. Cincinnati. Ohio 45229

xv

K e n n n e t h S. Zaret (301 ) Cell and Developmental Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111


About the Editors

J a ne t Rossant Dr. Janet Rossant is Joint Head of the Program in Development and Fetal Health at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto and University Professor and Professor in the Department of Molecular and Medical Genetics and the Department of Obstetrics/Gynaecology, University of Toronto. Her research interests center on understanding the genetic control of normal and abnormal development in the early mouse embryo. She uses the powerful techniques available to genetically manipulate the mouse genome to address these problems. Recently, her research has moved in two new directions. First, stem cell research, with her discovery of a novel placental stem cell type, the trophoblast stem cell. Second, genome-wide functional genomics. She directs the Centre for Modelling Human Disease in Toronto, which is undertaking genome,wide mutagenesis in mice to develop new mouse modet~:iof 9human disease. Dr. Rossant trained at the Universities of Cambridge and Oxford, United Kingdom and has been in Canada since 1977, first at Brock University and then in Toronto. She is a Fellow of both the Royal Societies of London and Canada, an International Scholar of the Howard Huges Medical Institute, and a Distinguished Scientist of the Canadian Institutes of Health Research. Dr. Rossant is actively involved in the international developmental biology community. She was Editor of the jour-

xvii

nal Development for many years. She has organized a number of international developmental biology meetings, including the International Developmental Biology Cong e s s in 1997. She was President of the Society for Development Biology in 1996/97. She has also been actively involved in public issues related to developmental biology, most recently serving as Chair of the Canadian Institutes of Health Research working ~ o u p on stem cell research.

•

About the Editors Patrick P. L. T a m

Dr. Patrick R L. Tam is a Senior Principal Research Fellow of the National Health and Medical Research Council (NHMRC) of Australia. He is the Head of the Embryology Research Unit at the Children's Medical Research Institute in Sydney and holds a conjoint appointment of Senior Principal Research Fellow in the Faculty of Medicine. University of Sydney. His research focuses on the elucidation of the cellular and molecular mechanisms of body patterning during mouse development. Dr. Tam pioneers the application of micromanipulation and embr)'o culture for analyzing tissue potency and lineage specification in normal and mutant embryos. He studies the morphogenetic role of the gastrula organizer in axis formation, and the developmental processes leading to the regionalization of the neural tube. the paraxial mesoderm, and the embryonic gut. His other current research is on the pathogenesis of X-linked diseases using mouse models generated by transgenesis, gene targeting, and chemical mutagenesis. Dr. Tam received his training in mammalian embr?ology in Hong Kong. London. and the United States. took a faculty appointment in Anatomy at the Chinese University of Hone, Kong. and has been in Australia since 1990 to establish his research laboratory, in Sydney. He is an honorary consulting scientist of the Children's Hospital at Westmead. Australia and an Honorary Professor at the University of Hong Kong. He was a recipient of the S,'mington Memorial Prize in Anatomy of the Anatomical Society of Great Britain and Ireland and the Croucher Foundation Fellowship at Oxford University, and has been holder of the prestigious NHMRC Research Fellowship since 1996.

Dr. Tam was an instructor and lecturer of the teaching course on molecular embryology of the mouse at the Cold Spring Harbor Laboratory. served on numerous grant and fellowship review panels, and was involved with the organization of international conferences on cell and developmental biology. He was an Editor of Anatomy and Emb 9 and is currently a member of the editorial boards of Devel-

opmental Biology Mechanisms of Development, International Journal of Developmental Biology, Genesis, Differentiation, The Scientific World, the FacuI~, of 1000, and the Highlights Advisor3" Board of Nature Reviews Neuroscience.

Establishment of Body Patterns

It has been long held that, in contrast to lower vertebrate and invertebrate embryos, the mouse embryo develops from the zygote without any endowment of positional information from the mother or early zygote to specify the orientation and polarity of the prospective embryonic axis. Experimental manipulation of the early preimplantation embryo revealed that early blastomeres are remarkably plastic in their lineage potency and that the assembly of cells into an embryo is highly regulative. These findings lent staunch support to the concept that the preimplantation embryo is a "tabula rasa.'" Morphological study of the fertilized oocytes of other mammalian embryos, such as the rat and the goat (De Smedt et al., 2000), however, has shown that localization of cytoplasmic inclusions or cellular organelIes occurs, which seems to be consistent with the concept of localized morphogenetic determinants with long lasting impact on the patterning of the embryo. Some hint of as~'mmeto" is present in the fertilized mouse oocyte. The second polar body, which contains the haploid set of maternal genome and associated cytoplasm, is extruded from the animal pole of the fertilized oocyte. The animal-vegetal axis as marked by the position of the polar body is found to be aligned with the plane that is orthogonal to the embryonic-abembryonic axis of the blastocyst (Chapter 2). The other landmark, albeit transient, is the localized protrusion on the oocyte surface known as the fertilization cone where sperm fusion occurs. It has been found that

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the meridional plane defined by the circumference passing through the polar body and the sperm entry point is frequently aligned with the plane of the first cleavage of the fertilized oocyte. The membrane of the sperm entry site also stays with the blastomere that preferentially contributes to the inner cell mass and the mural trophectoderm, in other words, cells on the embryonic half of the blastocyst (Piotrowska and Zernicka-Goetz, 2001; Piotrowska et a!., 2001 ). The position of the polar body and the sperm entry point in the fertilized oocyte therefore marks the animal-vegetal axis and the border of the embryonic and abembryonic compartments. It is not known whether the extrusion of polar bodies always happens at a defined site in the oocyte, but the position of the sperm entry site relative to the polar body may vary considerably. This raises the intriguing possibility that there may be no predetermined polarity in the unfertilized oocyte, and the information is acquired on sperm-egg association. The challenge in the future is to identi~, the subcellar reorganization (e.g., cytoskeletal remodeling and membrane stabilization), trafficking, and localization of morphogenetic determinants either preexisting in the oocyte or encoded by the activated zygotic genome (Chapter 1) that underpin this patterning process. Preimplantation mouse development is preoccupied with the generation of asymmetry and the delineation of the embryonic and extraembryonic cell lineages (Chapter 8). Recently, results of cell tracing experiments performed on periimplantation mouse embryos (i.e., embryos at blastocyst to

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I Establishment of Body Patterns pregastrula stages) points strongly to a relationship between the axes of asymmetr3 of the blastocvst and the primao" body axes of the early-organogenesis-stage embryo (Chapter 2). The most intriguing finding is the directional coherent movement of the visceral endoderm from the rim of the inner cell mass first to the extraembryonic and then to the posterior region of the gastrula. In the embryonic region, the endoderm at the most distal tip of the embryo seems to be displaced toward the prospective anterior region. This overall anterior shift of the endodermal population is reminiscent of that in the avian embryo, which has been shown to determine the orientation of the body axes (Stem. 2001 ). In contrast to the increasing knowledge of how cells move and how the embry, o changes its shape and tissue architecture, amazingly; little is known of the driving forces underlying these developmental phenomena. Some of the factors that might contribute to the morphogenetic forces are the differential rates of cell proliferation in the epiblast and the visceral endoderm. the kinetics of recruitment of cells from the epiblast to the visceral endoderm, the trafficking of cells between the visceral and parietal endoderm, the differential growth of the germ layers, localized epithelial expansion and selective tissue adhesion. In addition, the impact of the uterine environment. with respect to the space constraints in the implantation site and the physical and inductive interactions between the implanting embryo and the surrounding uterine tissues. should not be ignored. Chimera and cell marking experiments have shown extensive mixing of clonal descendants from the inner cell mass to the epiblast, ruling out the possibility that any positional information conferred on inner cell mass cells could have been maintained and utilized for later patterning of the embryonic tissues. The visceral endoderm, where interclonal mixing is more restricted, is a likely repositoo" of patterning information. Experimental evidence obtained from embryological experiments (Chapter 2) and analysis of mouse mutant (Chapter 3) has presented a compelling case for a critical role of the visceral endoderm in supporting the differentiation of the epiblast, the initiation of gastrulation, and the induction of anterior cell fates. The anterior region of the visceral endoderm (AVE) is postulated to be a particularly important source of patterning activity (Chapter 3). Whether AVE is absolutely required for the formation of anterior structures has yet to be tested critically, since the findings to date are largely indirect or circumstantial (Chapter 2). Other populations of the visceral endoderm, such as those associated with the primitive streak, might also have roles in patterning (Kalantry et al., 2001). Some indication of the critical role of posterior visceral endoderm is highlighted by the impact of loss of Wnt3 or Hnf3fl (Foxa2) activity in this tissue (Chapter 3). During gastrulation, the visceral endoderm is displaced by the definitive endoderm recruited from the epiblast. This raises the question of how the patterning information may be relayed to other germ layer tissues after the

departure of the initial signaling tissue. Analysis of the expression of genes like Hesxl points to possible crosstalk between the visceral endoderm and the epiblast, such that the epiblast retains and executes the patterning instruction acquired from the endoderm. The role in body patterning of the extraembronic ectoderm, which is derived from the polar trophectoderm and is closely associated with the proximal region of the embwo until the appearance of the extra~ embryonic mesoderm, is not known. However, the inductive activity mediated by bone morphogenetic proteins expressed in the extraembryonic ectoderm and the adjacent visceral endoderm is found to be critical for the formation and maintenance of the primordial germ cells (Chapter 9). Recently, activity of Arkadia. which may modulate Nodal signaling in the extraembryonic tissues, has been shown to be critical for the specification of the gastrula organizer in the mouse (Niederlander et al.. 2001) Extraembryonic tissues are not the only source of patterning activity in gastrulation. Indeed, until recently, they were not thought to be a major source of signals at all. The gastrula organizer, equivalent to the Spemann's organizer in the frog, was considered paramount. The gastrula organizer is defined by its fate to form the axial mesoderm (prechordal mesoderm and notochord), the expression of a set of genes that are common to those expressed by the organizers of fish, frog, and bird gastrula and, unique to this group of cells, the ability to induce the formation of a partial embryo in the host embryo after transplantation (Camus and Tam. 1999). However. it has been found that induction of anterior characteristics in the new body axis requires more than the organizer and involves the synergistic interaction with the anterior visceral endoderm and the associated anterior epiblast (Chapter 3" De Souza and Niehrs. 2000). Both the organizer and its axial derivatives are also essential for establishing left-fight asymmetry (Chapter 4). Embryos harboring mutations of genes that affect the formation or differentiation of the organizer (Chapter 3) are found to display abnormal specification of left-fight body asymmetry (Chapter 4). The node in particular plays a central role as the source of signals initiating the cascade of signaling activity involving fibroblast ~ o w t h factors (FGFs), Sonic hedgehog (SHH) protein, Nodal, EGF-CFC, and the asymmetrical activation of transcription factors that determine the sidedness of the body and handedness of visceral organs (Chapter 4). A strong correlation is seen between the ciliary activity of cells in the node to the generation of left-fight body asymmetry, but it is yet unclear whether this leads to a tidal flow for distributing morphogenetic factors or instead reflects a more global pattern of the cytoskeletal architecture that underpins the lateral asymmetry. The development of the neural axis begins with the induction of the neural primordium by the combined activity of the mesoderm and endoderm and the organizer (Chapter 3, Camus and Tam. 1999). The morphogenesis of the major re-

I Establishment of Body Patterns gions of the emb~,onic brain requires the continuous inductive activin" of the axial mesoderm, especially the prechordal mesoderm and the anterior notochord (Chapter 5). The delineation of the major brain segments is accomplished by regionalized gene activity and the consolidation of the expression domains defining the boundary between the segments (Chapter 6). Finer subdivision of the neural tube results in the delineation of the neuromeres (e.g., prosomeres in the forebrain and rhombomeres in the hindbrain) along the anterior-posterior axis (Chapters 5 and 6). This is accomplished primarily by the patterning activity of the organizing centers in the neural tube at the anterior neural ridge. the diencephalon-mesencephalon junction and the midbrainhindbrain junction (Chapters 5 and 6). The brain organizer acts mainly via FGF signaling, which controls cell proliferation and the activation of segment-specific transcriptional activity determining neuronal fates. How the brain organizers are formed is not known, but surgical ablation of the axial mesoderm results in the loss from the brain of the genetic activity that is characteristic of the organizers (Camus et al., 2000). Whether this mirrors the normal role of the axial mesoderm in the establishment of the brain organizers is not known. In addition to the brain organizers, the paraxial mesoderm of the avian embryo has been shown to impart an instructive effect on the expression of rhombomere-specific Hox genes in the hindbrain. This may suggest that the paraxial mesoderm not only influences the development of the peripheral nervous system by determining the segmental migratory paths of the neural crest cells (Chapter 7), but it may also provide the signal that specifies the segmental characteristics of the neural tube. The concept of organizers as key elements in pattern formation is not restricted to the main body axis. The formation and patterning of the limbs also involves localized sources of signals m the apical ectodermal ridge and the zone of polarizing activitymand some of the same signaling pathways including FGE WNTs. and SHH (Martin, 1999: Dudley and Tabin, 2000; Schaller et al., 2001). Besides the neuromeric pattern of the cephalic neural tube, the meristic organization of the paraxial mesoderm into somites in the trunk and tail and the presence of somitomeres in the cranial region and the presomitic mesoderm highlights the segmental characteristics of the body plan of the mouse embryo. During gastrulation, cells destined for the paraxial mesoderm are allocated to the anterior-posterior axis in the temporal order of their recruitment from the somitic progenitor pool and incorporation to the caudal end of the paraxial mesoderm (Kinder et al., 1999). Oscillatory genetic activity that regulates Notch signaling provides the molecular control of the timing of segmentation of the presomitic mesoderm and the positioning of the intersegmental and half-segment boundary of the somite (Chapter 7). Patterning of the tissues in the dorsoventral plane of the embo'o is accomplished by competing inductive signals

from the axial mesoderm and the floor plate of the neural tube, and from the roof and dorsal plate of the neural tube (Chapter 6; JesseI1, 2000). The same signals are also involved with the specification of the ventromedial (sclerotome) and dorsolateral (dermomyotome) compartments of the somites (Chapter 7). An additional source of regionalization signal emanating from the lateral plate mesoderm further specifies the medial (epaxial) and lateral (hypaxial) myotome of the somites (Chapters 7 and 13). The axial mesoderm also acts to compartmentalize the embryonic gut and its accessory organs into the dorsal (closer to the axial mesoderm) and the ventral (farther from the axial mesoderm) primordia primarily through SHH signaling activity (Chapter 15: Wells and Melton, 1999). The contents of the seven chapters in this section summarize our current understanding of the cellular and molecular aspects of embryonic patterning from fertilization to early organogenesis. Despite significant gaps in our knowledge about the transition of embryonic architecture and cell lineages from blastocyst to the gastrula and details of the functional interactions of the signaling molecules that leads to the organization of the body plan, several themes of the developmental process can be recognized: I. The blastocyst architecture and the pattern of allocation of the progeny of blastomeres to the embryonic and extraembryonic lineages may be founded on the asymmetry of the zygote established epigenetically at fertilization. 2. The asymmetry of the blastocyst as defined by the animal-vegetal and embryonic-abembryonic axes may have a consequential relationship to the orientation of three primary embryonic axes. 3. The translation of the blastocyst asymmetry to the polarity and orientation of the embryonic axes is influenced by morphogenetic tissue movements driven by physical constraints and expediency within the embryonic and the uterine confine. 4. There are likely multiple sources of patterning activity from the extraembryonic tissues, intraembryonic sources, and the gastrula organizer. Embryonic patterning is the result of the interplay between synergistic and antagonistic actions of morphogenetic factors from these sources. 5. The patterning activity of the organizer is not restricted to the various forms of gastrula organizer but also their derivatives. 6. Finer patterning of the body plan requires more than the activity of the organizer derivatives and involves other tissues that provide the supplementary patterning instructions. 7. Organizer activities are not restricted to the body, but are also involved in the axes of appendages. The final outcome of these patterning activities is the establishment of a basic body plan with defined anteriorposterior polarity, the segmentally organized neural tube and

I Establishment of Body Patterns paraxial mesoderm, a distinct laterality of organ primordia, and the dorsoventrally compartmentalized neural tube. somites, and endodermal structures.

References Camus. A., Davidson, B. R. Billiards. S.. Khoo. R. Rivera-Perez. J. A.. Wakamiva. M.. Behringer. R. R.. and Tam. R R (2000). The morphogenetic role of midline mesendoderm and ectoderm in the development of the forebrain and the midbrain of the mouse embo'o. Development 127. 799-813. Camus. A.. and Tam. R R L. (1999). The organizer of the gastrulating mouse embryo. Curr. Topics Dev. Biol. 145. 117-153. De Smedt. V.. Szollosi. D.. and Kloc. NI. (2000). The Balbioni bod`. : Asvmmeterv in the mammalian oocvte. Genesis 26, 268-312. De Souza. F. S.. and Neihrs. C. (2000). Anterior endoderm and head induction in early vertebrate embryos. Cell Tissue Res. 300, 207-217. Dudley. A. T.. and Tabin. C. J. (2000). Constructive antagonism in limb development. Curr. Opin. Genet. Dev. 10. 387-392. JesselI. T. M. (2000). Neuronal specification in the spinal cord: Inductive signals and trasnscriptional codes. Nature Rev. Genet. 1, 20-29. Kalantrv. S.. Mannin,z_. S.. Haud. O.. Tomihoara-Ne~vberoer C.. Lee. H.-G.. Fangman. J.. Dieteche. C. M.. Manova. K.. and Lacy. E. (2001). The

arnnionless gene. essential for mouse gastrutation.encodes a visceralendoderm-specific protein with an extracetlular cysteine-rich domain. Nature Genet. 27, 4 1 2 - 416. Kinder. S. J.. Ysang. T. E.. Quinlan. G. A.. Hadjantonakis. A. K.. Yagy. A.. and Tam, R P. L. (1999). The orderly allocation of mesodermal cells to the extraembr?;onic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126, 4691-4701. Martin. G. R. (1999L The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 157 I - I 5 8 6 . Niederlander. C.. Walsh. J. L., Episkopou. 'v~ and Jones. M. J. (2001). Arkadia enhances nodal-related signalling to induce mesendoderm. Nature 410, 830-834. Piotro~ska. K.. and Zernicka-Goetz. M. (12001). Role for sperm in spatial patterning of the early mouse emb~;o. Nature 409, 517-52 I. Piotro~vska. K.. Wianny. F.. Pedersen, R. A.. and Zernicka-Goetz. M. (2001). Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development. Development 128, 3 7 3 9 3748. Schaller. S. A.. Li. S.. Ngo-Muller. 'v:. Han. M. J.. Omi. M.. Anderson. R.. and Muneoka. K. (200 t). Cell biology of limb patterning. Int. Rev. Cvtol. 203. 483 -517 Stern. C. D. (2001). Initial patterning of the central nervous system: How man,, oro_anizers? Nature Rev. Neurosci. ~ 92- 98. Wells. J. M.. and Melton. D. A. (1999). Vertebrate endoderm development. Annu. Rev. Cell Dev. Biol. 15, 3 9 3 - 4 1 0 .

Fertilization and Activation of the Embrjonic Genome D a v o r Solter,*-t W i l h e l m i n e N. d e Vries, t A l e x e i V. Evsikov, t A n n e E. Peaston,t Frieda H. C h e n , * a n d Barbara B. K n o w l e s t *Mcc~ Planck .Institute o f Irnmunobiology, 79108 Freiburg, Germany § The Jackson Laboratory, Bar Harbor. Maine 04609

I. Introduction Ii. Oogenesis !!1. Meiosis and the Beginning of Oocyte Asymmetry IV. Fertilization V. Transcription and Its Control VI. mRNA Utilization during Oocyte Maturation and Preimplantation Development VII. Gene Expression in the Early Mouse Embryo VIII. Functional Analysis References T

T

I. I n t r o d u c t i o n The full-grown oocyte, arrested in prophase of the first meiotic division, contains all of the molecules that will be utilized to bridge the period of transcriptional silence that begins with the completion of oocyte growth. Under hormonal stimulation the full-grown oocyte begins maturation, completing the first meiosis and the first half of the second

Mouse Development

meiotic division before arresting in metaphase of the second meiotic division. During this period the extensive stores of maternal messages are selectively utilized, which can result in the synthesis of a new" and perhaps different set of proteins. Simultaneously, preexisting maternal proteins can undergo post-translational modification and degradation. These programmed events result in an oocyte that is ready for fertilization. Fertilization initiates a cascade of events, also dependent on protein modifications and on the timely synthesis of new" proteins from maternal mP,NA stores, that leads to completion of the second meiotic division, remodeling of egg and sperm chromatin, DNA synthesis, entry into the first mitosis, and activation of the embryonic genome. The molecular control of the oocyte-to-embryo transition is underexplored. However, novel strategies are now being employed to identify the genes and their function at the initiation of development. This chapter covers what is known about the crucial molecular events and processes that lead to the activation of the embryonic genome and the beginning of development. We do not delve into these processes in extensive detail. For details, the reader should consult the following excellent and extensive reviews: Johnson (1981), Kidder (I 993), Latham (1999), Nothias et al. (1995), Schultz (1999), Telford et al. (1990), and Thompson et al. (1998). Copyright 9 2002 Academic Press All rights of reproduction in any form reset'red.


!i. Oogenesis The initial stages of oogenesis take place during fetal development in mammals. After entering the genital ridge, the germ cells begin to divide mitotically' and thus create a large supply of oogonia, v~hich then enter into meiosis. A large proportion of these primary oocytes degenerates and only some of them start forming primary follicles. At birth a finite number of primary follicles containing primary oocytes arrested in the prophase of the first meiotic division is available to provide the female with functional gametes for her entire reproductive life. Most of these oocytes also degenerate: only a small proportion ever develops into mature fertilizable ova. The oocytes continue to grow within the follicle. thus gradually creating in their cytoplasm a molecular environment that contains all nutritive and informational elements necessary to start and support the initial stages of development. Cellular organization and structure are largely dictated by function, thus it is to be expected that cells performing the same function in widely distant species are structurally very similar. However. development in different species can vary greatly; and thus it is not surprising that oocytes display a wide variety in size and molecular content. Despite these real and important differences, certain basic principles operate equally in sea urchin, frog. and ostrich oocytes, with diameters ranging from 100/am to a score of centimeters. One basic but trivial principle is that the content of nutrients in each oocyte is commensurate with the length of time the developing embryo needs to be supported without access to an external food source. The size of oocyte in each species is largely determined by this specific demand. Another much more interesting basic principle is that each oocyte contains a certain amount of morphogenetic information. Cytoplasmic regionalization and organization are often obvious by inspection alone (Davidson. 1986). but our understanding of the molecular organization of an oocyte and its role in further development has been dramatically expanding in recent years. It is well beyond the scope of this chapter to detail the tremendous amount of genetic and biochemical information as it relates to the molecular biology of the morphogenetic information in the Drosophila or Xenopus egg, the most intensely studied models. It is clear that correct spatial and temporal control of distribution and utilization of maternal mRNA and protein molecules is the prerequisite for normal embryonic development in these species and in many other nonmammalian species. One example of a mechanism establishing cytoplasmic regionalization involves the regulation of transport and anchorage of specific mRNA molecules to defined positions in the eo,,.......The specific motif in the mRN'A is recognized by a chaperone protein with dual or multiple specificities, which recognize both the target mRNA molecule and part of the cellular cytoarchitecture

where this particular mRNA molecule is to be localized. Another set of controlling mechanisms most probably involves specific and selective mRNA translation-inhibition in all parts of the eo,,~=except where the protein product is spatially and temporally required. For further examples and details, the reader is referred to numerous articles and reviews on the subject (e....,,, Danpure, 1995" Gavis.. 1997; Holliday, 1989 King et al., 1999" Schnapp, 1999; Solter and Knowles. 1999). Do these or similar mechanisms also operate during mammalian oogenesis? That is, can we detect cytoplasmic localizations with morphogenetic consequences in the mammalian egg? One should bear in mind that, although the mouse is the most intensely studied of all eutherian species and a large amount of information has been accumulated on mouse development, the question remains To what extent is early mouse development the universal model for that in all mammals? Nevertheless, to our admittedly limited observational capacities, the fully grown mouse oocyte is radially symmetrical and without obvious morphological differences throughout. After the oocyte matures and progresses through meiosis (see Section III), certain obvious morphological changes take place. However, can there be any elaboration of cytoplasmic diversity during oocyte growth and before maturation in the mouse? If so, does this have any functional significance? In many invertebrate and vertebrate species the initial expansion phase of gametogenesis (before the initiation of meiosis) involves a series of divisions of a single progenitor cell whose progeny remain connected by intercellular bridges, thus forming a syncytium. Such a group of cells is called a germline cyst, and synchronous divisions and an exchange of material between the cells in the cyst are probably essential for normal gametogenesis. A typical example is Drosophila oogenesis, during which 1 of the 16 interconnected cells becomes an oocyte and the remaining 15 are localized on one side of the egg and serve as nurse cells supplying the developing,_ eoo~, with nutrients and localization signals. Pepling and Spradling (1998) observed premeiotic germ cells in clusters in fetal mouse ovaries; these cells were connected by intercellular bridges often containing mitochondria, which would suggest active intercellular transport as observed in the germline cysts of Drosophila. They also observed cell cycle synchronization within the cluster. In addition, the number of cells in the cluster frequently corresponded to powers of 2, again suggesting their origin from a single cell. The existence of several mouse mutants involving genes like Daxl (Yu et al., 1998) or rjs/jdf-2 (Lehman et al., 1998), which result in the formation of follicles containing more than one oocyte, suggests the possible presence and important functional role for the germline cyst. Germline cysts are an essential feature of gametogenesis in vertebrate males and in higher insect females where they can easily be observed, since ga_metogenesis takes place continu-

1 Fertilization and Activation of the Embryonic Genome ously during adult life (Pepling et aL, 1999). If germline cysts are formed during oogenesis in mammals, then it is possible that the primary oocyte was one of several sister cells and the remaining cells would be the equivalent of nurse cells. The presence of germline cysts in the ovaries of mammalian females has not yet been established beyond doubt and their possible function is even less clear.

i11. Meiosis and the Beginning of Oocyte Asymmetry Mammalian oocytes are arrested in the diplotene stage of the first meiotic prophase and they continue growing and accumulating RNA, protein, and other molecules until they reach their full size (----80/xm in diameter in the mouse). Fully grown oocytes [also known as germinal vesicle (GV) oocytes because their nuclear membrane is still intact] are able to reinitiate meiosis and undergo maturation, which makes them capable of being fertilized. Follicle cells normally maintain the oocyte in a state of meiotic arrest as evidenced by the observation that oocytes released from follicular cells in vitro spontaneously initiate maturation and resumption of meiosis (Edwards. 1965). Maturation in vivo is initiated by a surge of luteinizing hormone that binds to receptors on the follicular cells. Follicular cells signal the oocyte through junctional complexes formed between them and oocyte microvilli. The molecular aspects of maturation and control of meiosis are fairly well understood and. though obvious differences exist between different species, certain basic elements are the same. The central component of the entire process is the M-phase or maturation promoting factor (MPF) (Masui and Markert. 1971). which is composed of protein kinase p34 ~J~2and the regulatow protein cyclin B. Germinal vesicle breakdown (GVBD) and reinitiation of meiosis are independent of protein synthesis, indicating that the MPF components are present, but inactive, in the GV oocyte. Inactivation is likely mediated by phosphorylation at tyrosine 15 of p34 ~d~2 through the action of WEE 1 kinase (Mitra and Schultz, 1996). The p34 cat2 is then dephosphorylated, most likely by a mouse homologue of yeast cdc25 kinase, two of which have been described in the mouse oocyte and early embryos (Wickramasinghe et al., 1995). In addition to phosphorylation-dephosphorylation of p34 cat2, its rapid accumulation at the end of oocyte growth probably also contributes to the acquisition of meiotic competence (de Vant~ry et al., 1996). The synthesis and accumulation of the regulatory subunit of MPE cyclin B (de Vant6ry et aL, 1997: Polanski et aL, 1998), represent another controlling factor in oocyte maturation. Because the level of cyclin B mRNA is essentially identical in meiosis-incompetent and meiosis-competent oocytes (de Vant~ry et al., 1997), the amount of cyclin B is determined by c o n -

trolled translation. Translational control of cyclin B synthesis (Tay et al., 2000) involves cytoplasmic polyadenylation of dormant mRNA. This control mechanism is of great significance during the period of transcriptional silence between the cessation of oocyte growth and the activation of the embryonic genome (see Section VI). Several other molecules such as MOS (Gebauer et al., 1994) involved in progression through meiosis are similarly regulated. In mice, MOS is necessary not for GVBD and initiation of meiosis as it is in X e n o p u s (Sagata et al., 1988), but for progression from meiosis I to meiosis II. MOS kinase is an essential part of the cytostatic factor (CSF), which is necessary to stabilize the high level of MPF activity that leads to arrest in metaphase II representing the end of oocyte maturation. In M O S -/mice, the oocytes complete meiosis I but instead of going into meiosis II. the nuclei are reformed and the oocytes can undergo parthenogenetic activation (Colledge et aL, 1994: Hashimoto et al., 1994). The cytoplasmic polyadenylation and translation of c-mos mRNA in X e n o p u s are regulated through binding of its cytoplasmic polyadenylation element (CPE) by the CPE binding factor (CPEB); phosphorylation of CPEB by Eg2 kinase is essential for it to bind to the CPE of MOS in the X e n o p u s oocyte (Mendez et aI., 2000). Two kinases, which share high amino acid homology with Eg2, have recently been described in mouse (AIE1) and human (AIE2) (Tseng et al .... 1998). AIE1- related kinases (STK-1 and IAK1/Aykl) were identified in mouse oocytes (Tseng et aL, 1998). In addition, expression sequence tags (ESTs) corresponding to AIE1 and AIE2 were found among the 15,000 ESTs in the KnowlesSolter two-cell stage library (B. B. Knowles and D. Solter, unpublished results). These data suggest that M O S may also be regulated during mouse oocyte maturation by a complex sequence of phosphorylation reactions that regulates translational control of its mRNA. Although we have identified some of the molecular events that occur during mouse oocyte maturation and regulate progression through meiosis. many more questions remain. The radial symmetry of the GV fully grown oocyte is lost during oocyte maturation. A detailed discussion about the possible functional consequences of this loss is provided in Chapter 2. It has recently been shown that even GV oocytes display a nonsymmetrical distribution of leptin and STAT3, both members of the transcriptional activator cascade (Antczak and Van Blerkom, 1997). Both leptin and STAT3 were localized within a subpopulation of follicular cells and a corresponding portion of the oocyte, again suggesting differences within the follicular cell population and the possible existence of nurse cells and germline cysts in mouse oogenesis(see Section II). On completion of oocyte maturation, the location of the first polar body denotes the animal pole and also marks part of the e~,o,, surface lar~ely.~ devoid of microvilli (Evans et aI., 2000: Van Blerkom and Motta, 1979). It has been demonstrated that cortical endoplasmic reticulum

i Establishment of Body Patterns accumulates in every part of the cortex except in the microvilli-free area around the metaphase spindle, that is. the area tu which the second polar body is released (Kline et al., 1999). In mice. sperm entry is restricted to the e_..~ surface containing microvilli, although the functional significance of these domains is open to question since human eggs lack completely a nonmicrovillar region (De Smedt et al.. 2000). Another possible example of asymmetry in the eggs of some mammalian species involves the movement of the so-called Balbiani body, which in X e n o p u s localizes to the vegetal pole and likely participates in the formation of germinal plasma (Kloc and Etkin. I995). It is possible that a similar structure also localizes to the vegetal pole of some mammalian oocytes (De Smedt et aI.. 2000). Currently, there is little evidence for the existence of functionally relevant cytoplasmic localization in the mammalian egg although the question may be clarified as more candidate genes expressed during oogenesis become known and available for investigation.

IV. Fertilization Only a mature egg arrested in metaphase of the second meiotic division is capable of being fertilized. Fertilization initiates numerous complex changes in all compartments of the resulting conceptus (Snell and White. 1996: Wassarman. 1999) involving all components of the egg as well as the sperm-derived chromatin (Wright. 1999). Although mammalian eggs can be activated by many divergent stimuli. natural fertilization cannot be reproduced in full by artificial activation (Fissore et al., 1999). This certainly contributes to the poor success rate of cloning by nuclear transfer (Solter. 2000). For example, incorporation of the sperm membrane into the oolemma results in an e.._~ membrane block to polyspermy, whereas oocytes activated by intracytoplasmic sperm injection (ICSI) and parthenogenetically activated eo,,s can be penetrated by additional sperm (Maleszewski et al., 1996). Following fertilization the arrested meiosis resumes, the second polar body is soon extruded, and the female pronucleus is formed to join the already formed male pronucleus. This process is controlled by the inactivation of MPF, which has been stabilized in oocytes by CSF composed of MOS and other MAP (mitogen-activated protein) kinases. Fertilization results in a rapid decrease in MPF activity followed by a slower decrease in MAP kinase activity (Moos et al., 1995). The role of inactivation of these kinases in completing meiosis and initiating interphase is further demonstrated by the ability of protein kinase inhibitors to initiate the same events (Sun et al.. 1998). Changing the metaphase spindle into an anaphase configuration requires the presence of calcium/calmodulin-dependent protein kinase II (CaM kinase II), which is associated with the metaphase spindle: following fertilization calmodulin is veQ quickly colocalized, presumably leading to the activation of CaM kinase II (Johnson

et aI., 1998). Another molecule associated with the meiotic spindle is spindlin (Oh et aI., 1997). which is also phos-

phorylated, depending on the cell cycle stage, and whose phosphorylation is at least in part mediated by the M O S MAP kinase pathway (Oh et al., 1998). In M O S -'~- mice, spindlin is hypophosphorylated and. although abundantly present in the cytoplasm, it does not bind to the spindle (Oh et al.. 1998). As mentioned before, the eggs of M O S - / - females undergo spontaneous activation, and it is possible that the lack of spindlin phosphory,lation is one of the elements that destabilizes metaphase II arrest. Fertilization triggers a series of signaling events accompanied by a series of Ca-'- transients (waves of increased Ca-" concentration passing through the egg cytoplasm). Subsequently, the cytoplasm and the nuclear compartments of the egg are remodeled suggesting that Ca z- transients may play a crucial role in the initiation of transcription. Calcium transients following fertilization may be mediated by the soluble sperm protein oscillin (Parrington et aI., 1996), though its mechanism of action is not clear. The presence of this protein may explain oocyte activation following ICSI. and it may be possible to use this soluble protein to "normalize" egg activation in nuclear transfer experiments. One analysis of Ca z- transients demonstrated that they initially originate at the point of sperm penetration, perhaps an immediate effect of oscillin, and their later origin from a site opposite the second polar body (Deguchi e t al., 2000). Ca:- waves continue until pronuclear formation and possibly. at a low frequency, until the formation of two-cell embryos. Ca2- transients could have many important and long-range consequences. It was shown that the number of cells in the inner cell mass was higher in embryos parthenogenetically activated by Sr- (Ca:- transients present) as compared with those activated by ethanol (Ca 2" transients absent) (Bos-Mikich et aI., 1997). Ca:- oscillations reduce the effective Ca :~ threshold necessary for the activation of transcription factors (Dolmetsch et al., 1998). and it was also suggested that the frequency of oscillation may determine which factors are activated (Dolmetsch et al., 1998). If one extrapolates these results to the fertilized e,,o one can envision all kinds of regulatory events that might proceed in waves directionally through the cytoplasm. Temporal changes in Ca 2- transients (Tang et al., 2000), and a positional change in their origin could provide both sequential and spatial information.

V. Transcription and Its Control The period of intensive transcriptional activity during mouse oogenesis ceases when the egg is arrested at the GVintact, full-grown oocyte stage and only gradually resumes after fertilization. Until the emb~onic genome is activated, the processes of e,,o==maturation, completion of meiosis and

1 Fertilization and Activation of the Embryonic Genome initial postt%rtilization events are controlled by maternal molecules (proteins and RNAs) accumulated during oogenesis. These maternal molecules are also responsible tbr proper embryonic genome activation. This period of development has been extensively studied and certain basic mechanisms are being elucidated (e.g.. Latham. 1999: Nothias et al.. 1995: G. A. Schultz, 1986: R. M. Schultz. 1999; Telford et al.. 1990: Thompson et at., 1998). In perusing the literature on transcription control in early mouse embryos the reader should be aware of a possible source of confusion. Certain authors (Stein and Schultz. 20,00: Wiekowski et al., 1997) describe two-cell stage chromatin as being in a transcriptionally, repressive state because at that stage transcription is *br the first time dependent on the presence of enhancers. Others (Forlani et aI., 1998) talk about a transcriptionally repressive state when describing the genome of the early one-cell stage, that is, at a time when there is no observable transcription. This duplication of usage (though both can be justified) is unfortunate and should be resolved, or one could end up saying that DNA synthesis in the zygote leads to the establishment (first definition) or abolishment (second definition) of the transcriptionally repressive state. It is not entirely clear how transcription is suspended in the full-grown and maturing oocyte and immediately after fertilization, but the absence and/or inhibition of the functional basic transcription machinery may be responsible. Transcription was not observed when transcriptionally competent nuclei were transferred into the cytoplasm of the early zygote, but was detected following transfer into the cytoplasm of the late zygote (Latham et al., 1992). The largest subunit of RNA polymerase II is hyperphosphorylated in the oocyte but becomes dephosphorylated following fertilization (Bellier et aI., 1997). The hyperphosphorylated form is thought to be unable to initiate transcription, explaining at least in part the transcriptional silence of the full-grown oocyte. It is unclear whether its dephosphowlation, which takes place several hours after fertilization, is responsible for the short burst of transcription in the late zygote. A gTadual reestablishment of the "somatic"'-type phosphowlation pattern of RNA polymerase II, and its translocation to the nucleus, takes place in the two-cell stage embwo concomitant with complete embryonic genome activation (Bellier et al., 1997: Worrad et al., 1995). The mechanism that blocks transcription immediately after fertilization and throughout the first cell cycle is multifactorial and complex. In addition to the changes in RNA polymerase II discussed above, the chromatin status of both the male and female pronucIeus is initially incompatible with transcription. The transcription that occurs in the late zygote is more prominent in the male than in the female pronucleus. This disparate rate of transcription has been observed for endogenous genes (Aoki et at.. 1997) and also for injected plasmid constructs (Wiekowski et at., 1993). Differential active demethylation of the paternal genome, while the mater-

nal genome remains methylated (Mayer et al., 2000: Oswald et al., 2000), may be one of the factors that aft%cts this differential transcription rate. but this would affect only endogenous genes. Transcription of both endogenous and injected constructs is probably also affected by the structure of chromatin and the changing rates of histone synthesis. Hvperacetvlated histone H4 is preferentially associated with the male pronucleus in the early zygote, and only at the end of the first cell cycle is this tbrm of H4 associated with all chromosomes (Adenot et aI., 1997). Histones are obviously translated from maternal mRNA: however, while H3 and H4 are continuously synthesized, synthesis of HI, H2A, and H2B starts only in the late one-cell stage (Wiekowski et al., 1997). Acetylated histone H4 (and also RNA polymerase II) becomes localized to the nuclear periphery in the two-cell stage embryo, and this restricted localization is no longer visible in four-cell or later embryos (Worrad et al., 1995). It is likely that the availability of acetylated histones and their specific localization within the nucleus controls to some extent the nature of genome transcription in the two-cell-stage embryo. Other structural components of chromatin that may be relevant for the control of transcription are high-mobilitygroup (HMG) proteins. HMG1 (Spada et al., 1998) and HMG-I/Y (Beaujean et al., 2000; Thompson et al., 1995) have been observed in early mouse embryos. H M G - I / Y translocates to the pronuclei and its accumulation therein is associated with embryonic genome activation. Microinjection of antibody to HMG-I/Y delays the onset of transcription while injection of purified HMG-I/Y protein advances it (Beaujean et al., 2000). In addition, injection of HMGI/Y also modifies the structure of chromatin as demonstrated by increased DNaseI sensitivity (Beaujean et al., 2000). In addition to the presence or absence of the basic transcriptional apparatus and chromatin state, other general and specific transcription factors play a role in activation of the emb~'onic genome, mRNAs for Spl and TBP (TATA box binding protein) are present during oocyte maturation and their amount decreases especially in the two-cell stage. A steady increase in abundance follows due to the increase in transcription from the embryonic genome (Worrad and Schultz. 1997). Considering global transcription factors, it is interesting to note that, at least for one gene, the utilization of the TATA-Iess promoter in preference to the TATA-containing promoter has been observed at the time of genome activation (Davis and Schultz, 2000). This may indicate that the overall transcriptional control at the beginning of genome activation could be subtly different from that usually observed in somatic or later embryonic cells, mTEAD-2 mRNA is present in the oocyte, decreases significantly in abundance at the two-cell stage, and then gradually increases (Kaneko and DePamphilis, 1998: Kaneko et al., 1997). However, the activity of mTEAD was detected only in two-cell-stage embryos at the time of embryonic genome

i Establishment of Body Patterns

10 activation (Kaneko et al.. 1997). The most likely explanation for this apparent discrepancy is that mTEAD maternal mRNA is stored in a dormant state and is activated for translation by polysomal recruitment at the two-cell stage (Wang and Latham. 2000). Recruitment to polysomes, resulting in translation initiation, is one of several post-transcriptional controlling mechanisms significant at this time in development (see Section VI). The uncoupling of transcription and translation of endogenous genes at the beginning of embryonic genome activation is an obvious feature of stored maternal mRNA utilization, but it has also been observed with genes transcribed for the first time only after fertilization. It is at present unclear whether endogenous genes, transcribed at the beginning of embryonic genome activation, are immediately translated. This postfertilization uncoupling phenomenon was described only when transgenes (Matsumoto et al.. 1994) or injected constructs (Nothias et al.. 1996) were analyzed. Analysis of other transcription factors present in the embryo at the time of transition from maternal to zygotic control is just beginning, and as yet only a small number have been described (Bevilacqua et aI., 2000: Parrott and Gay. 1998). However. EST analysis of the available cDNA libraries derived from oocyte and earl}: embyos (see Section VII) should proceed rapidly, enabling identification of more transcription factors and the subsequent determination of their targets and functional roles (Bevilacqua et al.. 2000). Another group of transcription factors, which could play a role in controlling gene expression in the perifertilization period. is represented by the gene M a i d (Hwang er al.. 1997). Basic helix-loop-helix (bHLH) factors are present in the oocyte and during early development (Domashenko er al.. 1997). and their main function is to activate the genes involved in various differentiation programs. Premature activation of such genes might be detrimental at specific developmental stages. It is possible that Maid. which belongs to the Id family of proteins (Hwang er al., 1997). interacts with bHLH factors during oogenesis and in early preimplantation embryos, preventing the premature activation of differentiation-related genes (Norton et aI., 1998). As is the case with several other members of the Id gene family (Afouda et al., 1999), M a i d translation is translationally controlled (Hwang et al., 1997).

VI. mRNA Utilization during Oocyte Maturation and Preirnplantation Development The proper utilization of maternal mRNAs is the key to molecular control of early development. In the development of any multicellular organism, there exists a period of transcriptional silence between the time when the oocyte

achieves full growth and the activation of the embryonic genome. This period can either be short, in the range of hours as in the sea urchin, fruit fly. or frog, or several days as is the case in mammals. During this period of transcriptional silence all processes in the egg and embryo are carried out by the molecules stored during oocyte growth. The sequestration of maternal mRNA molecules to within the ooptasm and their activation for translation in precise spatial and temporal sequences suggest their central rote in controlling early development (Wickens et al.. 19961). The role of cytoplasmic polyadenylation in controlling the translation of mammalian maternal mRNAs has been extensively documented using tissue plasminogen activator (tPA) as a model (Strickland er al.. 1988: Vassalli et al., 1989). In the full-grown oocyte deadenylated tPA mRNA is abundant, stable, and not translated. During oocyte maturation tPA mRNA is polyadenylated, translated, and degraded so that there is no detectable tPA mRNA in the zygote. A short motif in the 3' untranslated region (3'UTR), the CPE, first identified in the frog (for review, see Richter, 1999) and then in the mouse (Verrotti et al.. 1996), was shown to be essential for these changes (Strickland et al., 1988; Vassalli et al.. 1989). tPA is only one of many genes whose mRNA is deposited in the growing oocyte and whose translation depends on processes regulating cytoplasmic polyadenylation (Oh er al.. 2000). We are just beginning to realize that translational control based on 3'UTR sequences is complex and could be responsible for the precise timing of maternal mRNA translation. CPEB is a protein that binds to CPE in the X e n o p u s oocyte and is essential for polyadenylation. A similar mouse protein, mCPEB, has been identified (Gebauer and Richter. 1996). In Xenopus, CPEB binds Maskin, a protein that could interact with the 5' translation initiation complex, specifically with elF-4E, thus preventing initiation of translation (Stebbins-Boaz et al., 1999). Thus CPEs, and possibly other motifs in the 3'UTR. could have a dual role: first by ensuring the dormancy of maternal mRNA in the full-grown oocyte and. subsequently, by promoting polyadenylation and translation (Simon and Richter, 1994). The presence of a silencing factor was implicated in the repression followed by translation of cyclin B 1 and tPA in X e n o p u s and mouse oocytes, respectively (Barkoff et al., 2000: Stutz et al., 1998). However. derepression and translation of tPA required only the displacement of the putative silencing factor but not the extension of the short polyA tail. We investigated in detail the timing of polyadenylation of mRNAs of spindlin, a gene abundantly expressed in the mouse oocyte and zygote (Oh er aI.. 1997. 1998). Transcription of Spin results in mRNAs of three different sizes, each with the same open reading frame but differing in the length of the 3'UTR. Two of these messages undergo differential cytoplasmic polyadenylation and translation during oocyte maturation and postfertilization (Oh et al.. 2000). The longest Spin message is polyadenylated, but not translated in the full-grown

1 Fertilization and Activation of the Embryonic Genome

11

VII. G e n e Expression in the Early Mouse E m b r y o

oocyte. Following initiation of maturation the message is deadenylated and still not translated, However, after fertilization the message is polyadenylated and translated. It has been suggested that ongoing polyadenylation and not a static polyA tail can displace the factors that prevent translation, although the exact mechanism is not fully understood (Stebbins-Boaz et al., 1999). It is thus possible that only deadenylation followed by readenylation can ensure the eventual translation of a long Spin message. CPEB-mediated control of translation is crucially important in regulating gene expression during oocyte-to-embryo transition but this mechanism also functions in adult tissues (Wu et aI., 1998). Substantial scope for control is provided by the understanding that translation depends on assembly of multicomponent complexes at the 3' and 5' ends of the mRNA molecule. which then leads to RNA unwinding, recruitment to polysomes, and initiation of translation (Coller et aI., 1998: Craig et aI., 1998: Davis et al., 1996: Deo et aI., I999: Gao et aI., 2000: Jacobsen et al.. 1999: Laroia et at., 1999: Paynton, 1998). Novel 3' and 5' sequence-specific RNA binding proteins can be identified in mammalian oocytes and early embryos. Considering the tremendous importance of these mechanisms in controlling the early development of Drosophila and Xenopus, it would be surprising if similar molecules and mechanisms were not identified in mammals.

Analysis of gene expression and gene discovery using early mammalian embryos was, until recently, seriously limited by the scarcity of experimental material. Analysis of protein biosynthesis in oocytes, zygotes, and cleavage-stage mouse embryos by computerized 2-D gel methods suggested that gene expression patterns change substantially during the oocyte-to-embryo transition (Latham et al., 1991). The improvement of molecular biology techniques eventually led to the development of cDNA libraries representing nearly all stages of preimplantation mouse development. Several sets of such libraries are available today (Table I) (Ko et al., 2000: Rothstein et al., 1992, 1993" Sasaki et al., 1998). This material has enabled us to initiate the search for novel genes expressed at this time in development and to attempt to gain some global insight into the control of gene expression. Three major sets of cDNA libraries representing preimplantation development have been described and a number of ESTs from these libraries have been sequenced (Table I). These sequences are available through several Internet resources (see, for example, http://www.ncbi.nim.nih.gov/ dbEST/index.html: http://www.ncbi.nim.nih.gov/UniGene /

Table I Preirnplantation Embryo Libraries with Sequenced ESTs

Library name Knowles-Solter Knowles-Solter Knowles-Solter Knowles-Solter Mouse Mouse Mouse Mouse Mouse Mouse Mouse

unfertilized e,,o mouse 2 cell mouse blastocyst B 1 mouse blastocyst B3

unfertilized egg cDNA fertilized l-cell-emb~'o cDNA 2-cell-stage embryo cDNA 4-cell-embryo cDNA 8-cell-stage e m b ~ o cDNA 16-cell-emb~'o cDNA 3.5-dpc blastocyst cDNA

Unigene identification number

DbEST identification number

Total number EST

Reference

89 88 85 94

867 862 850 875

403 14813 12955 2499

a a b b

I51 t06 t49 175 150 176 102

t389 1119 I382 I524 I381 I532 1021

3096 3314 3687 3011 3'443 3196 5692

c c c c c c c

Mouse early blastocvst

134

1310

1161

d

RIKEN full-length enriched, in vitro fertilized eggs RIKEN full-length enriched. 2-ceil eggs

319 414

2589 Notavailable

7664 6315

e e

"Rothstein et al. (1992) "S.-Y. Hwang. D. Solter and B. B. Knowles (unpublished results). These blastocyst libraries were made from the mRNA from 800 blastocysts using a similar approach as before (Rothstein et at.. 1992). Briefly. first-strand cDNA synthesis was primed using SalI-dT ( 5 ' - C G G T C G A C C G T C G A C c G T T T T T T T T T T T T T T T - 3 ' ) primer, dscDNAs were sized: insert > 1500 bp and inserts 1500-1000 bp were used to make B1 and B3 libraries, respectively, cDNAs were cloned into NotI/SalI sites of pSPORT vector. ~Ko et al. (2000) o'Sasaki et al. (1998) Relatively little information is provided concerning the construction and analysis of these libraries. For the available information consult http:/!www.ncbi.nim.nih.gov/UniGene/Ibrowse.cgi?ORG= M m

12


Mm.Home.html: and h t t p : / / w w ~ v . l g s u n . g r c . n i a . n i h . g o v ) , thus enabling clustering and library comparison. Onlv a few attempts have been made to explore this material in depth and even these attempts were limited either to only one library (Sasaki et al.. 1998)orto libraries (Ko et al.. 2000)from one laboratory. The confidence level of these analyses is obviously influenced by the reliability and quality of the respective libraries and. because the amount of material used was minimal, all preimplantation embryo libraries probably suffer from a variety of inherent deficiencies. For example, an abundant EST cluster is derived from a bacterial contaminant of the E s c h e richia coli tRNA carrier used in the two-cell-stage library from our laboratory (Rothstein et al., 1992). Two of the three most highly represented EST clusters (3.1 and 1.8% of all ESTs in a 3.5-dpc blastocyst library) correspond to adult hemoglobin chains (Ko et al.. 2000). which must be due to blood contamination of the original RNA sample. There is no hemoglobin in another, much larger, blastocyst library (Rothstein et al., 1992). This type of contamination is of serious concern because any other cDNAs from mouse erythrocytes or other somatic cells cannot be distinguished from those of the blastocyst. The oocyte and preimplantation libraries produced in our laboratory were made by standard cloning procedures, the RNA was DNAse treated and the RNA was not PCR amplified (Rothstein et al., 1992). The other published libraries (Ko et al.. 2000 Sasaki et al.. 1998) and presumably those produced by RIKEN (http://www.ncbi. nim.nih.gov/UniGene/lbrouse.cgi?org=Mm) were made without pronase treatment to remove the zona pellucida (ZP). Removal of the ZP eliminates any possible contamination by adherent somatic cells and also eliminates fragmented oocytes that could otherwise be included in samples of cleavage-stage embryos. Likewise. the libraries described by Ko et al. (2000). Sasaki et al. (1998). and the RIKEN set were not subjected to DNAse treatment and PCR was used to ampli~, dsDNA. As a consequence, several clusters containing a large number of ESTs are apparently derived from genomic DNA representing repetitive elements. In addition, the number of ESTs in the clusters is not always a true indication of mRNA abundance, as several abundant clusters contain ESTs, which are all obviously derived from a single amplicon, that is. all sequences have exactly the same 5' start site. Considering the difficulties involved in making these libraries, it is not surprising that some artifacts are present. This does not diminish the value of the information we can derive from them but the results from in silico analysis must be rigorously checked using freshly isolated embryos. Despite these caveats, analysis of the libraries provides some initial information about gene expression in preimplantation embryos. A large number of abundantly expressed genes, especially in oocyte and two-cell-stage emb~os, are unknown (Ko et al., 2000). A significant proportion of highly abundant mRNAs at these stages contain CPEs and the translation 4

of these CPE-containing mRNAs appears to be regulated (Oh et al., 2000). The power and possible pitfalls of an in silico mining approach for characterizing genes that are important for preimplantation development are exemplified below. We have clustered all the ESTs from our two-cell cDNA library and performed BLAST searches for 500 clusters. Each cluster was tentatively identified to represent a known mouse gene, the mouse homologue of a known gene from another species, or a novel gene. More than half of these were completely unknown or they were previously named genes of unknown function. The most abundant 26 EST clusters from this library were used to construct a "virtual Northern blot" by on-line (http://www.algenes.org/) library analysis (Table II). Four of them. OM2, Pc3B, and two unknowns comprised of 39 and 23 ESTs. were only found in the oocyte and cleavagestage libraries, suggesting they are products of genes of limited tissue expression. Eight of these gene clusters were not found in the available oocyte and zygote libraries. It may be that these represent the first transcripts from the embryonic genome but more likely it is a reflection of the small library size, the small insert size. and the nonrepresentative nature of the available oocyte and zygote libraries (Table II). Eighteen of these 26 EST clusters (70%) contain CPEs, suggesting that their translation is controlled during the oocyte-toembryo transition. To refine the picture of the genes expressed at this time in development additional techniques must also be employed. For example, quantitative amplification and dot blotting have been employed to measure the abundance of transcripts of specific genes throughout early development (Rhambhatla et al., 1995). cDNA arrays may also be employed because it is now possible to produce informative probes from a very limited amount of material ( 10-100 ng of total RNA) (Luo et al., 1999). Another alternative is to prepare better cDNA libraries. Sequenced ESTs from libraries of growing and full-grown oocytes are needed to represent the pattern of gene expression at the beginning of the period of transcriptional silence. Existing libraries can be used not only for the global analysis of gene expression and for gene discovery in silico (Hwang et al., 1999. 2001), but for more conventional approaches of identi~;ing novel genes. Subtraction hybridizations have been used to identi~ several previously unknown genes (Temeles et al., 1994), which have not been characterized further, and to identify' Spin (Oh et al., 1997)and M a i d (Hwang et al., 1997). Another tool for gene discovery, differential display, has been used on cDNA libraries from embryos or on embryo-isolated RNA material (Zimmermann and Schultz. 1994). The translation initiation factor eIF-4C that is transiently expressed in two-cell-stage embryos and could have a significant role in the activation of the embryonic genome was demonstrated by this technique (Davis et al., 1996) as was Melk. a novel member of the Snfl/ AMPK kinase family (Heyer e t al., 1997. 1999).

13

1 Fertilization and Activation of the Embryonic G e n o m e Table !1 EST Assemblies That Contain at Least 20 Sequences from Knowles, Solter Two-Cell Embryo cDNA Library" GenBank

Number Cluster designation

of E S T s

113

OM2b

45

Unknown Unknown Spin Unknown

40

M - p h a s e phosphoprotein. M P P 6

39

Unknown

36 34

Siah-2

48 47 45

accession number $81935

Presence of C P E within 120 nt upstream of poly(A) signal ~' Oc~cyte

Similar ESTs found in other libraries Zygote

Morula

Blastocyst

Other

Yes

+

No No

* -

+

--

4-

+

U48972

Yes

+

§

4-

4-

4-

X98263

"yes "yes

+ 4-

§

--

_

§

4-

§

--

Yes

+

.+_

_

__

§

§

--

_

--

+

4-

ZI9581

Pc3b

A J0()5120

Yes

+

§

I3 protein

AF106967

Yes

*

--

31

Ubiquitin-conjugating e n z y m e , ubc4

U62483

No

+

28

EI24

U41751

27

Unknown Nuclear export factor (exportin t) Ornithine decarboxvlase Unknown

No Yes

+

-r-

+

--

4-

Y08614

Yes

-

_

_

§

§

M 10624

Yes

-

--

§

+

§

Yes

+

+

+

--

§

_

+

§

+

+-

4-

--

§

4-

26 25 25 24

Hmg4

AF022465

Yes

-

_

23

CD63

23

Unknown Unknown

Yes Yes Yes

4+

--

23

D16432 AB045323

23

Cdr2

22

Ingl Bmi-1 Unknown Tcllbl DBF4-related protein

U88588 AF177757

Yes No

-

M64067

No Yes

+ +

§

--

4-

+

+

+

--

§

AF195488

No

+

§

--

_

§

AJ003132

Yes

+

+

--

+

+

21 2O

"Mitochondrial, viral, and contaminant clusters were excluded, *'For derails, see Oh er al. (2000).

"Combined 8- and 16-cell librarx~ data.

Analysis of EST libraries demonstrated that a large proportion of abundantly expressed genes, especially in twocell-stage embryos, is un'l~nown and of unknown function. The characterization and especially the establishment of the functional role of these genes will represent a major effort in the future. In addition to this gene discovery effort, preimplantation embryos are being analyzed for the expression of known genes in order to expand our understanding of their possibly multiple functions. A few examples of such work include the analysis of G proteins (Rambhatla et al., 1995" Williams et al., 1996). the establishment of the role of protein kinase C in preimplantation development (Gallicano et al., 1997; Pauken and Capco, 2000), and the examination of the expression of the genes involved in apoptotic pathways (Jurisicova et al., 1998). An excellent site to find all published information about the genes expressed during mouse preimplantation development is maintained by The Jackson Laboratory (http : l / w w w . i n f o r m a t i c s . j ax. org/menus l expression_menus.html).

These few, briefly discussed examples indicate that mammalian preimplantation embryos can be successfully analyzed using all the standard tools of molecular biology and. moreover, that such tools must be applied even when the scarcity of material makes such an application difficult (Oh et al., 1999). It is certainly true that most molecular processes can be analyzed and explained using more accessible models, but the complete understanding of mammalian development cannot be accomplished without the molecular analysis of embryos and the understanding of the function of the genes expressed in embryos.

VIII.

Functional Analysis

The loss of the ability of the zygote cytoplasm to reprogram somatic nuclei introduced by nuclear transfer dramatically illustrates its functional distinction from the ooplasm. However, the molecular changes that accompany this func-

14


tional differentiation are totally unknown. The usual methods used to find mutant genes that function at this time of development, for example, that affect fertility, are impractical. Simple knock-out studies are complicated: namely, if the gene in question is not uniquely' expressed in the oocyte and zygote, null mutants may die as embryos, or the females may,' fail to reach sexual maturity. This is a problem for the mutation. which must be studied in ovulated e....s~g and embryos. . However. there are now methods to engineer the loss or gain of expression of specific genes, and to follow the effect of mutation on timely progression through meiosis/mitosis into preimplantation embryogenesis.

A. In Vitro Approaches These approaches are primarily aimed at inhibiting the translation of mRNAs present in the oocyte, or those that are transcribed following activation of the embryonic genome. Antisense oligonucleotides or antisense RNA directed against coding or 3'UTR sequences have been used to eliminate the expression of a number of genes (Ao et al.. 1991 O'Keefe et al., 1989 Strickland et al., 1988: Tay et al., 2000). However. the results obtained from these experiments vary widely in both the level of elimination of gene expression as well as the confidence level of the results. Recently the use of morpholino-oligonucleotides seems to have produced more reliable results (Summerton and Weller. 1997). Morpholino-oligonucleotides seem to act by preventing translation rather than causing RNase H-mediated mRNA cleavage and they must be targeted to a narrow area around the translation start site. Morpholino-oligonucleotides have been used successfully in the analysis of early events during X e n o p u s development (Heasman et al., 2000), but their utility in the analysis of early mammalian development awaits experimental confirmation. Double-stranded RNA (dsRNA). can produce potent, specific, post-transcriptional gene silencing (Birchler et al.. 2000 Bosher and Labouesse. 2000: Fire. 1999) and this method is known as RNA-mediated interference (RNAi). Microinjection of dsRNA has been used to determine specific gene function in disparate animal species, such as nemat o d e s m C a e n o r h a b d i t i s elegans (Fire et al., 1998), D r o s o p h i l a (Kennerdell and Carthew, 1998), and zebrafish (Li et al., 2000). The mechanism is incompletely understood, but the intracellular introduction of double-stranded sense/antisense RNA corresponding to a single gene results in degradation of its homologous RNA. thereby silencing expression of the cognate gene (Montgomery et al., 1998 Plasterk and Ketting. 2000; Tuschl et al., 1999; Zamore et al., 2000). The effects of dsRNA appear to be nonstoichiometric in invertebrates, occurring after injection of only a few molecules. The effect seems to be passed from cell to cell and may persist through several mitoses, suggesting replication of some RNAi effector molecules (Birchler et al., 2000 Fire, 1999). Microinjection

of dsRNA was recently documented to downregulate specific gene expression in the mouse oocyte and preimplantation embuo. demonstrating in principle that RNAi may be a useful tool to investigate vertebrate oocyte and early zygotic gene product function (Svoboda et al., 2000: Wianny and Zernicka-Goetz. 2000). However, the use of dsRNA injection in studying development has not been tested extensively in vertebrates, and several practical problems remain to be addressed. It is not yet clear whether RNAi is as specific in vertebrates as it appears to be in invertebrates. Some investigators have noted indiscriminate downregulation of gene expression after microinjection of dsRNA in zebrafish embryos (Oates et al., 2000) and mammalian cell lines (Caplen et al., 2000). The RNAi in mouse oocytes appears to require injection of a greater quantity of dsRNA than in invertebrate cells and it may be involved in a stoichiometric process. Microinjection of proteins is another means of studying specific gene product function, by augmenting the protein content of the cell. by replacing a missing protein, or by inhibiting its activity with inactivating antibody. For example, the function of MPF has been studied by microinjection of purified MPF in mouse oocytes (Nakano and Kubo, 20-00): and microinjection of antibodies directed against specific histones has been used to study the relationship between transcriptional control and chromatin structure in mouse oocytes and e m b ~ o s (Adenot et al.. 2000: Beaujean et al., 2000). These types of approaches are suitable for diverse biochemical investigations, despite the demanding technical nature of microinjection-based methods, and the difficulties associated with collection of sufficient material to enable adequate interpretation of the results. However. methods targeting genes with maternal stores of both mRNA and protein may not effectively eliminate gene function because of persistence of the protein product. The many in vitro manipulations (injections of oocytes, in vitro fertilization, growth in culture to blastocyst, transfer to the uterus) and the time of exposure of the oocyte and embwo to culture conditions can by themselves affect normal development. This makes attribution of the effect of any perturbation of development, other than blatant lethality, difficult. Furthermore, the experimental manipulations must be repeated each time a new permutation is investigated.

B. In IAvo Approaches The ideal way of initiating a study to find the function of a gene is to eliminate it. or to inhibit its function in a heritable fashion, and then study the phenotype in the intact organism. A tissue-specific gene deletion paradigm is possible in the oocyte, and in the embryo before transcription initiates. Expression of the zona pellucida 3 gene (Zp3), which encodes the egg glycoprotein to which the sperm binds (Bleil et al., 1988), begins early in oogenesis and is oocyte restricted (Epifano et al., 1995). Z p 3 mRNA is synthesized as

1 Fertilization and Activation of the Embryonic Genome oocytes enter the growth phase about day 8 after birth, reaching a peak at days 1 0 - 1 4 after birth. By the time oocytes are fully grown and transcription has ceased, Zp3 message is at background levels. Study of transgenic mice carrying the Z p 3 promoter driving expression of antisense RNA directed against maternal transcripts of tPA demonstrated oocytespecific transgene expression, although the maternal message was not completely eliminated (Richards et al., 1993). A further elaboration of tissue-specific gene targeting is provided by the recently developed Cre-loxP approach. The Cre recombinase of the P 1 bacteriophage mediates site-specific recombination between loxP sites, the 34-bp sequence at which recombination takes place (Sternberg and Hamilton, 1981). The Cre-IoxP recognition system was shown to function in euka,wotes in vitro (Sauer and Henderson, 1989) and in vivo (Medberry et al.. 1995), where it is now routinely used for tissue-specific gene targeting. Mice with a homozygous floxed allele (a gene flanked by loxP sites), prepared by standard methods in ES cells, can be crossed to a transgenic line expressing the Cre recombinase under the control of any tissue-specific promoter and the gene will be deleted in Cre-expressing but not in Cre-negative cells (Gu et al., 1994). A Z p 3 - C r e transgenic FVB (FVB mouse strain) lineage has been reported (Lewandoski et al., !997) and we have made several C 5 7 B L / 6 J Z p 3 - C r e transgenic lineages. These Z p 3 - C r e transgenic mice can be used to specifically eliminate a target gene only in the oocyte, thus providing the means to study the role of the maternal m R N A of a specific gene (de Vries et al., 2000). This experimental approach enables us for the first time to explore the oocyte-specific function of otherwise lethal mutations. An alternative approach is to prevent the normal function of the gene coding for maternal m R N A without an actual mutation. As discussed before, it would be ideal if this could be accomplished by transgenic means so that the experimental material does not depend on repeated injections of interfeting molecules with concomitant experimental variations. One possible way to accomplish this is to adopt PCNAi methodology to the transgenic situation. RNAi preventing expression of transgenes and their homologous endogenous genes has been observed in transgenic n o n m a m m a l i a n model organisms (Bosher and Labouesse, 2000), and silencing of a transfected gene and its endogenous homologue by a mechanism closely resembling RNAi has been observed in rodent cells (Bahramian and Zarbl, 1999). These observations raise the notion that RNAi might be amenable to deliberate induction by transgenic m e t h o d s indeed, heritable, inducible specific downregulation of gene expression has recently been documented in several invertebrates carrying a transgene expressing dsRNA (Bastin et aI., 2000" Kennerdell and Carthew. 2000, Lam and Thummel. 2000: Shi et al., 2000; Tavernarakis et al., 2000). Such strategies are conditional in time and can be made tissue-type specific (i.e., oocyte specific by using the Zp3 promoter) so that the experiment

15

can be repeated in vivo time after time. The mutants can be cryopreserved individually, and if it becomes interesting to study whether a particular gene product functions in a pathway, combinations of mutants can be made. The m R N A s essential for early development are now being identified and we are just beginning to address their function and role in this process. Recent technological advances hold the promise that we will soon have an enhanced ability to understand their function.

Acknowledgments D.S. and B.B.K. dedicate this chapter to the memory of G. Christian Overton who worked in concept and in fact on the problem of the initiation of embryogenesis with us. We thank Dr. Christian Stoeckert at the Center for Bioinformatics at the University' of Pennsylvania and Dr. Webb Miller. Penn State University. for their helpful advice. Original work presented herein was supported by grants from NIH-NICHD and the Lalor Foundation.

References Adenot, R G.. Mercier. Y.. Renard, J.-R, and Thompson, E. M. (1997). Differential H4 ace~lation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of t-celt mouse embryos. Development (Cambridge, UK) 124, 4615-4625. Adenot, R G., Campion. E., Legouy, E.. Allis. C. D.. Dimitrov, S., Renard. J.-R. and Thompson, E. M. (2000). Somatic linker histone H1 is present throughout mouse emb~ogenesis and is not replaced by variant H1 degrees. J. Ceil Sci. 113, 2897-2907. Afouda. A. B., Reynaud-Deonauth, S.. Mohun. T., and Spohr. G. (1999). Localized XId3 mRNA activation in Xenopus emb~'os by cytoplasmic polyadenylation. Mech. Dev. 88, 15-31. Antczak, M., and Van Blerkom, J. (I997). Oocyte influences on early development: The regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embrs'o. Mol. Hum. Reprod. 3, 1067-t086. Ao. A.. Erickson. R. R. Bevilacqua, A., and Karolyi, J. (1991). Antisense inhibition of/3-glucuronidase expression in preimplantation mouse embryos: A comparison of transgenes and oligodeoxynucleotides. Antisense Res. Dev. 1, 1-10, Aoki, F., Worrad. D. M., and Schultz, R. M. (1997). Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse emb~,o. Dev. Biol. 181,296-307. Bahramian, M. B., and Zarbl, H. (1999). Transcriptional mad posttranscriptional silencing of rodent alpha 1(I) collagen by a homologous transcriptionally self-silenced transgene. Mol. Cell. Biol. 19, 274-283. Barkoff, A. E. Dickson, K. S.. Gray, N. K., and Wickens, M. (2000). Translational control of cyclin B 1 mRNA during meiotic maturation: Coordinated repression and cytoplasmic polyadenylation. Dev. Biol. 220, 97-109. Bastin, R, Ellis. K., Kohl, L., and Gull, K. (2000). Flagellum ontogeny in t~panosomes studied via an inherited and regulated RNA interference system. J. Cell Sci. 113, 3321-3328. Beaujean, N.. BounioI-Baly. C., Monod. C., Kissa, K., Jullien, D., Aulner. N., Amirand. C., Debey, R, and Kas, E. (2000). Induction of early transcription in one-cell mouse emb~'os by microinjection of the nonhistone chromosomal protein HMG-I. Dev. Biol. 221, 337-354. Bellier, S., Chastant. S.. Adenot. E G.. Vincent, M., Renard, J.-R, and Bensaude. O. (1997). Nuclear translocation and carboxyl-terminal domain

! Establishment of Body Patterns

16 phosphorylation of RNA polymerase II delineate the two phases of zygotic gene activation in mammalian embry'os. EMBO J. 16, 62506262. Bevilacqua, A., Fiorenza, M. T., and Mangia, F. (2000). A developmentally regulated GAGA box-binding factor and Spl are required for transcription of the hsp70.] gene at the onset of mouse zygotic genome activation, Development (Cambridge. (.,:Kj 127, 1541 -1551. Birchler, J. A., Bhadra. M. R, and Bhadra. U. (20001. Making noise about silence Repression of repeated genes in animals. Curt. Opm. Gener Dev. 10, 21 1-216. Bleil, J. D., Greve. J. M., and Wassarman. P. M. (1988~. Identification of a secondary sperm receptor in the mouse egg zona pellucida: Role in maintenance of bindino_,_ of acrosome-reacted sperm to e_=s.oo,,Dev. Biol. 128, 376-385. Bosher, J. M., and Labouesse, M. (2000). RNA interference: Genetic wand and genetic watchdog. Nar Cell Biol. 2, E31-E36. Bos-Mikich. A.. Whittingham, D. G.. and Jones, K. T. (1997 ). Meiotic and mitotic CA:- oscillations affect cell composition in resulting blastocvsts. Dev. Biol. 182, 172-179. Caplen, N. J.. Fleenor. J., Fire. A.. and Morgan. R. A. (2000). dsRNAmediated gene silencing in cultured drosophila cells: A tissue culture model for the analysis of RNA interference. Gene 282, 95-105. Colledge, W H., Carlton. M. B. L.. Ud,v, G. B.. and Evans, M. J. (1994). Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature (London) 370, 65-68. Coller. J. M.. Gray. N. K.. and Wickens, M. R (1998). mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation. Genes Dev. 12, 3226-3235. Craig, A. W.. Haghighat, A., Yu, A. T.. and Sonenberg. N. (1998). Interaction of polyadenylate-binding protein with the elF4G homologue PAIP enhances translation. Nature (London) 392, 520-523. Danpure, C. J. (1995). How can the products of a single gene be localized to more than one intracellular compartment'? Trends Cell Biol. 5, 230-238. Davidson. E. H. (1986). "'Gene Activity in Early Development." Academic Press, New York. Davis. W., Jr.. and Schultz. R. M. (2000). Developmental change in TATAbox utilization during preimplantation mouse development. Dev. Biol. 218, 275 -283. Davis, W:, Jr.. De Sousa, R A.. and Schultz, R. M. (1996). Transient expression of translation initiation factor eIF-4C during the 2-cell stage of the preimplantation mouse embD'o: Identification by mRNA differential display and the role of DNA replication in zygotic gene activation. Dev. Biol. 174, 190-201. Deguchi, R.. Shirakawa. H., Oda. S.. Mohri, T.. and Miyazaki. S. (2000). Spatiotemporal analysis of Ca:- waves in relation to the sperm entry site and animal-vegetal axis during Ca:- oscillations in fertilized mouse eoos Dev. Biol. 218, 299-313. Deo, R. C., Bonanno. J. B., Sonenberg. N., and Burley. S. K. (1999). Recognition of polyadenylate RNA bv the poly(A)-binding protein. Cell (Cambridge, Mass.)98, 835-845. De Smedt, V., Sz6116si, D., and Kloc, M. (2000). The Balbiani body: Asymmetl"y- in the mammalian oocyte. Genesis 26, 208-212. de Vantdw, C.. Gavin, A. C., Vassalli, J. D.. and Schorderet-Slatkine, S. (1996). An accumulation of p34 ~': at the end of mouse oocyte growth correlates with the acquisition of meiotic competence. Dev. Biol. 174, 335-344. de Vant&y', C., Stutz, A.. Vassalli. J. D., and Schorderet-Slatkine, S. (1997). Acquisition of meiotic competence in growing mouse oocytes is controlled at both translational and posttranslational levels. Dev. Biol. 187, 43 -54. de Vries, W. N.. Binns, L. T., Fancher. K. S.. Dean, J.. Moore, R., Kemler, R.. and Knowles, B. B. (2000). Expression of Cre recombinase in mouse oocytes" A means to study" maternal effect genes. Genesis 26, 110-112. ~ .

9

Dolmetsch. R. E., Xu, K., and Lewis, R. S. (11998). Calcium oscillations increase the efficiency and specificity of gene expression. Nature (London) 392, 933-936. Domashenko, A. D., Latham, K. E., and Hatton. K. S. (1997). Expression of myc-family, myc-interacting and myc-target genes during preimplanration mouse development. Mol. Reprod. Dev. 47, 57-65. Edwards, R.G. (1965). Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature (London) 208, 349-351. Epifano, O.. Liang, L.-F.. Familari. M., Moos. M. C., Jr.. and Dean, J. (1995). Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development (Cambridge, UK) 121, 1947-1956, Evans. J. R. Foster. J. A.. McAvey. B. A., Gerton, G. L.. Kopf, G. S., and Schultz, R. M. (2000). Effects of perturbation of cell polarity on molecular markers of sperm-egg bindin~ sites on mouse ec,os Biol. Reprod. 62, 76-84. Fire. A. (1999). RNA-triggered gene silencing. Trends Gener 15, 358363. Fire, A., Xu. S., Montgomery, M. K.. Kostas, S. A., Driver. S. E., and Mello. C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature (London) 391, 806-811. Fissore. R. A.. Long, C. R., Duncan, R. R, and Robl, J. M. (1999). Initiation and organization of events during the first ceil cycle in mammals: Applications in cloning. Cloning 1, 89-100. Forlani, S.. Bonnerot, C., Capgras, S.. and Nicolas. J.-F. (1998). Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication. Development (Cambridge. UK) 125, 3153-3166. Gallicano, G. I.. McGaughey, R., W., and Capco, D. G. (19971). Activation of protein kinase C after fertilization is required for remodeling the mouse egg into the zygote. Mol. Reprod. Dev. 46, 587-601. Gao, M., Fritz, D. T., Ford, L. R, and Wilusz, J. (2000). Interaction between a poly(A)-specific ribonuclease and the 5' cap influences mRNA deadenylation rates in vitro. Mol. Cell S, 479-488. Gavis. E. R. (1997). Expeditions to the pole: RNA localization in Xenopus and Drosophila. Trends Cell Biol. 7, 485- 492. Gebauer, F., and Richter. J. D. (1996). Mouse cytoplasmic polyadenylylation element binding protein: An evolutionarily conserved protein that interacts with the cytoplasmic polyadenylylation elements of c-mos mRNA. Proc. Natl. Acad. Sci. g(S.A. 93, 14602-14607. Gebauer, F., Xu, W., Cooper, G. M.. and Richter, J. D. (1994). Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse. EMBO J. 13, 5712-5720. Gu. H., Marth, J. D., Orban, R C., Mossmann, H., and Rajewsky, K. (1994). Deletion of a DNA polymerase/3 gene segment in T cells using ceil typespecific gene targeting. Science 268, 103-106. Hashimoto, N., Watanabe, N.. Furuta, Y., Tamemoto. H., Sagata, N., Yokoyama. M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa. Y., and Aizawa, S. (1994). Parthenogenetic activation of oocytes in c-mos-deficient mice. ,Nature (London) 370, 68-71. Heasman, J., Kofron, M., and Wylie, C. (2000)./3-catenin signaling activity dissected in the early Xenopus embry'o: A novel antisense approach. Dev. Biol. "~2"~ 124-134. Heyer, B. S., Warsowe, J., Solter, D., Knowles, B. B., and Ackerman, S. L, (1997). New member of the Snfl/AMPK kinase family, Melk, is expressed in the mouse egg and preimplantation embryo. Mol. Reprod. Dev. 47, 148-156. Hever, B. S., Kochanowski, H., and Solter, D. (1999). Expression of Melk. a new protein kinase, during early mouse development. Dev. Dyn. 215, 344-351. Holliday, R. (1989). A molecular approach to the problem of positional information in eggs and early emb~os. New Biol. 1 (3), 337-343. Hwang, S. Y., Oh, B., Fiichtbauer, A., F/,ichtbauer, E. M., Johnson, K. R,, Solter, D., and Knowles, B. B. (1997). Maid: A maternally transcribed novel gene encoding a potential negative regulator of bHLH proteins in the mouse egg and zygote. Oev. Dyn. 2tl9, 217-226.

I Fertilization and Activation of the Embryonic Genome Hwang. S.-Y.. Oh. B.. Zhang. Z.. Miller. W., Solter, D.. and Knowles. B. B. (1999). The mouse cornichon gene family,. Dev. Genes Evol. 209, 1 2 0 - 125.

Hwang, S. Y.. Oh. B.. Knowles. B. B.. Solter. D., and Lee, J-S. (2001). Expression of genes involved in mammalian meiosis during the transition from e....S2to embryo., Mol. Reprod. Dev. 59, 14-4-t50. Jacobsen. S. E.. Running. M. P.. and Meyerowitz. E. M. (1999). Disruption of an tL\'A helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems. Development (Cambridge. UK) 126, 5231-5243. Johnson. J.. Bierle. B. M.. Gallicano. G. I.. and Capco. D. G. (t998). Calcium/Calmodulin-Dependent protein kinase II and calmodulin: Regulators of the meiotic spindle in mouse eggs. Dev. Biol. 204, 464-477. Johnson. M. H. (19811). The molecular and cellular basis of preimptantation mouse development. Bio. Rev. Cambridge Philos. Soc. 56, 463-498. Jurisicova. A.. Latham. K. E.. Casper. R. F.. and Varmuza. S. L. (1998). Expression and regulation of genes associated with celt death during murine preimplantation embryo development. Mot. Reprod. Dev. 51, 243-253. Kaneko. K. J.. and DeParnphilis. M. L. (1998). Regulation of gene expression at the beginning of mammalian development and the TEAD family of transcription factors. Dev. Genet. 22, 43-55. Kaneko. K. J.. Cullinan. E. B.. Latham. K. E.. and DePamphilis. M. L. (1997). Transcription factor mTEAD-2 is selectively expressed at the beginning of zygotic gene expression in the mouse. Development (Cambridge, UK) 124, 1963 - 1973. Kennerdell. J. R.. and Carthew. R. W: (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell (Cambridge. Mass.) 95, t017-1026. Kennerdell. J. R.. and Carthew. R. W\ (2000). Heritable gene silencing in drosophila using double-stranded RNA. Nat. Biotechnol. 18, 896898. Kidder. G. M. (1993). Genes involved in cleavage, compaction, and blastocvst formation. In "'Genes in Mammalian Reproduction" (R. B. L Gwatkin. ed.), pp. 45-71. Wiley-Liss. New York. King. M. L.. Zhou. Y.. and Bubunenko, M. (1999). Polarizing genetic information in the e....:,.ogRNA localization in the frog oocyte. BioEssays 21,546-557. Kline. D., Mehlmann. L., Fox. C.. and Terasaki. M. (1999). The cortical endoplasmic reticulum (ER) of the mouse e...,:ggLocalization of ER clusters in relation to the generation of repetitive calcium waves. Dev. Biol. 215,431-442. Kloc, M.. and Etkin. L. D. (1995). Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. Development (Cambridge. UK) 121, 287-297. Ko. M. S. H., Kitchen. J. R., Wang. X., Threat. T. A.. Wang. X., Hasegawa. A., Sun. T.. Grahovac. M. J.. Kargul, G. J., Lira. M. K.. Cui. Y.. Sano, Y.. Tanaka. T.. Liang, Y.. Mason, S., Paonessa. R D., Sauls. A. D.. DePalma. G. E.. Sharara. R.. Rowe. L. B.. Eppig, J.. Morrell. C.. and Doi. H. (2000). Large-scale cDNA analysis reveals phased gene expression patterns during preimplantation mouse development. Development (Cambridge. UK) 127, 1737-1749. Lam. G.. and Thummel, C. S. (2000). Inducible expression of doublestranded RNA directs specific genetic interference in Drosophila. Curt. Biol. 10, 957-963. Laroia. G.. Cuesta. R.. Brewer. G.. and Schneider. R. J. (1999). Control of mRNA decay by" heat shock-ubiquitin-proteasome pathway. Science 9,284, 499-502. Latham. K. E. (1999). Mechanisms and control of embwonic genome activation in mammalian embryos. Int. Rev. Cytol. 193, 7I-124. Latham. K. E.. Garrels. J. I.. Chang. C.. and Solter. D. ( 1991 ). Quantitative analysis of protein synthesis in mouse emb~'os. I. Extensive reprogramming at the one- and two-cell stages. Development (Cambridge, UK) 112, 921-932.

17

Latham. K. E.. Solter, D.. and Schuttz. R. M. (1992). Acquisition of a transcriptionally permissive state during the 1-cell stage of mouse embwogenesis. Dev. Biol. 149, 457-462. Lehman. A. L., Nakatsu. Y.. Ching, A.. Bronson. R. T.. Oakey. R. J., KeiperHunko, N.. Finger. J. N.. Durham-Pierre. D., Horton, D. B., Newton, J. M.. Lyon. M. F.. and Brilliant, M. H. (1998). A yew large protein with diverse functional motifs is deficient in rjs (runty. jerky, sterile) mice. Proc. Natl. Acad. Sci. U.S.A. 95, 9436-9441. Lewandoski. M., Wassarman. K. M.. and Martin, G. R. (1997). Zp3-cre, a transgenic mouse line t'or the activation or inactivation of loxP-flanked taget genes specifically in the female germ line. Curt Biol. 7, 148-15 I. Li. Y.-X.. Farrell. M. J.. Liu. R., Mohanty. N.. and Kirby. M. L. (2000). Double-stranded RNA injection produces null phenotypes in zebrafish. Dev. Biol. 217, 394-405: erratum: 1bid. 220, 432 (2000). Luo. L.. Salunga. R. C.. Guo. H.. Bittner. A.. Joy. K. C., Galindo. J. E.. Xiao. H.. Rogers. K. E.. Wan. J. 'v\[. Jackson, M. R.. and Erlander. M. G. (I999). Gene expression profiles of laser-captured adjacent neuronal subtypes. :Vat. Med. 5, 117-122. Maleszewski. M.. Kimura, Y.. and Yanagimacht, R. (1996). Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: Evidence based on intracytoplasmic sperm injection experiments in the mouse. Mol. Reprod. Dev. 44, 256-259. Masui. Y.. and Markert, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. 3'. Exp. ZooL 177, 129-145. Matsumoto, K.. Anzai. M.. Nakagata. N.. Takahashi. A.. Takahashi. Y., and Miyata. K. (I 994). Onset of paternal gene activation in early mouse embry,os fertilized with transgenic mouse sperm. MoL Reprod. Dev. 39, I36-140. Mayer. W.. Niveteau. A.. Walter. J.. Fundele. R.. and Haaf, T. (2000). Demethylation of the zygotic paternal genome. Nature (London) 403, 501-502. Medberry. S. L.. Dale. E., Qin. M.. and Ow, D. W. (1995). Intra-chromosomaI rearrangements generated by Cre-lox site-specific recombination. Nucleic Acids Res. 23, 4 8 5 - 490. Mendez. R.. Hake. L. E.. Andresson. T., Littlepage. L. E., Ruderman, J. V., and Richter, J. D. (2000). Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature (London) 404, 302-307. Mitra. J., and Schultz. R. M. (1996). Regulation of the acquisition of meiotic competence in the mouse: Changes in the subcellular localization of cdc2. cyclin B 1, cdc25C and wee 1, and in the concentration of these proteins and their transcripts. J. Ceil Sci. 109, 2407-2415. Montgome~. M. K., Xu. S.. and Fire, A. (1998). RNA as a target of doublestranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 95, 15502-15507. Moos. J.. Visconti, P. E., Moore, G. D.. Schultz, R. M.. mad KopL G. S. (I995). Potential role of mitogen-activated protein kJnase in pronuclear envelope assembly and disassembly following fertilization of mouse eggs. Biol. Reprod. 53, 692-699. Nakano. H., and Kubo. H. (2000). Study of the in vitro maturation of mouse oocytes induced by microinjection of maturation promoting factor (MPF). J. Assist. Reprod. Genet. 17, 67-73. Norton, J. D., Deed. R. ~:, Craggs, G., and Sablitzky, E (I998). id helixloop-helix proteins in celt growth and differentiation. Trends Cell Biol. 8, 58-65. Nor.hias, J.-Y.. Majumder, S., Kaneko. K. J., and DePamphilis, M. L. (1995). Regulation of gene expression at the beginning of mammalian development. J. BioL Chem. 270, 22077-22080. Nothias. J.-Y.. Miranda, M., and DePamphilis, M. L. (1996). Uncoupling of transcription and translation during zygotic gene activation in the mouse. EMBO J. 15, 5715-5725. Oates, A. C.. Bruce, A. E.. and Ho. R. K. (2000). Too much interference: Injection of double-stranded RNA has nonspecific effects in the zebrafish embp,,o. Dev. Biol. 224, 20-28.

18 Oh, B.. Hwang, S.-Y.. Solter, D.. and Knowles, B. B. (1997). Spindlin. a major maternal transcript expressed on the mouse during the transition from oocyre to embryo. Development (Cambridge. UK) 124, 493-503. Oh. B., Hampl, A.. Eppig. J. J., Solter, D., and Knowles. B. B. (1998). SPIN. a substrate in the MAP kinase pathway in mouse oocytes. ,Viol. Reprod. Dev. 50, 240-249. Oh. B.. Hwang, S.-Y.. De Vries, W. N., Solter. D.. and Knowles. B. (1999). Identification of genes and processes guiding the transition between the mammalian gemete and embrs,'o. In "'A Comparative Methods Approach to the Study of Oocytes and Embr?'os'" (J. D. Richter. ed.), pp. 101-126. Oxford University Press. New York. Oh. B Hwang. S.-Y.. McLaughlin. J.. Solter, D.. and Knowles. B. B. (2000). Timely translation during the mouse oocyte-to-embr2:'o transition. Development (Cambridge. UK) 127, 3795-3803. O'Keefe, S. J., Wolfes. H.. Kiessling, A. A., and Cooper. G. M. (11989). Microinjection of antisense c-mos oligonucleotides prevents meiosis II in the maturin~ mouse eoo Proc. Natl. Acad. Sci. 8:S.A. 86, 70387042. Oswald, J., Engemann, S., Lane, N., Mayer, W., Olek. A., Fundele, R., Dean, Vv:, Reik, W:, and Walter, J. (2000). Active demethylation of the paternal genome in the mouse zygote. Curr. Biol. 10, 475-478. Parrington, J., Swarm, K., Shevchenko. V. I.. Sesay. A. K., and Lai, A. F. (1996). Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature (London) 379, 364-368. Parrot-t, J. N.. and Gay. N. J. (1998). Expression and subcellular distribution of rel/NF~cB transcription factors in the preimplantation mouse embrT'o: Novel ~ forming unit-granulocyte, e~'throid, macrophage, megarkar?ocyte), CFU-GM (colon> forming unit-granulocyte, macrophage), BFLr-E (burst forming unit-e~'throid), CFU-E (colony forming unit-e~throid), and CFU-MEG (colon): forming unit-megakaryocyte). The gray box represents the immature progenitor compartment that has yet to be fully characterized.

the dorsal aorta, gonads, and mesonephroi (AGM). In vivo transplantation, the experimental forte of mouse, revealed that the AGM is the first site of HSC initiation and is a potent source of adult hematopoietic cells. New and evolving concepts regarding the hematopoietic potentials of stem cells from other organ systems are now leading to notions of reprogramming, dedifferentiation, and/or maintenance of ontogenic potential (plasticity). This chapter presents the concepts and data past and present that lead to our current understanding of the developmental aspects of hematopoiesis.

II. Cellular Aspects of Blood Development in the Mouse Embryo

A. Adult Hematopoietic Hierarchy The adult hematopoietic system is composed of many cells positioned in a complex differentiation hierarchy. At least eight different lineages of functional hematopoietic

cells are formed from a small population of HSCs that reside mainly in the bone marrow of the adult and serve as the renewable source of the adult blood system. The terminally differentiated hematopoietic cells consist primarily of erythrocytes, monocytes, platelets, neutrophils, mast cells, eosinophils, B lymphocytes, and T lymphocytes (Fig. 1). These functional blood cells differentiate from the HSC through a series of committed progenitor cells. Progenitors are classified retrospectively by the functional blood cells they generate, using in vivo and in vitro clonal assays (Metcalf, 1984). Some of the more frequently used assays that detect the presence of progenitors and/or HSCs are indicated in Table I. These assays yield readouts for progenitors at different stages of commitment, including progenitors that give rise to only one lineage (e.g., CFU-E, CFU-Meg), multilineage progenitors (cells with potential for two or more lineages such as CFU-GEMM and CLP (Kondo et aI., 1997) and pluripotent HSCs.

B. Identification of Hematopoietic Progenitors and Stem Cells Unfortunately, no single descriptive characteristic can identify progenitors or stem cells in the absence of functional assays. Cells that are functionally distinct have overlapping cell surface phenotypes, responses to growth factors, homing abilities, and potential for self-renewal. For example, adult HSCs express Sca-I. c-kit, CD34, CD31 and AA4.1 (Ikuta et al., 1990: Ikuta and Weissman, 1992; Jordan et al., 1990; Spangrude et al., 1988; van der Loo et al., 1995) (see Table II), but these markers are not exclusively expressed on HSCs. Activated T cells and some epithelial cells express Sca-1 (Sinclair and Dzierzak. 1993); c-kit is expressed by hematopoietic progenitors and primordial

10 Vertebrate Hematopoietic System

193

Table I Cell type Hematopoietic stem cell

H e m a t o p o i e t i c Activities

Potency

Assay

Cell at the foundation of the adult differentiation hierarchy,

Neonatal repopulating cell

Pre-HSC or cell at the foundation of a neonatal/fetal hierarchy

Multipotential cell

Cell with ery'throid-myeloidlymphoid lineage multipotency

CFU-S (colony forming unit-spleen)

Immature progenitor with euthroid and myeloid activity

CFU-C (colony, forming unit culture)

More mature progenitor with erythroid and myeloid activity

B-lymphoid progenitor

B tymphocytes

T-lymphoid progenitor

T lymphocytes

germ cells (PGCs) (Keshet et at., 1991" Morro et al., 1991): CD34 is expressed by hematopoietic progenitors, endothelial cells, and muscle cells (Wood et al.. 1997; Young et al., 1995); and AA4.1 is expressed by pre-B cells (Cumano et al., 1992). Generally, HSC isolation is performed using a combination of markers to first deplete cell populations of more mature, committed hematopoietic cells (negative selection), then to enrich for the HSCs (positive selection). Adult HSCs do not express a number of cell surface markers characteristic of mature hematopoietic cells, including B220 (B cells). CD3, CD4, and CD8 (T cells), Gr-1 (granulocytes), and Mac 1 (monocytes and granulocytes). These markers are often used for negative selection of differentiated blood cells

Table II Hematopoietic stem cells Positive

Negative

AA4.1 CD34 c-kit Sca- 1 CD31

B220 CD3 CD4 CD8 Gr- 1 Mac 1

Cell Specificity Markers Endothelial cells

Hematopoieticl endothelial cells in arterial clusters

Positive

Positive

CD34 Tie-I Tie-2 VE-cadherin ilk- 1 CD31 SCL AA4.1

AML 1/CB F/3 CD3I CD34 c-kit GATA-2 ilk- 1 SCL AA4.1

In vivo transplantation into hematopoietic-ablated adult recipients leading

to long-term, high-level, multilineage repopulation. Such stem cells are capable of self-renewal. In vivo transplantation into hematopoietic-ablated newborn (within 1 day of birth) recipients leads to long-term, moderate level, multitineage repoputation. These cells are unable to directly repopulate adult recipients. Two-step in vitro a~ssay in which cells are clonally expanded in culture. After clonal expansion cells are tested under three sets of conditions leading to B-lymphoid, T-lymphoid. or eo'throid-myeloid differentiation. These cells cannot in vivo repopulate adult recipients. In vivo transplantation into lethally irradiated adult recipients leads to the short-term (within 9 - I 4 days after transplantation) formation of macroscopic colonies. bz vitro 5 to I4-day culture in semisolid medium with hematopoietic growth factors leading to the production of colonies containing one or mixed lineages of differentiated erTthroid and myeIoid cells. Coculture with S17 stromaI line and IL-7 leads to differentiation of B cells. Fetal thymic organ culture (FTOC). Fetal thymic rudiments ablated of all progenitors are seeded with test cells in a hanging drop culture. All CD4 and CD8 subsets of T cells are differentiated in such in vitro cultures.

from the HSC population followed by positive marker selection (Sca-1, c-kit, CD34, and/or AA4.1). Recently, it was shown that the chemical Hoechst 33342 can be used in flow cytometfic sorting to enrich HSCs (Goodell et al., 1996) as well as stem cells for other tissues (Gussoni et al., I999: Jackson et al., 1999). Hoechst 33342 fluorescent marking identifies cells that rapidly efflux small molecules and promises to be an extensively used research tool in stew, cell analyses. Responsiveness to hematopoietic growth factors has also been used to categorise hematopoietic cells (Metcalf, 1984, 1998). Specific growth factors affect distinct classes of progenitor cells in vitro. For example, interleukin 7 (IL-7), erythropoietin, granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (MCSF) promote the growth of pre-B lymphocytes, CFU-E, CFad-GM, and CFU-M, respectively. Hematopoietic growth factors thought to affect HSCs and progenitors include IL-3, SCE IL-6, and thrombopoietin. Cocktails of such factors are often used in in vitro cultures to maintain the growth of hematopoietic progenitors and stem cells (Breems et aL, 1998). Although certain combinations of growth factors promote the expansion and differentiation of progenitor cells, no special combination has been found that expands the starting stem ceil pool, suggesting that there may be separate, yet to be described factors involved in stem cell growth. Targeted disruption of many individual (and in combination) growth factor and growth factor receptor genes has very little effect on the in vivo growth and differentiation of hematopoietic

194 cells, suggesting functional redundancy (Lieschke et al., 1994: Nilsson et al., 1995). However. some growth factors do provide function in distinct hematopoietic cell subsets in vivo. For example, thrombopoietin is used at both ends of the hematopoietic hierarchy--for the maintenance of HSCs as well as for the terminal differentiation of megakaryocytes (Kimura et aI.. 1998). Thus, growth factor regulation of hematopoiesis is complex and the role of specific growth factors or combinations of growth factors in HSC and hematopoietic progenitor function during adult and embryonic stages, particularly in vivo, is unclear. It has been proposed that the pattern of gene expression could be used to determine a cell's position within the hematopoietic hierarchy. For example, expression of hematopoietic growth factor receptors such as the CSF-1 receptor might characterize a macrophage progenitor while expression of the IL-7 receptor might be used to identi~ a lymphoid progenitor. A single-cell nested polymerase chain reaction (PCR) approach has recently shown that all progenitor cells may express a whole spectrum of receptors and are thus "primed" for a stochastic event leading to commitment (Hu et al., 1997). As the cell progresses down a particular differentiation pathway, expression of the unnecessary receptors is dow,nregulated. The ability to study global gene expression patterns through the use of gene chip technology could eventually establish a gene expression profile for each class of hematopoietic progenitors and fully differentiated blood cells (Phillips et al., 2000). In summary, the adult hematopoietic hierarchy is a functional continuum of differentiating cells as defined by in vitro and in vivo assays with many overlapping and redundant features.

C. Hematopoietic Cells in the Conceptus 1. Differentiated Hematopoietic Cells How similar is the mammalian hematopoietic system of the embryo to that of the adult? The appearance of functional hematopoietic cells, hematopoietic progenitors and stem cells has been examined both temporally and spatially in mouse (reviewed in Dzierzak et al., 1998) and human embwos (Huyhn et aI., 1995, Tavian et al., 1999). Table III summarizes the major landmarks in mouse and human development. Most of the differentiated hematopoietic lineages found in the adult can also be found in the embryo. However. some clear differences exist between embryonic and adult cell types especially in the erythroid lineage. Historically, embryonic erythrocytes that retain their nuclei and express embryonic globin genes were called "primitive" to distinguish them from the enucleated "definitive" erythrocytes that appear somewhat later in development and express fetal and/or adult globin genes. Acknowledgment of these differences has led to a general but confusing terminology

il Lineage Specification and Differentiation with the term "primitive" referring to the early embwonic hematopoietic system and "definitive" referring to the adult hematopoietic system. Many difficulties in the use of this terminology arise during the intermediate fetal and neonatal stages, and in particular when it is used for other lineages of hematopoietic cells. These difficulties reflect our lack of knowledge concerning the lineage relationships and embryonic origins of many of the hematopoietic cells. The first and most numerous differentiated hematopoietic cells in the mammalian embryo, primitive erythrocytes, are found in the yolk sac (Fig. 2 and Table III). As the developing intraembryonic vitelline and umbilical vessels link to the yolk sac vessels and the placenta, respectively, primitive erythrocytes begin to circulate throughout the embryo. These cells predominate in the yolk sac until a switch to definitive erythropoiesis occurs. The first cells of the macrophage lineage appear in the yolk sac of the mouse conceptus soon after the primitive erythrocytes. These primitive macrophages are able to colonize other embryonic tissues through the circulation. However, definitive macrophages found during fetal and adult stages do not circulate, and arise from monocytic progenitors that begin to appear slightly later in the fetal liver and yolk sac. Thus, there appear to be two separate waves of erythroid and macrophage production. The production of lymphoid lineage cells occurs only during the later stages of gestation when the fetal liver is the predominant hematopoietic site (Table III). B-lymphoid precursors in the mouse embryo seed the fetal liver after E11, T-lymphoid precursors are found in the thymus beginning at E 12-13. and the circulation of mature lymphoid cells begins at E17 (Delassus and Cumano, 1996). The order in which various hematopoietic cells appear in the human embryo and fetus is similar to that in the mouse. The timing of human hematopoietic development is different due to the longer gestation period, and is summarized in Table III. It is unclear whether all lineages of differentiated hematopoietic cells in the embryo and the adult are functionally equivalent (Bonifer et al., 1998). In fact, at least three functional cell types are unique to the embryo or fetus. The first example is the primitive erythrocyte. In mouse and human embryos, primitive erythrocytes are larger than adult er,.~rocytes, retain their nuclei, and express embwonic globin genes. In contrast, adult erythrocytes are small, enucleated, and express adult globin genes (Russell, 1979). The functional outcome of these differences is that embryonic hemoglobin has an increased affinity for oxygen as compared to adult hemoglobin, thereby facilitating the transport of oxygen through the placenta to the conceptus. Another example is the TcRT3 subset of lymphoid cells, that is thought to be important in mucosal immunity, and is produced during fetal but not the adult stages of development (ILkuta et al., 1990). Likewise, the CD5 subset of B lymphocytes is produced predominantly during fetal stages and is also thought to serve a special immune function as compared to adult B cells (Her-

10 Vertebrate Hematopoietic System Table III Gestation day E7.0 E7.5 to E8.0

195

Landmarks in Mouse (and Human) Hematopoietic Development

Developmental stage Early primitive streak Advanced primitive streak

Landmarks (human)

E 10.5

35-39 Somite pairs

Gastrulation. mesoderm formation ":folk sac blood islands with primitive erythrocytes appear (primitive ewthrocytes appear in human at E16-20) Cells of primitive circulating macrophage lineage appear in yolk sac (first cells of the human monocytic macrophage lineage appear at 4-5 w'eeks) Yolk sac vasculature and paired dorsal aortae form Onset of circulation, intraembwonic vitelline and umbilical vessels link to yolk sac vessels and placenta, respectively Pronephric duct and nephric vesicle formation Urogenital ridges and liver form Liver rudiment contains first ery.'throbiast beginning at 28 somite pair stage Switch from primitive to definitive e~'thropoiesis (in humans this switch is visible at 7-10 weeks in the blood and slightly earlier in the fetal liver) Monocytic progenitors appear in yolk sac arid fetal liver (human primitive macrophages in circulation until 14 weeks and definitive monocytes appear meter I 1 weeks) HSC generation commences in AGM

E11

> 40 Somite pairs

HSCs appear in yolk sac and fetal liver

1- 2 Somite pairs E8.5

8-12 Somite pairs

E9.0 E9.5

15-20 Somite pairs 20-29 Somite pairs

EIO to El2

El2 to El3

El4 to El6

B-lymphoid precursors found in fetal liver, Tlymphoid precursors found in thymus (in the human embryo, production of lymphoid cells begins at 7-I0 weeks, with small lymphocytes in the circulation beginning at 9 10 weeks) Spleen, omentum, and bone marrow begin to serve as hematopoietic sites

zenberg e t a l . , 1986). In addition, during mid- and late gestation some l y m p h o c y t e s are thought to contribute to the maturation of secondary lymphopoietic organs and microenvironments through inductive signals and " c r o s s t a l k " (van Ewijk e t al., 2000). In summary, and as s h o w n in Fig. 2, hematopoietic cells are harbored in several sites in the m a m m a l i a n e m b r y o and include the yolk sac, fetal liver, and circulation (Delassus and C u m a n o , 1996). Slightly later in the m o u s e embryo, beginning at E 1 4 - 1 6 and thereafter, the thymus, spleen, o m e n turn, and bone marrow also serve as hematopoietic sites (Godin e t a l . , 1999).

2. Hematopoietic Progenitors and Stem Cells The locations of precursors for differentiated h e m a t o p o i etic cells, that is, the hematopoietic progenitors and stem cells, have been mapped temporally and spatially in the

References Kaufman (1992) Russel and Belmstein ( t 966): Keleman et al. (1979): Palis et al. (1999~: Naito (1993): Naito et al. (1996): Kaufman (1992)

Garcia-Porrero et al. (1995): Kaufman (1992) Kaufman (I992) Kaufman (1992): Johnson and Jones (1973) Keleman et al. (1979): Naito (I993): Nai1:oet at. (1996)

Muller et at. ( 1994): Medvinsky and Dzierzak (1996) Muller et al. (1994): Medvinsky" and Dzie~ak (1996) Delassus and Cumano ( 1996): Keleman et aL (1979)

Godin et al. (1999)

m o u s e conceptus. Recent studies show that the earliest onset of h e m a t o p o i e s i s occurs in the proximal regions of the egg cylinder at midprimitive streak stage (E7.5) (Palis e t a l . , 1999) with the production of primitive erythroid and macrophage precursors. T h e erytt'a'oid and m a c r o p h a g e precursors increase in n u m b e r in the yolk sac until E8.25 and then sharply decline by E9.0. At no time are these progenitors found in the e m b r y o body. Hence, the yolk sac appears to be the exclusive source o f the first wave of primitive erythroid and m a c r o p h a g e progenitors. M u l t i p o t e n t C F U - C progenitors ( g r a n u l o c y t e and macrophage c o l o n y - f o r m i n g cells), C F U - S , arid H S C s were first d e m o n s t r a t e d to appear within the yolk sac o f the m o u s e conceptus beginning at E7, E8, and E11, respectively, by M o o r e and M e t c a l f (1970). The appearance o f these hematopoietic activities in the yolk sac was always followed by their a p p e a r a n c e I - 2 days later in the circulation as well as

!1 Lineage Specification and Differentiation

196 in the fetal liver and led to the general notion of the yolk sac origins of adult hematopoiesis (reviewed in Medvinsky et al., 1993). The use of fetal thymic organ cultures (FTOCs) and B-ceil stromal cultures in the 1980s enabled other researchers to find T- and B-lymphoid progenitors in the yolk sac beginning at E8.5 (Eren et al., 1987) and E l 0 (Ogawa et al., 1988), respectively. Although B-lymphoid progenitors were also detected in the embryo body at E9.5 (Ogawa et al., 1988), the preliver embryo body was generally, ignored as a hematopoietic site for many years. In 1993 a potent preliver site of hematopoietic progenitor and stem cell activity was uncovered in the mouse embryo body (Godin et al.. 1993 Medvinsky et al.. 1993). Dissected caudal regions of embryos, which included the dorsal aorta. gonads, and pro/mesonephroi ( A G M see Fig. 2). contained the more complex and potent hematopoietic progenitor and stem cells characteristic of those found in an adult-type hematopoietic system. A number of different in vivo and in vitro assays (Fig. 3), together with the careful dissection of the AGM or its earlier ontogenic form. the para-aortic splanchnopleura (PAS) (both are derived from the intraembryonic splanchnopleura: see Kaufman. 1992) at E 8 . 5 - 9 revealed the presence of CFU-S erythroid-myeloid progenitors (Medvinsky et al.. 1993), B-lymphoid progenitors (Godin et al., 1993), multipotential erythroid-myeloid-lymphoid progenitors (Godin et al., 1995). and multipotential cells capable of repopulating neonatal recipients on transplantation (Yoder et al., 1997). These activities generally appeared simultaneously in both the yolk sac and PAS/AGM. and before the onset of fetal liver hematopoiesis. Quantitative comparisons of progenitor numbers in the yolk sac and PAS/AGM revealed that while the number of neonatal repopulating cells was higher in the yolk sac. the number of CFU-S was higher in the AGM (Fig. 4). Furthermore, with the exception of CFU-S. none of these hematopoietic progenitors was capable of giving rise to in vivo short-term or long-term hematopoietic repopulation of adult mice. Efforts to determine when and where long-term adult repopulating HSCs first appear revealed their presence in the AGM region (Muller et al., 1994) and the vitelline and umbilical vasculature (de Bruijn et al., 2000b) at El0.5 ( > 3 4

somites) before their appearance in the yolk sac. Furthermore, at later time points the AGM was shown to contain HSCs at a higher frequency than the yolk sac (Muller et at., 1994). While these results demonstrated that the AGM region harbors adult-type HSCs. whether they originate in the AGM region and the vitelline and umbilical arteries remains controversial. This is due in part to the fact that blood freely circulates between the embryo body and the yolk sac starting at E8.5 (Garcia-Porrero et aI., 1995, see Fig. 2). In addition, interstitial migration of cells may play a role in hematopoietic cell dispersion thoughout the mouse conceptus as observed in amphibian embryos (Turpen et al., 1981).

D. Origins of Embryonic and Adult Hematopoietic Compartments 1. H e m a t o p o i e t i c O r i g i n s in the Avian and

Amphibian Species The yolk sac origin for the adult hematopoietic system was first called into question by findings from embryo culture and grafting experiments performed in nonmammalian vertebrate species. In the mid-1970s, elegant studies on cell fate. morphogenesis, and organogenesis in birds were performed through the use of interspecific grafts (between quail and chick embryos) or intraspecific grafts (between different strains of chicks). Grafting was used to create chimeras in which the embryonic origins of adult blood cells were identified using nucleolar or immunochemical markers to determine from which tissue (endogenous or ~afted) the differentiated adult blood cells were derived (Dieterlen-Lievre, 1975: Dieterlen-Lievre and Le Douarin, 1993). For example, a quail embryo body was grafted onto the extraembr?'onic area of a chick blastodisc, and blood cells in the chimeric fetus or adult animal were examined to determine if they were of quail or chick origin. The combined results of many such experiments (Beaupain et at., 1979; Dieterlen-Lievre, 1975: Dieterlen-Lievre and Martin. 1981" Martin et al., 1978) showed that the first emerging hematopoietic cells are derived from the extraembryonic yolk sac. Slightly later in development, hematopoietic cells derived from both extra-

Figure 3 Explant culture experiments. Schematic of the strategy and results of three experiments examining the embrT,onic sites where the first hematopoietic progenitors and stem cells are produced. (a) ClzU-C production was found in E7 yolk sac explants cultured for 2 days but not in cultured E7 emb~'o body (EB) explants. The presence of CFU-Cs in the EB of the cultured intact conceptus suggeststhat CFUCs are produced in the yolk sac and subsequently migrate and colonize the EB (Moore and Metcalf. 1970). (b) Multipotential progenitors for the ewthroid, myeloid, and lymphoid lineages are first generated in the precirculation (<E8.5) intraembr~'onic splanchnopleura. After the circulation is established between the yolk sac (YS) and PAS. multipotential progenitors are found in both sites (Cumano et al.. 1996). (c) CFUS and HSC production. Explant cultures of EI0 YS and AGM reveal that both tissues contain CFU-S but only the AGM can autonomously produce HSCs. At E11 both cultured tissues contain CFIS-S and HSCs. suggesting that AGM-generated HSCs colonize the YS or that the YS can automously produce HSCs at this later time (Medvinsky and Dzierzak. 1996).

10 Vertebrate Hematopoietic System

197

198

I! Lineage Specification and Differentiation

Figure 4 Waves of hematopoietic activity in the mouse embry'o. At least three waves of hematopoietic activity are thought to occur in the mouse embryo. The embr~onic wave consists of the exclusive production of primitive erythrocytes and macrophages by the yolk sac. The fetal wave of hematopoiesis includes the production of several committed and multilineage progenitors and neonatal repopulating cells from the YS and PAS/AGM. Quantitative levels of progenitors in these two sites of production are compared. The adult wave of hematopoiesis includes the generation of the first HSCs in the AGM region, and the vitelline and umbilical arteries. Abbreviations: ys, yolk sac, PAS. para-aortic splanchopleura" AGM. aorta-gonads-mesonephros, th. thymus: sp, spleen bm, bone marrow.

and intraembryonic sites were found in the fetus. The permanent adult hematopoietic system is derived almost entirely from intraembryonic sites. Thus it appears that cells derived from the extraembryonic yolk sac contribute only transiently to embryonic hematopoiesis and become extinct. Further experimentation suggested that the adult hematopoietic system is derived more specifically from the region containing the dorsal aorta (Cormier and Dieterlen-Lievre. 1988: Dieterlen-Lievre. 1975). More recent experiments indicate that the allantois is also a potent source of adult blood cells in the chick (Caprioli et aL, 1998). Amphibian embryo grafting experiments, in which DNA content was used as the donor graft marker, also demonstrated independent intraembryonic and extraembryonic sites of hematopoiesis (Kau and Turpen, 1983" Maeno et al., 1985" Turpen et al., 1981). Chimeric frog embryos were generated by reciprocal grafting of the ventral blood island region (VBI, yolk sac analog) and the dorsal lateral plate region (DLR intraembryonic region analog) from diploid and triploid embryos. These experiments revealed that the VBIs produce the first hematopoietic cells, and the DLP subsequently generates adult hematopoietic cells. Intrabody hematopoiesis was specifically localized to the developing excretory system near the aortic region, with the most abundant

hematopoiesis found in the pronephros (Turpen and Knudson, 1982). In contrast to the avian species, some VBIderived cells are thought to persist into the adult stage in amphibians and contribute to both red and white blood cell populations (Kau and Turpen, 1983; Maeno et aI., 1985; Turpen et aI., 1997). Recent lineage tracing experiments have shown that the DLP and the VBIs are derived from distinct blastomeres of the 32-cell-stage embryo, thus clearly demonstrating two distinct early origins for hematopoietic cells (Ciau-Uitz et aI., 2000). 2. Hematopoietic Origins in Mammals Explant cultures have been used to study the origins of hematopoietic progenitors and stem cells in the mouse embryo. Moore and Metcalf (1970) performed the first study using E7 mouse embryos. After 2 days of culture, abundant granulocyte-macrophage progenitors (CFU-Cs; colony forming units-culture) were detected in yolk sac explants, whereas embryo body explants were devoid of this activity (Fig. 3a). If, however, whole E7 embryos were cultured in vitro for 2 days before separating them into embryo bodies and yolk sacs, CFU-C activity was also detected in the embryo body. These results suggested that the first CFU-Cs eme~e from the yolk sac and migrate to the embryo proper

10 Vertebrate Hernatopoietic System between E7 and E9. While these studies examined only one progenitor cell type, the results contributed to the general notion that the yolk sac served as the embryonic source of entire adult hematopoietic system of mammals. More recently, the source of the first multipotential hematopoietic progenitors was tested by explant cultures of mouse embryonic tissues both pre- and postcirculation (Cumano et al.. 1996). An initial organotypic culture step with isolated yolk sac and intraembryonic splanchnopleura explants was followed by a two-step clonal in vitro culture system with a readout for erythroid-myeloid, B- and T-lymphoid cells (Fig. 3b). It was found that the intraembryonic splanchnopleura autonomously produces such progenitors beginning at E7.5. one full day earlier than they appear in the yolk sac. These studies not only showed that multipotential progenitors are present first in the embryo body, but also suggested that a more complex hematopoietic progenitor (one with potential for the lymphoid lineage, which is considered to be a more adult-type activity) is present at very early stages of mouse development. This progenitor activity could be demonstrated only in in vitro assays. No hematopoietic activity was detected after in vivo transplantation, demonstrating that these cells are not fully potent adult-type HSCs. As shown in Fig. 3c, organotypic cultures were also used to identify the first source of definitive CFU-S and HSCs (Medvinsky and Dzierzak, 1996). Yolk sac, AGM. and liver tissues from E9. E 10. and E 11 conceptuses were cultured as explants at the air-medium interface for 2 - 4 days and assayed by in vivo transplantation for short-term and long-term hematopoietic repopulation. At early El0 (32-33 somites), CFU-S appear simultaneously in both yolk sac and AGM explants. However, HSCs are detected only in AGM explants from mid-E10 embryos (>34 somites). No HSCs are found in El0.5 yolk sac explants or E9 AGM or yolk sac explants. Thus, the AGM region autonomously generates the first HSCs. Interestingly, while the frequency of HSCs in directly transplanted El0.5 AGM regions is very low [only 3 of about 100 recipients receiving one embryo equivalent of AGM cells is repopulated by HSCs (Muller et aL, 1994)], it dramatically increases after explant culture (24 out of 27 recipients are repopulated by HSCs). Therefore. HSCs continue to be induced or expand during the culture period independently of influx from other embryonic tissues. HSCs are found in explants of yolk sac and liver at E 1 I, suggesting that these tissues may be colonized by AGM-generated HSCs. Alternatively, HSCs may emerge autonomously in the yolk sac, but one day later than the AGM. In summary, the PAS/AGM region in the mouse is the most potent and first autonomous source of multipotential hematopoietic progenitors and adult-type HSCs, strongly suggesting that the mammalian PAS/AGM, like that of nonmammalian vertebrates, is a major source of cells leading to adult hematopoiesis. Hence, strong evolutionary conserva-

199 tion in the origins of embryonic and adult hematopoietic cells exists across vertebrate species (Dzierzak et al., 1998; Zon. 1995).

3. Embr?'onic to Definitive Hematopoietic HierarchymEvidence for Multiple Hematopoietic Births In the early/midgestation mammalian embryo, terminally differentiated hematopoietic cells appear temporally before hematopoietic progenitors, and these progenitors in turn appear before HSCs (Fig. 4). Although the adult hematopoietic system is thought to derive solely from the HSCs (Fig. 1), the hematopoietic system in the mammalian embryo must have alternative pathways leading to the rapid and early production of functionally differentiated blood cells. Indeed, primitive erythroid cells are found in the yolk sac of the mouse embryo within 1 day of gastrulation and generation of the first mesoderm (Kaufman, 1992; Russell, 1979), r~aling out the possibility that they differentiate through a conventional HSC. Thus, our concept of the a:dult hematopoietic hierarchy as a continuum from HSCs to functional blood cells does not provide a logical framework for hematopoietic cell development in the early embryo. Instead. hematopoietic development in the embryo appears to progress via successive births of multiple independent lineages of cells with progressively more complex hematopoietic activities (Fig. 4). Depending on the position and timing of birth, properties such as high proliferative potential, the presence of certain homing receptors, and the ability to self-renew may be sequentially and/or differentially attained. In other words, between E7.5 when the first primitive erythrc,cytes appear, and El0.5 when the first HSCs appear, the successive waves of hematopoietic progenitors Chat emerge seem to be less "differentiated" or "less mature," as defined by the adult hematopoietic scheme (Fig. 1). Progenitors committed to a single lineage appear first in development followed by bipotential progenitors, then multilineage progenitors, CFU-S, neonatal repopulating cells, and finally at E 10.5 adult HSCs. There is strong evidence for at least tvvo independent origins of hematopoiesis in nonmammalia:n and mammalian vertebrates (yolk sac and PAS/AGM). In addition, the vitelline and umbilical arteries in the mouse may be additional sites from which hematopoietic ceils are autonomously generated. This is based on the recent demonstration that CFUS and HSCs are present in these vessels beginning at E10.5 (de Bruijn et al., 2000a,b). In the avian embryo it was recently demonstrated that the allantois is also a hematopoietic site (Caprioli et al., 1998). It is speculated that a close association of mesoderm and endodermal cells is necessary for hematopoietic generation (Pardanaud and Dieterlen-Lievre, 1999). Using a transgenic mouse explant culture system, it was shown that primitive endoderm adjacent to embryonic ectoderm or nascent mesoderm leads to the production of hematopoietic cells and cells with endothelial markers

200 (Belaoussoff et al., 1998). If indeed a close association of certain mesodermal components in direct or indirect contact with certain endodermal components can lead to the generation of the hematopoietic lineage, it is possible that more sites of hematopoietic birth in the mammalian embryo remain to be uncovered. In summary, unlike the adult hematopoietic system that is harbored in mainly one site. the bone marrow, the embryonic hematopoietic system appears to emerge through multiple births in anatomically distinct sites. 4. Mesodermal Precursors to the

Hematopoietic Lineage The hemangioblast theory of hematopoietic cell generation is a long-held notion of a common mesodermal source for both the hematopoietic and endothelial lineages in the yolk sac blood islands (Murray'. 1932: Sabin. 1920). At present, embryonic stem (ES) cells are the best experimental system for studying hemangioblasts (Choi et al.. 1998). Differentiating ES cells contain a unique precursor population, which in response to vascular endothelial growth factor (VEGF) gives rise to blast colonies in semisolid culture medium. Cells from blast colonies can generate both hematopoietic and endothelial cells in Htro. suggesting that blast colony-forming cells are hemangioblasts. It is interesting to speculate that hemangioblasts exist not only in the yolk sac but also in PAS/AGM. However unlike the yolk sac in which endothelial cells and primitive erythrocytes appear almost simultaneously, in the PAS/AGM endothelial cells differentiate and form functional arteries before hematopoietic progenitors such as CFU-S and HSCs emerge. Nevertheless, a close developmental link is seen between endothelial cells and hematopoietic cells in PAS/AGM. Clusters of hematopoietic cells are found on the ventral wall of the dorsal aorta in the AGM region, and in the vitelline and umbilical arteries in close association with the endothelium, at the time when CFU-S and HSCs appear in these sites (Cormier and Dieterlen-Lievre, 1988: Garcia-Porrero et al., 1995" Tavian et al., 1996: Wood et al.. 1997: Medvinsky et al., 1993" Muller et al., 1994: de Bruijn et al., 2000a,b). These clusters have been seen in many vertebrate species ranging from sharks to humans. The hematopoietic clusters are arranged in 1-2 rows along the lumenal endothelial lining of these vessels, and some cells at the base of the cluster form tight junctions with the endothelial cells (GarciaPorrero et al.. 1995). Studies in avian and amphibian embryos suggest that the intra-aonic clusters and endothelial cells on the floor of the dorsal aorta share a common precursor in the splanchnopleural mesoderm (Pardanaud et al., 1996) and are derived from the same early stage blastomeres (Ciau-Uitz et al.. 2000), respectively:. The regulation of endothelial and hematopoietic cell surface markers on endothelial cells and cells in the clusters also suggests a precursorprogeny relationship. For example, before the hematopoietic

II Lineage Specification and Differentiation clusters appear in the chick, all the endothelial cells lining the dorsal aorta express VEGF-R2, the receptor for the vascular endothelial growth factor. As clusters appear, the cells in the clusters no longer express VEGF-R2, but instead express the pan-leukocyte marker CD45. while the endothelial cells in the dorsal aspect of the aorta continue to express VEGF-R2 (Eichmann et al., 1996: Jaffredo et al., 1998 Pardanaud er aI., 1987, 1989). Speculation on these descriptive data have led to the notion that hematopoietic cells bud from the endothelium rather than develop directly from a hemangioblast. In this scenario hemangioblasts could adopt an endothelial fate but retain plasticity for the hematopoietic lineage. An alternative hypothesis is that mesenchymal cells/ hemangioblasts located between the endothelial cells or beneath the aorta may be the direct precursors to the emerging hematopoietic clusters. Two recent experiments support the hypothesis that hematopoietic cells can differentiate directly from endothelial cells. Fate mapping experiments in avian embryos suggest that the hematopoietic clusters on the ventral wall of the dorsal aorta differentiate from hemogenic endothelium (Jaffredo et al.. 1998, 2000). Low-density lipoprotein (LDL) linked to the lipophilic fluorescent dye DiI was injected into the heart cavity of chick embryos in order to specifically mark the endothelial cells lining the vascular tree. The LDL-DiI injections were performed at Hamburger and Hamilton (HH) stage 13, 1 day before hematopoietic clusters appear. DiIpositive cells were found in hematopoietic clusters 2 4 - 4 8 hr after the endothelial cells were labeled, suggesting that the hematopoietic clusters are the progeny of the marked endothelial cells. CD45-positive cells that have ingressed into the mesenchyme ventral to the dorsal aorta were also DiI positive, suggesting that endothelial cells are capable of generating cells that migrate from the endothelium both into and away from the aortic lumen. It was demonstrated previously that chick embryos contain diffuse hematopoietic foci in the mesentery underlying the ventral part of the aorta (Dieterlen-Lievre and Martin, 1981). Diffuse loci of cells that express the hematopoietic markers GATA-3, LMO2, and AA4.1 were also found in the mesenchyme ventral and lateral to the aorta in the early stage mouse embryo (Manaia et al., 2000: Petrenko et al., 1999). At present, the function and significance of the para-aortic loci expressing these markers in the mouse are unknown. The second experiment that supports the notion of a hemogenic endothelium is that VE-cadherin-positive endothelial cells, when sorted from mouse embryos and plated on OP9 stromal cells, yield hematopoietic cells in vitro (Nishikawa er al., 1998). VE-cadherin is expressed at the adherens junctions that connect adjacent endothelial cells and is thought not to be expressed on hematopoietic progenitor cells. Finally, the expression of common markers on endothelial cells and hematopoietic cells supports the notion of a close developmental link between these lineages in both hu-

10 Vertebrate Hematopoietic System man and mouse embryos (Table II). The cell surface sialomucin CD34. adhesion molecule CD31. and the ilk-1 receptor tyrosine kinase are expressed by both hematopoietic clusters and endothelial cells but generally not by mesenchyreal cells (Wood et al., 1997: Tavian et al., 1996. 1999: Labastie et al., 1998: Cai et aI., 2000). The transcription factors SCL and GATA-2 are also expressed by hematopoietic cell clusters and endothelial cells (Tavian et aI., 1996, 1999 Labastie et al., 1998). Expression of the extracellular matrix glycoprotein tenascin C in the human embryo is concentrated along the ventral endothelium as well as in hematopoietic clusters (Marshall et al., 1999). In the mouse, one recently described marker is expressed only by the endothelial cells in the ventral wall of the dorsal aorta where hematopoietic clusters are localized, in some of the mesenchymal cells underlying this region, and in the hematopoietic cell clusters themselves (North et al., 1999). This marker is the AML1 (Runxl or CBFa2)transcription factor that was shown to be required for definitive adult hematopoiesis (Okuda et al., I996" Q. Wang et al., 1996a). AML1 is spatially and temporally expressed in sites with demonstrated functional HSCs, suggesting that HSC activity is derived from the hematopoietic clusters, the mesenchymal cells and/or endothelial cells on the ventral part of the dorsal aorta (North et al., I999). Furthermore, AML1 is required for the formation of the intra-aortic hematopoietic clusters (North et al., 1999). The study of AML 1 expression, in combination with that of other more general markers, appears to be promising in deciphering the precursor-progeny relationships of the cells within this complex region. What types of hematopoietic activities do lumenal clusters contain? In the chick, in vitro CFU-Cs were demonstrated to be present in the para-aortic cluster region (Cormier and Dieterlen-Lievre. 1988). While the grafting experiments revealed that the avian adult hematopoietic system originates from the region of the dorsal aorta, the exact location of the the HSCs within the aortic region was not shown. Recent subdissections of the mouse AGM region reveal that the dorsal aorta and its surrounding mesenchyme are indeed the first source of adult-type HSCs (de Bruijn et al., 2000b). As described above, the marker expression patterns attributed to HSCs can be found in the aortic hematopoietic clusters. However, marker expression within the cell clusters is heterogeneous at least for one molecule, glycoprotein IIb-IIIa, suggesting that the clusters contain several types of hematopoietic cells (Ody et al., 1999). Until unique and specific markers for the endothelium, mesenchyme, and cells in the aortic clusters in the AGM region become available, it will not be possible to determine which of these cells types is the source of HSCs.

5. Colonization Theol. of Hematopoiesis How do secondary tissues such as the thymus, spleen, liver, and bone marrow become hematopoietic? Much of our

201 current thinking is influenced by the observations made in I967 by Moore and Owen. Using parabiosed chick embryos, these investigators found that hematopoietic tissues were colonized by cells delivered through the blood. Definitive proof that hematopoietic cells in adult tissues are extrinsically derived came from grafting studies where it was found that waves of hematopoietic activity entered and colonized thymus and spleen rudiments of avian embryos during receptive periods (Dieterlen-Lievre, 1975 Martin et al., 1978). Thus thymus and spleen provide the microenvironment for the seeding and differentiation of extrinsic precursors. The sites of de n o v o hematopoietic cell emergence ','ere found to be both the yolk sac and the intraembryonic region containing the dorsal aorta. Interestingly, during embryonic stages a small number of intrabody-derived hematopoietic cells can be found in the yolk sac and a small number of yolk sacderived macrophage-like (microglial) cells can be found intraembryonically (localized to the eye and brain) (Pardanaud et al., 1987). Thus, migration is not unidirectional and colonization is thought to occur through the circulation of small populations of hematopoietic cells. Similarly, recent experiments in which the prevascutarized allantoic bad from quail embryos was grafted into the coelom of chick: embyros resulted in hematopoietic and endothelial cells of quail origin in the bone marrow of the chick host (Caprioli et al., 1998). These data suggest that the adult hematopoietic bone marrow is also colonized by precursors that arise in situ in the allantois. In mammals, Cudennec and colleagues (1981) performed one of the experiments that strongly suggested the liver is colonized by cells from the yolk sac. The inclusion of a cell nonpermeabte filter between yolk sac and live.r explants in culture blocked the influx of cells between the tissues and resulted in no erythropoietic activity in the liver. Colonization was also demonstrated in ma in vivo mouse model system in which fetal liver rudiments were implanted under the kidney capsule of adult mice. After several days the liver rudiments were examined and found to be colonized by adult hematopoietic cells, thus demonstrating the liver's receptivity to exogenous hematopoietic cells (Johnson and Moore, 1975). These early experiments indicate that hematopoietic colonization of secondary territories most likely plays an important rote in mammalian development. A summary of the possible colonization events occurring within the mammalian embryo during ontogeny is shown in Fig. 4.

6. Fate Mapping Largely absent from all of the studies performed on mammalian embryonic hematopoietic cells is a cleax demonstration of precursor-progeny relationships. As emphasized in the previous sections, fate mapping is necessary to determine the true precursor(s) of hematopoietic cells, whether they are hemangioblast, mesenchymal, endothelial, or even other hematopoietic cells. It is possible, for example, that CFU-S at

202

II Lineage Specification and Differentiation

E 1 1 and E12 are derived from HSCs, whereas CFU-S at E9 and E 10 are derived from mesenchymal, hemangioblast, or endothelial cells (de Bruijn et al., 2000a). Without fate mapping we cannot unambiguously determine if the first HSCs observed at E 11 in the fetal liver are derived from cells that originated in the PAS/AGM region or from yolk sac hematopoietic progenitors that acquired HSC characteristics in the fetal liver. Injections of cells into embryo or adult recipients reveal the potential of cells, but do not illuminate the developmental process as it occurs in the embryo. For example, it was shown that E8 yolk sac cells injected transplacentally (Toles et al., 1989) or into the amnionic cavity (Weissman et al.. 1978) of early stage mouse embryos results in contribution of yolk sac donor cells to the adult hematopoietic system. These experiments demonstrate that E8 yolk sac cells have the potential to contribute to adult hematopoiesis in these experimental settings, but they do not show that E8 yolk sac cells normally contribute to the adult hematopoietic system in an intact organism. While injection of E9 yolk sac cells into the hernatopoietically active livers of neonatal mice uncovers their long-term multilineage potential (Yoder et al., 1997), without fate mapping we cannot conclude that the fetal liver plays a role in the maturation of yolk sac progenitors into HSCs. Similarly, direct injection of AGM cells into adult recipients demonstrates that the AGM harbors HSCs. However. it remains possible that the adult hematopoietic system is de n o v o generated by colonization of the bone cavities at later stages of gestation by hemangioblasts that emerge from the AGM or from elsewhere in the embryo. Fate mapping will help resolve issues such as precursorprogeny relationships, homing, colonization, and transient versus long-lived functional hematopoiesis. Cre-LoxP fate mapping may help to resolve this issue.

III. Molecular Genetic Aspects of Blood Development in the Mouse Embryo

A. Developmental Regulation of Hematopoiesis The molecular genetic basis of hematopoietic development has been of great interest starting from the time when the human/3-globin gene was cloned. Since then a wealth of genes important for various aspects of hematopoiesis has been isolated. These include genes encoding g o w t h factors and their receptors, signaling molecules, transcription factors, homing/adhesion molecules, as well as proteins like the globins and immunoglobulins that are involved in specific erythroid and lymphoid cell functions, respectively. Much insight and many general molecular genetic paradigms have emerged from the studies of lineage-specific transcription factors and. in particular, the developmental regulation of the globin gene locus.

The globin gene program exemplifies a tightly controlled system of developmentally expressed genes. The human/3globin locus on chromosome 11 is organized as a linear array of the emb~'onic E, fetal cy and AT and adult/3 genes ordered along the DNA in the sequence in which they are developmentally expressed (Grosveld et al., 1993). Analyses of promoter regions and enhancers have revealed consensus sequences for putative DNA binding proteins. Many erythroid-specific DNA binding proteins such as GATA-1, EKLE and Sp-1 have been isolated and shown to be important for the expression of these globin genes and in some cases other erythroid-specific genes (reviewed in Orkin, 1995). Moreover. the strict developmental regulation of embryonic, fetal, and adult globin genes in e~thropoiesis is controlled by a powerful regulatory element called the locus control region (LCR). The globin gene expression program may be determined intrisically by the hematopoietic precursor/stem cell or may be influenced by the surrounding microenvironment or both. Today these issues are still controversial and efforts are under way to link gene activity in the nucleus with signals emanating from the inside or outside of the ceil. Genetic studies on the macromolecular level are a focus of attention in the hematopoietic lineages. Chromatin and chromosome structure is known to have important consequences for gene activity. For example, the globin LCR has been implicated in the formation of a holocomplex of transcription factors that maintains the chromatin accessibility of the globin locus in erythroid cells (Wijgerde et al., 1995; Fraser and Grosveld. 1998). It is thought that the chromatin structure is important in the commitment or maintenance of plasticity in specific lineages of cells. Finally, metaphase spreads of chromosomes from leukemic cells were instrumental in revealing macromolecular changes in genes important in hematopoiesis. The most frequent types of leukemias, acute myelogenous leukemias, were characterized by several common chromosomal translocations/inversions in hematopoietic progenitor cells, for example, t(15" 17), t(8" 21), inv(16) (Rowley, 1999). The chromosomal breakpoints were mapped and cloned and provided a means by which genes such as the A~v~I/CBFc~ transcription factor, involved in both normal and abnormal hematopoiesis, could identified and characterized further (Miyoshi et aL, 1991 Liu et aL, 1993). Thus, these molecular genetic studies performed at many levels have contributed enormously to our current understanding of developmental hematopoiesis.

B. Genetic Program for Hematopoiesis The genetic program of hematopoiesis is thought to involve a progressive cascade of gene expression starting from the first signals for hematopoietic lineage specification and proceeding through the final stages of functional blood cell

10 Vertebrate Hematopoietic System Table IV Target

Mesoderm

203

Genetic Requirements in Hematopoietic Development

YS hematopoiesis and endothelium

Fetal and adult hematopoiesis

Time

E7

E9.5-11.5

ElO-16

Gene

FGF TGF-/3:

TGF-/3: flk-! VEGF VE-cadherin SCL LMO2 c-mvb

SCL GATA-2 RunxI CBFb c-kit SCF

production. This cascade has many gene products that serve functions in several stages of hematopoiesis, and also in nonhematopoietic lineages. Examples of genes required for hematopoiesis are provided here (also see Table IV).

1. Genes Involved in Mesoderm Formation and Hematopoietic Specification A graded expression pattern of many genes acting as positive and negative signals (Stennard and Gurdon, 1997) is thought to define the normal spatial borders of hematopoiesis in different mesodermally derived regions of the conceptus. The transforming growth factor 13 (TGF-/3) and fibroblast growth forming (FGF) families of genes are pivotal in mesoderm and blood formation in X e n o p u s embryos (Dale et al., 1992: Smith and Albano. 1993). BMP4, a TGF-/3 family member, acts as a ventralizing molecule and induces the expression of Mix. 1, a gene that induces hematopoiesis in the Xenopus animal cap assay (Mead et al., 1996; Turpen et al., 1997). Mice deficient for BMP4 suffer embryonic lethality at the time of gastrulation, and have little or no mesoderm (Winnier et al., 1995). The few BMP4-deficient embryos that do survive to postgastrulation stages show profound decreases in mesoderm formation and erythropoiesis in the yolk sac, demonstrating that B MP4 is strictly required for the formation of ventral mesoderm. Indeed, when BMP4 is administered in vitro to presumptive anterior headfold tissue from the epiblast, hematopoietic cell production (in CFU-C assays) is induced (Kanatsu and Nishikawa, 1996). Overlapping with early mesodermal induction events and/ or slightly downstream, other gowth factors, receptors, and transcription factors were found to be critical for hematopoietic specification. Both endothelial cells and hematopoietic cells require TGF-/31. Perinatal lethality occurs in TGF-/3~deficient embryos between E9.5 and 11.5 (Dickson et al., 1995). Although initial endothelial cell differentiation begins, these cells do not form a vascular network. Mice null for the receptor tyrosine kinase ilk-1 (Kdr) also suffer early embryonic lethality between E8.5 and 9.5 (Shalaby et al., 1995). Flk-l-deficient embryos are defective in

Lineage specification

GATA-I (erythrocytes) Jak2 (erythrocytes) EKLF (er?throcytes) Ikaros (T lymphocytes) Pax5 (B lymphocytes)

Migration

/3~integrin c~ intergrin

Proliferation

LIF IL-6 flk-2/flt3

yolk sac blood island and vessel formation and hematopoietic progenitor numbers are reduced. Similar defects (although with slightly later embryonic lethality at E 11) are observed in mice null for the ilk-1 ligand, VEGF (Carme!iet et al., 1996; Ferrara et aI., 1996). Knock-in of a l a c Z marker gene into the ilk-1 locus revealed that l a c Z - e x p r e s s i n g cells accumulated in the amnions mad never entered into the areas of putative blood island formation in flk-I null embryos. FIk-1 is also required for formation of the adult hematopoietic system as revealed by chimeras :made from ilk-1 homozygous null ES cells (Shalaby et at., 1997). Targeting of the Sct gene demonstrated a requirement for the production of all hematopoietic lineages in the embryo (Robb et at., 1995; Shivdasani et al., 1995) and the adult (Porcher et aI., 1996; Robbet al., 1996). SCL is also required in the endothelial lineage. It is not required for formation of the small yolk sac vessels. However, it is required for angiogenesis and the formation of vitelline vessels connecting the embryo with the yolk sac (Visvader et al., 1998). Absence of the partner protein of SCL, LMO2, results in an identical phenotype (Warren et aL, 1994; Yamada et aI., 1998).

2. Gene Affecting Definitive and Fetal Liver Hematopoiesis After specification of cells to the hematopoietic lineage, genes involved in the generation, maintenance, self-renewal, and/or differentiation of definitive hematopoietic progenitors and stem cells come into play. The G ATA-2 transcription factor is important for the proliferation of hematopoietic progenitors (Tsai et aL, 1994). GATA-2-deficient embryos generate primitive erythrocytes but in low number, and definitive progenitors (CF1.J-Cs) in the yolk ,;ac are decreased 100-fold. GATA-2 null embryos exhibit severe fetal liver anemia and embryonic lethality at E 10.5. No contribution of GATA-2 null ES cells to the adult hematopoietic system of chimeric mice was observed, suggesting that GATA-2 acts in a cell autonomous fashion in hematopoietic progenitors at all stages of fetal and adult hematopoiesis.

204

Two genes in the family of core-binding factors (CBFs) are the most frequent targets of chromosomal rearrangements in human leukemias. AML1 (Runxl) and CBF/9 form a heterodimeric transcription factor that binds the core enhancer motif present in a number of genes expressed in hematopoietic cells. Both AML1 and CBF/3 were shown to be required for definitive hematopoiesis by' gene targeting experiments (Okuda et aI.. 1996: Sasaki et al.. 1996: Wang e ; al.. 1996a: Q. Wang et al.. 1996b). AML1 and CBF/3 null embryos die at E12.5 and suffer from severe fetal liver anemia. and HSCs and definitive hematopoietic progenitors are absent. Furthermore. a hemizygous dose of AML1 affects both the timing and spatial distribution of HSCs and CFU-S within the AGM and yolk sac at El0 and E l l (Cai et al.. 2000). AML1 is expressed in hematopoietic clusters, endothelial cells, and mesenchymal cells in the ventral aspect of the dorsal aorta, and in endothelial cells and hematopoietic clusters in the vitelline and umbilical arteries (North et al.. 1999). The AML 1CBF/3 heterodimer appears to be required for the generation, proliferation, and/or maintenance of the first HSCs as they emerge in the embryo, and perhaps for the differentiation of HSCs to committed definitive hematopoieric progenitors. Targeted disruption of the c - m v b proto-oncogene transcription factor also affects definitive fetal liver hematopoiesis but not yolk sac hematopoiesis (Mucenski et al.. 1991). Multipotential granulocyte-macrophage progenitors and adult erythrocytes are decreased ( L i n e t al.. 1996) but other hematopoietic lineages and hematopoiesis in adult chimeric mice must be examined to determine whether a progenitor or stem cell is affected. The available data suggest that c-mvb acts downstream of the GATA-2 and A M L I CBF/3 transcription factors in definitive hematopoiesis. Two of the most widely used naturally occurring mouse hematopoietic mutants are the dominant white spotting (W) and Steel (S1) mouse strains. W mice are affected in CFU-S and mast cell production. The most severe W alleles result in embryonic lethality at around El6. While the S1 mouse strain has virtually the same phenotype, transplantation studies originally showed that these strains complement each other for CFU-S development. The W strain is defective for the c-kit receptor tyrosine kinase, which is expressed on HSCs, and the mutation in the S1 strain disrupts the gene encoding the c-kit ligand [also called steel or stem cell factor (SCF)], which is expressed by stromal cells (Morro et al., 1991 Keshet er al., 1991 and reviewed in Bernstein, 1993: Fleischman. 1993).

3. Genes Affecting Lineage Specification and/or Differentiation One of the major branch points of the hematopoietic hierarchy is where the lymphoid lineage diverges from the e~throid-myeloid lineage (Fig. 1). Two transcription factors near this branch point are Ikaros and Pax5, which are impli-

!i Lineage Specification and Differentiation cated in the specification of the T- and B-lymphoid lineages, respectively. Ikaros null mice display defects that differentially affect the development of fetal and adult lymphocytes, in that T lymphocytes are absent from the fetus but present in the adult (J. H. Wang et al., 1996). B lymphocytes on the other hand. are absent from both the fetus and the adult. The T lymphocytes in Ikaros null mice that are found postnatally display defects in 2/6 T cells and thymic dendritic cells. ProB cells from Pax5 null mice are capable of differentiating into many hematopoietic lineages on transplantation into recipient mice. but do not give rise to B-lymphoid cells, suggesting that Pax5 plays an essential role in B-lineage commitment by' suppressing alternative lineage choices (Nutt e ; al., 1999). Gene targeting has identified many genes essential in the erythroid lineage differentiation program. The most essential erythroid-specific transcription factor is GATA-1 (Fujiwara e t al.. 1996: Pevny et al., 1991). which is required for the production of both primitive and adult erythrocytes. The GATA- I null defect has been localized to the proerythroblast stage of differentiation, leading to anemia and embryonic lethality around El0. The Jak2 kinase signal transduction molecule is required for definitive but not primitive erythropoiesis. Targeted disruption of Jak2 results in embryonic lethality at E 12.5. with no detectable BFU-E or CFU-E in the fetal liver. Targeted disruptions of the EKLF transcription factor, erythropoietin growth/differentiation factor, and the erythropoietin receptor genes result in embryonic lethality due to the lack of definitive erythropoiesis. These defects manifest themselves slightly later than those in Jak2 mutants in that lethality due to fetal liver anemia occurs at E14-16, at the time of the switch from fetal to adult erythropoiesis.

4. Genes Affecting Hematopoietic Cell Migration Integrins have been found to play a role in the differentiation and migration of hematopoietic cells, presumably by promoting adhesive interactions between hematopoietic cells and supportive stromal cells. Hematopoietic ceils generated in intra- and extraembryonic sites during midgestation are thought to seed the secondary hematopoietic territories: fetal liver, spleen, thymus, and bone marrow. Gene targeting revealed an important role for the/3~ integrin in seeding the fetal liver. Since/31 integrin null embryos die during preimplantation stages (Fassler and Meyer, 1995 Stephens et al., 1995) embryos chimeric for/3~ integrin null ES cells were generated. These experiments revealed that although a normal number of/3~ integrin null hematopoietic cells were found in the yolk sac and circulation, none were found in the fetal liver, thymus, or bone marrow, suggesting that/9~ integrin is necessary for seeding these secondary hematopoietic sites. Likewise, chimeric mice made with o~aintegrin null ES cells revealed that c~: integrin is required for the homing of B and T lymphocytes to adult hematopoietic territories, but not to the fetal liver (Arroyo et at., 1996).

10 Vertebrate Hematopoietic System 5. Genes Thought to Affect the Proliferative Potential of Hematopoietic Progenitor/Stem Cells The adult hematopoietic system can arise from a single clonally marked transplanted HSC (Lemischka et at., 1986) and hence does not absolutely require the simultaneous function of all HSCs. It appears that a balance between selfrenewal and proliferation of HSCs is maintained. Several genes have been implicated through targeted gene disruption to affect the balance between HSC proliferation and selfrenewal. Mice lacking the growth factors IL-6 or LIE or the flk-2/flt-3 receptor tyrosine kinase do not suffer from embryonic lethality or the absence of definitive hematopoietic cells. However. subtle phenotypes are observed after transplantation into normal adult recipients. LIF-deficient mice have decreased numbers of CFU-S and other clonogenic progenitors (Escary et al., 1993), while IL-6 deficient mice have a decrease in CFU-S progenitor numbers and potency of HSCs (Bernad et al., 1994). In contrast, flk-2/flt-3 null mice are defective in myeloid and lymphoid reconstitution but not in CFU-S or pre-CFU-S numbers (Mackarehtschian et al.. 1995). Thus. these genes affect maintenance, proliferative potential, and/or self-renewal of hematopoietic progenitors and/or stem cells.

IV. Current Cellular and Molecular Conceptual Frameworks for Hematopoietic Ontogeny A. Origins General concepts conserved throughout evolution are evident from the experimental studies of hematopoiesis in vertebrate animal models. The vertebrate hematopoietic system originates within the mesodermal germ layer and its progeny. This property is conserved in invertebrates as well. Drosophila embryos have two major types of hematopoietic cells, plasmatocytes and crystal cells, and both cell types differentiate from hemocyte precursors located in the head mesoderm of the Drosophila embryo (Tepass et al., 1994). In vertebrates, multiple births of the hematopoietic system occur in anatomically distinct mesodermal sites of the embryo, including the yolk sac, PAS/AGM, and the allantois (the last has been demonstrated in avian but not in murine embryos). The vitelline and umbilical vasculature may also generate hematopoietic cells, and it is speculated that other yet unknown sites may be hemogenic. At this time, most researchers in the field believe that hemangioblasts are a common precursor for at least some hematopoietic and endothelial lineages. But there may be several types of hemangioblasts (or hemogenic precursors) that originate in different anatomical sites during ontogeny. Indeed experiments in avian and mouse embryos have strongly suggested that some en-

205 dothelial cells are direct precursors of hematopoietic cells. It is also possible that hemogenic potential :is harbored in mesodermal/mesenchymal cells. It is important to identify the immediate precursors of hematopoietic cells, and to determine if hematopoietic activities are sequestered in some unexpected sites in the adult.

B. Colonization Colonization of secondary hematopoietic organs is an important feature of developmental hematopoiesis. Sequential colonization events suggest that the growing embryo needs additional territories to expand hematopoietic cells. Colonization of the fetal liver has been a focus of interest in early stages of hematopoiesis since the appearance of the different hematopoietic activities in this tissue parallels their birth in other sites in the mouse embryo. The first cells colonizing the fetal liver are primitive erythroid cells, followed by committed progenitors, CFU-S, multipotential progenitors, and finally HSCs. In general, these activities appear in the fetal liver I - 2 days after they are detected in the yolk sac and PAS/AGM. These observations strongly suggest that waves of hematopoietic activity seed the liver after migrating from their originating source(s). While it is generally accepted that the secondary hematopoietic tissues are colonized by hematopoietic cells generated elsewhere in the embryo, in vivo clonal fate mapping has not been performed. It remains possible that within hematopoietic sites that appear later in development, such as the bone marrow, commitment to the hematopoietic lineage may occur from mesodermal cells or hemangioblasts seeding this tissue rather than from the definitive HSCs generated in the AGM.

C. Genetic Programming Gene targeting studies in the mouse have revealed a complex genetic cascade that can be generally classified into important developmental milestones. These milestones include formation of the mesoderm, hematopoietic specification, the emergence of primitive and definitive hematopoietic progenitors, and the generation of complex hematopoietic characteristics such as multilineage capability, self-renewing ability, homing to specific hematopoietic territories, and/or high proliferative potential. A major decision point appears to be at the crossroads of primitive embryonic hematopoiesis and definitive adult hematopoiesis as the PAS/AGM becomes maximally active and the fetal liver becomes a secondary hematopoietic territory. Clearly, the initial molecular programs of primitive and definitive hematopoietic cells overlap in their requirements for genes inducing mesoderm formation, some hematopoietic genes, and even some genes for terminal differentiation (particularly in the erythroid lineage). However, the definitive hematopoietic program is much more complex, requiring an abundance of unique

!i Lineage Specification and Differentiation

206

genes that promote the establishment of the complete multilineage adult hematopoietic hierarchy. Do these genetic programs have a basis in the anatomically separate and independent sites of hematopoietic generation in the conceptus, are they intrinsic to the mesodermal precursors, or are they regulated by the microenvironment? Further studies of the embryonic localization and specific microenvironments required for hematopoietic progenitor/stem cells should allow for the isolation of novel genes and molecules that may be useful for manipulating human hematopoietic cells in a clinical setting. In the future, a database of all expressed sequences in HSCs isolated from bone marrow, fetal liver, and AGM promises to further our understanding and manipulation of the genetic program of HSCs throughout ontogeny (Phillips et al., 2000).

D. Redirecting the Fate of Stem Cells Only a few years ago it was accepted that stem cells for tissues such as blood, skin. gut. muscle, and neurons in the adult vertebrate were restricted in fate. and able to contribute through differentiation to only' one somatic lineage. This paradigm was recently challenged by demonstrations that stem cells isolated from one tissue can form functional cells of another tissue. For example, it has been found that bone marrow-derived cells can contribute to a phagocytic cell type in the brain called microglia (Theele and Streit. 1993) with a small percentage of cells also developing into astrocytes (Eglitis and Mezey, 1997). The reciprocal experiment, testing whether neural stem cells could change fate. found that indeed neural stem cells injected into the circulation of irradiated adult mice contributed to blood cell formation (Bjornson et al., 1999). Thus, the blood cell microenvironment appears capable of redirecting the program of neural stem cells to the blood cell fate. Furthermore. human mesenchymal stem cells, which are thought to be multipotent cells in the adult marrow, replicate as undifferentiated cells in culture but can be induced to differentiate into adipocytic, chondrocytic, or osteocytic lineages (Pittenger et al., 1999). Most recently, bone marrow cells have been shown to take on muscle characteristics (Ferrari et al., 1998; Gussoni et al., 1999) and muscle cells to take on hematopoietic characteristics (Jackson et al., 1999) when transplanted in vivo into adult recipient mice. Despite the use of cell populations in these studies, it is interesting to speculate that what were formerly thought to be tissue-restricted stem cells are in fact multipotential cells. Perhaps common features in the genetic programming of the many types of stem cells (hematopoietic stem cells, neural stem cells, mesenchymal stem cells, muscle stem cells) will be found and reveal genes that maintain these cells in an uncommitted state. In such a scenario, plasticity of all these stem cells is maintained and it is the inductive microenvironment that determines the ultimate cell

fate. The precise mechanisms of stem cell maintenance and induction remain to be determined.

V. Future Directions The future of developmental hematopoietic studies in mammals will rely on fate mapping linked to functional hematopoietic readouts. Until now only avian and amphibian embryo grafting methods were efficient for fate mapping. However. whole mouse embryo culture is improving and can be used in DiI marking studies and clonal retroviral marking. Transgenic mouse approaches hold promise for both fate mapping studies and deleting genes in specific cell lineages at defined developmental time points. The cre-lox recombination system together with the induced expression of the mutated ligand binding domain of the estrogen receptor is a two-tiered method of controlling recombination in the targeted population and requires no in ~,itro manipulation. Thus. targeted hematopoietic cells can be followed through all developmental stages in vivo, from their sites of origin to sites of colonization. The genetic programming of hematopoietic cells at all stages of development is only in its infancy. The complex levels of interacting gene programs will become increasingly apparent as more genes are cloned and as more hematopoietic mutant mice are generated. Furthermore, the genes that retain the self-renewing properties and plasticity of stem cells will be important for understanding how stem cells are sequestered throughout life. In summary, the study of hematopoietic development has contributed to many paradigms in stem cell biology and will continue to be on the forefront of stem cell biology through its focused examination of a complex developmental problem. Clinical applications are sure to follow.

References Arroyo. A. G.. Yang. J. T., Rayburn. H.. and Hynes, R. O. (1996). Differential requirements for alpha4 integrins during fetal and adult hematopoiesis. Cell (Cambridge. Mass.) 85, 997-1008. Beaupain, D.. Martin. C.. and Dieterlen-Lievre.F. ( 1979)..awedevelopmental hemoglobin changes related to the origin of stem cells and site of ery'thropoiesis? Blood 53, 212-225. Belaoussoff. M.. Farrington, S. M.. and Baron, M. H. (I998). Hematopoieric induction and respecificationof A-P identity by visceral endoderm signaling in the mouse embry'o. Development (Cambridge, UK) 125, 5009-5018. Bernad. A.. Kopf, M.. Kulbacki. R.. Weich. N.. Koehler. G., and GutierrezRamos. J. C. (1994). Interleukin-6is required in vivo for the regulation of stem cells and committed progenitors of the hematopoieticsystem. Immuni~" 1,725-731. Bernstein. A. (1993). Receptor tyrosine kinases and the control of hematopoiesis. Semin. Dev. Biol. 4, 351-358.

10 Vertebrate Hematopoietic System Bjornson. C. R.. Rietze. R. L.. Reynolds, B. A.. Magli, M. C.. and Vescovi. A. L. (1999). Turning brain into blood: A hematopoietic fate adopted by adult neural stem cells in vivo. Science 283, 534-537. Bonifer. C.. Faust. N.. Geiger. H.. and Muller. A. M. (1998). Developmental changes in the differentiation capacity of haematopoietic stem cells, lmmunol. Today 19, 236-241. Breems. D. A.. Blokland. E. A.. Siebel. K. E., Mayen, A. E.. Engels. L. J., and Ploemacher. R. E. (1998). Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34 + mobilized peripheral blood stem cells. Blood 91, 111 - 117. Cal. Z. L.. de Bruijn. M., Ma, X.. Dortland. B.. Luteijn, T.. Downing. J. R.. and Dzierzak. E. (2000). Haploinsufficiency of AML1/CBFA2 affects the temportal and spatial generation of hematopoietic stem cells in the mouse embr)'o. Immunity" 13, 4 2 3 - 431. Caprioli. A.. Jaffredo, T.. Gautier. R.. Dubourg. C., and Dieterlen-Lievre. E (1998). Blood-borne seeding by hematopoietic and endothelial precursors from the allantois. Proc. NatL Acad. Sci. UIS.A. 95, 164I-I646. Carmeliet. R. Ferreira. V.. Breier. G., Pollefeyt. S.. Kieckens, L.. Gertsenstein. M.. Fahrig. M., Vandenhoeck. A.. Harpal, K.. Eberhardt. C.. Declercq. C.. Pawling. J., Moons. L.. Collen, D., Risau. W.. and Nagy, A. (1996). Abnormal blood vessel development and lethality in emb&'os lacking a single VEGF allele. Nature (London) 380, 435- 439. Choi. K.. Kennedy. M., Kazarov, A.. Papadimitriou. J. C.. and Keller. G. (1998). A common precursor for hematopoietic and endothelial cells. Development (Cambridge. UK) 125, 725-732. Ciau-Uitz. A.. Walmsley. M.. and Patient. R. (2000). Distinct origins of adult and embr?'onic blood in Xenopus. Ceil (Cambridge, Mass.) 102, 787-796. Cormier, F.. and Dieterlen-Lievre. E (1988). The wall of the chick emb~'o aorta harbours M-CFC. G-CFC. GM-CFC and BFU-E. Development (Cambridge, UK) 102, 279-285. Cudennec. C. A.. Thief. J. P., and Le Douarin, N. M. (1981). In vitro induction of adult e~thropoiesis in early mouse yolk sac. Proc. NatL Acad. Sci. U.S.A. 78, 2412-2416. Cumano. A.. Paige. C. J.. Iscove. N. N., and Brady, G. (1992). Bipotential precursors of B cells and macrophages in murine fetal liver. Nature (London) 356, 612-615. Cumano. A.. Dieterlen-Lievre. F.. and Godin. I. (I996). Lymphoid potential. probed before circulation in mouse, is restricted to caudal intraemb~'onic splanchnopleura. Cell (Cambridge, Mass.) 86, 907-916. Dale. L.. Howes. G.. Price, B. M., and Smith. J. C. (1992). Bone morphogenetic protein 4: A ventralizing factor in early Xenopus development. Development (Cambridge. UK) 115, 573-585. de Bruijn. M. R. T. R.. Peeters. M. C. E., Luteijn. T.. Visser, P., Speck, N. A., and Dzierzak. E. (2000a). CFU-S~ ~activity does not localize solely with the aorta in the AGM region. Blood 96, 2902-2904. de Bruijn. M. R. T.. R.. Speck. N. A.. Peeters. M. C. E.. and Dzierzak. E. (2000b). Definitive hematopoietic stem cells first emerge from the major arterial regions of the mouse embu'o. EMBO J. 19, 2465-2474. Delassus, S., and Cumano. A. (1996). Circulation of hematopoietic progenitors in the mouse emb~'o. Immunio" 4, 97-106. Dickson. M. C.. Martin, J. S.. Cousins, F. M., Kulkarni, A. B., Karlsson. S.. and Akhurst. R. J. (1995). Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 "knock out mice. Development (Cambridge. UK) 121, 1845-1854. Dieterlen-Lievre. E (1975). On the origin of haemopoietic stem cells in the avian emb~o: An experimental approach. J. Embo'oL Exp. MorphoL 33, 607-619. Dieterlen-Lievre. F.. and Le Douarin, N. M. (1993). Developmental rules in the hematopoietic and immune systems of birds: How general are they? Semin. Dev. Biol. 4, 325-332. Dieterlen-Lievre. E, and Martin, C. (1981 ). Diffuse intraembryonic hemopoiesis in normal and chimeric avian development. Dev. Biol. 88, 180191.

207 Dzierzak, E., Medvinsky. A., and de Bruijn, M. (t998). Qualitative and quantitative aspects of haemopoietic cell development in the mammalian embryo. Immunot. Today 19, 228-236. Eglitis, M. A., and Mezey, E. (i997). Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc. Natl. Acad. Sci. U.S.A. 94, 4080-4085. Eichmann, A., Marcelte. C.. Breant, C., and Le Douarin. N. M. (1996). Molecular cloning of Quek I and 2, two quail vascular endothelial m'owth factor (VEGF) receptor-like molecules. Gene 174,13-8. Erem R., Zharhar3", D.. Abel. L.. and Globerson. A. (t987). Ontogeny of T cells: Development of pre-T cells from fetal liver and yolk sac in the thymus microenvironment. Cell ImmunoL 108, 76--84. Escary. J. L.. Perreau. J., Dumenil, D.. Ezine, S., and Brulet, R (1993). Leukaemia inhibitop," factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation. Nature (Loz~don) 363, 361-364. Fasster, R.. and Meyer. M. (1995). Consequences of lack of beta I integrin gene expression in mice. Genes Dev. 9, I896-1908. Ferra_ra. N., Carver-Moore, K.. Chen. H.. Dowd, M.. k u, L., O'Shea. K. S.. Powell-Braxton. L.. Hillan. K. J.. and Moore, M. W. (I996). Heterozygous emb~'onic lethality induced by targeted inactivation of the VEGF gene. Nature (London) 380, 439-442. Ferrari. G.. Cusella-De Angelis. G.. CoIetta, M.. Paolucci. E.. Stornaiuolo. A.. Cossu, G., and MaviIio, E (1998). Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279', 1528-1530. Fleischman. R. A. (I993). From white spots to stem cells: The role of the Kit receptor in mammalian development. Trends Genet. 9, 285-290. Fraser, R and Grosveld, F. (t998). Locus control regions, chromatin activation and transcription. Curr. @in. Cell Biol. 10,1361-365. Fujiwara. Y.. Browne, C. P.. Cunniff, K.. Goff, S. C.. and Orkin. S. H. (t 996). Arrested development of embryonic red cell precursors in mouse embD'os lacking transcription factor GATA-1. Proc. Natl. Acad. Sci. U.S.A. 93, 12355-12358. Garcia-Porrero. J. A.. Godin. I. E.. and Dieterlen-Lievre. E (1995). Potential intraembD'onic hemogenic sites at pre-liver stages in the mouse. Anat. Embr3'oL 192, 425-435. Godin, I. E.. Garcia-Porrero, J. A., Coutinho. A., Dieterlen-Lievre, E, and Marcos, M. A. (i993). Para-aortic splanchnopleura from early mouse embryos contains B I a cell progenitors. Nature (London) 364, 67-70. Godin, I. E., Dieterlen-Lievre. F:. and Cumano, A. (1995). Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embr3~os, beginning at 8.5 days postcoitus. Proc. Natl. Acad. Sci. UIS.A. 92, 773-777. Godin, I. E.. Garcia-Porrero, J. A.. Dieterlen-Lievre. E. and Cumano, A. (I999). Stem cell emergence and hemopoietic acthity are incompatible in mouse intraembu'onic sites. J. Exp. Med. 190, 42;-52. Goodell, M. A.. Brose, K.. Paradis. G., Connen A. S.. and Mulligan. R. C. (1996). Isolation and functional properties of m,arine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 1183, t797-I 806. Grosveld. E. Dillon, N.. and Higgs. D. (1993). The regulation of human globin gene expression. In "Bailli~re's Clinical Haematology: The Haemoglobinopathies'" (D. R. Higgs and D. J. W~atheralI. eds). Vol. 6, no. 1, pp. 31-66. Balliere Tindall. London. Gussoni, E., Soneoka, Y.. Strickland, C. D.. Buzney, E. A.. Khan, M. K., Flint. A. E, Kunkel, L. M., and Mulligan, R. C. (1 c,O9). Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature (London) 401, 390-394. Herzenberg, L. A., Stall, A. M., Lalor, R A., Sidman. C.. Moore. W. A., Pai'ks, D. R., and Herzenberg, L. A. (11986). The Ly-I B ceil lineage. Immunol. Rev. 93, 81 - 102. Hu, M.. Krause, D., Greaves, M., Sharkis. S.. Dexter, M., Heyworth. C.. and Enver. T. (1997). Multilineage gene expression precedes commiunent in the hemopoietic system. Genes Dev. 11,774-785. Huyhn, A., Dommer~es, M., Izac, B., Croisille. L.. Katz, A., Vainchenker, W:, and Coulombel, L. (I995). Characterization of hematopoie-

208 tic progenitors from human yolk sacs and emb~'os. Blood 86, 44744485. Ikuta. K.. and Weissman. I. L. (1992). Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc. Natl. Acad. Sci. U.S.A. 89, 1502-1506. Ikuta, K., Kina. T.. MacNeil. I.. Uchida, N.. Peault. B.. Chien. Y. H.. and Weissman. I. L. (1990). A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell (Cambridge. Mass. i 62, 863 - 874. Jackson. K. A.. Mi. T.. and Goodell. M. A. (1999). Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc. :Vatl. Acad. Sci. U.S.A. 96, 14482-14486. Jaffredo. T., Gautier. R.. Eichmann. A., and Dieterlen-Lievre. F. (1998). Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny. Development (Cambridge. UK) 125, 4575 - 4583. Jaffredo. T.. Gautier. R.. Brajeul. V. and Dieterlen-Lievre. F. (2000). Tracing the progeny of the aortic hemangioblast in the avian emb~'o. Dev. Biol. 224, 204-214 Johnson, G.R. and Jones. R.O. (1973). Differentiation of the mammalian hepatic primordium in vitro. I. Morphogenesis and the onset of haematopoiesis. J. Embrvol. Exp..t4orphol. 30. 83-96. Johnson. G. R.. and Moore. M. A. (1975). Role of stem cell migration in initiation of mouse foetal liver haemopoiesis. Nature (Londonj 258, 726 -728. Jordan. C. T.. McKearn. J. R. and Lemischka. I. R. (1990). Cellular and developmental properties of fetal hematopoietic stem cells. Cell (Cambridge. Mass.) 61,953-963. Kanatsu. M.. and Nishikawa. S. I. (1996). In vitro analysis of epiblast tissue potency for hematopoietic cell differentiation. Development (Cambridge. UK) 122, 823-830. Kau. C. L.. and Turpen. J. B. (1983). Dual contribution of embDonic ventral blood island and dorsal lateral plate mesoderm during ontogeny of hemopoietic cells in Xenopus laevis. J. Immunol. 131, 2262-2266. Kaufman. M. (1992). "'The Atlas of Mouse Development." Academic Press, London. Kelemen. E.. Calvo. W., and Fliedner. T. (1979). "'Atlas of Human Hemopoietic Development." Springer-Verlag, Berlin. Keshet, E.. Lyman. S. D.. Williams. D. E.. Anderson. D. M.. Jenkins. N. A.. Copeland. N. G.. and Parada. L. F. (1991). Embryonic tCNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development. EMBO J. 10, 2425-2435. Kimura, S.. Roberts. A. W. Metcalf. D.. and Alexander. W. S. (1998). Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin. Proc. Natl. Acad. Sci. U.S.A. 95, 1195-1200. Kondo. M.. Weissman. I.L.. and Akashi. K. (1997). Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell (Cambridge. Mass.) 91, 661 - 672. Labastie. M. C., Cortes, F., Romeo. R H., Dulac. C.. and Peault, B. (1998). Molecular identity of hematopoietic precursor cells emerging in the human embDo. Blood 92, 3624-3635. Lemischka. I. R.. Raulet, D. H.. and Mulligan, R. C. (1986). Developmental potential and dynamic behavior of hematopoietic stem cells. Cell (Cambridge..Mass.) 45, 917-927. Lieschke. G. J., Stanley, E., Grail. D., Hodgsom G., Sinickas, V., Gall, J. A.. Sinclair, R. A.. and Dunn, A. R. (1994). Mice lacking both macrophageand granulocyte-macrophage colony- stimulating factor have macrophages and coexistent osteopetrosis and severe lung disease. Blood 84, 27-35. Lin, H. H., Sternfeld, D. C., Shinpock. S. G.. Popp. R. A.. and Mucenski. M. L. (I996). Functional analysis of the c-myb proto-oncogene. Curr. Top. Microbiol. Immunol. 211, 79-87. Liu, R, Tarle, S. A., Hajra, A.. Claxton. D. F.. Marlton. R. Freedman. M.. Siciliano. M. J.. and Collins. F. S. (1993). Fusion between transcription factor CBF beta/PEBF'2 beta and a myosin heavy chain in acute myeloid leukemia. Science 261, 104 I - 1"044.

II Lineage Specification and Differentiation Mackarehtschian. K., Hardin. J. D.. Moore, K. A., Boast, S., Goff, S. R, and Lemischka, I. R. (t995). Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors, bnmuniu 3, 147-161. Maeno, M., Tochinai. S., and Katagiri. C. (1985). Differential participation of ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus, as revealed in diploid-triploid or interspecific chimeras. Dev. Biol. 110, 503 -508. ManNa. A.. Lemarchandel. V.. Klaine, M.. Max-Audit. I., Romeo, R, Dieterlen-kievre. F.. and Godin. I. (2000). Lmo2 and GATA-3 associated expression in intraembr?'onic hemogenic sites. Development (Cambridge. UK) 127, 643-653. Marshall, C. J.. Moore. R. L., Thorogood, R. Brickell. R M., Kinnon. C., and Thrasher. A. J. (1999). Detailed characterization of the human aortagonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer. Dev. Dvn. 215, 139-147. Martin. C.. Beaupain. D.. and Dieterlen-Lievre. F. (1978). Developmental relationships between vitelline and intra-embryonic haemopoiesis studied in a`,ian "yolk sac chimaeras'. CelI Differ. 7, 115-130. Mead. R E.. Brivanlou. I. H.. Kelley. C. M.. and Zon, L. I. (I996). BMP-4responsive regulation of dorsal-ventral patterning by the homeobox protein Mix. 1. Nature (London) 382, 357-360. Medvinskv. A. L.. and Dzierzak. E. A. (1996). Definitive hematopoiesis is autonomously initiated by the AGM region. Cell (Cambridge, Mass.) 86, 897-906. Medvinsky, A. L., Samoylina. N. L., Muller. A. M.. and Dzierzak, E. A. (1993). An early pre-liver intraemb~onic source of C ~ - S in the developing mouse. Nature (London) 364, 64-67. Medvinsky. A. L.. Gan. O. I.. Semenova. M. L., and Samoylina, N. L. (1996). Development (Cambridge, UKr of day-8 colony-forming unitspleen hematopoietic progenitors during early murine emb~'ogenesis: spatial and temporal mapping. Blood 87, 557-566. Metcalf. D. (1984). "'The Hemopoietic Colony Stimulating Factors." Elsevier. Amsterdam. Metcalf. D. (11998). The molecular control of hematopoiesis: Progress and problems with gene manipulation. Stem Cells 16, 314-321. Miyoshi, H.. Shimizu. K.. Kozu. T., Maseki. N.. Kaneko, Y.. and Ohki, M. (1991 ). t(8,21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene. AML1. Proc. .Natl. Acad. Sci. U.S.A. 88, 10431-10434. Moore, M. A., and Metcalf, D. (11970). Ontogeny of the haemopoietic system: Yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br. J. Haematol. 18, 279-296. Moore. M. A., and Owen, J. J. (1967). Experimental studies on the development of the thymus. J. Exp. Med. 126, 715-726. Morro, B.. ,,'an der Kooy. D.. Rossant, J.. Reith. A., and Bernstein. A. (1991). Contiguous patterns of c-kit and steel expression: Analysis of mutations at the W and S1 loci. Development (Cambridge, UK) 113, 1207-122I. Mucenski, M. L., McLain. K.. Kier, A. B., Swerdlow. S. H.. Schreiner, C. M., Miller. T. A., Piet~ga, D. Vv:, Scott, W. J., Jr., and Potter, S. S. ( 1991 ). A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell (Cambridge, .Mass.) 65, 677-689. Muller, A. M.. Medvinsky, A.. Strouboulis, J., Grosveld, E, and Dzierzak, E. (1994). Development of hematopoietic stem cell activity in the mouse embryo. Immunio" 1,291-301. Murray, R (1932). The development in vitro of the blood of the early chick embrT'o. Proc. R. Soc. London 11, 497-521. Naito, M. (1993). Macrophage heterogeneity in development and differentiation. Arch. Histol. CvtoI. 56, 331-351. Naito, M., Umeda, S., Yamamoto, T.. Moriyama, H.. Umezu. H., Hasegawa, G.. Usuda. H.. Shultz. L. D., and T'~ahashi. K. (t996). Development, differentiation, and pheno~'pic heterogeneity of routine tissue macrophages. J. Leukocyte Biol. 59, 133 - 138. Nilsson, S. K.. Lieschke. G. J., Garcia-Wijnen, C. C.. Williams, B., Tzele-

10 Vertebrate Hematopoietic System pis. D.. Hodgson. G.. Grail. D.. Dunn. A. R.. and Bertoncelto. I. (t995). Granulocyte-macrophage colony-stimulating factor is not responsible for the correction of hematopoietic deficiencies in the maturing op/op mouse. Blood 86, 66-72. Nishikawa. S. I.. Nishikawa. S.. Kawamoto. H.. Yoshida, H.. Kizumoto. M.. Kataoka. H.. and Katsura. Y. (1998). In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embwos. lmmuni~" 8, 761-769. North, T.. Gu. T.-L., Stacv. T.. Wang. Q., Howard. L., Binder. M., MarinPadilla. M., and Speck. N. (1999). Cbf is required for the formation of intraaortic hematopoietic clusters. Development (Cambridge. UK) 126, 2563-2575. Nutt. S. L.. Heavey. B.. Rolink. A. G.. and Busslinger, M. (1999). Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature (London) 401,556-562. Od``'. C.. Vaigot. R. Quere. P., Imhof. B. A.. and Corbel. C. (1999). Glycoprotein lib-Ilia is expressed on avian multitineage hematopoietic progenitor cells. Blood 93, 2898-2906. Ogawa. M.. Nishikawa. S.. Ikuta. K.. Yamamura, E. Naito. M.. Takahashi. K.. and Nishikawa. S. (1988). B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone. ST2 B cell progenitor develops first in the embwonal body rather than in the yolk sac. EMBO J. 7, 1337-1343. Okuda. T.. ,,'an Deursen. J.. Hiebert. S. W.. Grosveld. G.. and Downing, J. R. (19961). AML1. the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Celt (Cambridge. Mass.) 84, 321-330. Orkin. S.H. (1995). Regulation of globin gene expression in ewthroid cells. Eur. J. Biochem. 15, 271-281. Palis. J., Robertson, S.. Kennedy, M.. Wall. C.. and Keller, G. (1999). Development of erythroid and myeloid progenitors in the yolk sac and embr?o proper of the mouse. Development (Cambridge. UK) 126, 50735084. Pardanaud. L.. and Dieterlen-Lievre. E (1999). Manipulation of the angiopoietic/hemangiopoietic commitment in the avian emb~o. Development (Cambridge, UK) 126, 617-627. Pardanaud. L., Altmann. C.. Kitos. R. Dieterlen-Lievre. F.. and Buck. C. (1987). Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development (Cambridge. UK) llR), 339-349. Pardanaud. L.. Yassine. F.. and Dieterlen-Lievre. F. (1989). Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development (Cambridge. UK) 105, 473-485. Pardanaud. L., Luton, D.. Prigent, M., Bourcheix. L. M., Catala. M., and Dieterlen-Lievre, F. (I996). Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. Development (Cambridge. UK) 122, 1363-1371. Petrenko. O.. Beavis. A.. Klaine, M.. Kittappa. R.. Godin, I., and Lemischka. I. R. (1999). The molecular characterization of the fetal stem cell marker AA4. Immunio" 10, 691-700. Pevny. L.. Simon. M. C.. Robertson. E.. Klein, W: H., Tsai. S. F., "4, D. A., Orkin. S. H.. and Costantini. F. ( 1991 ). Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA- 1. Nature (London) 349, 257-260. Phillips, R. L., Ernst, R. E.. Brunk. B.. Ivanova. N., Mahan, M. A., Deanehan, J. K.. Moore. K. A.. Overton, G. C.. and Lemischka. I. R. (2000). The genetic progam of hematopoietic stem cells. Science 288, 1635-11640. Pittenger. M. F., Mackay. A. M.. Beck. S. C.. Jaiswal. R. K, Douglas, R., Mosca. J. D.. Moon'nan. M. A.. Simonetti. D. WL. Craig. S.. and Marshak. D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 2,84, 143-147. Porcher, C.. Swat, W., Rockwell. K.. Fujiwara. Y.. Alt. F. W., and Orkin. S. H. (I 996). The T cell leukemia oncoprotein SCLltal- 1 is essential for development of all hematopoietic lineages. Cell (Cambridge. Mass.) 86, 47-57.

209 Robb. L.. Lyons. I.. Li. R.. Hartley. L., Kontgen. E. Harvey. R. R. Metcaif. D.. and Begley. C. G. (1995). Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scI gene. Proc. Natl. Acad. Sci. U'.S.A. 92, 7075-7079. Robb. L.. Elwood. N. J., Elefanty. A. G.. Kontgen. F., Li. R., Barnett. L. D.. and Begley. C. G. (1996). The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J. 15, 4123-4129. Rowley. J. D. (t999). The role of chromosome transtocations in leukemogenesis. Semin. HematoL 36(4. Suppl. 7). 59-72. Russell. E. S. (1979). Hereditary, anemias of the mouse: A review for geneticists. Adv. Genet. 20, 357-459. Russell, E. S.. and Bernstein, S. E. (1966). Blood and bl,:u:u:t formation. In "'Biology of the Laboratou Mouse" (E. L. Green. ed.). pp. 351-372. McGraw-Hilt, New York. Sabin. F. (1920). Studies on the origin of blood vessels and of red blood corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Carnegie Inst. Washington Publ. 272 Contrib. Embo'ol. 9, 214. Sasaki. K.. Yagi, H.. Bronson. R. T.. Tominaga. K.. Matsunashi. T.. Deguchi. K., Tani, Y., Kishimoto. T., and Komori. T. (1996). Absence of fetal ti``,er hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta. Proc. Natl. Acad. ScL U.S.A. 93, 12359-12363. Shalaby, E. Rossant, J., Yamaguchi. T. R. Gertsenstein, M., Wu. X. F.. Breitman, M. L.. and Schuh, A. C. (1995). Failure of blood-island formation and vasculogenesis in Flk-l-deficient mice. Nature (Lzmdon) 376, 62-66. Shalaby. F.. Ho. J., Stanford, W. L.. Fischer. K. D.. Schuh. A. C., Schwartz.L, Bernstein, A., and Rossant. J. (I997). A requirement for Flkl in primitive and definitive hematopoiesis and vasculogenesis. Cell (Cambridge, Mass.) 89, 981-990. Shivdasani. R. A.. Mayer, E. L., and Orkin. S. H. (1995). Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature (London) 373, 432-434. Sinclair, A. M., and Dzierzak. E. A. (1993). Cloning of the complete Ly6E. i gene and identification of DNase I hypersensitive sites corresponding to expression in hematopoietic cells. Blood 82, 305;2-3062. Smith. J. C.. and Albano. R. M. (1993). Mesoderm induction and ewthroid differentiation in early vertebrate development. Semin. Dev. Biol. 4, 315-324. Spangrude. G. J., Heimfeld. S., and Weissman. I. L. (1988). Purification and characterization of mouse hematopoietic stem cells. Science 241, 58-62. Stennard F. R. K.. and Gurdon, J. B. (19971). Markers of vertebrate mesoderm induction. Curr. Opin. Genet. Dev. 7, 620-627. Stephens, L. E.. Sutherland, A. E.. Klimanskaya, I. V.. Andrieux, A., Meneses. J., Pedersen, R. A.. and Damsky. C. H. (1995). Deletion of beta l integrins in mice results in inner cell mass failure and periimplanatation lethality. Genes Dev. 9, 1883-1895. Tavian, M., CoulombeI, L.. Luton, D.. Clemente, H. S.. Dieterlen-Lievre, E. and Peault, B. (1996). Aorta-associated CD34 a hematopoietic cells in the early human embryo. Blood 87, 67-72. Tavian, M.. Hallais. M. E, and Peault, B. (1999). Emergence of intraembwonic hematopoietic precursors in the pre-liver human embryo. Development (Cambridge, UK) 126, 793-803. Tepass, U., Fessler. L. I.. Aziz, A.. and Hartenstein, V. (1994)Embryonic origin of hemocytes and their relationship to cell death in Drosophila. Development (Cambri~ee, UK) 1120, 1829-I837. Theele. D. R. and Streit. ~ : J. (1993). A chronicle of microglial ontogeny. Glia 7, 5-8. Toles, J. E. Chui. D. H.. Belbeck. L. W:, Starr. E., and Barker. J. E. (I989). Hemopoietic stem cells in murine embwonic yolk sac and peripheral blood. Proc. Natl. Acad. Sci. U.S.A. 86, 7456-7459. Tracey, Wl D., Jr.. Pepling, M. E., Horb. M. E., Thomsen. G. H., and Gergen. J. E (1998). A Xenopus homologue of aml-I reveals unexpected

210 patterning mechanisms leading to the formation of embr~onic blood. Development (Cambridge. UK) 125, 1371-1380. Tsai, F. Y., Keller, G., Kuo, F. C., Weiss. M.. Chen, J., Rosenblatt, M., Alt. F. W., and Orkin, S. H. (1994). An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature (London) 371, 221226. Turpen, J. B., and Knudson, C. M. (1982). Ontogeny of hematopoietic cells in Rana pipiens: precursor cell migration during embryogenesis. Dev. Biol. 89, 138-151. Turpen, J. B., Knudson, C. M.. and Hoefen, R S. ( 1981 ). The early ontogeny of hematopoietic cells studied by grafting cytogenetically labeled tissue anlagen: Localization of a prospective stem cell compartment. Dev. Biol. 85, 99-112. Turpen, J. B.. Kelley, C. M.. Mead, R E.. and Zon, I~. I. (1997). Bipotential primitive-definitive hematopoietic progenitors in the vertebrate emb~o. Immunity 7, 325-334. Van der Loo. J. C. M.. Slieker. W. A. T.. Kieboom. D.. Ploemacher. R. E. (1995) Identification of hematopoietic stem cell subsets on the basis of their primitiveness using antibody ER-MP12. Blood 85, 952-962. van Ewijk. W.. Hollander, G., Terhorst. C.. and Wang. B. (2000). Stepwise development of thymic microenvironments in vivo is regulated by thvmocyte subsets. Development (Cambridge. UK~ 127, 1583-1591. Visvader, J. E.. Fujiwara, Y., and Orkin. S. H. (1998). Unsuspected role for the T-cell leukemia protein SCL/taI-1 in vascular development. Genes Dev. 12, 473-479. Wang. J. H., Nichogiannopoulou. A., Wu, L., Sun. L.. Sharpe, A. H., Bigby, M., and Georgopoulos, K. (1996a). Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5, 537-549. Wang. Q., Stacy, T., Binder, M., Marin-Padilla, M., Sharpe, A. H.. and Speck, N. A. (1996a). Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc. Natl. Acad. Sci. G{S.A. 93, 3444-3449. Wang, Q., Stacy. T., Miller, J. D., Lewis, A. F., Gu. T. L., Huang, X.. Bush-

il Lineage Specification and Differentiation weller, J. H., Bories. J. C., Alt, E W., Ryan, G., Liu, R R. WynshawBoris. A.. Binder, M., Marin-Padilla, M., Sharpe. A. H., and Speck, N. A. (1996b). The CBFbeta subunit is essential for CBFalpha2 (AML 1 ) function in vivo. Cell (Cambridge. Mass.) 87, 697-708. Warren. A. J., Colledge. W. H.. Carlton. M. B.. Evans. M. J., Smith, A. J., and Rabbitts. T. H. (1994). The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erT'throid development. Cell (Cambridge. Mass. ) 78, 45-57. Weissman, I., Papaioannou, V., and Gardner, R. (1978). Fetal hematopoietic origins of the adult hematolymphoid system. In "'Differentiation of Normal and Neoplastic Hematopoietic Cells'" (B. Clarkson, R A. Marl:s, arid J. E. Till. eds), Conf. Cell Proliferation. Vol. 5. pp. 33-47. Cold Spring Harbor Lab.. Cold Spring Harbor. NY. Wijgerde. M.. Grosveld. E. and Fraser, R (1995). Transcription complex stability and chromatin dynamics in vivo. Nature (London) 21,209-213. Winnier, G.. Blessing, M., Labosky, R A.. and Hogan, B. L. (1995). Bone morphogenetic protein4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105-2116. Wood, H. B.. May. G.. Healy, L., Enver, T.. and Morriss-Kay. G. M. (1997). CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis. Blood 90, 2300-2311. Yamada. Y.. Warren, A. J., Dobson, C., Forster. A., Pannell, R., and Rabbitts, T. H. (1998). The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis. Proc. Natl. Acad. Sci. U.S.A. 95, 3 8 9 0 3895. Yoder, M. C., Hiatt, K., Dutt, R, Mukherjee, R, Bodine, D. M., and Orlic, D. (1997). Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunio" 7, 335-344. Young, R E., Baumhueter, S., and Lasky, L. A. (1995). The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during routine development. Blood 85, 96 - 105. Zon, L. I. (1995). Developmental biology of hematopoiesis. Blood 86, 2876-2891.

Vasculogenesis and Angiogenesis Thomas N. Sato a n d Siobhan L o u g h n a The Universi O, of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390

i. Introduction

II. O v e r v i e w

of Vascular Development

II. Overview of Vascular Development Iil. Generation of Endothelial Cells IV. Vascular Morphogenesis V. Concluding Remarks References

!. I n t r o d u c t i o n Formation of the vascular system is recognized as one of the most important events in development and has been a subject of intensive investigations. Although modem analyses of vascular development date back to the beginning of the 20th century, the primary focus was to provide morphological description of the process (Evans, 1909; His, 1900; Sabin, 1917). Two sequential key morphogenic processes underlie vascular development: vasculogenesis (Box 1) and angiogenesis (Box 2). Recently, significant advancement in our understanding of molecular mechanisms for these processes, as well as the emergence of new concepts, has been revolutionizing the field. Application of many mouse genetics tools have played a pivotal role in this recent revolution of the field. In this chapter, we discuss many of the key principles of vascular development with a primary emphasis on the studies accomplished by the use of contemporary mouse genetics and embryological tools.

Mouse Development

2!1

Development of virtually all organs is heavily dependent on sufficient nutrient feeding mad oxygenation, processes primarily accomplished by the vascular system during embr-yogenesis. Thus, the normal patterned formatiion of a functional vascular system is one of the most critical and earliest events to occur during emb~ogenesis. The sequences leading to the formation of the vascular system have been classically divided into multiple phases (Fig. 1). The first vascular structure can be identified as early as the gastrulation stage. At this embryonic stage, a subset of resident mesodermal cells differentiates to endothelial cells, which assemble to form an initial vascular network. This vascular network is referred to as the primary capillary plexus (RSsau and Flamme, 1995). At this early stage, the entire vascular network is composed of primzwily one cell type, the endothelial cell. In both the yolk sac and embryo, subsets of mesodermal cells differentiate to endiothelial cells and form cell clusters referred to as blood islands (Box 1). These clusters subsequently become connected to each other to form an intricate network of vessels. This initial vessel network (i.e., primary capillary plexus) is characterized by its relatively uniform honeycomb-like capillary channel network (Fig. 1). This process of primary capillary plexus formation from in situ differentiating endothelial cells is referred to as vascutogenesis (Box 1). Subsequently, the primary capillary plexus expands by forming additional branches and remodeling its network Copyright 9 2fX~2 Academic Press All rights of reproduction in any form reserved.


212

Box 1: Vasculogenesis a n d Blood~ .....

Islands~,

:~

Vasculogenesis define:: the first morphogenic process during vascular development. Clusters of mesoderm~erived an~oblastic cells differentiate to blood and endothelial cells, to form a structure referred to as a blood island. In this structure, clusters of blood cells are surrounded by a single layer of endothelial cells. The endothelial cells of these blood islands subsequently coalesce to form a humbet of initial vascular channels called the pr/mary capillary plexus. This process, the formation of the primary capillary plexus, is referred to as vasculogenesis. The primary capillary plexus is characterized by a honeycomb-like network of vascular channels of uniform diameter. This vascular network can be clearly identified in the yolk sac and embryo, such as the cephalic r e . o n , by 8.0 dpc.

to assume its final form (Risau, 1997). During this later phase, the vessels form a more complex network and cells other than endothelial cells become involved in the process (Fig. 1). This later phase of vascular development is referred to as angiogenesis (Box 2). Two distinct mechanisms are proposed for the formation of additional vessel branches

Box 2" Angiogenesis Angiogenesis was classically defined as a process involving sprouting from preexisting vessels, such as the primary capillary plexus. However, as we learned more details of vessel formation, this process was found to be far more complex than just sprouting of vessels. It has been proposexl that the primary capillary plexus expands its network by both sprouting and nonsprouting processes. Furthermore, this newly expanded vascular network is suggested to undergo "'pruning," "remodeling," and "maturation" to complete the whole process. At this point, the morphogenic processes followhag vasculogenesis seem to be complex, and are not critically well defined. Therefore, the authors would like to use the term angiogenesis to cover the entire vessel formation process ~following vasculogenesis including pruning, remodeling, and maturation. .

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from the primary capillary plexus: sprouting and nonsprouting (Fig. 2). In the sprouting process, an endothelial cell that is already part of a continuous vessel transforms to an elongated shape, invades nearby tissue, and forms an additional vascular channel from the existing one. In the nonsprouting process, surrounding cells of an existing vascular channel invade and intercept the vessel, thus leading to the splitting of one vascular channel into two. Combining both of these processes, the primary' capillary, plexus transforms to more complex vascular channels. Finally. these primarily endothelial-based processes are further integrated with a process involving nonendotheliaI vascular cells during angiogenesis. "vascular channels formed by endothelial cells are now being invested by smooth muscle cells. Smooth muscle cells infiltrate the vascular channels to provide more rigid inte~ity to the vessels as well as contractility. In many organs, the vessel phenotypes are further modified. Various organs require distinct morphological and phenotypical characteristics of the vessels in order to support specific developmental processes and physiological functions. This leads to significant variations and specifications among the vessels of different organs and parts of the body. In the following sections, each process leading to a mature vessel is described in detail. Both cellular and molecular mechanisms are emphasized, with an in-depth discussion of some critical questions.

III. G e n e r a t i o n of Endothelial Cells As briefly outlined in Section II, endothelial cells are the primary cell type involved in the initial phases of vascular development. Generation of endothelial cells is regulated via two processes: differentiation and expansion (Fig. 3). By means of a differentiation pro~am, the endothelial ceil lineage is specified. By means of an expansion program, both proliferation and survival of progenitors and endothelial cells are tightly controlled to establish the normal size of the endothelial cell population. In this section, the mechanistic basis of these programs underlying endothelial cell generation is described in detail.

A. Endothelial Cells Are Mesodermal in Origin All endothelial cells originate from mesoderrn during embryogenesis (Figs. 1 and 3). In mice, at around 7.0-7.5 days postcoitus (dpc), the lateral mesoderm produces a population of progenitors called hemangioblasts (Box 3) from which endothelial cells are differentiated. Endothelial cells generated from hemangioblasts at this emb~onic stage in mice contribute primarily to the vasculature of extraembryonic yolk sac membrane. Later (7.5-8.0 dpc), as embryonic structures becomes more discrete, scattered clusters

213

11 Vasculogenesis and Angiogenesis

Figure 1

Over~,iew of vascular development. See accompanying text for the description.

of hemangioblasts derived from intraembryonic mesodermal cells become identifiable, and these cells contribute to the intraemb~onic vasculature.

B. Regulation of Endothelial Cell Lineage Specification Molecular mechanisms of neither hemangioblast nor endothelial specification from multipotential mesodermal cells

during mouse emb~ogenesis remain elusive at this time. However, studies with several zebrafish mutants provided some insight into the specific genetic pathways controlling this cell type specification process. A series of genetic studies in zebrafish resulted in an emerging paradigm defining how hemangioblast lineage may be established. Zebrafish c l o c h e is a mutation identified in zebrafish, and no endothelial or hematopoietic cells exist except in the dorsal tip region in this mutant (Stainier et al., 1995). Further-

214


F i g u r e 2 Sprouting and nonspmuting angiogenesis. There are two distinct mechanisms for angiogen~esis(i.e., formation of new vessel channels): sprouting and nonsprouting. In sprouting angiogenesis, an endothelial cell (EC) transforms to an elongated shape and invades the irranediately proximal space. This involves loosing interactions of surrounding nonendothelial cells such as mesenchymal cells, which normally function as supporting cells for EC. Each EC sprout eventually interacts with another EC sprout and forms a new vascular channel. In nonsprouting angiogenesis, surrounding mesenchymal cells push each EC inward, and the ECs eventually divide one vascular channel into two. These two processes are considered to be the main mechanisms for angiogenesis based on discontinuous and non-real-time histological analyses of vessels undergoing angiogenesis. Definitive description of cellular processes underlying angiogenesis waits for future documentation of angiogenesis in real time.

more, genetic studies in zebrafish showed that the cloche function is u p s t r e a m to the expression of one of the earliest h e m a n g i o b l a s t markers, VEGF-R2/flk-1 (Kdr) (Box 4) (Liao et al., 1997). Therefore, these studies suggested that the putative cloche gene is critical for establishing hernangio-

blast lineage. Cloche was also s h o w n to be u p s t r e a m of t w o other genes critical for h e m a n g i o b l a s t formation: Scl (tall) (Box 5) and Hhex (Box 6) (E. C. Liao et al., 1998; W. L i a o et al., 2000). In a cloche mutant, neither Scl nor Hhex expression is detected. F u r t h e r m o r e , lack of endothelial cells


215

Figure 3 Multilevelregulation for establishingnormalsize of endothelial population. The final population size of ECs is determinedat the multiple level. A subset of mesodermalcells is committedto the hemangioblastlineage, which determinesthe initial number of hemangiobl~Lsts.These hemangioblasts undergo an expansion process that is regulatedby subtle balances between the regulator" mechanismsunderlying EC survival and proliferation. As a subset of hemangioblastsdifferentiates to ECs. this process determines the initial number of endothelial cells. The final number of ECs is determined by the subtle balance between EC survival and proliferation signals.

in developing e m b ~ o s can be rescued by forced expression of either Scl or Hhex in the cloche mutant. Interestingly, forced expression of Scl or Hhex also results in the expression of the other, suggesting a mutual coregulation of these two transcription factors. Another potential regulator of endothelial cell lineage specification is bFGF (Fgf2). It has been suggested that bFGF plays a role in endothelial and hematopoietic cell differentiation, at least in vitro (Flamme and Risau, 1992). Uncommitted mesodermal cells in quail epiblasts were successfully induced to form both hematopoietic and endothelial cells. This was based on the staining of these cells with lineage-specific monoclonal antibodies. However, the precise differentiation stages of mesodermal cells stained by these antibodies were not clearly defined. Therefore, at present, it is difficult to define the mode of bFGF action for endothelial and hematopoietic differentiation. An alternative interpretation of this in vitro result is that there are cells already committed to differentiating to the hematopoietic and endothelial cells that do not stain with these lineage-specific antibodies in the epiblast preparation. In any event, further investigation is required to determine whether bFGF :itself or other related factors are in fact inducer(s) for endothelial and hematopoietic cell differentiation during normal devel-

opment. It is also important to keep in mind that this is an in vitro system and therefore needs confi~nation by in vivo analyses.

C. Regulation of Normal Endothelial Cell Number The number of hemangioblasts affects the final size of endothelial cell population and is tightly controlled during vascular development. A reduced number of hemangioblasts (i.e., lack of sufficient number of endothelial cells) leads to undervascularization. The presence of too many hemangioblasts leads to abnormal vascular development. One of the most extensively studied pathways controlling the endothelial population size is a VEGF receptor family (Box 4). Two receptors among this receptor family seem to be critical in controlling the number of endothelial cells, and they do so in an elegantly orchestrated manner. Hemangioblasts were found to express VEGF-R2 (Kdr), one of the receptors for vascular endothelial growth factor (VEGF, Vegf). Furthermore, the formation of hemangioblasts was found to be dependent on VEGF in a culture medium. However, VEGF-R2-'- ES cells were able to focm hemangioblast

Ii Lineage Specification and Differentiation

216

Vascular endothelial growth factor (VEGF) is a family of soluble glycoproteins. The first family member, VEGF-A, was originally isolated as vascular permeability factor (VPF) based on its activity to induce vascular leakiness. Subsequently, it was also shown to induce proliferation of cultured endothelial cells in a cell type specific manner. The gene for VEGF-A encodes three isoforms of VEGF-A, VEGF-164, VEGF120, and VEGF-188 in mice. All of these isoforms are generated by alternative splicing of the same gene. VEGF-A 164 is the most predominant form. Functional differences among these three isoforms remain to be determined. Subsequently, three additional VEGF family members have been identified based on hi~a sequence homology. They are now referred to as VEGF-B, VEGF-C, and VEGF-D, and are all encodexl by distinct genes. In addition, placenta growth factor (Pgf) is also included in this family as the fifth member. Recently, VEGF related gene was also discovered in Off virus, a member of the poxyvirus family that produces a pustular dermatitis in sheeps, goats, and humans.

VEGFs bind specitieally to a f a m i l y o f receptors, VEGF receptors (VEGF-Rs): Cu_rrently, lthere i i ;I are three VEGF-Rs, VEGF-RI (Fltl), V E G F - ~ ( K d r [ for mouse gene and KDR for R3 (Flt4), VEGF-Rs ~ l o n g ~ rosine kinases, ~ extracellu consists of seven immunogh modular strucaa~ is also fou growth factor (PDGF) recept( also distinct receptor-ligand binding ~ f i c i t y exists: : VEGF-A binds specifically VEGF-RI and V E G F - ~ ~ VEGF-B binds to V E G F - R 1 VEGF-C (Vegfc) binds to VEGF-R2 and VEGF-R3. VEGF-D (Figf) binds to VEGF-R2 and VEGF-R3. Recently, another receptor that is not related to VEGF-Rs was discovered a n d shown to interact with VEGF-A. This receotor. pilin- 1, was originally behavior, particularly lin-1 was shown to inu

A164 isoform.

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~~ .. ~~!i!:i~~!~i i,~ ~i~:ii~il yolk sac vessels. However, the observed defects were ...... ~ ~I The Scl/tall gene was originally identified through the level of endothelial cell organization rather than en- ii its translocation in acute T-cell lymphoblastic leukemia. dothelial cell differentiation, : : : ~::::~ It encodes a basic helix-loop--helix transcription factor In addition, forced expression of wild-type Scl, ::>:::::~tall ..... :: and is expressed specifically in hematopoietic and enin Cloche mutant zebrafish (see above) r e s c u ~ b o t h l ~ ::: dothelial lineages, as well as in the developing brain. matopoietic and endothelial defects m this n retain, . . . . . . .sug . . >::: Null mutant embryos for the Scl/tall gene exhibit de:lies:~downsi gesting that the. .Scl/tall . . . . . . . function ....:~ l~:th( ~ fective erythropoiesis and Scl/tall -/- embryonic stem cells do not contribute to any hematopoietic lineages in : cloche function a n d iscdtic~ chimeric mice. ~ f o r e , it is: prot~sed that the Scl/tall : l i ~ cell lineage,:: This : :~ fimction is critical for hematopoietic fineage regulation. for ::~:Scl/tall transca-iptilon volved in endothelial . . . . . ......................... I Rescue of the ScUtall function in the hematopoiexic ingly in this respect, it has al:so re~nflybe~n s h o . lineage was accomplished by expressing the wild-type .the function of ScUtall in re gulatingbeIr:mtopoir Scl/tall gene specifically in this lineage in a null back.... ...... endothelial lineages may be!, indepr ~.......... n t from it,' ~ound. Although the hematopoietic defect was combinding activity, indic~gnovel:::::mech pletely rescued, these embryos exhibit abnormal yolk function. sac vascular development. Furthermore, Scl/tall -lL ~fill:!!~i>~)fill!i endothelial cells failed to contribute to developing

11 Vasculogenesis and Angiogenesis .....

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was : ~ t i ~ y i icatb pressed-::: gene,: H e x :was :: lyi tiffed as being expres~ inboth:he efic~c endothelial lineages. It' has beer " ..... :i. ::sho~ . . . . . . ~::thatHe~ ... works as a transcfiptional:repressor:torc~~ an: terior and dorsoventral patterning.: It i~: als6:

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colonies in a VEGF-dependent manner in vitro (Schuh et al., 1999). In this in vitro study, a unique system called blast colonyf o r m i n g cell (BL-CFC) assay (Box 7) was used. In this assay, a unique clonal population of transitional precursor cells from the ES cell-derived embryoid bodies was identified. This clonal population of cells was induced to differentiate into both hematopoietic and endothelial cells when cultured in the presence of VEGE This provided the first proof of the existence of a bipotential hemangioblast population at least in vitro. In vivo, VEGF-R2 -j- embryos exhibit complete lack of blood cells and endothelial cells (Shalaby et al., 1995).

.... ~ :i/~/

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suspension :i~

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VEGF-R2 -/- ES cells were used in the study of chimeric embryos, and it was found that these mutant ES cells do not contribute to either endothelial or hematopoietic cells in vivo (Shalaby et al., 1997). Based on these results, it has been proposed that VEGF-R2 is probably dispensable for hemangioblast differentiation. However, it may play a critical role in survival and expansion of the hemangioblast population. In addition, VEGF-R2 may be required for the directed migration of hemangioblasts to receive a cue(s) from the appropriate microenvironment, which seems to be essential for hematopoietic differentiation (Hidaka et aL, 1999; Shalaby et al., 1997). In addition to VEGF-R2, another VEGF receptor, VEGFR1 (Fltl), has also been implicated in hemangioblast development. Lack of VEGF-R2 expression results in an increased number of endothelial cells both in vitro and in vivo (G.-H. Fong et al., 1996; G.-H. Fong et at., 1999). This phenotype was proposed to be a result of increased commitment of mesodermal cells to the hemangioblastic lineage (G.-H. Fong et aI., 1999). Interestingly, the lack of a cytoplasmic tyrosine kinase domain for VEGF-R1 does not seem to be critical for its function in vivo (Hiratsuka et al., 1998). Thus, it was suggested that VEGF-R1 acts as a "VEGF sink" by binding to extracellular VEGF and makdng only the optimal concentration of VEGF available to the cells (G.-H. Fong et al., 1999). This particular function of VEGF-R 1 is mediated in a cell nonautonomous fashion (G.-H. Fong et al., 1999). Based on this model, VEGF is considered to be a hemangioblast differentiation inducer. However, in vitro differentiation studies have clearly shown that VEGF-R2 -/- ES cells can be induced to form hemangioblasts, albeit less efficiently, in a VEGF-dependent manner (Schuh et al., 1999). Therefore, it is possible that VEGF acts through another class of VEGF receptor to regulate hemangioblast formation. Alternatively, it is possible that VEGF acts through VEGF-R2 in normal hemangioblast formation in vivo and a putative compensatory mechanism is switched on when VEGF-R2 is absent in vitro. It is also possible that VEGF/ VEGF-R2 function is to regulate migration and expansion of hemangioblasts, rather than the formation of these cells. Interestingly, aberrant migration of the VEGF-R2 -/- ES cells that would normally have expressed this receptor was observed in vivo (Shalaby et al., 1997). This suggested that VEGF-R2 controls the directed migation of such cells. This migration enables these cells to receive an appropriate signal from the microenvironment that is necessar:y for their differentiation. With respect to the possibility that VEGF may regulate the expansion of the hemangioblas1: population, one needs to also consider the fact that the regulation of hemangioblast formation via VEGF-R1 is independent from cell proliferation and survival. Clearly, furdaer studies are required to fully understand how endothelial lineage specification arid expansion are or-

218


chestrated by VEGF and VEGF receptors. Furthermore, it would be essential to understand how the VEGF pathway interacts with other pathways such as Cloche and bFGF in determining the final size of the endothelial population.

D. Diversity of Endothelial Cells Recently, the existence of many molecularly distinct subclasses of endothelial cells has become evident. Some of them are genetically preprogrammed and the phenotypes of the others are determined bv their microenvironment. In this section, representative classes of endothelial cells are described. 1. Arterial versus Venous Endothelial Cells Although blood vessels have been conventionally classified as arteries or veins based primarily on physiological parameters, it has recently become clear that this distinction between arteries and veins is genetically encoded (Gerety et al., 1999; Wang et al., 1998). Furthermore, this distinction seems to be already established at the v e ~ beginning of blood vessel formation during embryogenesis (Gerety et al., 1999: Wang et aI., 1998). It has been found that arterial and venous endothelial cells can be defined by their specific expression of ephrin-B2 ( E f n b 2 ) and EphB4 ( E p h b 4 ) , respectively (Box 8) (Gerety et al.. 1999: Wang et al., 1998). This distinctive expression pattern was detected early, when initial blood vessel formation begins during embryogenesis. This indicates that a molecular distinction between arterial and venous endothelial cells is established during the initial phase of blood vessel formation.

Box 8: Eph and Ephrin Family in Vascular Development Eph is a class of transmembrane receptors that belongs to a family of receptor tyrosine kinases. The extracellular portion of the receptor consists of an N-terminal globular domain, a cysteine-rich r e , o n , and two fibronectin type IH domains. The intracellular portion has intrisic tyrosine kinase activity. Ephrins are a family of specific ligands for the Eph receptors' Ephrins are expressed on the cell surface, either by GPI linkage to the membrane or tra_nsmembrane domain. This ligand-receptor family has been known to critically regxllate developmental processes such as axon guidance and neural cell migration. Recently, some members of this ligand-receptor family were shown to participate in vascular development.

Although this surprising finding is fascinating, many important questions remain unsolved. Assuming that the initial endothelial cells immediately following the differentiation of hemangioblasts do not exhibit a distinctive expression pattern of ephrin-B2 and EphB4, how is this expression pattern subsequently established? Are there any local cues that turn on the expression of ephrin-B2 and/or EphB4? What are the mechanisms underlying this mutually exclusive expression pattern? Alternatively, it is possible that all of the initial endothelial cells generated from hemangioblasts already express either ephrin-B2 or EphB4, with subsets subsequently expressing the other gene. For example, all of the initial endothelial cells may express EphB4 (i.e.. they are, in essence, venous type) and, later, subsets of them begin to express ephrin-B2 and assume an arterial phenotype while the remainder continue to express EphB4 to maintain a venous phenotype. It is also possible that there are two distinct sets of hemangioblasts, one expressing ephrin-B2 and the other expressing EphB4. Distinctive expression pattern of epbxinB2 and EphB4 seems to persist following the completion of blood vessel formation. Therefore. it would be of interest to investigate the mechanisms that underlie the maintenance of this mutually exclusive expression pattern and its relationship to the initial induction of the expression of such markers. 2. Lymphatic Endothelial Cells

Most of the studies of vascular development have focused on blood vessels. However, another circulatory system, the lymphatic vascular system, provides important vessels for cellular waste drainage and circulation of lymphocytes. VEGF-R3 (Flt4) was found to be expressed in venous endothelial cells during early embryonic development, but the expression becomes gradually restricted to lymphatic endothelial cells as the lymphatic vasculature is formed (Kaipainen et al., 1995). This expression study suggested a close lineage relationship between venous and lymphatic endothelial cells. Furthermore, one of the specific ligands for VEGF-R3, VEGF-C (Vegfc), was shown to induce lymphatic angiogenesis when ectopically overexpressed in vivo (Jeltsch et al., 1997). This finding indicates that the VEGF-C/VEGF-R3 pathway may be important for lymphatic vessel development. However, VEGF-R3-'- embryos die before the first lymphatic vessels form, precluding a possibility to test the potential involvement of this ligand-receptor system in lymphatic vessel formation (Dumont et al., 1998). Future development and analysis of conditional knock-outs for these genes may allow us to test this possibility directly. More recently, it has been found that one of the homeobox genes, P r o x l , is also expressed by developing lymphatic endothelial cells in the embryo (Wigle and Oliver, i999). P r o x l -/- embryos exhibit significant retardation of the lymphatic vessel formation, suggesting that this putative tran-

219

11 Vasculogenesis and Angiogenesis scription factor may be involved critically in this process (Wigle and Oliver. 1999).

3. Organ-Specific Endothelial Types As blood vessels form and mature, it is essential that they meet the specific demands of the particular organs being vascularized. It is speculated that this process requires very sophisticated cell communications between vascular cells and surrounding nonvascular organ-specific cells. Therefore, each organ presumably requires a specific input from its vasculature that exhibits a unique structure and physiological function. Although this is an important aspect of vascular development, very little information is available. a. Brain. Brain vascular endothelial cells form complex tight junctions and also develop specialized transporter systems (Risau et al.. 1986a,b: Risau and Wolburg, 1990). These structural and biochemical barriers formed by the brain vascular endothelial cells permit the selective trafficking of chemicals between the circulation and nervous system. Therefore, this unique blood-brain barrier (BBB) protects the nervous system against toxic insults from the circulation. This barrier system has important physiological and therapeutic implications. However. very few molecular mechanisms underlying BBB formation or maintenance are known. In vitro, coculturing endothelial cells with astrocytes was shown to induce the tight-junction phenotype of endothelial cells found at the BBB (Neuhaus et al., 1991). Obviously, further studies are necessary to decipher the unique molecular nature of endothelial cells at the BBB. b. Heart. The heart may require a specialized circulation that is essential for its physiological functions. This requirement may be met by reciprocal cellular and chemical communications between cardiac vessel endothelial cells and the myocardium. While this area has obvious therapeutic importance, it has not been explored extensively at a molecular level. One study has suggested that cardiac vessel endothelial cells are molecularly distinct from other endothelial cell types. A part of the upstream promoter sequences of the yon Willebrand factor ( V w f ) gene was shown to confer cardiac vessel endothelial specific expression when studied in transgenic mice (Aird et al., 1997). This putative unique cardiac vessel endothelial expression was shown to be regulated, in part, by the PDGF-B (PdgfO) pathway (Edelberg et al., 1998). Sufficient evidence has recently accumulated to further suggest that the distinct nature of cardiac vessel endothelial cell type reflects its unique developmental origin. Chimera and retroviral cell lineage analyses in chick embryos identified epicardial cells as the origin of many of the coronary vessel endothelial cells (Dettman et al., 1998; Mikawa and Fischman, 1992; Mikawa and Gourdie. 1996, Perez-Pomares et al., 1998: Vrancken Peeters et al., 1999). During heart

development, a subset of epicardial cells immediately surrounding the outside of the myocardium migrates into the subepicardiai space, a space between the epicardium and myocardium. Following the migration, these epicardialderived cells undergo epithelial-mesenchymal transformation, arid subsequently differentiate to hemangioblasts, endothelial cells, and smooth muscle cells, all of which contribute to coronary vessel formation. In mice, this principle seems to hold true as knock-out mice for genes such as VCAM1 ( V c a m l ) , c~ integrin (ltga4), and erytt-n-opoietin (Epo) exhibit the failure of epicardial development, resulting in the lack of a coronary vasculature (Kwee et al., 1995; Wu et al., 1999, "fang et aL, 1995). Recently, a novel cofactor of GATA transcription factors, FOG-2 (Zfpm2), was also show'n to be critical for the formation of coronary vessels (Tevosian et al., 2000). In the FOG-2 -/- embryo, no coronary vessels (neither endothelial nor smooth muscle cells) were formed. Because epica_rdium formation in this knock-out embryo is normal, it is suspected that FOG-2 plays a critical role in the epithelialmesenchymal transformation of the epicardial cells. Interestingly, forced expression of FOG-2 in the myocardium was found to rescue the coronary vessel phenotype in the FOG2 -/-. Therefore, it is proposed that FOG-2, together with GATA factors, regulates the expression of a set of genes whose functions are important for paracrine regulation between epicardial cells and myocardial cells to regulate the epithelial-mesenchymal transformation. c. Eye. Some of the vascular beds form only transiently during development and will eventually recess. Prominent examples are the hyaloid and papillary membrane vessels of the developing eye. These vessels form during embryonic stages but regress postnatally. While the mechanisms underlying regression of these vessels are not clear, it has been suggested that macrophages may participate in this process (Diez-Roux et al., 1999; Lang and Bishop, 1993" Meeson et at., 1996, 1999). In one experiment, macrophages were specifically ablated by directing diphtheria toxin expression by using macrophage-specific promoter elements in transgenic mice (Lang and Bishop, 1993). In these mice, persistent hyaloid arteries were observed, suggesting that macrophages are required for normal hyaloid artery recession.

4. Hemogenic Endothelial Cells Hemogenic endothelial cells (see Chapter 10) represent a unique class of progenitors (Smith and Glomski, 1982). It has been suggested that some of the hematopoietic cells in embryos originate from endothelial cells. These hematopoietic cells originate from endothelial cells located at specialized vascular beds, such as the ventral portion of the dorsal aortae, the aorta-genital ridge-mesonephros (AGM) region, and umbilical vessels (Dieterlen-Lievre and Martin, 1981"

!i Lineage Specification and Differentiation

220 Godin et al., 1995: Medvinsky and Dzierzak. 1996: Medvinsky et al., 1993 Tavian et al., 1996). In these vascular beds, subsets of the endothelial cells were shown to "bud out" and become hematopoietic cells (Smith and Glomski, 1982). One of the most critical genes in this process was recently discovered. Cbfa2 and Cbfb encode two subunits of corebinding factor (CBF), which is required for definitive hematopoiesis during an early embryonic stage (Niki et al.. 1997: Okuda et al., 1996: Sasaki et al., 1996 Wang et aI., 1996a,b). Cbfa2 was shown to be expressed by a subset of endothelial cells budding and differentiating to hematopoietic cells (North et al., 1999). Furthermore. Cbfa2 -j- embryos exhibit a lack of hematopoietic emergence from hemogenic endothelial cells (North et al., I999). Therefore. this stud5, suggested that Cbfa2 function is to suppress endothelial phenotype and/or induce hematopoietic differentiation. However. a more definitive conclusion may rely on systematic dissection of genetic pathways regulated downstream from Cbfa2.

5. Blood-Borne Circulating Endothelial Progenitor Cells in the Adult Recently. cells that can differentiate to endothelial cells have been isolated from adult blood. These cells were referred to as "'blood-borne'" endothelial progenitors (Asahara et al., 1997). Subsequently. bone marrow transplantation experiments suggested that these progenitors could be bone marrow derived (Asahara et al.. 1999). Furthermore. they were found to participate in blood vessel formation in the adult (Asahara et al.. 1999). Although these findings are intriguing and provide a novel concept in the field, so far none of these findings have been confirmed independently. Further investigation is required to confirm the existence of physiologically relevant endothelial progenitors in the adult circulation. One of the first challenges is to define these putative endothelial progenitors at the molecular level. It is also important to reevaluate the source of these putative progenitors. One of the most difficult tasks is to eliminate potential problems resulting from the contamination from tissuederived endothelial and/or endothelial progenitor cells, and to show that the circulation of such progenitors is physiological.

E. Current View of Endothelial Cell

Type Specification

Based on the discoveries outlined in Section III. the current view of endothelial cell type specification is schematically shown in Fig. 4. One new principle in this field learned during the 1990s is that endothelial cells are a highly heterogeneous population of cells. We are just beginning to understand their origins and the regulatory mechanisms that underlie the establishment of this diversity among the endothelial cells. Many of the specific questions for future chal-

lenges are already discussed in this section. However, one of the most urgent goals is to identify further molecular markers for a variety of endothelial cell types that possibly exist and remain to be defined. The ability to separate and characterize one type of endothelial cell from another based on the expression of such marker(s) certainly facilitates the identification of even further heterogeneity of endothelial cells. The regulatory mechanism underlying the establishment and maintenance of heterogeneous endothelial types is virtually unknown and is certainly an area for future challenge.

IV, Vascular Morphogenesis Establishment of a blood vessel network is a dynamic process. The initial phase is accomplished primarily by endothelial cells. In later phases, other cell types such as smooth muscle cells participate in further shaping up the vessel network. Vascular morphogenesis is operationally divided into two phases" vasculogenesis and angiogenesis. In this section, mechanisms underlying each phase are discussed.

A. Vascuiogenesis The first blood vessel network is formed by assembling primarily endothelial cells into a channel-like structure (Box 1). These vascular channels fuse to each other to form an interconnected network of blood vessels. As described in Section II, this initial vessel network is referred to as the primary capillary plexus (Risau and Flamme. 1995). It is thought that this process involves morphological changes of endothelial cell shape, cell-cell adhesion, cell-matrix interactions, and perhaps some d e g e e of endothelial cell migration. Numerous studies have led to the implication that formation of this initial network is an intrinsic property of differentiated endothelial cells. Endothelial cells in culture are known to spontaneously form tubule-like structures that resemble the primary capillary plexus observed in vivo (Folkman and Haudenschild, 1980). In an explant culture system, bFGF-induced endothelial cell differentiation from mesodermal precursors is always accompanied by the formation of blood islands (Box 1), a precursor structure toward the primary capillary plexus (Flamme and Risau, 1992). The precisely controlled number of existing endothelial cells seems to be essential for producing a normal network of primary capillary, plexus (G.-H. Fong et al., 1999). As described in Section III, VEGF-R 1 knock-out embryos exhibit a dramatically increased number of endothelial cells. This resulted in an abnormal patterning of the primary capillary plexus in the yolk sac, an extraembryonic membrane where these first vessels form.


221

Figure 4 Current model of endothelial ceil types specification. (A) Many endothelial cells originate from emb~onic mesodermal cells and are generated by sequential cell specification processes. The details are described in the text. (B) In heart, coronary" vessel endothelial cells seem to originate from epicardium. A subset of epicardial cells undergoes epithelial-mesenchymal transformation, and these mesenchymal cells contribute to cell types that participate in the formation of coronary vessels including endothelial cells. It is proposed that myocardial cells send a critical paracrine signal(s) to epicardium for its transformation to mesenchymal cells. (C) In the adult, the presence of circulating endothelial cell progenitors, possibly derived from bone marrow, is proposed.

11Lineage Specification and Differentiation

222 In addition to these cell-biological parameters, it is known that the transforming growth factor/3 (TGF-/3) (Tgfol) signaling pathway may also play an important role in vasculogenesis. ES cells that overexpress the kinase-deficient type II TGF-/3 receptor (Tgfor2) failed to contribute to a normal primary capillary plexus in the yolk sac of chimeric embryos (Goumans et al., 1999). Similar results were also obtained by studying the differentiation of embryoid bodies from these ES cells in vitro (Goumans et aI.. 1999). This vasculogenic defect was correlated with a significant reduction in the production of extracellular matrix proteins such as fibronectin and laminin (Goumans et al., 1999). These results led to the conclusion that the TGF-/3 signaling pathway is critical for vasculogenesis, and that this process is either directly or indirectly mediated by controlling the production of extracellular matrix proteins such as fibronectin and laminin. Alternatively. it is also possible that expression of the dominant-negative type II TGF-/3 receptor leads to buffering of extracellular factors that are not the authentic ligands for this receptor. As a consequence, this may result in the modulation of other signaling pathways that are not related to the authentic TGF-/3 signaling pathway, but which are critical for vasculogenesis. At this point, very little is known about the specific factors that control cell-biological processes underlying vasculogenesis. None of the gene knock-out lines analyzed so far exhibit a complete lack of vasculogenesis. This may be due in part to the fact that these morphogenic processes are critically regulated by factors that control fundamental aspects of cell migration, survival, and cell shape control in a wide range of cell types. This problem could be addressed by examining the functions of such factors that control these fundamental cell biological processes during vasculogenesis, via either endothelial-cell-specific gene knock-out or inducible knock-out strategies.

B. Angiogenesis In contrast to vasculogenesis, the mechanisms of angiogenesis (Box 2) have been more extensively investigated, since it is considered to be a primar?' process involved in the pathogenesis of many human diseases.

1. Models for Angiogenesis Subsequent to vasculogenesis, the vascular network expands and remodels. This process is referred to as angiogenesis and is thought to involve multiple related but distinguishable phases (Risau. 1997). However. no reliable documentation regarding how the primary, capillary plexus expands and remodels its network is currently available. This is in part due to the fact that no studies have recorded ongoing angiogenesis at the real timescale with the single-cell resolution in vivo.

In this respect, a useful transgenic model was recently developed. In this transgenic mouse liner green fluorescent protein (GFP) was specifically expressed in virtually all endothelial cells using an endothelial specific promoter/enhancer expression vector (Motoike et al., 2000). This resulted in the labeling of endothelial cells by GFP and the visualization of live fluorescent endothelial cells without any complex pretreatment. This transgenic line of embryo may eventually be useful to visualize and follow the behavior of each endothelial cell with sophisticated imaging methods in combination with an in vitro emb~'o culture system to achieve real-time documentation of angiogenesis. Although no real-time recording of angiogenesis has been documented so far, substantial information is available regarding how angiogenesis proceeds based on conventional histological examinations. By these studies, multiple distinct processes underlying angiogenesis were identified and each process is discussed in this section. a. Formation of New Vascular Channels. As discussed in Section II, new vascular channels are formed by both sprouting and nonsprouting mechanisms (Fig. 2). These processes are mediated by dynamic remodeling of the extracellular environment and changes in endothelial motility and shape. Endothelial cells secrete various proteases that digest extracellular matrices. These endothelial extracellular matrices usually keep endothelial cells in place by providing structural integrity and cell survival signals. However, in order to generate new vascular channels, endothelial cells are required to digest and remodel these extracellular matrices to create a microenvironment that allows increased motility. Endothelial extracellular matrix components and their remodeling, which are involved in the formation of new vessels during angiogenesis, have been extensively studied. Many of these components are not unique to endothelial cells, but rather play a general role in morphogenesis for many different cell types and organ systems. Therefore, matrix remodeling is a common biological strategy utilized among many biological systems that require dynamic changes such as angiogenesis. Here, some of the specific components that belong to this category and play roles in new vessel formation are discussed. Fibronectin (Fnl) is one of the major extracellular matrix components (Hynes, 1985). It plays important roles in cell survival, migration, and shape control (Hynes, 1985). Fibronectin null mutants exhibit abnormal formation of blood vessels during mouse embryonic development (George et aL, 1997). However, because fibronectin is expressed in many different cell types during early embryonic development, it is difficult to determine which of these defects is directly caused by the absence of fibronectin function. It would be important to address this question by a more sophisticated genetic manipulation method, such as cell type specific and/ or inducible knock-out of the fibronectin gene.

11 Vasculogenesis and Angiogenesis Integrins are a family of transmembrane proteins that are specific receptors for extracellular matrix proteins such as fibronectin, laminin, and vitronectin (Hynes, 1992; Hynes et al., 1999). They mediate intracellular signaling pathways that lead to the control of cell survival, migration, and morphogenesis (Hynes, 1992: Hynes et al., 1999). The ~ , ( I t g a 4 ) f l . , ( h g b 3 ) , an integrin complex expressed by endothelial cells, has been suggested to play a key role in angiogenesis (Brooks et al., 1994). The main evidence for this comes from an experiment using the specific peptide inhibitor for this integrin (Brooks et al., 1994: Drake et al., 1995). This peptide inhibitor has been shown to interfere with angiogenesis in the chick embryo, in tumor formation models, and in in vitro angiogenic assays (Brooks et al., 1994: Drake et al., 1995). However, mouse genetic experiments have not supported such a specific role for this integrin in angiogenesis. Both c~,-/- and/33-;- embryos exhibit extensive normal vasculogenesis and angiogenesis, suggesting an alternative explanation for the inhibitow peptide experiment (Bader et aI., 1998 Hodivala-Dilke et al.. 1999). It is possible that the inhibitor; peptide binding to c~,/33 integrin complex leads to the modulation of signaling pathways that are not normally under the control of this integrin complex. This ectopic signaling pathway in endothelial cells may be inhibitory for angiogenesis, although the cz,./33-integrin complex may not be directly involved in angiogenesis. Further studies will be required to sort out this controversy. Extracellular proteinases modulate the extracellular microenvironment of vascular cells (Haas and Madri, 1999; Nagase and Woessner. 1999; Werb. 1997; Werb and Chin, 1998). This modulation may provide a permissive environment for angiogenesis. Matrix metalloproteinases represent a family of extracellular proteinases that have been implicated in angiogenesis (Haas and Madri, 1999; Nagase and Woessner, 1999: Werb, 1997; Werb and Chin, 1998). MMP9/gelatinase B ( M m p 9 ) has been shown to play an important role in angiogenesis during bone development (Vu et al., 1998). MMP-9/gelatinase B -/- mice exhibit aberrant angiogenesis, which was shown to be rescued by transplantation with gelatinase-B-expressing wild-type bone marrow cells (Vu et al., 1998). Furthermore, growth plates from MMP-9/ gelatinase-B -/- mice were shown to exhibit delayed release of an angiogenic activator (Vu et at., 1998). These results suggest that gelatinase-B produced by bone-marrow-derived cells allows the release of angiogenic factors from these cells that would otherwise be sequestered as an inactive form. Matrix metalloproteinases have their specific endogenous inhibitors (Haas and Madri, 1999; Nagase and Woessner, 1999" Werb, 1997; Werb and Chin, 1998). Functions of these factors have been studied in vivo by generating null mutant mice (Shapiro, 1997). However, it is difficult to assess their specific role in the developing vasculature due to their expression by multiple cell types. Important insights into

223 the roles of such family of factors in angiogenesis may be gained by generating and analyzing vascular-cell-typespecific knock-out mice in the future. The matrix remodeling discussed above provides a permissive microenvironment for endothelial cells to form new vascular channels. However, it is believed that subsequent local inductive signals are required to ultimately make endothelial ceils form a new vessel. It is proposed that VEGF (Box 4) is one class of soluble factors that may take part in this process. Much evidence supports the notion that VEGF is capable of inducing an angiogenic response by endothelial cells. VEGF was shown to induce endothelial cell proliferation in vitro (Leung et aL, 1989). Overexpression of VEGFA in developing skin during postnatal stages results in increased skin vascularization (Thurston et ai., I999). Overexpression of VEGF-C was also shown to :result in ectopic angiogenesis of both lymphatic and blood vessels in vivo (Jeltsch et aI., i997). Furthermore, VEGF-A gene knockout mice exhibit severely decreased angiogenesis (Carmeliet et at., 1996; Ferrara et al., 1996). Interestingly, reduced angiogenesis was also observed in mice that are heterozygous for the VEGF-A gene mutation, suggesting that a subtle titration of VEGF-A activity is critical for normal angiogenesis (Carmeliet et al., 1996; Ferrara et al., 1996). Multiple receptors are suspected to mediate the angiogenic function of VEGFs. VEGF-R1 and VEGF-R2 knock-out mice die before active angiogenesis occurs during embryogenesis (Fong et aL, 1995" Shalaby et al., 1995), precluding the possibility of using these knock-out mice to study the roles of VEGF in angiogenesis. However, it was shown that overexpressing soluble VEGF-R1 (i.e., blocker of VEGFR1 binding to VEGF) during postnatal mouse development leads to insufficient blood vessel development and growth retardation of many organs (Gerber et al., 1999). Moreover, it was reported that VEGF-R3 knock-out embryos exhibit retarded embryonic angiogenesis at 9.5 dpc (Dumont et aI., 1998). In addition to the classic VEGF receptor family, it was shown that neuropilin-1 (Nrp), one of the factors that mediate neuronal cell guidance, also serves as an isoform specific receptor for one alternatively spliced form of VEGFA, VEGF~65 (Soker et aI., 1998). To investigate the possibility that this unconventional VEGF receptor may also be involved in angiogenesis, neuropilin-1 knock-out mice were generated and characterized (Kawasakj et al., 1999). In this study, neuropilin-1 -/- embg,~os were shown to exhibit defective aortic arch formation (Kawasaki et at., 1999). Although the study failed to pinpoint the exact process that is affected in the formation of the aortic arch vessels, it is possible that the defect may be in the angiogenic response by aortic arch endothelial cells (Kawasak5 et al., 1999). Another class of soluble factors that may be involved in angiogenesis is fibroblast growth factor (FGF). FGF-1 ( F g f l ) and FGF-2 ( F g f 2 ) were found to induce angiogenesis in vivo (Jouanneau et al., 1995" Klagsbrun, 1991" Schelling,


224 1991- Seghezzi et al., 1998). Therefore. this suggested that exogenous FGF can induce angiogenesis by itself or in combination with other endogenous factor(s). However, there are no studies clearly showing that "endogenous" FGFs or FGF receptors are involved in angiogenesis during normal development. The third class of soluble factors that induces the formation of new vascular channels is the angiopoietins (Box 9) (Davis and Yancopoulos, 1999). Currently, several available data suggest that angiopoietins may be involved in angiogenesis. Ectopic expression of angiopoietin-1/Ang-1 (Agpt) in vivo is sufficient to induce new blood vessels (Suri et aI., 1998). Furthermore. mouse embryos deficient in Ang-1 or its specific receptor. Tie2/Tek (Tek), were shown to exhibit retarded angiogenesis (Dumont et al., 1994: Sato et al.. 1995 Suri et al., 1996). In addition, ectopic expression of the inhibitory ligand for the Tie2 receptor, angiogpoietin-2/ Ang-2 (Agpt2), was shown to cause inhibition of embryonic angiogenesis (Maisonpierre et al., 1997). Another member of the Tie receptor family. Tiel (Tie 1), was also shown to be important for an angiogenic response of endothelial cells during development (Puri et al., 1995 Sato et al., 1995).

Box 9: Angiopoietin and

While no ligand(s) have been identified for this receptor, Tiel -/- embryos exhibit severe hemorrhaging and edema (Puri et al., 1995; Sato et aL, 1995). Tie 1-:- ES cells contributed normally to many vessels except the capillaries of the brain and kidney (Partanen et al., 1996). In these organs, vascularization is thought to be primarily accomplished by sprouting of preexisting vessels (Risau, 1991a,b). These studies suggest that Tie l may also be involved in angiogenesis in an endothelial cell autonomous manner during embryonic development. In addition, functional interactions have been proposed between Tie 1 and Tie2 receptors during blood vessel formation, based on the analyses of double knock-out and compound heterozygous mice for these two related genes (Puff et al., 1999). Spatial and temporal expression patterns of endogenous angiopoietins suggest an interesting correlation with their potential role in angiogenesis (Holash et al., 1999; Maisonpierre et al., 1997). Ang-1 expression is primarily associated with vessels that have completed their formation and become stable. In contrast, Ang-2 expression is associated with vessels that are either just beginning to form or regessing. When Ang-2 expression is codetected with VEGE these ves-

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in the formation of blood vessels during the early s t a g e s : Tie receptors were originally discovered as a family of vascular development. :~: ::~:::: ~, of novel receptor tyrosine kinases that is preferentially As suggested from the unique d o ~ expressed in endothelial cells. Two related receptors bethe extracellular :part : o f the receptor, a unique ...... ........... cla of :::..:. long to this class of receptor tyrosine kinases: Tiel (also known as Tie) and Tie2 (also known as Tek). Both resoluble ligandsfor ~Tie2~recepl ceptors exhibit a tmique setof domain structures in ~ i r `~ There': :::: = iiUi[ extraceHular region. It consists of two immuno~obulinAng- 1, angaol;::>oietin-2lAng-2~:":~: an~opo~e~-3/Ang-~ (identified only in human so far), and angaopoietin-41 like domains flanking three EGF-like repeaxs, which are Ang-4 (Agpt4). They are all secreted ~ycoproteins:::~;~ followed by three fibronectin type HI repeats. Both of possess a unique domain structure consisfingi::of::::::~ the receptors were found to be expressed mainly in vas. . . . . C~ coiled-coil domain followed by: a fibrinog ' en-like cular endothelial cells during development and in the main. It has been shown that this coiled-coil::..... me adult. Subsequently, these receptors were also shown to diates the m u l t i m e f i z ~ o n of angiopoietins that, seem '~t(:~:~ be expressed in hematotxfietic lineages. This relatively specific expression in endothelial cells suggested impormediate bioactivities. :of the . proteins, . . All' of ; tam roles in the vascular system. To confirm ~ s pospoietins were shown tobind t o . T i e 2 /n sibility, knock-out mice forthe genes encodin'g T i e l and but failed to b i n d t o ~the:Tieli:i h Tie2 were generate~i and characteri_z~. Tiel knock-out ~ : these : : mice died between 14'5 dpc and PO and exhibited severe .......... ofthe : also: :: :F~ existence of~afeWa vascular: hemorrhage. This phenotype and the expres.... teins has been reported. However, ~ s e angi sion of the receptor in endothelial cells suggested that a related proteins do not exhibit specific binding to the Tie ~:::::::::i::1 primary role for Tie1 is to maintain the integrity of receptors. The functional significance of bot.h:::::splice: :: :::] blood vessels. Tie2 knock-out mice died between 9.5 variants of the angiopoietins:and the: and 10.5 dpc, and exhibited severe vascular malformation. This phenotype suggested a primary role for Tie2 .

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11 Vascuiogenesis and Angiogenesis sels seem to be actively engaged in vessel formation. However. when Ang-2 expression is not associated with VEGE these vessels tend to be regressing. These studies suggest that Ang-2 may serve as a local angiogenic signal for endothelial cells. Blocking Ang-1 signaling by Ang-2 in an endothelial cell may create a permissive environment for the cell to respond to other angiogenic signals such as VEGF. Intracellular signaling pathways that may be subjected to this "ying-yang" regulation of the angiogenic phenotypes of endothelial cells by angiopoietins was investigated. It was recently shown that activation of the Tie 2 receptor by Ang-1 leads to activation of the PI3 kinase and Akt/protein kinase B pathway in vitro (Kontos et al.. 1998: Papapetropoulos et al., 1999, 2000). It is possible that fundamental cell-biological processes directly controlled by this signaling pathway may underlie the determination of the endothelial cell phenotype regarding angiogenic versus nonangiogenic states. In addition to regulation by soluble factors, certain physiological conditions also play important roles in inducing new vessel formation by endothelial cells. One such condition is hypoxia. One of the most important physiological functions of the circulatory system is to deliver sufficient oxygen to organs during development and in the adult. This leads to a notion that organs that are not fed with sufficient oxygen need more blood vessels. Thus, it is conceivable to imagine that a hypoxic environment induces angiogenesis. Recently, significant advances have been made in understanding the molecular mechanisms underlying hypoxic regulation of angiogenesis. A family of nuclear receptors, collectively referred to as bHLH/PAS transcription factors. is involved in the hypoxic responses by a variety of cells (Crews, 1998 Crews and Fan, 1999; Semenza, 1999). They have two key domains, a basic helix-loop-helix (bHLH) domain that mediates the interaction with another member of this family and a PAS domain, which seems to be a key element for transcriptional target specificities. This family of transcription factors has been shown to be critically involved in many biological systems including neurogenesis, xenobiotic metabolism, and angiogenesis (Crews, 1998" Crews and Fan, 1999). Two key bHLH/PAS transcription factors have been clearly shown to be involved in angiogenesis HIF-lc~ (Hifla) and ARNT (Arnt). HIF-lc~ -/- mouse embryos die by E11.5 due to several morphogenic defects, including cardiovascular malformations (Iyer et al., 1998; Ryan et al., 1998). Furthermore, HIF-lc~ -/- ES cells failed to upregulate VEGF in response to hypoxic conditions (Iyer et al., 1998). In addition, HIF-1 c~-/- tumors failed to support ag~essive tumor angiogenesis (Carmeliet et al., 1998" Ryan et at., 1998). It has been suggested that hypoxia-mediated tumor angiogenesis is, in part, regulated by the selective de~adation of HIF-lc~ by p53 (Trp53) (Ravi et aI., 2000). A more detailed analysis of HIF-lc~ -/- emb~,os and ES cells in vitro suggested that the lack of HIF-Ic~ leads to the death of mesen-

225 chymal cells that surround developing embryonic vessels (Kotch et al., 1999). This in turn leads to the failure of normal vessel development in a VEGF-independent manner. perhaps due to the lack of other mesenchyme-derived angiogenic factor(s) (Kotch et al., 1999). These m vivo and in vitro results suggest that a hypoxic environment is translated to upregulation of angiogenic factors that are required for angiogenesis during development and tumor growth. In addition, HIF-lc~ forms a functional heterodimer with ARNT. ARNT -/- mouse embryos do not survive past 10.5 dpc, with detective angiogenesis in the yolk sac and placenta (Koza_k et al., 1997; Maltepe et aL, 1997). These analyses support the notion that both HIF-lc~ and ARNT play a critical role in embryonic angiogenesis, perhaps as a heterodimer. b. Segregation o f Arteries and Veins. As discussed in Section III.D. 1, arterial arid venous endothelial identities are established prior to angiogenesis. This fact implies that arterial endothelial cells assemble among themselves to form arteries, and venous endothelial cells do so among themselves. Therefore, two mechanisms are expected to be in place to achieve this segregation process: a mechanism that allows homophilic interactions among each endothelial cell type and a mechanism that prevents interaction of these two endothelial types. Two molecular pathways are proposed to be critically involved in these processes. Arterial endothelial cells express ephrin-B2 and venous endothelial cells express its receptor, EphB4 (Wang et aL, 1998). This transmembrane ligandreceptor pathway has been suggested to operate as a cellcell repulsion signal (Cook et al., 1998; Mellitzer et al., 1999; Wilkinson, 2000a,b; Xu et al., 1999). Therefore, it is possible that this specific segregation of ephrin-B2 and EphB4 expression pattern ensures the segegation of arterial and venous vessels during angiogenesis. To support this possibility, knock-out mice lacking either ephrin-B2 or EphB4 exhibit abnormal angiogenesis (Gerety et al., 1999, Wang et al., 1998). Most interestingly, it is reported that the borders between arterial and venous channels become less clear in these knock-out mice (Gerety et al., 1999; Wang et al., 1998). Furthermore, it is reported that ectopic expression of epl~-'in-B2 or dominant negative EphB4 in Xenopus embpjos results in the ectopic formation of veins into a region where these vessels do not normally invade (Helbling et al., 2000). These experimental results seem to strongly support the idea that this ligand-receptor pathway plays a unique role in segregation of arteries and veins by mediating the repulsion between these two types of vessels during anNogenesis. In addition to this Iigand-receptor pathway, the TGF-/3 pathway is also shown to be involved in this process. Activin receptor-like kinase-1 (Acvrll) encodes a type I receptor for the TGF-/3 ( T g ~ r l ) superfamily of growth factors, and the knock-out of the A c v r l l gene in mice resulted in the downregulation of ephrin-B2 (arterial-specific marker) arid the


226 subsequent failure of shunting between dorsal aortae (artery) and cardinal vein (Unless et al., 2000). This study suggests that the Acvrll-mediated signal transduction pathway is critical for establishing the identity of arterial endothelial cells and/or segregating arteries and veins. The third pathway that seems to be involved in this process is the Notch pathway. Notch is a family of transmembrane receptors whose function is primarily regulated by a specific family of ligands (Gridley, 1997). One of the Notch family,' members. Notch4, was identified as an endothelialspecific Notch in mouse development and in adult (Shirayoshi et al., 1997: Uyttendaele et aI.. 1996). Notch4 is very similar to other mammalian Notch proteins because it contains conserved motifs: however. Notch4 has fewer EGF-like repeats and a shorter intracellular domain than other mouse Notch homologs. These structural differences, along with the endothelial-specific expression of Notch4. suggest that this Notch protein plays a unique role in vascular development. To support this notion, double knock-out embryos for Notch4 and N o t c h l genes result in vascular malformation (Krebs et al., 2000). Furthermore. a novel Notch ligand. Delta-Like Ligand 4 (Dll4) was found to show an expression pattern that is consistent with the vascular phenotype of the Notch l~ Notch4 double knock-out (Krebs et al., 2000: Shutter et al., 2000). Interestingly. the expression of Dll4 is relatively restricted to arterial endothelial cells (Shutter et aI.. 2000). Because Notch receptors do not exhibit such specificity, it is possible that arterial expression of Dl14 may elicit specific signals in the regulation of angiogenic pathways in both venous and arterial endothelial cells in a paracrine and autocrine manner, respectively. c. Establishment

o f Endothelial

Cell-Cell

Junctions.

As new vascular channels are formed, it becomes essential to establish "'leakage-proof'" endothelial cell-cell junctions during angiogenesis. Endothelial cells establish these cellcell junctions, and it is assumed that most of the general cell-junction-forming mechanisms found in other cell types such as epithelial cells are at work. In addition to these general mechanisms, endothelial cells utilize cell type specific mechanisms. Cadherins are a family of transmembrane proteins localized specifically at the cell-cell junction, and mediate Ca2--dependent homophilic interaction between the cells (Urushihara and Takeichi. 1980). Endothelial cells express a cell type specific cadherin, called VE-cadherin (Cdh5) (Breier et al., 1996: Wu and Maniatis, 1999, 2000). A critical role for VE-cadherin in angiogenesis was recently shown by studying VE-cadherin-"- mice (Carmeliet et al., 1999). VE-cadherin-;- embryos failed to form a normal vascular network at 9.5 dpc, Furthermore, deletion of the cytoplasmic portion of VE-cadherin was sufficient to produce a similar phenotype in vascular network formation (Carmeliet et al., 1999). Therefore, this study suggested an essential role for VE-cadherin in angiogenesis, which may be

regulated by its interaction with cytoplasmic components that lead to various intracellular signaling events in endothelial cells. d. Recruitment o f Smooth M u s c l e Cells and Pericytes.

During angiogenesis, endothelial cells recruit other cell types into the vascular channels. Smooth muscle cells and pericytes become invested around the endothelial cells of medium to large sized vessels and capillaries, respectively. Smooth muscle cells form continuous layers around the vessels. but pericytes are invested around the capillaries only in a discontinuous manner. Smooth muscle cells and pericytes are considered to be related. Both of them provide structural integrity and contractility of the vessels, two hallmarks of the mature vessel. For recruitment of smooth muscle cells/ pericytes, reciprocal communications between these cells and endothelial cells are critical. Endothelial cells secrete PDGF-B (Pdgfb). which binds and activates the specific receptor PDGFR-/3 (Pdgfrb) expressed on the surface of smooth muscle/pericyte precursors. This paracrine pathway mediates the migration of these precursors into the immediate proximity of the endothelial cells that are already a part of the vessel. This initial recruitment of smooth muscle/pericyte precursors seems to result in the clustering of these cells at the ventral abluminal surface of the vessels (Hungerford et al.. 1997: Lee et aI.. 1997 Sato, 2000). This model is supported by the fact that both PDGF-B -~- and PDGFR-/3 -~- emb~'os lack pericytes along the endothelial vascular channels and vessel integrity was impaired, resulting in microaneuw s m (Hellstrom et aI., 1999" Lindahl et ai., 1997). Differentiation of these precursors to more mature smooth muscle cells/perictyes is mediated by the TGF-/3 pathway. It has been shown that smooth muscle progenitorlike cell lines or multipotential mesodermal-derived cell lines can convert to smooth muscle cells on treatment with TGF-/3 in vitro (Hirschi et aI., 1998). In contrast to this in vitro result, a lack of TGF-/3 in mice does not seem to interfere with the differentiation of smooth muscle cells (Dickson et al., 1995; Kulkarni et al., 1993). This may be due to the multiplicity of receptor specificity and other factors that belong to the TGF-/3 family acting in a compensator'2," manner. In support of this possibility, knock-out mice for endoglin (Eng), a gene encoding an extracellular TGF-/3 binding protein. were shown to exhibit reduced numbers of smooth muscle cells and lacked normal endothelial/smooth muscle cell interaction (Li et al., 1999). The TGF-/3 signaling pathway is mediated by a family of signal transduction proteins called SMAD (Derynck et al., 1998: Heldin et al.. 1997: Whitman, 1997). Among them, Smad5 has been shown to be critically involved in embryonic vascular development (Chang et al., 1999" Yang et al., 1999)" it is thought to transduce signals of the bone morphogenic protein (BMP) pathway (Miyazono, 1999: Raftery and Sutherland, 19991). Smad5 (Madh5) null mutant mice exhibit

11 Vasculogenesis and Angiogenesis abnormal vascular development, although they seem to have differentiated smooth muscle cells (Chang et al., 1999; "fang et al., 1999). It is possible that Smads have overlapping functions and therefore a single gene knock-out does not cause complete absence of smooth muscle cell differentiation. Such compensato~ mechanisms are likely since various TGF-/3 and related signaling pathways are regulated by numerous and complex interactions involving Smad family members. Although pericytes are often localized as a single cell on top of the endothelial cells in a discontinuous manner, smooth muscle cells form a complete layer around the vessels. This requires the upward migration of smooth muscle cells initially localized to the ventral abluminaI surface along the vessels. This step seems to be mediated by a class of glycolipids-mediated-signaling pathway. Sphingosine phosphate- 1 binds and activates a family of G-protein-coupled receptors referred to as the Edg family (Hla et al., 2000). In mice lacking one of this receptor family members, Edgl ( E d g l ) , smooth muscle cells remain clustered at the ventral abluminal surface of the vessels and fail to surround the vessels (Liu et al., 2000). Therefore, it is proposed that this novel ligand-receptor pathway is involved in this later phase of smooth muscle cell investment of the vessel wall. e. Regression o f Vascular Channels. During angiogenesis, some of the vascular channels undergo regression. As discussed in Section III.D.3.c, hyaloid and papillary" membrane vascular systems in the eye are the most extensively studied, at least at the morphological level. The molecular basis for the regression of specific vascular channels during vascular development is completely unknown at this time and awaits future investigation.

f. Establishment of Vascular Polarity. The vascular network is a highly polarized structure. Branching points distributed along the vascular channel and the directionality of the new branches form a basis for the polarity. Furthermore, this polarized formation of new branches together with regression of specific vascular branches during embryonic angiogenesis leads to the establishment of a left-right asymmetry of the network. One pathway has been implicated to play a critical role in establishing this left-right asymmetry of the vascular network. Mice doubly mutated for Ang-1 and Tie 1 have been shown to exhibit the lack of fight-handside cardinal veins, but normal left-hand-side cardinal veins (Loughan and Sato, 2001). This suggested that the combinatorial role of Ang-1 and Tie l is required in the formation of specifically the right-hand-side cardinal veins. Furthermore, this asymmetrical phenotype was shown to correlate with the polarized expression of Ang-1 in the sinus venosus from which the cardinal veins branch. Based on this study, it is anticipated that other pathways may also regulate the establishment of vascular polarity.

227 2. Clinical Implications Although the main theme of this chapter is vascular development in mice, many of the findings described above are leading the way to potential therapeutics for human diseases. Tumor angiogenesis is one of these areas. In addition to its developmental roles, the VEGF pathway is implicated in tumor angiogenesis. This pathway has been one of the most popular drug development targets for antiangiogenic therapy in an effort to starve tumor cells to death by cutting the blood vessel supply fT. A. Fong et al., 1999; Millauer et al., 1994, 1996" Witte et al., 1998). In addition, in an experimental animal model for tumor formation, blocking Tie2 receptor function prevented tumor angiogenesis and consequently retarded tumor growth significantly (Lin et al., I998). Another area is proangiogenic therapy. The objective of this therapy is to induce blood vessel formation in damaged organs such as after heart failure. Recent studies such as gene transfer, recombinant protein injections, and transgenic analyses have suggested that VEGF and angiopoietins may be potentially useful for this type of therapy (Peters, 1998; Suri et al., 1998; Thurston et al., 1999). Furthermore, several of the genes described above have been implicated in human genetic diseases related to vascular malformations. Tie2 receptor mutations have been linked to venous malformation disease (Vikkula et al., 1996). Endoglin mutation has been linked to vascular malformation disease associated with hereditary hemorrhagic telangiectasia (HHT1) as discussed above (Guttmacher et aI., 1995" McAllister et aL, 1994). VHL tumor suppressor gene (Vhlh), a critical angiogenesis regulator, is associated with von Hippel-Lindau disease (Gnarra et al., 1996; Linehan et al., 1995). Notch pathways axe involved in several human disease conditions (Gridley, 1996, 1997; Joutel and TournierLasserve, 1998). As we learn more about the mechanisms of vascular development and human genetics in the future, the list of factors and pathways that are critical for both normal development and human disease formation is expected to expand. This may certainly lead to the invention of novel and more effective therapies for many human diseases.

3. Major Questions on Angiogenesis Based on the most current description of angiogenesis, it is clear that our current "knowledge of angiogenesis is composed of only fragmentary information. Furthermore, the detailed biochemical and molecular mechanisms of the biological functions of each factor discussed in this section remain highly speculative. Angiogenesis is clearly a complex and heterogeneous process. Angiogenesis at different developmental stages and in different organs may involve distinct mechanisms. It is likely that even a single process during angiogenesis involves multiple factors and signaling pathways. The specific factors and the physiological envi-


228 ronment discussed in this section are only a fraction of the continuously growing list of angiogenic factors. Therefore, it will be our future task to decipher precise mechanisms by which multiple factors and signaling pathways interact to establish new blood vessels. It is also important to understand how angiogenesis utilizes differential mechanisms at each developmental stage and in each organ.

V. Concluding Remarks As outlined in this chapter, it is clear that the vascular development is regulated by a complex network of gene functions and signaling pathways. The use of modem mouse genetics and embryo manipulation technologies has significantly contributed to our advancement of this field. However, key questions regarding several fundamental aspects of vascular development still remain unanswered. We would like to discuss a couple of them as our concluding remarks. As discussed in Section III. we are beginning to realize the complexity and heterogeneity of vascular cell types. Our knowledge of the exact origins of various vascular cell types and their eventual fates remains at the preliminary and inconclusive level. Conventionally, this area was studied by using classic chimera and cell transplantation approaches as well as a retrovirus-mediated cell marking system (Mikawa and Fischman, 1992; Mikawa and Gourdie, 1996; Perez-Pomares et al., 1998: Yamashita et al., 2000). These methods will certainly continue to be useful however, the complex nature of vascular morphogenesis may require more sophisticated methods. Recently, recombinase-based cell fate mapping methods have been applied to study cell lineage regulation in the mammalian system (Chai et al., 2000: Dymecki and Tomasiewicz, 1998: Jiang et al., 2000: Kimmel et al., 2000: Zinyk et al., 1998). This system relies on the cell type specific recombinase (such as Cre and Flp) mediated permanent cell marking. The recombinase can be expressed transgenically by using a promoter that can drive the expression specifically in a progenitor cell population. This recombinase transgenic line is crossed to a Cre-excision reporter transgenic line in which the excision of the sequences flanked by two pairs of l o x P (for Cre) or Frt (for Flp) elements leads to the permanent reporter such as l a c Z expression in all the descendent cells during development. One advantage of this method is the ability to permanently mark the specific cell lineage of mammalian embryos, which are quite inaccessible to retrovirus marking system. Furthermore, recombinasebased methods allow the analysis of "true" cell fate regulation during normal and physiological development, as opposed to the chimera and cell transplantation methods, which can only measure the "ability" of transplanted cells to contribute to various lineages (Yamashita et al., 2000). Application of such a novel in vivo cell fate mapping system to the

problem in vascular cell lineage regulation is expected to contribute significantly to the advancement of the field. The second area is that of understanding how multiple pathways regulate vascular development. This is a very cornplex and challenging subject. However, the recent advent of new mouse genetic manipulation methods is expected to aid this problem. We can now knock out genes in an inducible manner (Rossant and McMahon, 1999). This possibility certainly allows us to address the function of specific genes in developmental stages where conventional gene knock-out strategies could not be applied due to the earlier embryonic lethality. Furthermore, several useful transgenic expression systems now exist that allow the targeting of gene expression in a specific vascular cell type (Kappel et al., 1999; Korhonan et al., 1995: Li et aI., 1996; Schlaeger et al., 1995, 1997). The availability of these methods and reagents is expected to facilitate the dissection of complex pathways underlying vascular development by knocking out or overexpressing specific genes in a specific vascular cell type at various developmental stages transiently or permanently. New genetic and embryo manipulation tools are continuing to be invented in the mouse system. Continuing improvements in our understanding of vascular cell lineage regulation not only contribute to our fundamental understanding of vascular development, but also facilitate the invention of new reagents that may allow us to manipulate the expression of specific genes in a specific vascular cell type. Many intriguing possibilities clearly lie ahead of us in this field and we all look forward to it.

References Aird. W. C.. Edelberg. J. M., Weiler-Guettler,H.. Simmons, W. ~:, Smith, T. W.. and Rosenberg, R. D. (1997). Vascularbed-specific expression of an endothelial cell gene is pro~ammed by the tissue microenvironment. J. Cell Biol. 138, 1117-1124. Asahara, T., Murohara. T., Sullivan, A., Silver, M., van der Zee, R., Li, T., Witzenbichler, B., Schatteman. G., and Isner, J. M. (1997). Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964-967. Asaharm T.. Masuda. H.. Takahashi, T., Kalka, C., Pastore, C., Silver, M., Kearne. M., Magner, M., and Isner, J. M. (1999). Bone marrow origin of endothelial progenitor cells responsible for postnatal vascutogenesis in physiological and pathological neovascularization. Circ. Res. 85, 221-228. Bader, B. L., Rayburn, H., Crowley, D., and Hynes, R. O. (I998). Extensive vasculogenesis, angiogenesis, and organogenesisprecede lethality in mice lacking all alpha v integrins. Cell (Cambridge. Mass.) 95, 507519. Breier. G., Breviario. F., Caveda, L., Berthier, R., Schnurch, H., Gotsch, U., Vestweber. D., Risau. W.. and Dejana, E. (1996). Molecularcloning and expression of murine vascular endothelial- cadherin in early stage development of cardiovascularsystem. Blood 87, 630-64 I. Brooks. E C., Clark. R. A., and Cheresh, D. A. (1994). Requirement of vascular integrin alpha v beta 3 for angiogenesis.Science 264, 569-571. Carmeliet. E, Ferreira. V.. Breier, G., Pollefeyt, S., Kieckens, L.. Gertsen-

11 Vasculogenesis and Angiogenesis stein, M., Fahrig, M., Vandenhoeck. A., Harpal, K.. Eberhardt, C.. Declercq, C., Pawling. J.. Moons. L., Collen. D.. Risau. W\. and Nagy, A. (1996). Abnormal blood vessel development and lethality in emb~'os lacking a single VEGF allele. Nature (London.) 380, 435-439. Carmeliet. R, Dor. Y., Herbert, J. M.. Fukumura, D.. Brusselmans, K.. Dewerchin. M.. Neeman. M., Bono. E, Abramovitch. R.. Maxwell. R, Koch. C. J.. Ratcliffe. R. Moons. L.. Jain, R. K.. Collen. D., and Keshet, E. (1998). Role of HIF-1 alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature (London) 394, 485-490. Carmeliet. R. Lampugnani, M. G., Moons, L.. Breviario. E, Compernolle. V.. Bono. F.. Balconi, G., Spagnuolo. R.. Oostuyse, B.. Dewerchin. M.. Zanetti. A.. Angellilo. A.. Mattot. V., Nuyens. D., Lutgens, E.. Clotman. F., de Ruiter, M. C., Gittenberger-de Groot. A.. Poelmann. R.. Lupu. E. Herbert. J. M.. Collen. D.. and Dejana. E. (i999). Targeted deficiency' or cvtosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell (Cambridge, Mass.) 98, 147-157. Chai. Y.. Jiang. X.. Ito, Y., Bringas. R, Hart. J.. Rowitch. D. H.. Soriano. R, McMahon. A. R. and Sucov. H. M. (2000). Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development (Cambridge. UK) 127, 1671-1679. Chang. H., Huylebroeck. D.. Verschueren. K.. Guo, Q.. Matzuk, M. M.. and Zwijsen, A. (1999). Smad5 knockout mice die at mid-gestation due to multiple embry'onic and extraembryonic defects. Development (Cambridge. UK) 126, 1631-t642. Cook. G., Tannahill, D.. and Keynes. R. (I998). Axon guidance to and from choice points. Curr. Opin. Neurobiol. 8, 64-72. Crews, S. T. (1998). Control of cell lineage-specific development and transcription by bHLH- PAS proteins. Genes Dev. 12, 607-620. Crews. S. T.. and Fan. C. M. (1999). Remembrance of things PAS Regulation of development by' bHLH-PAS proteins. Cur~ Opin. Genet. Dev. 9,580-587. Davis. S., and Yancopoulos. G. D. (1999). The angiopoietins: Yin and Yang in angiogenesis. Curr. Top. Microbiol. lmmunol. 237, 173-185. Der?'nck. R.. Zhang, Y., and Feng. X. H. (I998). Smads: Transcriptional activators of TGF-beta responses. Cell (Cambridge, Mass..) 95, 737740. Dettman. R. W.. Denetclaw. W:, Jr.. Ordahl. C. R, and Bristow. J. (I998). Common epicardial origin of coronary' vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev. Biol. 193, 169-181. Dickson. M. C., Martin, J. S., Cousins. E M.. Kulkarni. A. B.. Karlsson. S.. and Akhurst. R. J. (1995). Defective haematopoiesis and vasculogenesis in transforming growth factor-/31 knock out mice. Development (Cambridge, UK) 121, 1845-1854. Dieterlen-Lievre, F.. and Martin, C. (1981). Diffuse intraembwonic hemopoiesis in normal and chimeric avian development. Dev. Biol. 88, 18019I. Diez-Roux, G.. Argilla. M.. Makarenkova. H., Ko, K.. and Lang. R. A. (1999). Macrophages "kill capillar?' cells in G1 phase of the cell cycle during programmed vascular regression. Development (Cambridge, UK) 126, 2141-2147. Drake, C. J.. Cheresh. D. A.. and Little, C. D. (I995). An antagonist of integrin alpha v beta 3 prevents maturation of blood vessels during embry,onic neovascularization. J. Cell Sci. 108, 2655-2661. Dumont. D. J.. Anderson. L.. Breitman. M. L.. and Duncan. A. M. V. (1994). Assignment of the endothelial-specific protein receptor tyrosine kinase gene (TEK) to human chromosome 9p21. Genomics 23, 512513. Dumont, D. J., Jussila, L., Taipale. J., Lymboussaki, A., Mustonen. T., Pajusola, K., Breitman, M., and Alitalo, K. (1998). Cardiovascular failure in mouse embuos deficient in VEGF receptor-3. Science 282, 946-949. Dymecki, S. M.. and Tomasiewicz, H. (I998). Using Flp-recombinase to characterize expansion of Wntl-expressing neural progenitors in the mouse. Dev. Biol. 201, 57-65.

229 Edelberg, J. M., Aird. W. C.. Wu. W.. Rayburn, H., Mamuya, W. S.. Mercola. M.. and Rosenberg. R. D. (1998). PDGF mediates cardiac microvascular communication. J. Clin. Invest. 102, 837-843. Evans. H. M. (i909). On the development of the aortae, cardinal and umbilical veins, and the other blood vessels of vertebrate embryos from capillaries. Anat. Rec. 3, 598-519. Ferrara. N., Carver-Moore. K., Chen. H., Dowd. M., Lu, L.. O'Shea. K. S., PowelI-Braxton, L.. Hillan. K. J.. and Moore. M. W. (1996). Heterozygous embwonic lethality induced by targeted inactivation of the VEGF gene. Nature (London) 381), 439-442. Flamme. I.. and Risau. W: (1992). Induction of vasculogenesis and hematopoiesis in vitro. Development (Cambridge. UK) 116, 435-439. Folkman, J., and Haudenschild. C. (1980). Angiogenesis in vitro. Nature (London) ~ 8 , 551-556. Fong. G.-H.. Rossmnt. J.. and Breitman. M. L. (t995). Role of the Flt-1 receptor ~:rosine kinase in regulating the assembly of vascular endothelium. Nature (London) 376, 66-70. Fong, G.-H.. Klingensmith, J., Wood, C. R.. Rossant, J.. and Breitman. M. L. (1996). Regulation offlt-1 expression during mouse emb~ogene sis suggests a role in the establishment of vascular endothelium. Dev. Dyn. 207, 1- I 0. Fong. G.-H., Zhang. L.. B~ce. D. M., and Peng. J. (1999). Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-I knock-out mice. Development (Cambridge, UK) 126, 3015-3025. Fong, T. A., Shawver, L. K., Sun, L.. Tang. C., App, H., PoweI1, T. J., Kim. Y. H.. Schreck, R., Wang, X.. Risau, W:. Uilrich. A.. Himh. K. R, and McMahon. G. (1999). SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-I/KDR) that inhibits ~rosine k_inase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 59, 99-106. George. E. L., Baldwin. H. S.. and Hynes, R. O. (1997). Fibronectin are essential for heart and blood vessel morphogenesis but are dispensable for initial specification of precursor ceils. Blood 90, 3073-308 I. Gerber. H. R, Hiilan, K. J., Ryan, A. M., Kowalski, J., Keller, G. A., Rangell. L., Wright, B. D., Radtke, F., Aguet, M., and Ferrara, N. (1999). VEGF is required for growth and survival in neonatal mice. Development (Cambridge. UK) 126, 1149-1159. Gerety, S. S.. Wang, H. U., Chen, Z. E, and Anderson, D. J. (I999). Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephvin-B2 in cardiovascular development. MoI. Cell 4, 403-414. Gnarra, J. R., Duan. D. R., Weng, Y., Humphrey, J. S.. Chen, D. Y., Lee, S., Pause. A., Dudley, C. F., Latif. E, Kuzmin, I., Schmidt, L., Duh. F. M., Stackhouse, T.. Chen. E, Kishida. T.. Wei, M. H.. Lerman, M. I.. Zbar. B., Klausner, R. D., and Linehan. ~ : M. (1996). Molecular cloning of the yon Hippet-Lindau tumor suppressor gene and its role in renal carcinoma. Biochim. Biophys. Acre 1242, 201-210. Godin, I.. Dieterlen-Lievre, E, and Cumano. A. (1995). Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse emb~os, beginning at 8.5 days postcoitus. Proc. Natl. Acad. Sci. U.S.A. 92, 773-777: erratum: Ibid., p. 10815. Goumans. M. J., Zwijsen. A., van Rooijen. M. A., Huylebroeck, D., Roelen, B. A., and Mumme~, C. L. (1999). Transforming growth factor-beta signalling in extraemb~'onic mesoderm is required for yolk sac vasculogenesis in mice. Development (Cambridge, UK) 126, 3473-3483. Gridley, T. (1996). Human genetics. Notch, stroke and dementia. Nature (London) 383, 673. Gridley, T. (I 997). Notch signaling in vertebrate development and disease. Mot. Cell Neurosci. 9, 103-108. Guttmacher, A. E., Marchuk, D. A.. and White, R. I., Jr. (1995). HereditmW hemorrhagic telangiectasia. N. Engl. J. Med. 333, 918-924. Haas. T. L., and Madri, J. A. (1999). Extracellular matrix-driven matrix metalloproteinase production in endothelial cells: Implications for angiogenesis. Trends Cardiovasc. Med. 9, 70-77.

230 Helbling. R M.. Saulnier, D. M.. and Brandli. A. W. (2(X)0). The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus iaevis. Development (Cambridge, UK) 127, 269-278. Heldin. C. H.. Miyazono. K., and ten Dijke. R (1997). TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature (London) 390, 4 6 5 - 471. Hellstrom, M., Kal, M., Lindahl. R. Abramsson. A.. and Betshohz. C. (1999). Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during emb~'onic blood vessel formation in the mouse. Development (Cambridge. UK) 126, 3047-3055. Hidaka, M.. Stanford. W L.. and Bernstein. A. (1999). Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells. Proc. Natl. Acad. Sci. ~,IS.A. 96, 7370-7375. Hiratsuka. S.. Minowa. O.. Kuno, J.. Noda, T., and Shibuva. M. (t998). Flt-1 lacking the t)'rosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc..\:atl. Acad. Sci. LIS.A. 95, 9349 -9354. Hirschi. K. K.. Rohovsky, S. A.. and D'Amore. P. A. t 1998). PDGF. TGFbeta, and heterotypic cell-cell interactions mediate endothelial cellinduced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J. Cell Biol. 141. 805 - 814: erratum: Ibid.. p. 1287. His. W. (1900). Lecithoblast und Angioblast der Wirbelthiere. Abh. K. Sitzungsber Ges. Wiss.. Math.-Phvs. 22, 171-328. HIa, T.. Lee, M. J., Ancellin. N.. Thangada. S.. Liu. C. H., Kluk. M., Chae. S. S.. and Wu, M. T. (2000). Sphingosine-1-phosphate signaling via the EDG- 1 family' of G-protein-coupled receptors. Ann. eV. E Acad. Sci. 905, 16-24. Hodivala-Dilke, K. M., McHugh. K. R, Tsakiris, D. A., Rayburn, H., Crowley, D., Ullman-Cullere, M.. Ross, F. R, Coller. B. S.. Teitelbaum. S.. and Hynes, R. O. (1999). Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia shorting placental defects and reduced survival. J. Clin. Invesr 103, 229-238. Holash, J., Maisonpierre, R C., Compton. D., Boland, R, Alexander, C. R., Zagzag, D., Yancopoulos, G. D., and Wiegand. S. J. (1999). Vessel cooption, regression, and growth in tumors mediated by' angiopoietins and VEGF. Science 284, 1994 - 1998. Hungerford, J. E., Hoeffler, J. R, Bowers. C. W., Dahm. L. M., Falchetto, R., Shabanowitz, J., Hunt, D. F., and Little, C. D. (1997). Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells. J. Cell Biol. 137, 925-937. Hynes, R. (1985). Molecular biology of fibronectin. Annu. Rev. Cell Biol. 1,67-90. Hy'nes, R. O. (1992). Integrins: Versatility', modulation, and signaling in cell adhesion. Cell (Cambridge, Mass.) 69, I 1-25. Hynes, R. O., Bader, B. L., and Hodivala-Dilke, K. (1999). Integrins in vascular development. Brae,. J. Med. Biol. Res. 32, 501-510. Iyer, N. V., Kotch, L. E., Agani, F., Leung, S. W:, Laughner, E., Wenger, R. H.. Gassmann. M.. Gearhart. J. D.. Lawler, A. M.. Yu. A. Y.. and Semenza, G. L. (1998). Cellular and developmental control of 02 homeostasis by hypoxia- inducible factor 1 alpha. Genes Dev. 12, 149162. Jeltsch, M.. Kaipainen, A., Joukov. V., Meng, X., Lakso, M.. Rauvala. H., Swartz. M., Fukumura, D.. Jain. R. J.. and Alitalo, K. (1997). Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276, 1423-1425. Jiang, X.. Rowitch, D. H.. Soriano, R, McMahon. A. R. and Sucov, H. M. (2000). Fate of the mammalian cardiac neural crest. Development (Cambridge. UKj 127, 1607-1616. Jouanneau, J.. Moens, G., Montesano, R., and ThierT'. J. R (1995). FGF-1 but not FGF-4 secreted by carcinoma cells promotes in vitro and in vivo angiogenesis and rapid tumor proliferation. Growth Factors 12, 37-47. Joutel. A., and Tournier-Lasserve. E. (1998). Notch signalling pathway and human diseases. Semin. Cell Dev. Biol. 9, 619-625.

ii Lineage Specification and Differentiation Kaipainen, A., Korhonen. J., Mustonen, T., van Hinsbergh, V. Wi. M.. Fang. G.-H., Dumont. D.. Breitman. M., and Alitalo, K. (1995). Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic eondothelium during development. Proc. Natl. Acad. Sci. &:S.A. 92, 3 5 6 6 3570. Kappel. A.. Ronicke. V.. Damert. A.. Flamme. I.. Risau. W., and Breier. G. (1999). Identification of vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) promoter/enhancer sequences sufficient for angioblast and endothelial cell-specific transcription in transgenic mice. Blood 93, 4284 -4292. Kawasaki, T.. Kitsukawa. T.. Bekku. Y.. Matsuda. Y.. Sanbo. M.. Yagi, T.. and Fujisawa. H. (1999). A requirement for neuropilin-I in embry'onic vessel formation. Development (Cambridge, UK) 126, 4895-4902. Kimmel, R. A.. Turnbull. D. H.. Blanquet. V., Wurst. W., Loomis. C. A., and Joyner. A. L. (2000). Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation. Genes Dev. 14, 1377-1389. Klagsbrun. M. ( 1991 ). Angiogenic factors: Regulators of blood supply-side biology. FGF. endothelial cell growth factors and angiogenesis: A keystone symposium. Keystone. CO. USA. April 1-7. 1991. New Biol. 3, 745 -749. Kontos. C. D.. Stauffer. T. R. Yang. W. R. York. J. D.. Huang, L.. Blanar. M. A., Meyer. T.. and Peters. K. G. (1998). Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt. Mol. Celt Biol. 18, 4131-4140. Korhonan. J.. Lahtinen. I.. Halmekyto, M.. Alhonen, L.. Janne, J., Dumont. D.. and Alitalo, K. (1995). Endothelial-specific gene expression directed by the tie gene promoter in vivo. Blood 86, 1828-1835. Kotch. L. E., Iyer, N. V.. Laughner, E., and Semenza, G. L. (1999). Defective vascularization of HIF-lalpha-null embryos is not associated with VEGF deficiency but with mesenchymal cell death. Dev. Biol. 209, 254 -267. Kozak. K. R.. Abbott. B.. and Hankinson. O. (1997). ARNT-deficient mice and placental differentiation. Dev. Biol. 191,297-305. Krebs. L. T.. Xue. Y.. Norton, C. R.. Shutter. J. R., Maguire, M., Sundberg, J. R. Gallahan, D.. Closson, V., Kitajewski. J.. Callahan, R.. Smith, G. H., Stark. K. L., and Gridley, T. (2000). Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 14, 1343-1352. Kulkarni, A. B.. Huh, C. G., Becker. D.. Geiser. A., Lyght, M., Flanders, K. C.. Roberts, A. B.. Sporn, M. B., Ward, J. M.. and Karlsson, S. (1993). Transforming growth factor beta 1 null mutation in mice causes exces, sive inflammatory response and early death. Proc. Natl. Acad. Sci. 6(S.A. 90, 770-774. Kwee. L.. Baldwin. H. S.. Shen. H. M., Stewart, C. L.. Buck, C., Buck, C. A., and Labow. M. A. (1995). Defective development of the embryonic and extraembr?onic circulatory' systems in vascular celt adhesion molecule (VCAM-1) deficient mice. Development (Cambridge, UK) 121,489-503. Lang, R. A.. and Bishop, J. M. (1993). Macrophages are required for ceil death and tissue remodeling in the developing mouse eye. Cell (Cambridge. Mass. ) 74, 453 -462. Lee, S. H.. Hungerford, J. E.. Little, C. D., and Iruela-Arispe, M. L. (1997). Proliferation and differentiation of smooth muscle cell precursors occurs simultaneously during the development of the vessel wall. Dev. Dyn. 209, 342-352. Leung. D. W., Cachianes. G.. Kuang. W: J., Goeddel, D. V., and Ferrara. N. (1989). Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246, 1306-1309. Li. D. Y., Sorensen. L. K., Brooke. B. S., Urness. L. D.. Davis. E. C., Taylor, D. G., Boak. B. B.. and Wendel. D. R (1999). Defective angiogenesis in mice lacking endoglin. Science 284, 1534-1537. Li. L.. Miano. J. M.. Mercer. B.. and Olson. E. N. (1996). Expression of the SM22a promoter in transgenic mice provides evidence for distinct transcriptional regulatory' programs in vascular and visceral smooth muscle cells. J. Cell Biol. 132, 849-859. Liao, E. C., Paw, B. H.. Oates, A. C.. Pratt. S. J.. Postlethwait. J. H.. and

11 Vasculogenesis and Angiogenesis Zon. L. I. (1998). SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes Dev. 12, 621- 626. Liao. W., Bisgrove. B. ~:. Sawyer. H., Hug. B.. Bell, B., Peters, K., Grunwald. D. J.. and Stainier, D. Y. R. (1997). The zebrafish gene cloche acts upstream of a ilk-1 homologue to regulate endothelial cell differentiation. Development (Cambridge, UK) 124, 381-389. Liao. W.. Ho, C.. Yan. Y. L.. Postlethwait. J.. and Stainier. D. Y. (2000). Hhex and scl function in parallel to regulate early endothelial and blood differentiation in zebrafish. Development (Cambridge, UK) 127, 43034313. Lin. R. Buxton. J. A., Acheson, A.. Radziejewski. C., Maisonpierre. R C.. Yancopoulos. G. D.. Channon, K. M.. Hale, L. R. Dewhirst. M. W, George, S. E.. and Peters, K. G. (1998). Antiangiogenic gene therapy' targeting the endothelium-specific receptor tyrosine kinase Tie2. Proc. Natl. Acad. Sci. U.S.A. 95, 8829-8834. Lindahl, R. Johansson. B. R.. Leveen, R. and Betsholtz. C. (1999). Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277, 242-245. Linehan. W: M.. Lerman. M. I.. and Zbar. B. (I 995). Identification of the yon Hippel-Lindau (VHL) gene. Its role in renal cancer. JAMA, J. Am. Med. Assoc. 273, 564-570. Liu, Y.. Wada. R.. Yamashita. T., Mi. Y.. Deng. C.-X., H0bson. J. R. Rosenfeldt. H. M.. Nava. V. E.. Chae, S.-S.. Lee, M.-J., Liu. C. H.. Hla, T.. Spiegel, S.. and Proia. R. L. (2000). Edg-1, the G-protein-coupled receptor for sphingosine- 1-phosphate. is essential for vascular maturation. J. Clin. Invest. 106, 951-961. Loughan. S.. and Sato. T. N. (2001). A combinatorial role of angiopoietin-1 and orphan receptor Tie 1 pathways in establishing vascular polarity during angiogenesis. Mol. Ceil 7, 233-239. Maisonpierre. R C.. Suri. C.. Jones. R E, Bartunkova. S., Wiegand. S. J.. Radziejewski, C.. Compton. D.. McCIain. J.. Aldrich, T. H., Papadapoulos, N., Daly. Y. J.. Davis. S., Sato, T. N., and Yancopoulos. G. D. (1997). Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55-60. Maltepe. E.. Schmidt. J. V.. Baunoch. D.. Bradfield. C. A., and Simon. M. C. (1997). Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature (London) 386, 403-407. McAllister. K. A., Grogg. K. M., Johnson, D. W., Gallione. C. J.. Baldwin, M. A.. Jackson. C. E.. Helmbold. E. A.. Markel, D. S.. McKinnon, W. C., Murrell. J.. McCormick, M. K.. Pericak-Vance, M. A., Heutink, R. Oostra. B. A.. Haitjema. T.. Westerman, C. J., J., Porteous. M. E.. Guttmacher. A. E.. Letarte. M.. and Marchuk. D. A. (I994). Endoglin. a TGF-/3 binding protein of endothelial cells, is the gene for hereditary' haemorrhagic telangiectasia type 1. Nat. Genet. 8, 345-351. Medvinsky, A., and Dzierzak, E. (1996). Definitive hematopoiesis is autonomously initiated by the AGM region. Celt (Cambridge. Mass.) 86, 897-906. Medvinsky. A. L.. Samoylina, N. L.. Muller. A. M.. and Dzierzak, E. A. (1993). An early pre-liver intraembwonic source of CFU-S in the developing mouse. Nature (London) 364, 64-67. Meeson, A. R. Palmer, M.. Calfton, M.. and Lang, R. A. (1996). A relationship between apoptosis and flow during programmed capillary regression is revealed by vital analysis. Development (Cambridge, UK) 122, 3929-3938. Meeson, A. R. Argilla, M.. Ko, K., Witte. L., and Lang. R. A. (1999). VEGF deprivation-induced apoptosis is a component of pro~arnmed capillary,, regression. Development (Cambridge, UK) 126, 1407-1415. Mellitzer, G.. Xu. Q.. and Wilkinson. D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication. Nature (London) 400, 77-81. Mikawa. T., and Fischman. D. A. (1992). Retroviral analysis of cardiac morphogenesis: Discontinuous formation of coronaxy vessels. Proc. Natl. Acad. Sci. U.S.A. 89, 9504-9508.

231 Mikawa. T.. and Gourdie. R. G. (1996). Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ. Dev. Biol. 174, 221-232. Millauer, B.. Shawver. L. K.. Plate, K. H., Risau. W., and Ullrich. A. (1994). Glioblastoma growth inhibited in vivo by a dominant-negative Flk- I mutant. Nature (London) 367, 576-579. Millauer, B., Longhi. M. R, Pilate. K. H.. Shawver, L. K., Risau, W.. Ullrich. A.. and Straw'n, L. M. (1996). Dominant-negative inhibition of Flk-t suppresses the growth of many tumor types in vivo. Cancer Res. 56, I615-1620. Miyazono, K (1999). Signal transduction by bone morphogenetic protein receptors: Functional roles of Smad proteins. Bone 25, 91-93. Motoike, T., Loughna, S., Perens. E.. Roman, B. L., Liao. W.. Chau, T. C.. Richardson. C. D., Kawate. T.. Kuno, J.. Weinstein. B. M.. Stainier, D. Y.. and Sato. T. N. (2000). Universal GFP reporter for the study of vascular development. Genesis 28, 7 5 - 8 I. Nagase. H., and Woessner. J. F.. Jr. (1999). Matrix metalloproteinases. J. Biol. Chem. 274, 21491-21494. Neuhaus. J.. Risau, W, and Wblburg, H. (I991). Induction of bto~d-brain barrier characteristics in bovine brain endothelial cells by rat astroglial cells in transfilter coculture. Ann. N. Y Acad. Sci. 633, 578-580. Niki, M.. Okada, H.. Takano, H.. Kuno. J.. Tani. K., Hibino, H.. Asano. S., Ito, Y.. Satake. M.. and Noda. T. (1997). Hematopoiesis in the fetal liver is impaired by targeted mutagenesis of a gene encoding a non-DNA binding subunit of the transcription factor, polyomavirus enhancer binding protein 2/core binding factor. Proc. Natl. Acad. Sci. U.S.A. 94, 56975702. Nor,&, T.. Gu. T. L., Stacy, T.. Wang. Q.. Howard, L., Binder, M., MarinPadilla. M.. and Speck. N. A. (1999). Cbfa2 is required for the formation of intra-aortic hematopoietic clusters. Development (Cambridge, UK) 126, 2563-2575. Okuda. T.. van Deursen. J.. Hiebert, S. W., Grosveld, G., and Downing, J. R. (I9%). AML1. the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell (Cambridge. Mass.) 84, 321-330. Papapetropoulos, A., Garcia-Cardena, G., Dengler. T. J.. Maisonpierre, R C.. Yancopoutos. G. D., and Sessa. W. C. (1999). Direct actions of angiopoietin-I on human endothelium: Evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab. Invest. 79, 213-223. Papapetropoulos. A., Fulton, D., Ma_hboubi. K.. Kalb. R. G.. O'Connor. D. S., Li. E, Altieri, D. C.. and Sessa. Vvl C. (2000). Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survMn pathway. ,/. Biol. Chem. 275, 9102-9105. Partanen. J.. Puff, M. C., Schwartz. L., Fischer, K.-D.. Bernstein, A.. and Rossant. J. (t 996). Cell autonomouse functions of the receptor D'rosine TIE in a late phase of anNogenic capillary growth and endothelial cell survival during murine development. Development (Cambridge. UK) 122, 3013-3021. Perez-Pomares. J. M., Macias, D., Garcia-Garrido, L., and Munoz-Chapuli. R. (1998). The origin of the subepicardial mesenchyme in the avian embD'o: An immunohistochemical and quail-chick chimera study. Dev. Biol. 200, 57-68. Peters, K. G. (1998). Vascular endothelial ~owth factor and the angiopoietins: Wbrking together to build a better blood vessel. Circ. Res. 83, 3 4 2 343. Puri, M. C., Rossant, J., Alitalo. K., Bernstein, A., and Partanen, J. (1995). The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells. EMBO Z 14, 5884-5891. Puri, M. C.. Partanen, J., Rossant, J., and Bernstein, A. (1999). Interaction of the TEK and TIE receptor tyrosine "kinases during cardiovascular development. Development (Cambridge, UK) 126, 4569-4580. Rafte~'. L. A.. and Sutherland. D. J. (1999). TGF-beta family signal transduction in Drosophila development: From Mad to Smads. Dev. Biol. 21(t, 25 t-268.

232 Ravi, R.. Mookerjee, B., Bhujwalla, Z. M., Sutter, C. H., Artemov, D., Zeng, Q., Dillehay. L, E., Madan, A., Semenza, G. L., and Bedi, A. (2000). Regulation of tumor angiogenesis by p53-induced degradation of hypoxia- inducible factor l alpha. Genes Dev. 14, 3 4 - 4 4.~, Risau, * : (199Ia). Embryonic angiogenesis factors. PharmacoI. Ther, 51, 371-376. Risau. V~,.(1991b). Vasculogenesis, angiogenesis and endothelial cell differentiation during embr?onic development. In "'The Development of the Vascular System" (R. N. Feinberg. G. K. Shere, and R, Auerbach, eds.), pp. 58-68. Krager, Basel. Switzerland. Risau, W. (1997). Mechanisms of angiogenesis. ~'~rure (London) 386, 671 674. Risau, W:, and Flamme, I, (19951). Vasculogenesis. Annu. Rev. Cell Dev. Biol. 11, 73-91. Risau. W., and Wolburg, H. (1990). Development of the blood-brain barrier. Trends Neurosci. 13, 174 - 178. Risau. W., Haltmann. R.. and Albrecht. U. (1986a). Differentiationdependent expression of proteins in brain endothelium during developmerit of the blood-brain barrier. Dev. Biol. 117, 537-545. Risau, W., Hallmann, R., Albrecht, U.. and Henke. F. S. (1986b). Brain induces the expression of an early cell surface marker for blood-brain barrier-specific endothelium. EMBO J. 5, 3179-3183. Rossant. J., and McMahon, A. (1999). "'Cre"-ating mouse mutants~a meeting review on conditional mouse genetics. Genes Dev. 13, 142-145. Ryan, H. E.. Lo, J., and Johnson, R. S. (1998). HIF-1 alpha is required for solid tumor formation and embr'vonic vascularization. EMBO J. 17, 3005-3015. Sabin, F. R. (1917). Origin and development of the primitive vessels of the chick and of the pig. Carnewie Contrib. EmbrYol. 6, 61-124. Sasaki, K., Yagi, H., Bronson, R. T., Tominaga. K.. Matsunashi, T.. Deguchi, K., Tani, Y.. Kishimoto, T.. and Komori. T. (1996). Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta. Proc. Natl. Acad. Sci. C~S.A. 93, 12359-12363. Sato, T. N. (2000). A new role of lipid receptors in vascular and cardiac morphogenesis. J. CIin. Invesr 106, 939-940. Sato, T. N., Tozawa, Y.. Deutsch. U.. Wolburg-Buchholz, K., Fujiwara. Y., Gendron-Maguire, M.. Gridley, T.. Wolburg, H., Risau, W., and Qin. Y. (1995). Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature (London) 376, 70-74. Schelling. M. E. (1991). FGF mediation of coronary' angiogenesis. Ann. N.Y. Acad. Sci. 638, 467-469. Schlaeger, T. M.. Qin, Y.. Fujiwara. Y.. Magram. J., and Sato, T. N. (1995). Vascular endothelial cell lineage-specific promoter in transgenic mice. Development (Cambridge. UK) 121, 1089-1098. Schlaeger, T. M., Bartunkova, S., Lawitts. J. A., Teichmann. G., Risau, W., Deutch, U., and Sato, T. N. (1997). Uniform vascular-endothelial-cellspecific gene expression in both emb~onic and adult transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 94, 3058-3063. Schuh, A. C., Faloon, R, Hu, Q. L., Bhimani, M., and Choi, K. (I 999). In vitro hematopoietic and endothelial potential of ilk-l(-/-) embDonic stem cells and embrvos. Proc. Natl. Acad. Sci. U.S.A. 96, 2159-2164. Seghezzi, G., Patel, S., Ren, C. J., Gualandris, A., Pintucci, G., Robbins, E. S., Shapiro, R. L., Galloway, A. C., Rifkin, D. B., and Mignatti, R (1998). Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries" An autocrine mechanism contributing to angiogenesis. J. Cell Biol. 141, 1659-1673. Semenza, G. L. (1999). Perspectives on oxygen sensing. Cell (Cambridge. Mass.) 98, 281-284. Shalaby, F., Rossant, J., Yamaguchi, T. R, Breitman, M.. and Schuh, A. C. (1995). Failure of blood island formation, vasculogenesis, and hematopoiesis in ilk-1 deficient mice. Nature (Londonj 376, 62-66. Shalaby, F., Ho, J., Stanford, W. L., Fischer, K.-D., Schuh, A. C., Schwartz, L., Bernstein, A., and Rossant, J. (1997). A requirement for Flkl in

il Lineage Specification and Differentiation primitive and definitive hematopoiesis and vasculogenesis. Cell (Cambridge, Mass. ) 89, 981-990. Shapiro. S. D. (1997). Mighty' mice: Transgenic technology "knocks out" questions of matrix metalloproteinase function. Matrix Biol. 15, 527533. Shirayoshi, Y.. Yuasa, Y., Suzuki, T., Sugaya, K., Kawase, E., Ikemura, T., and Nakatsuji, N. (1997). Proto-oncogene of int-3, a mouse Notch homoIogue, is expressed in endothelial cells during early emb~;ogenesis. Genes Cells "~ 213-224. Shutter. J. R., Scully, S., Fan, W., Richards, W G., Kitajewski, J., Deblandre, G. A., Kintner, C. R., and Stark, K. L. (2000). DlI4, a novel notch ligand expressed in arterial endothelium. Genes Dev. 14, 1313 - 1318. Smith. R. A., and Glomski, C. A. (19821). "'Hemogenic endothelium" of the embryonic aorta: Does it exist'? Dev. Comp. Immunol. 6, 359-368. Soker, S., Takashima. S.. Miao, H. Q,, Neufeld, G., and Klagsbrun, M. (1998). Neuropilin-1 is expressed by" endothelial and tumor cells as an isoform- specific receptor for vascular endothelial growth factor. Cell (Cambridge. Mass. j 92, 735 - 745. Stainier, D. Y.. Weinstein. B. M., Detrich, H. W.. 3rd, Zon, L. I., and Fishman, M. C. (1995). Cloche. an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development (Cambridge. UK) 121, 3141-3150. Suri, C.. Jones. P. F., Patan. S., Bartunkova, S.. Maisonpierre. R C., Davis, S., Sato, T. N.. and Yancopoulos, G. D. (1996). Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell (Cambridge. Mass.) 87, 1171 - 1180. Suri, C., McClain, J., Thurston, G., McDonald, D. M., Zhou, H.. Oldmixon, E. H., Sato, T. N., and Yancopoulos, G. D. ( 1998 ), Increased vascularization in mice overexpressing angiopoietin-I. Science 282, 468- 471. Tavian, M., Coulombel. L., Luton, D., Clemente, H. S., Dieterlen-Lievre, F., and Peault. B. (1996). Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood 87, 67-72. Tevosian, S. G., Deconinck, A. E., Tanaka, M., Schinke, M., Litovsky, S. H., Izumo, S.. Fujiwara, Y., and Orkin. S. H. (2000). FOG-2. a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary," vessels from epicardium. Cell (Cambridge. Mass.) 101,729-739. Thurston, G., Suri, C., Smith, K., McClain, J., Sato, T. N., Yancopoulos, G. D.. and McDonald, D. M. (1999). Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286, 25112514. Urness, L. D., Sorensen, L. K.. and Li, D. Y. (2000)..~teriovenous malformations in mice lacking activin receptor-like kinase-1. Nar Gener 26, 328-331. Urushihara, H.. and Takeichi, M. (1980). Cell-cell adhesion molecule: Identification of a glycoprotein relevant to the Ca2 +-independent aggregation of Chinese hamster fibroblasts. Cell (Cambridge, Mass.) 20, 363-371. Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D., and Kitajewski, J. (1996). Notch4/int-3, a mammary, proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development (Cambridge, UK) 122, 2251 - 2259. Vikkula, M., Boon, L. M., Carraway, K. L., Calvert, J. T., Diamonti, A. J., Goumnerov. B., Pasyk, K. A., Marchuk, D. A., Warman, M. L., Cantley, L. C., Mulliken. J. B.. and Olsen, B. R. (1996). Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell (Cambridge. Mass. ) 87, 1181 - 1190. Vrancken Peeters, M. R, Gittenberger-de Groot, A. C., Mentink, M. M., and Poelmann, R. E. (1999). Smooth muscle cells and fibroblasts of the coronary" arteries derive from epithelial-mesenchymal transformation of the epicardium. Anat. Embrvol. 199, 367-378. Vu, T. H., Shipley, J. M.. Bergers, G., Berger, J, E., Helms, J. A., Hanahan, D., Shapiro, S. D., Senior, R. M., and Werb, Z. (1998). MMP-9/gelatinase B is a key regulator of gowth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell (Cambridge, Mass.) 93, 411-422.

1 1 Vascuiogenesis and Angiogenesis Wang. H. U., Chen, Z. F., and Anderson, D. J. (1998). Molecular distinction and angiogenic interaction between embr?'onic arteries and veins revealed by' ephrin-B2 and its receptor Eph-B4, Cell (Cambridge. Mass, ) 93, 741-753. Wang. Q., Stacy. T., Binder, M., Marin-Padilla, M,, Sharpe, A. H., and Speck, N, A. (1996a). Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc. NatL Acad. Sci. g~S.A. 93, 3444-3'449. Wang. Q.. Stacy. T., Miller. J. D.. Lewis. A. E. Gu, T. L.. Huang. X.. Bushweller. J. H.. Bones. J. C.. Att. F. W.. Ryan, G.. Liu, R R. WvnshawBoris. A.. Binder. M.. Marin-Padilta. M., Sharpe. A. H., and Speck. N. A. (1996b). The CBFbeta subunit is essential for CBFaiph~ (AML1) function in vivo. Cell (Cambridoe. Mass.) 87, 697-708. V~rb. Z. (1997). ECM and cell surface proteolysis: Regulating cellular ecology. Cell (Cambridge. Mass.) 91,439-443. Werb. Z.. and Chin. J. R. (1998). Extracellular matrix remodeling during morphogenesis. Ann. ~\(g Acad. Sci. 857, I i 0 - 1 i8. Whitman. M. (1997). Signal transduction. Feedback from inhibitory: SMADs. Nature (London) 389, 549-551. Wigle, J. T.. and Oliver. G. (1999). Prox 1 function is required for the development of the routine lymphatic system. Cell (Cambridge, Mass.) 98, 769-778. Wilkinson. D. G. (2000a). Eph receptors and ephrins: Regulators of guidance and assembly, lnt. Rev. Cvtol. 196, 177-244. Wilkinson. D. G. (2000b). Topographic mapping: Organizing by repulsion and competition'? Curr. Biol. 10, R447-R451. Witte. L.. Hicklin. D. J.. Zhu. Z.. F'y:towski, B.. Kotanides, H.. Rockwell. R, and Bohlen. R (1998). Monoclonal antibodies targeting the VEGF re-

233 ceptor-2 (Flkl/KDR) as an anti-angiogenic therapeutic strategy. Cancer Metastasis Rev. 17, 155 -161. Wu, H.. Lee, S. H., Gao, J.. Liu, X.. and Iraela-.a,-ispe. M. L. (1999). Inactivation of erythropoietin leads to defects in cardiac morphogenesis. Development (Cambridge, UK) 126, 3597-3605. Wu, Q., and Maniatis, T. (1999). A striking organization of a large family of human neural cadherin-like ceil adhesion genes. Cell (Cambridge. Mass.) 97, 779-790. Wu. Q . and Maniatis. T. (20001). Large exons encoding multiple ectodomains are a characteristic feature of protocadherin genes. Proc. NatL Acad. SoL G~S.A. 97, 3124 -3129. Xu. Q., Melli~er, G.. Robinson, V.. and Wilkinson. D. G. (1999). In vivo ceil sorting in complementary segmental domains mediated by Eph receptors and ephrins. Nature (London) 399, 267-27 I. Yamashita. J.. Itoh. H., Hirashima, M.. Ogawa. M.. Nishikawa, S., Yurugi. T.. Naito, M.. and Nakao. K. (2000). Flkl-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature (London) 408, 92-96. Yang. J. T., Rayburn. H.. and Hynes, R. O. (1995). Celt adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development (Cambridge. UK) 121,549-560. Yang. X., Castilla. L. H., Xu. X., Li. C.. Gotay. J.. Weinstein. M.. Liu. P. P.. and Deng. C. X. (I999). Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5. Development (Cambridge. UK) 126, t571-1580. Zinyk, D. L., Mercer. E. H., Harris. E., Anderson, D. J.. and Joyner. A. L. (t998). Fate mapping of the mouse midbrain-hindbrain constriction using a site- specific recombination system. Curt. Biol. 8, 665-668.


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Stem Cells of the Nera_Jous System S e a n J. M o r r i s o n Departments of Internal Medicine and Ceil and Developmental Biolog), Howard Hughes Medical Institute, Universin; of Michigan. Ann Arbor. Michigan. 48109

I. Introduction II. Lineage D e t e r m i n a t i o n of Neural Stem Cells iil. Do Stem Cells Retain Broad or N a r r o w Neuronal Potentials? IV. Regulation of Neural Stem Cell Self-Renewal V. Differences b e t w e e n Hematopoietic Stem Cells and Neural Stem Cells Vl. In Vivo Function of Neural Stem Cells VII. Surprising Potential of Neural Stem Cells VIII. Are Neural Stem Cells Involved in Disease? IX. O u t s t a n d i n g Issues References T

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I. I n t r o d u c t i o n

Like other tissues, the nervous system contains stem cells. Stem cells are self-renewing, multipotent progenitors with the broadest developmental potential in a given tissue at a given time (Morrison et aI., 1997a). Neural stem cells give rise to multiple different types of neurons and glia. Many different types of neural stem cells are probably present in different regions of the nervous system that differ in the types of cells they produce. Broadly speaking, neural stem cells can be considered to fall within two general classes:

Mouse Development

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central nervous system (CNS) stem cells (McKay, 1997; Gage, 1998; Temple and Atvarez-Buylla, 1999) and neural crest stem cells (NCSCs) (Anderson, 1989, 1997). These two classes of stem cells are present in different regions of the nervous system, have different developmental potentials, and give rise to different types of cells in vivo. CNS stem cells are born in the ventricular zone of the neural tube and give rise to neurons, astrocytes, and oligodendrocytes in the spinal cord and brain (Fig. i). Multipotent CNS progenitors self-renew in vitro (Davis and Temple, 1994; Gritti et al., 1996), and lineage marking experiments strongly suggest that these cells self-renew in vivo as well (Reid et aL, 1995; Morshead et at., 1998). NCSCs are also born in the neural tube but migrate throughout the embryo and give rise to the neurons and glia of the peripheral nervous system (PNS) as well as mesectodermal derivatives (such as vascular smooth muscle and bone) in other tissues (Fig. 1). NCSCs self-renew in vitro and in vivo (Stemple and Anderson, 1992; Morrison et aI., 1999). Although CNS stem cells and NCSCs are different types of neural stem cells, important principles in neural stem cell biology can best be illustrated by combining examples from both systems.

A. When and Where Are Neural Stem Cells Present? CNS stem cells persist throughout life in addition to participating in the formation of the CNS during embryonic Copyright 9 2002 Academic Press All rights of reproduction in any form reserved.

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F i g u r e 1 The types of lineages generated by neural stem cells. CNS stem cells and neural crest stem cells are different types of stem cells that can be distinguished based on differences in where they occur in vivo and in the types of cells they give rise to. CNS stem cells self-renew and also give rise to neurons. astrocvtes, and oligodenclrocytes.Astrocvtes and oligodendrocytesare the two types of glia from the CNS. Stem cells from different regions of the CNS and from different times during ontogeny give rise to different types of neurons. NCSCs self-renew and also give rise to neurons. Schwann cells, and rnesectoderm. Schwann cells are glia from the PNS. Mesectodermalcells include certain bones in the head, dermis in the head. and vascular smooth muscle around the great arteries near the heart. NCSCs give rise to different types of neurons in different regions of the PNS.

development. During fetal development, stem cells are present in the ventricular zone that lines the lumen of the neural tube. These cells are present throughout the neural axis. from regions fated to form the cortex (Davis and Temple. 1994) to regions fated to form the spinal cord (Kalyani et al., 1997: Palmer et al.. 1999). In the adult, stem cells continue to be present near the ventricles. The ventricular zone of fetal development is replaced in adults by the ependymal layer (immediately adjacent to the lumen of the ventricle) and the subependymal layer (Barres, 1999). There is evidence for the existence of stem cells in both the ependymal (Johansson et al., I999) and subependymal layers (Morshead et al., 1994: Doetsch et al., 1999) of the adult CNS. In addition to being present near the ventricles, some stem cells take up residence in the dentate gyms of the adult hippocampus (Palmer et aL, 1997). Indeed, there is evidence that neural stem cells can be generated from many different regions of the adult CNS by culturing cells in vitro (Weiss et al., 1996), even from regions of the CNS that show no evidence of cell turnover. One possibility is that rare, latent stem cells persist in a quiescent state throughout the CNS. Another possibility is that certain cells that do not have stem cell properties in vivo can sometimes acquire stem cell properties as a result of prolonged culture in high concentrations of mitogens like basic f i b r o b l a s t g r o w t h f a c t o r (bFGF or FGF-2) (Donovan, 1994: Gage, 1998). To confirm where and when CNS stem cells are present, it is necessary to identify them prospectively (identify markers that predict which cells are stem

cells and which are restricted progenitors) so that they can be studied in vivo. NCSCs are only known to exist during fetal development and there is not yet any convincing evidence for the persistence of NCSCs or the generation of new neurons in the adult PNS. Neural crest cells, including NCSCs, are born in the neural tube but migrate throughout the embryo in early to midgestation, depending on the species. Neural crest migration lasts only a short period of time, 2 4 - 4 8 hr in avians and rodents. Multipotent neural crest progenitors have been detected transiently in postmigratory sites such as the skin (Richardson and Sieber-Blum. 1993), the sensory ganglia (Duff et al., 1991" Deville et al., 1992; Hagedorn et aI., 1999). the gut (Deville et al., 1994; Lo and Anderson, 1995), and the sympathetic ganglia (Duff et al., 1991); however, multipotent neural crest cells progressively restrict their developmental potential while m i ~ a t i n g (Baroffio et aI., 1991), and many cells differentiate soon after reaching p o s t m i g a tory sites (Le Douarin and Dupin, 1992). Until recently there was no evidence for NCSC selfrenewal in vivo, and it had been thought that NCSCs differentiate within a few" days of migrating; however, NCSCs were recently observed to persist into late gestation by selfrenewing within peripheral nerves (Morrison et al., 1999). It remains to be determined whether NCSCs self-renew in other regions of the PNS. Nonetheless, the in vivo selfrenewal and persistence of NCSCs in nerves suggest that NCSCs may play a more dynamic role in PNS development

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12 Stem Cells of the Nervous System than previously thought. Even so, there remains no evidence that NCSCs persist or give rise to new neurons in the adult PNS.

B. What Do Stem Cells Do in the Adult CNS? Stem cells in the adult CNS give rise to neurons that may be involved in learning and memory. In adult mammals, neurons continue to be born in the olfactory bulb and hippocampus (Altman and Das. 1966 Altman. 1969: Kaplan and Hinds. 1977: Eriksson et al., 1998) and perhaps throughout the neocortex (Gould et al., 1999b). Stem cells that reside near the lateral ventricle (Lois and Alvarez-Buylla, 1993 Doetsch et al., 1999 Johansson et al., i999) undergo differentiation into neurons and glia in the subependymal zone. and then they migrate through what is "known as the rostral migratory stream into the olfactory bulb (Lois and AlvarezBuylla, 1994). Within the olfactory bulb the neurons integrate as inhibitory interneurons called granule cells. The exact function of the newly born granule cells is unknown but increased odorant stimulation promotes neurogenesis (Frazier-Cierpial and Brunjes, 1989: Rosselli-Austin and Williams. 1989 Corotto et al., 1994), and added granule cells may improve odorant discrimination by modulating the signals transmitted from the olfactory bulb to the cortex (Yokoi et al., 1995). In the hippocampus, stem cells reside in the dentate gyrus where they differentiate into granule neurons (Palmer et al., 1997). Again, the function of these new neurons is unknown but there is evidence that hippocampal neurogenesis is associated with environmental enrichment and learning (Kempermann er al., 1997, 1998: Gould et al., 1999a). The discovery, that some neurons born in the subventricular zone also incorporate into the neocortex has given further impetus to the idea that adult neurogenesis may be involved in learning and memory (Gould et aI., 1999b). The next step will be to determine whether the formation of new neurons in the hippocampus is required for learning or memory.

C. Do All Nervous System Cells Derive from Stem Cells? It is not yet clear whether all nervous system cells can be derived from stem cells. For example, certain lineages of nervous system cells may derive from progenitors that were born with restricted developmental potential rather than multipotency. Within the CNS. multipotent and restricted progenitors coexist in the ventricular zone (Luskin et al., 1988; Davis and Temple, 1994; Williams and Price, 1995). The simplest interpretation is that the restricted progenitors arise from stem cells, but we cannot refute the possibility that at least certain lineages of CNS cells arise from progenitors that were born committed (Barres, 1999). That is. such cells may have had restricted developmental potentials at the time they acquired neural potential.

Within the PNS there is evidence that some cells arise from committed progenitors that may be geneologically unrelated to NCSCs. The neural crest is composed of a heterogeneous collection of progenitors including multipotent as well as restricted progenitors (Le Douarin, 1986). At least some melanocytes may derive from progenitors that are already comrrfitted to this fate at the time they migrate from the neural tube (Erickson and Goins, 1995: Wakamatsu et al., 1998). Similarly, restricted progenitors of sensory neurons are observed among migrating neural crest cells (.Greenwood et al., 1999) as well as among developing sensory" ganglion cells (Frank and Sanes, 1991). Furthermore, even some premigratory neural crest progenitors (that still reside in the neural tube) are fated to give rise to only sensory ganglion or melanocyte lineage cells (Bronner-Fraser and Fraser, 1989; Serbedzija et al., 1994). These observations suggest the possibility that some committed progenitors may be born in the neural tube as a separate lineage from multipotent NCSCs, even though there is also evidence that multipotent progenitors can retain sensory (Fraser and Bronner-Fraser, 1991) and/or melanocytic (Sieber-Blum and Cohen, 1980; Baroffio et al., 1988 Sieber-Blum. 1989) potential. If certain lineages of cells do not arise from muitipotent stem cells, then controls on the overt differentiation of such cells might be quite different than on multipotent progenitors that must undergo lineage determination before differentiating.

II. Lineage Determination of Neural Stem Cells Environmental factors can promote the generation of a particular cell type from neural stem cells via two very different types of mechanisms: instruction and selection (Fig. 2). Instruction is the process by which a factor promotes differentiation into one lineage at the expense of other lineages by acting on the stem cells and causing them to preferentially undergo commitment. Selection does not act at the level of stem cells but rather affects the survival or proliferation of the committed cells that arise from the stem cells. Thus a factor can selectively promote the generation of a particular lineage by promoting cell survival or proliferation after they differentiate from stem cells (or by killing or impairing the proliferation of other lineages of cells after they arise from stem cells). In many systems, such as in hematopoiesis, it has been difficult to rigorously demonstrate whether particular factors act instructively or selectively because the stem cells cannot be studied in vitro with enough precision to determine the mechanism by which factors act. In contrast, outstanding tools are available to study the self-renewal and multilineage differentiation of individual neural stem cells in culture. Thus impressive progress has been made in understanding how lineage determination factors work in the

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F i g u r e 2 Lineage determination factors can act instructively or selectively. Instructive factors act at the level of stem cells to promote differentiation into one fate at the expense of other fates. For example. BMP2 instructs NCSCs to differentiate into neurons (Shah et al.. I996). Selective factors do not affect stem cell differentiation but increase the numbers of cells from one lineage by promoting their survival or proliferation after they differentiate, or by impairing the survival or proliferation of other lineages of cells after they differentiate. For example, neurotrophins often act selectively to regulate the survival of neurons after they differentiate.

nervous system. As described below, lineage determination factors in the nervous system can act instructively, selectively, or by a combination of those mechanisms. These mechanisms are best illustrated by examples taken from NCSC biology, but other growth factors are thought to have analogous effects on CNS stem cells.

A. Bone Morphogenetic Proteins Instruct Neuronal Differentiation Bone morphogenetic proteins (BMPs) instruct NCSCs to differentiate into autonomic neurons, thereby regulating the formation of the autonomic nervous system. At approximately E11 of mouse development, when autonomic ganglia are forming, BMP2 and/or BMP4 are expressed around the dorsal aorta, near where sympathetic neurons form, and in the heart and lung, near where parasympathetic neurons form (Bitgood and McMahon. 1995: Lyons et al., 1995; Reissman et al., 1996; Shah et al., 1996). Thus BMPs are expressed at the fight time and in the fight place to influence autonomic neuron differentiation. To demonstrate that B MPs are sufficient to cause autonomic neuron differentiation in vivo, BMP4 was ectopically expressed in the neural crest migration pathway of chick embwos (Reissman et al., 1996). This caused an increase in the number of sympathetic neurons that differentiated in the sympathetic ganglion as well as the formation of additional sympathetic neurons in locations where they do not normally form. such as in the neural crest migration pathway. To demonstrate that BMPs

are necessary, for autonomic neuron differentiation, Noggin, a protein that binds and inactivates BMPs (Smith and Harland. 1992: Zimmerman et al., 1996), was overexpressed in chick embwos (Schneider et at., 1999). This greatly impaired the differentiation of sympathetic neurons. Thus BMPs are necessary and sufficient for the generation of sympathetic neurons in vivo. Although in vivo studies have shown that BMPs are expressed at the fight time and in the fight place and are necessary and sufficient for sympathetic neuron differentiation, these studies did not tell us how BMPs regulate sympathetic neuron differentiation. Do BMPs instruct NCSCs to differentiate into neurons, or are they required for the survival of sympathetic neurons? Do BMPs act directly on NCSCs or indirectly by causing other cells in the environment to secrete secondary factors that affect NCSC differentiation? In vitro experiments were required to answer these questions. Purified recombinant BMP2 causes NCSCs to differentiate into neurons when added to cultures of purified NCSCs (Shah et al., 1996; Morrison et aI., 1999). Thus BMP2 acts directly on NCSCs or their progeny to cause neuronal differentiation. To determine whether B MP2 acts instructively or selectively, purified NCSCs were added to culture at clonal density (---20 stem cells/35-mm dish such that individual stem cells formed spatially separated clones in which the progeny of individual stem cells could be distinguished and observed separately). After 4 hr. when cells had attached to the plate, the live cells were circled by etching on the underside of the culture dish. Then B MP2 was added to some

12 Stem Cells of the Nervous System cultures. After only 4 days of incubation the cultures were stained with antibodies against the neuronal marker peripherin. In both control and BMP2-supplemented plates, around 90% of clones survived. In the absence of BMP2 no neurons differentiated (under control conditions neuronal differentiation is not evident for 13 days), but in the presence of BMP2 more than 80% of clones gave rise to neurons. Serial analysis of cultures every 24 hr indicated that only rare dead cells were observed within colonies exposed to BMP2, demonstrating that BMP2 could not be acting selectively in an intraclonal manner by killing cells within clones that did not differentiate into neurons (Shah et al., 1996). Thus B MP2 increases the rate and extent of neurogenesis by stem cells without killing cells and therefore acts instructively (Shah et al.. 1996: Morrison et al., 1999). B MP2 instruction promotes both sympathetic and parasympathetic neuron differentiation in culture, depending on culture conditions (Morrison et al., 2000). Under standard culture conditions BMP2 caused the differentiation of parasympathetic neurons, as judged by their expression of vesicular acetyl choline transferase (VAChT) and their lack of expression of sympathetic markers: however, when B MP2 was added along with forskolin to cultures in a reduced oxygen chamber that more closely approximates physiological oxygen levels, many neurons began expressing sympathetic markers (tyrosine hydroxylase, dopamine-/3-hydroxylase). These observations suggest that while B MP2 induces autonomic differentiation, other factors can determine the subtype of neurons that form. We must also remember that the effects of BMPs on cell fate determination are often highly concentration dependent (Mehler et al., 1997, Dale and Wardle. 1999). Therefore, it is possible that further work will show the sympathetic/parasympathetic lineage decision to be determined by different concentrations of B MPs in vivo. B MPs instruct the differentiation of autonomic neurons from NCSCs by inducing the expression of the transcription factor MASH1 (see Fig. 4 in a later section) (Sommer et al., 1995 Shah et at., 1996: Lo et aI., 1997). MASH I is a mammalian basic helix-loop-helix transcription factor with homology to the D r o s o p h i l a proneural a c h e t e - S c u t e genes (Johnson et al., 1990. 1992). MASH 1 is required for the generation of autonomic neurons in the PNS as well as adrenergic neurons of the CNS (Guillemot et al., 1993 Hirsch et at., 1998). MASH 1 initiates autonomic neuron differentiation by promoting the expression of pan-neuronal genes, like peripherin, as well as genes specific to autonomic neurons, like the transcription factor PHOX-2A (Sommer et al., 1995; Lo et al., 1998). In turn, PHOX-2A regulates the expression of additional neuronal genes such as c-Ret. Thus these data demonstrate how a cell-extrinsic factor, such as BMP2, can instruct stem cells to differentiate in a lineage-specific manner, by causing a hierarchy of genes to be expressed. In addition to instructing NCSCs to differentiate into autonomic neurons. BMP2 also causes CNS stem cells to dif-

239 ferentiate into neurons and astrocytes (Gross et aI., 1996; Li et aI., 1998: Mabie et al., 1999). Platelet-derived g o w t h factor (PDGF) has also been observed to instruct neuronal differentiation by CNS stem cells (Williams et al., 1997; Johe et al., 1996). The observation of neuronal differentiation in response to BMP2 by both CNS stem cells and NCSCs suggests that different types of neural stem cells can exhibit a similar response to the same lineage determination factor.

B. Neurotrophins Act Selectively to Regulate Neuronal Survival In contrast to the efl%cts of BMPs. neurotrophins promote the generation of neurons by acting selectively. Neurotrophins are often secreted by the target cells on which neurons synapse and are required for the survival of neurons. There are several neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and ,-,. All of these factors, alone or in comneurotrophin 3 ,.N.V. t~ ~-o). bination, have been implicated in regulating the survival of CNS and PNS neurons. For example, NGF and NT-3 are required for the survival of differentiated sympathetic neurons and their precursors in vivo (Francis et at., 1999). The demonstration that mature sympathetic neurons require target-derived NGF and NT-3 for survival indicates that these factors act selectively. NGF ai'id NT-3 do not promote neuronal differentiation by NCSCs (unpublished data) or oligopotent sympathoadrenal lineage progenitors (Anderson and Axel. 1986). This suggests that NGF and NT-3 act selectively but not instructively in promoting the differentiation of sympathetic neurons from neural crest progenitors. Although neurotrophins act selectively in promoting the generation of sympathetic neurons, they are also capable of acting instructively. NT-3 induces neuronal differentiation by CNS stem cells in vitro (Vicario-Abejon et al., 1995). This demonstrates that a cell-extrinsic factor can promote differentiation by acting selectively in one case, while acting instructively in another.

C. Neuregulin Acts Both Instructively and Selectively on Neural Crest Progenitors Neuregulin (also known as gliai growth factor; Marchionni et al., 1993) is expressed on motor axons in the nerve (Meyer and Birchmeier, 1994) and is required for Schwann cell development (Meyer and Birchmeier, 1995; Riethmacher et al., 1997). Neuregulin promotes the survival (Dong et al., 1995) and proliferation (Lemke and Brockes, 1984; Morrissey et aI., 1995; Rutkowski et aL, 1995) of Schwann cells and their progenitors. These observations suggested that Neuregulin was required to selectively increase the numbers of Schwann cells by promoting their survival and proliferation (Murphy et aI., 1996: Topilko et al., 1997b); however,


240 NCSCs have now been found within the fetal sciatic nerve when Schwann cells are differentiating (Morrison et al., 1999), and Neuregulin is capable of instructing NCSC to differentiate into glia (Shah et al., 1994). These latter observations suggested that Neuregulin might also be required to initiate glial differentiation by acting instructively on NCSCs. In fact, NCSCs in the sciatic nerve probably undergo differentiation within the nerve into both glia and myofibroblasts (Morrison et al.. 1999). By studying the effects of Neuregulin on different types of progenitors purified from sciatic nerve we have observed that Neuregulin has different effects on cells at different stages of development: Neuregulin promotes the survival of NCSCs and instructs them to differentiate into glia. Neuregulin promotes the survival and proliferation of glia, but only promotes the proliferation (not survival) of myofibroblasts (Morrison et al., 1999). Thus, Neuregulin can have both selective and instructive effects on the same cell and acts in different ways on cells at different stages of development. The genetic requirement for Neuregulin in nerve development development (Meyer and Birch-

meier, 1995 Riethmacher et al., 1997) may represent a cornplex combination of functions.

D. Neural Stem Cells Differentiate via Progressive Restrictions in Developmental Potential There are two alternative models by which stem cells can give rise to differentiated cells (Fig. 3). Stem cells can give rise directly to differentiated cells, such that different types of differentiated cells are generated from different mitoses. This mode of division is often observed in invertebrate stem cell lineages in which a stem cell goes through a stereotyped series of asymmetric divisions to generate a differentiated cell and a stem cell in each division [for example, in C a e n o r h a b d i t i s e l e g a n s germline specification (Mello et al., 1996) or in the D r o s o p h i l a neuroblast lineage (Li et al., 1997)]. Alternatively, stem cells can generate differentiated cells by undergoing an ordered pattern of progressive restrictions in

Figure 3 Directdifferentiation versus progressiverestrictions in developmentalpotential. Stemcells (uncolored) can either differentiate directly' into different types of mature cells (A) or can undergo progressive restrictions in developmental potential (B) by giving rise to oligopotent progenitors (brown and green), which in turn give rise to different types of mature cells. For example, work by Rao and colleagues (1998) has demonstratedthat stem cells in the neural tube give rise to a neuronal oligopotent progenitor and a glial oligopotent progenitor, which in turn give rise to different types of neurons and different types of glia respectively (Kalyani et al., 1997).

12 Stem Cells of the Nervous System developmental potential. For example, hematopoietic stem cells give rise to oligopotent lymphoid-committed (Kondo et al., 1997) or myeloid-committed progenitors that in turn become further committed to numerous individual lymphoid (B. T. or dendritic) or myeloid (macrophage, neutrophil, or euthroid) lineages (Morrison et al., 1995b). In this way stem cells give rise to differentiated cells by going through a series of progressive restrictions in developmental potential. At least some neural stem cells also give rise to differentiated cells by undergoing progressive restrictions in developmental potential. It has long been hypothesized that NCSCs might give rise to multiple lineages of differentiated cells by undergoing progressive restrictions in developmental potential (Anderson, 1989: Le Douarin and Dupin, 1992): however, direct evidence for progressive restrictions by NCSCs in vivo is lacking. In contrast. CNS stem cells derived from the neural tube give rise to neurons and glia by first giving rise to oligopotent restricted progenitors. Stem cells from the E 10.5 rat neural tube give rise to astrocytes, oligodendrocytes, and neurons with a variety of different neurotransmitter phenotypes (Kalyani et al., 1997). In vitro and in vivo the stem cells appear to give rise to E-NCAM § neuronal-committed progenitors as well as A2B5* N-CAMglial progenitors (Mayer-Proschel et al., 1997). The ENCAM -~ neuronal-committed progenitors retain the ability to give rise to multiple different classes of neurons (Kalyani et al., 1998). Similarly, the A2B5 "N-CAM- glial committed progenitors in turn can give rise to both oligodendrocytes and astrocytes (Rao et al., 1998). Thus stem cells from the developing spinal cord give rise to specific lineages of neuronal and glial cells by undergoing progressive restrictions in developmental potential marked by the generation of oligopotent progenitors.

II!. Do Stem Cells Retain Broad or N a r r o w N e u r o n a l Potentials? It is not yet clear whether neural stem cells tend to retain a broad potential to give rise to many different types of neurons, or whether neural stem cells tend to be restricted in the types of neurons they can form. After transplantation, progenitors derived from the telencephalon were able to give rise to neurons throughout the forebrain and midbrain while progenitors from the mesencephalon gave rise to neurons in the midbrain but not in parts of the forebrain (Campbell et al., 1995). Supporting this idea that neural progenitors are at least partially restricted in the types of neurons they can form. telencephalic progenitors differentiated appropriately when transplanted into different dorsoventral positions within the telencephalon, but did not differentiate appropriately when transplanted into different brain regions along the rostrocaudal axis (Fishell. 1995: Na et al., 1998). Even within the adult forebrain, stem cells from one location sometimes cannot give rise to normal neurons in other loca-

241 tions. Stem cells cultured from the hippocampus gave rise to neurons in both the hippocampus and the olfactory bulb on transplantation (Suhonen et aI., 1996). On the other hand, these stem cells gave rise to neurons in the retina but the neurons did not exhibit the normal phenotype of retinal neurons (Takahashi et al., 1998). NCSCs in the developing PNS may also be heterogeneous with respect to the types of neurons they can form. Postmigratory NCSCs isolated from sciatic nerve gave rise to parasympathetic and sympathetic neurons in vitro and in vivo but it is not yet clear whether such cells have sensory or enteric potential (Morrison et aI., I999. 2000). Indeed, migrating neural crest progenitors had sensory, pa_rasympathetic, and sympathetic potential upon transplantation, but postmigratory enteric neural crest progenitors had only parasympathetic potential (White and Andersom 1999). Together the data suggest that while stem cells often have the potential to give rise to multiple different classes of neurons they are at least partially restricted and cannot give rise to all classes of neurons. The corollary of the observation that different stem cells have different neuronal potentials is that different regions in the CNS and PNS must be formed by different types of stem cells (that are defined by different developmental potentials). Beyond the differences in neuronal potential cited above. NCSCs from different levels of the neural tube along the rostrocaudal axis differ in their potential to give rise to mesectodermal derivatives (Le Douarin, 1982, I986). Cephalic neural crest gives rise to dermis, cartilage, and bone, even when transplanted to the trunk level: however, trunk neural crest does not contribute to these tissues, even when transplanted to the cephalic level (Le Douarin and Dupin, 1992). These observations raise the possibility that the CNS and PNS may each include multiple classes of stem cells, in all cases these stem cells can give rise to neurons and glia but the types of neurons and glia may differ. The types of glia formed by stern cells from different levels of the neural axis may overlap broadly, with all CNS stem cells giving rise to astrocytes and oligodendrocytes, and all NCSCs giving rise to Schwann cells. The types of neurons formed by stem cells from different rostrocaudal levels may be more restricted. In many cases stem cells may be restricted to forming types of neurons that are specific to particular rostrocaudal regions. That is, although stem cells may be able to engraft in heterotopic regions of the nervous system, the neurons they produce may not exhibit the full phenotypic and functional properties of normal neurons from those regions. Beyond being a way to organize nervous system development, cellintrinsic differences between neural stem cells in neuronal potential may be a mechanism for generating neuronal diversity" Differences between stem cell populations may interact with environmental differences to cause different types of neurons to differentiate in different places. If neuronal identity were specified by both environmental and stem cellintrinsic determinants it would greatly simplify the problem of how such a vast diversity of neurons is generated.


242 IV. R e g u l a t i o n o f N e u r a l Self-Renewal

S t e m Cell

Self-renewal and differentiation are like opposite sides of a coin. They must be regulated in concert because in a given cell at a given time the execution of one excludes the other. Little is known about how" self-renewing divisions are regulated in mammalian stem cells. While transcription factors have been identified that are sufficient to cause the lineage-specific differentiation of stem cells, the regulation of self-renewal is much less well defined. Are there single factors that are sufficient to induce all of the machinery necessary for a stem cell to self-renew '~ A cell extrinsic factor. FGF-2. causes neural stem cells to self-renew in culture (Gritti et al., 1996). In contrast, no cell-intrinsic factors (such as transcription factors) have yet been identified whose expression is sufficient to cause the self-renewal of mammalian neural stem cells. The question of whether a single cell-intrinsic factor is sufficient to cause self-renewing divisions is important because it remains unclear whether the decision to divide in mammalian stem cells is regulated independently of the decision to remain undifferentiated. Although no genes have yet been shown to cause neural stem cell self-renewal in a cell-intrinsic manner, single gene products can promote the self-renewal of other stem cells. Constitutive activation of the Notch homolog Glp- 1 in C. eleg a n s germline stem cells is sufficient to cause indefinite self-

Autonomic neuron differentiation BMP2

cell-extrinsic ~trinsic

Olash-1

renewal (Wilson Berr?" et al., 1997). Overexpression of stabilized /3-catenin appears to promote the self-renewal o f epidermal stem cells (Zhu and Watt. 1999). Expression o f the homeodomain protein HoxB4 in mouse hematopoietic stem cells is sufficient to increase their numbers in v i v o (Sauvageau et aI., 1995); however, this example illustrates the challenges in demonstrating that a factor promotes m a m malian stem cell self-renewal because the mechanism by which HoxB4 acts is unknown. Rather than increasing selfrenewing divisions by stem cells, it might promote survival or impair differentiation. Nonetheless. if HoxB4,/3-catenin, or FGF-2 causes self-renewal by both promoting mitosis and impairing differentiation, then these factors may be a upstream of a genetic program that executes self-renewing divisions by stem cells. Presumably this p r o ~ a m would be composed of a hierarchy of genes similar to programs that execute lineage specific differentiation (Fig. 4). If so, one or a few gene products at the top of the hierarchy might be able to induce all of the other genes that are required to execute a self-renewing division. Perhaps some of the downstream gene products in the hierarchy regulate division while others inhibit differentiation. If so. then genes at the top of the hierarchy might promote mitosis as well as maintenance of the stem cell state, whereas downstream genes might regulate one or the other. This would be analogous to the regulation of neuronal differentiation by Mash-l, which couples the induction of pan-neuronal markers with subtype-specific

Neural stem cell self-renewal? FGF-2

cell-extrinsic tdnsic

1

.7 _

sufficient

for self-renewal

,r

Pan neuronal

Phox Za/2b

properties

- peripherin - NF1 6 0

Mitosis - cyclins

s u b t y p e specific

properties - c-Ret - neurotransrnitters

DNA replication

Maintenance of

multipotentiality

- repression of lineage commitment -induction of stem cell properties

Figure 4 Hierarchies of genes may control stem cell self-renewal as well as differentiation. The first panel depicts a schematic of autonomic neuron differentiation from NCSCs in which BMP2 induces Mash-1 expression. Mash-I is sufficient to cause autonomic neuron differentiation. The differentiation program involves inducing the expression of many genes, including those that regulate pan-neuronal properties and those that regulate autonomic-specific properties. Because the expression of pan-neuronal genes and subtype-specific genes is regulated differently it appears that Mash-1 must induce the expression of genes in multiple genetically independent pathways. Similarly, we might hypothesize that b-FGF leads to the expression of a transcription factor that is sufficient to cause neural stem cell self-renewal. Multiple genetic pathways may be induced by such a factor, including genes like cyclins that lead to DNA replication and mitosis, and genes that cause the cell to maintain the stem cell state. (The diagram describing neuronal differentiation was adapted from Lo et al., 1998.)

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12 Stem Cells of the Nervous System markers by inducing the expression of downstream genes that act through different pathways (Lo et aI., 1998). Maintenance of the stem cell state can be regulated independent of the decision to divide as demonstrated by the function of Pie-1 in the C. eIegans germline lineage. Pie-1 is a maternally expressed transcription factor that represses the transcription of embryonic genes associated with acquisition of somatic cell fates (Seydoux et al., 1996: Tenenhaus et al., 1998). In C. elegans embryos, totipotent germline blastomeres undergo a series of asymmetric divisions to yield one somatic daughter cell and one germline stem cell. Pie-I is asymmetrically localized to the germline stem cell in each division and appears to maintain totipotency by repressing the transcription of genes that confer a somatic fate (Mello et al.. 1996: Batchelder et al., 1999). Loss of Pie-1 function does not prevent mitosis, but both daughter cells assume somatic cell fates. It is not certain whether a mechanism analogous to Pie-1 operates in mammalian stem cells; however, a transcription factor called neuron restrictive silencing factor (NRSF) (Schoenherr and Anderson. 1995: Schoenherr et al., 1996) or REST (Chong et al., 1995) can inhibit neuronal differentiation by repressing the transcription of a range of neuron-specific genes (Chen et aI., 1998). Thus maintenance of the stem cell state can be regulated independently of the decision to divide, raising the possibility that stem cell selfrenewal may require the coordination of multiple genetic pathways. Few insights exist into which genes compose the selfrenewal program for neural stem cells, though some recently described mutations provide candidates. For example, deletion of the winged helix transcription factor B F-1 reduces the proliferation of undifferentiated neuroepithelial cells in the developing ventral telencephalon and leads to premature neuronal differentiation (Xuan et al., 1995). These observations strongly suggest that BF-1 is required for the normal self-renewal of CNS stem cells. Flat-top is a mutation (in an as yet unidentified gene) that was generated by ethyl-nitrosourea mutagenesis of mouse germ cells in a screen for mutations that affect CNS development (Hentges et al., 1999). The fiat-top mutation is associated with decreased proliferation of undifferentiated neural progenitors in the embryonic telencephalon, leading to a failure to form normal telencephalic vesicles. As genetic screens for mutations that affect nervous system development progress, and tools for studying stem cell function at the cellular level become increasingly sophisticated, the genetic regulation of neural stem cell self-renewal will be elucidated.

V. Differences between Hematopoietic Stem Cells and Neural Stem Cells Hematopoietic stem cells were first among stem cells to be purified and extensively characterized (reviewed by Morrison et al., 1995b) (see Chapter 13). As a result, they have

been a model on which predictions about the properties of other stem cells have been based. Many hypotheses about neural stem cell function have been inspired by analogy to hematopoietic stem cells. There are similarities between he, matopoietic stem cells and neural stem cells, but there are also fundamental differences in developmental strategy between the hematopoietic and nervous systems. If we are to create expectations of neural stem cell function based on the properties of hematopoietic stem cells, then it is critical m understand these parallels and differences.

A. Stem Cell Frequency The hematopoietic and nervous systems adopted a fundamental difference in strategy with respect to how they use stem cells. Hematopoietic stem cells are rare but give rise to massive numbers of progeny. For example, hematopoietic stem cells account for only 0.04% of mouse fetal liver cells around E12-14 (Morrison et al., 1995a), but in lineage marking experiments individual stern cells from around this stage of development gave rise to very large numbers of progeny by birth (Clapp et al., 1995). In contrast, when stem cells are present in the nervous system, large numbers of stem cells each give rise to only small numbers of progeny. For example, in the E 14 rat sciatic nerve, NCSCs account for around 15% of nerve cells (Morrison et al., 1999). But multipotent neural crest or CNS progenitors consistently give rise to fewer than 100 cells in the developing nervous system (Fraser and Bronner-Fraser, I991; Walsh and Cepko, t993; Henion and Weston, 1997). Thus the hematopoietic system forms few stem cells but causes them to give rise to enormous numbers of progeny, whereas the nervous system forms many stem cells that each give rise to few progeny. The hematopoietic and nervous systems may be subject to different developmental constraints that select for different strategies.

B. Regional Specialization A second difference in strategy between the hematopoieric and nervous systems is that while the hematopoietic system ~oes out of its way to avoid regional specialization, regional specialization is the rule in the nervous system. Hematopoiesis occurs in bone marrow that is distributed among many different bones throughout the body. Yet as far as we know the same cell types are produced in similar proportions by all bone marrow compartments. In addition, the hematopoietic system can be ablated with a lethal dose of radiation and then fully reconstituted by purified hematopoietic stem cells from any bone in the body (Spangrude et al., 1988; Uchida et al., 1994; Osawa et al., 1996: Morrison et al., 1997b). In contrast, even slight differences in position within the nervous system are associated with functional and phenotypic differences in the cell types that differentiate. This is


244 clearly demonstrated in the case of the neural crest, which gives rise to enteric neurons in the gut, sympathetic neurons in the sympathetic chain, parasympathetic neurons associated with organs, and sensory neurons in the spinal ganglia. Neural stem cells give rise to progeny that are often confined to a small area within the nervous system (Fraser and Bronner-Fraser. 1991 Reid et al., 1995). Because only a restricted subset of neural cells differentiates in any one location within the nervous system, it is likely that neural stem cells have less opportunity than hematopoietic stem cells to undergo multilineage differentiation in vivo. However. progenitors from one region of the developing nervous system sometimes fail to give rise to the neurons found in other regions of the nervous system on transplantation (Campbell et al.. 1995 Na et al.. 1998: Takahashi et al., 1998). This suggests that different lineages of cells within the nervous system derive from different types of stem cells. Indeed. while many if not most migrating neural crest cells are multipotent (Le Douarin and Dupin. 1992: Shah et al.. 1994), most neural crest progenitors are fated to give rise to only one type of differentiated cell (Henion and Weston. 1997). Thus regional specialization within the nervous system may prevent multipotent progenitors from exhibiting their full developmental potential.

VI. In I/Tvo Function of Neural Stem Cells These differences in strategy between the hematopoietic and nervous systems mean that we should be cautious about expecting neural stem cells to have properties analogous to hematopoietic stem cells. A single hematopoietic stem cell can engraft in lethally irradiated mice and give rise to all major lineages of blood cells for the life of the mouse (Morrison and Weissman. 1994: Spangrude et al., 1995 Osawa et al.. I996). This observation has caused some to propose that neural stem cells should also self-renew for long periods of time in vivo while giving rise to large numbers of progeny. But these may be unreasonable expectations, in part because most of the nervous system forms during a limited window of fetal and neonatal development, as discussed next.

A. Long-Term in Vivo Self-Renewal A self-renewing division is a mitosis in which the mother cell gives rise to at least one daughter cell that has a developmental potential that is indistinguishable from the mother cell. For example, if a multipotent mother cell gives rise to similarly multipotent daughter cell(s) then the mother cell is a self-renewing stem cell. In contrast, if a multipotent mother cell only gives rise to daughter cells with restricted developmental potentials, then it does not self-renew. Most neural stem cells probably persist for only short periods of time in vivo and therefore the criterion of long-term

self-renewal mav not be relevant. The PNS forms b e t w e e n E8 and P14 in mice and rats, in which gestation is 1 8 - 2 1 days (Pham et aI., 1991). NCSCs have only been shown to persist until E l 7 (Morrison et aI., 1999). Thus there is n o reason to believe that NCSCs should or can self'renew for more than a week or two in vivo even though they have the p o t e n t i a l to self-renew for considerably longer periods o f time in vitro. Most of the CNS also forms during fetal and neonatal life. Because CNS stem cells persist near the lateral ventricle and in the dentate gyms of adults, an opportunity may arise for some such cells to exhibit long term selfrenewal in those locations: however, like NCSCs, these cells appear to give rise to only small numbers of progeny in v i v o (Morshead et al., 1998: Doetsch et al., 1999: Johansson et al., 1999) despite their ability to self-renew and give rise to large numbers of progeny in vitro (Baroffio et al., 1988; Gritti et al.. 1996). Thus while these studies provided evidence that stem cells near the lateral ventricle undergo asymmetric self-renewing divisions in vivo, it is not clear whether individual stem cells repeatedly undergo such divisions over long periods of time or whether individual stem cells might only self-renew a few times before differentiating. The available evidence suggests that most neural stern cells self-renew to only a limited extent in vivo despite their capacity for extensive self-renewal in vitro. Therefore, the criterion of longterm self-renewal in vivo may be an unphysiological one for most neural stem cell populations. Unlike the hematopoietic system, there is no known circumstance in the nervous system in which neural stem cells are required to reconstitute most of the nervous system after injury. In vivo assays for hematopoietic stem cells depend on irradiating recipient mice to reduce competition from endogenous stem cells prior to reconstituting with transplanted stem cells. Mammals can live for around 2 weeks after a lethal dose of radiation that destroys most blood cells and the ability to make new blood cells. This provides an opportunity to test whether a small number of transplanted hematopoietic cells have the potential to self-renew, undergo multilineage reconstitution, and give rise to the massive numbers of blood cells that are required to reconstitute the hematopoietic system. While the number of stem cells in the subependymal layer of the lateral ventricle can be reduced by administering cytotoxic compounds prior to transplanting neural progenitors (Doetsch et aL, 1999), it is much more difficult in the nervous system to promote the engraftment and proliferation of transplanted progenitors by eliminating endogenous stem cells. Most of the nervous system cannot be experimentally ablated in viable animals or reconstituted by transplanted progenitors. It is possible that in the future an in vivo assay will be developed in which a single neural stem cell can be introduced into a tissue that is permissive for neural differentiation, but where there is little competition from endogenous neural progenitors. Only then are we likely to have an opportunity to demonstrate that single neu-

245

12 Stem Cells of the Nervous System ral stem cells consistently undergo multilineage differentiation and give rise to large numbers of progeny in vivo.

B. The Relationship between Fetal and Adult Neural Stem Cells It is sometimes assumed that stem cells in the adult CNS are equivalent to the stem cells that form the CNS during fetal development--that the stem cells in the adult brain are just "'left over" from fetal development. In fact, the relationship between stem cells in the adult CNS and stem cells in the fetal CNS is unknown. Stem cells in the adult CNS may be lineally unrelated to fetal CNS stem cells, or they may derive from fetal stem cells. Even if adult CNS stem cells derive from fetal CNS stem cells, the adult cells may be developmentally distinct. For example, fetal CNS stem cells may be able to give rise to certain classes of neurons that adult stem cells cannot. By analogy with the hematopoietic system such a possibility seems likely. Adult hematopoietic stem cells probably derive from fetal liver hematopoietic stem cells (Fleischman et at., 1982; Clapp et aI., 1995), but their properties differ in important ways. Certain lineages of lymphocytes are produced by fetal but not adult hematopoietic stem cells (Ikuta et al., i 990; Kantor et al., i992; Hardy and Hayakawa, 1994). More importantly, fetal hematopoietic stem cells have a greater self-renewal and proliferative potential than adult hematopoietic stem cells (Morrison et al., 1995a: Rebel et al., 1996). Additional work will be required to carefully compare the properties of stem cells from the fetal and adult CNS.

C. Cell Cycle Status The cell cycle distribution of neural stem cells is often hypothesized to be similar to hematopoietic stem cells. For example, adult CNS stem cells have been predicted to be quiescent in vivo by analogy to hematopoietic stem cells. Hematopoietic stem cells are relatively quiescent in normal C57BL adult mice: 75% of hematopoietic stem cells are in Go at any one time, and on average only 8% of stem cells divide each day (Cheshier et aI., 1999); however, this contrasts with hematopoietic stem cells from fetal liver, wt'fich undergo daily self-renewing divisions (Morrison et al., 1995a). Not only does the cell cycle status of hematopoietic stem cells change during ontogeny (Morrison et aI., 1996), but it changes in response to injury (Harrison and Lerner, 1991; Morrison et al., 1997c) and differs between mouse strains (deHaan et al., 1997). Thus we should be cautious when it comes to generalizing about the cell cycle regulation of neural stem cells. In fetal rat sciatic nerve, NCSCs are not quiescent but undergo rapid self-renewing divisions (Morrison et al., 1999). In the fetal CNS, analyses of the proliferation of ventricular zone progenitor cells suggest that fetal CNS stem cells are likely proliferative as well (Cai et al.,

1997), although this is not certain since stem cells compose a small minority of cells in the ventricular zone (Davis and Temple, 1994). Under normal conditions in the adult CNS there are quiescent as well as actively proliferating progenitors (Morshead et at., 1994, 1998; Johansson et al., 1999). These studies indicate that at least some stem cells in the adult CNS are quiescent but the precise relationship between the proliferating and the quiescent progenitors is uncertain because CNS stem cells have not yet been purified or studied directly. Although we have an incomplete understanding of the cell cycle regulation of neural stem cells it appears the analogy to hematopoietic stem cells is appropriate: fetal neural stem ceils tend to be proliferative, while adult CNS stem cells are often quiescent and can be activated in response to injury (Doetsch et al., 1999; Johansson et al., 1999).

VII. Surprising Potential of Neural Stem Cells A series of recent papers has suggested that cultured CNS stem cells have the potential to give rise to nonneural derivatives in tissues that have been thought to be developmentally unrelated. Other papers suggest that progenitors from tissues outside of the nervous system may retain the potential to make neurons and glia. Are classical lineage relationships incorrect? Or do stem cells retain potentials that they never exhibit during normal development? Or have we identified an intriguing phenomenon in which the developmental potential of cells can be reprogrammed under specific conditions in culture or after transplantation, without reflecting lineage relationships that exist in normal development? The surprising developmental potential of somatic stem cells suggests we may have to redefine our understanding of developmental potential. Stem cells cultured from the mouse or human CNS have the potential to give rise to nonneural derivatives. CNS stem cells are often isolated by culturing neurospheres, which are spheres of multipotent progenitors that g o w out of mixed populations of CNS cells in bFGF-containing media. Such neurospheres are thought of as CNS stem cells but because the neurosphere cells have only been studied in culture, it is not certain whether they have properties that are similar to normal CNS stem cells in vivo. Neurosphere cells have been observed to give rise to blood cells on transplantation into irradiated mice (Bjornson et al., 1999), skeletal muscle on coculture with a myogenic cell line or on transplantation into regenerating muscle in vivo (Galli et aI., 2000), or to colonize tissues of all three germ layers on injection into blastocysts or early chick embryos (Clarke et al., 2000). All of these papers concluded that CNS stem cells retain a much broader developmental potential than previously thought: however, there is concern that CNS progenitors can lose patterning information and acquire a broader developmental

246 potential as a result of being cultured in high concentrations of mitogens such as bFGF (Gage, 1998). Indeed, primordial germ cells have already been demonstrated to be reprogrammed in culture to acquire the properties of embwonic stem cells (Matsui et al., 1992: Donovan. 1994). As a result. it is unknown whether neurosphere cells correspond to normal CNS stem cells, or whether their developmental potential is broadened in culture. When it becomes possible to prospectively identity" and purify uncultured CNS stem cells, it will be important to test whether these cells still give rise to blood, muscle, and other somatic lineages on transplantation or whether they must be cultured first in order to exhibit these potentials. Another example of nervous system progenitors exhibiting unexpected developmental potentials comes from work on oligodendrocyte precursor cells (OPCs). OPCs are progenitors from the optic nerve that have been extensively studied and thought to be glial committed (.and therefore could be considered restricted progenitors rather than stem cells). The recent demonstration that OPCs can make neurons after being exposed in culture to a specific series of growth factors, including bFGF. is consistent with the idea that restricted neural progenitors can acquire neuronal potential in culture (Kondo and Raft, 2000). Indeed, the OPCs not only gave rise to neurons, but to neurospheres as well. This bolsters the concern that neurospheres can sometimes be produced in culture from cells that have properties in vivo that are different from multipotent CNS stem cells. The demonstration that OPCs can give rise to neurons is important because OPCs can be purified from uncultured postnatal optic nerve by immunopanning. Thus this is the first demonstration that a well-characterized, prospectively identified nervous system progenitor can acquire a broader developmental potential than previously thought in culture. This supports the idea that specific progenitors can exhibit unexpected developmental plasticity in culture but cautions us that such plasticity may not be exhibited during normal development. Not only can neural stem cells make non-neural cell types, but non-neural stem cells can make neurons and glia. Bone marrow progenitors, which are best known for making blood cells, have recently been observed to contribute to a variety of tissues on transplantation. Cells from mouse bone marrow have been observed to give rise to skeletal muscle (Ferrari et aI., 1998 Gussoni et al., 1999), hepatocytes (Peterson et aI., 1999: Lagasse et al., 2000), and neurons and glia (Kopen et al., 1999). The bone marrow is known to contain at least two different types of stem cells including hematopoietic stem cells and mesenchymal stem cells. Additionally. it is conceivable that stem cells from other tissues such as liver, nervous system, and muscle may circulate at low levels and be found in the bone marrow. So which stem cells gave rise to the unexpected derivatives'? The cells that gave rise to neurons and glia were from cultures of adherent bone marrow" stroma, suggesting that they included mesenchymal

Ii Lineage Specification and Differentiation stem cells (Kopen et al., 1999). Further work will be required to rigorously establish the identity and the origin o f the progenitors with neurogenic potential from bone marrow. With so many cells exhibiting unexpected developmental potentials, some have begun to question the classical germ layer origin of some tissues. Is it possible that CNS stern cells give rise to blood, muscle, and liver during normal development'? This question is testable by CRE-recombinase fate mapping, which can identi~' all of the progeny produced by a specific lineage of progenitors during normal development, as long as a promoter can be identified that is absolutely specific to that progenitor lineage. Several studies have used this approach to examine the progeny produced by cells that express Wnt-1, which include neural stem cells in the midbrain, dorsal neural tube. and neural crest. Wnt-1 expressing cells were observed to give rise to many expected CNS, PNS, and other neural crest derivatives, but have not yet been observed to give rise to blood, skeletal muscle, liver, "kidney. lung, or other unexpected derivatives (Chai et al., 2000: Jiang et al., 2000). This is not a perfect experiment in that not all neural stem cells are marked by W n t l expression, and it is possible that the CNS stem cells with unusually broad developmental potentials derive from lineages that never expressed W n t l . Nonetheless. the failure to yet observe any unusual derivatives from a wide cross section of neural progenitors in vivo during normal development contrasts with the spate of papers reporting the broad potential of a variety of cultured CNS stem cells after transplantation. This suggests that the potential exhibited by cultured CNS stem cells may not be exercised during normal development. Even if most of the surprising neural stem cell potential arises as a result of "de-differentiation" in culture and does not reflect potential that is normally exercised in vivo, it is still scientifically and clinically important to understand the phenomenon. On the other hand, if normal somatic stem cells routinely retain much broader developmental potential than they exhibit during normal development, this would pose the question as to whether differentiation is primarily environmentally regulated. That is, are the normal lineage relationships that exist between progenitors in different germ layers governed primarily by controlling the locations and interactions of such progenitors? Maybe the only reason why neural stem cells do not seem to make liver or blood during normal development is that such cells are prevented from accessing hepatic or hematopoietic microenvironments.

VIII. A r e N e u r a l Stem Cells I n v o l v e d in Disease?

A. NCSCs and Childhood Cancer There is no published evidence that NCSCs can persist postnatally, but if they do, it might have important implica-

12 Stem Cells of the Nervous System tions for understanding the origin of certain childhood cancers. A popular hypothesis is that many types of cancer cells derive from the transformation of normal stem cells (Sell and Pierce, 1994). A number of different types of tumors are thought to arise from neural crest derivatives including neuroblastomas, neurofibromas, and Ewing's sarcomas/peripheral neuroectodermal tumors (PNET). Ewing's sarcomas/ PNET are particularly primitive in appearance, containing cells that can express neuronal, glial, and mesectodermal markers. This has led to speculation that these tumors are derived from primitive neural crest progenitors (Fujii et at., 1989: Marina et al.. 1989). Indeed some speculate that PNS tumors not only derive from the transformation of neural crest progenitors but that the degree of differentiation exhibited by the tumor is determined by the stage of differentiation at which transformation of the normal progenitor occurred (Thiele, 1991 ). The obvious difficulty with this hypothesis is that. while these tumors occur predominantly in children and adolescents, there is yet no evidence for the persistence of uncommitted neural crest progenitors postnatally. If neural crest progenitors undergo terminal differentiation in midgestation, it is hard to imagine how there is an opportunity for such progenitors to be transformed in a way that leads to tumors that present late in childhood. If. on the other hand, rare NCSCs persist postnatally, then perhaps such cells represent the cellular locus for transforming events that lead to childhood cancers.

B. Adult Neurogenesis and Mental Illness Given the recent demonstration that neurogenesis continues in regions of the adult human brain that are involved in learning and memory (Eriksson et al., 1998), scientists have started to consider whether changes in neurogenesis might be associated with mental illness (Brown et al., 1999). It has been proposed that stress-induced changes in the hippocampus lead to the development of depression in at-risk individuals (Duman et al., 1997). Indeed, exposure to stress can reduce hippocampal neurogenesis, at least in adult monkeys (Gould et al., 1998). A possible mechanism that might link mental illness with altered neurogenesis comes from corticosteroids, which are elevated in patients with mood disorders (reviewed by Brown et al., 1999) and which can impair hippocampal neurogenesis (Cameron and McKay, 1999). Reduced hippocampal serotonin levels have also been associated with cognitive disorders such as depression and schizophrenia (Cross, 1990) and, when hippocampal serotonin levels are experimentally depleted, neurogenesis is reduced (Brezun and Daszuta, 1999). Much more work will have to be done to determine whether changes in neurogenesis are associated with mental illnesses, let alone to determine whether such changes are causative. Nonetheless these considerations illustrate the range of health issues that may be impacted by the discove~ of stem cells and neurogenesis in the adult human brain.

247 C. Do Neural Stem Cells Respond to Nervous System Injury? A number of neurodegenerative disorders that afflict older people are caused by the loss of certain types of neurons in the brain. Parkinson's disease is caused by the progressive loss of dopaminergic neurons from the substantia nigra in the striatum (Date, 1996). Huntington's disease is also caused by the loss of striatat neurons (Reddy et aI., 1999). Alzheimer's disease is caused by the loss of cholinergic as well as other neurons from the forebrain (Winkler et al., 1998). All of these neurodegenerative diseases have been treated by transplanting neural progenitor cells into the affected areas in an effort to regenerate the lost neurons (Winkler et al,, 1998; Bjorklund and Lindvall. 1999: Date and Ohmoto. 1999). Of these disorders, Parkinson's is thought to be the most promising clinical application for transplantation therapies, because the lesion is localized and because it may be sufficient to reintroduce dopaminergic cells without necessarily restoring their normal pattern of projections. Nonetheless, although the transplantation of neural progenitors has proven ve~' effective for ameliorating symptoms in Parkinsonian animal models (Date, 1996), success in patients has not been as consistent (Olanow et al., 1997). Improved techniques and more sophisticated strategies for generating neural progenitors for transplantation may lead to improvement in clinical outcomes. An outstanding question that has received little attention is whether there is any response of endogenous neural stem cells to neurodegeneration (Lowenstein and Parent, 1999). Do stem cells in the brain attempt to make new neurons to compensate for the neurons that die in Parkinson's, Huntington's, or Alzheimer's disease? At present there is no evidence of this, but dentate gyrus neurogenesis is accelerated in response to seizure and associated neuronal death (Bengzon et aI., 1997" Parent et al., 1997, 1998; Scott et al., 1998); furthermore, dentate gyms neurogenesis is also stimulated in response to mechanical lesions in the granule cell layer (Gould and Tanapat, 1997). If neurogenesis can be stimulated by excitotoxic or mechanical damage, it could also be stimulated by neural degeneration. If endogenous progenitors are activated by neurodegenerative disease then perhaps the clinical course of some diseases is slowed by regeneration. If not, is it because endogenous stem cells do not have the potential to make the types of neurons that are lost in most neurodegenerative disorders? Or are the stem cells in the wrong place and unable to make neurons that can migrate to the sites of the lesions? Could the response of endogenous stem cells to neurodegeneration be pharmacologically enhanced? Do existing pharmacological treatments for mental illness or neurodegeneration impact on the function of stem cells in the brain? These are fundamental questions that will be addressed as more sophisticated tools become available to study the in vivo functions of neural stem cells.

!! Lineage Specification and Differentiation

248

IX. Outstanding Issues Since the pioneering studies of Ram6n y CajS.1 (Ram6n y Caj~il and May. I959" cited in Lowenstein and Parent, 1999; and Rakic. 1985), the nervous system was thought to form during e m b r y o n i c development and then to remain invariant throughout adult life. with no capacity for neurogenesis. Starting in the 1960s with studies by Altman (1962. 1969) and Altman and Das (1966), neurogenesis in certain regions of the adult brains of rodents was recognized. However, it was not until 1992 that stem cells were discovered in the CNS (Reynolds and Weiss. 1992) and in the neural crest (Stemple and Anderson. 1992). Not until 1998 was neurogenesis demonstrated in the adult human brain (Eriksson et al., 1998). Thus the study, of neural stem cells is an exciting new field with many unanswered questions and surprises yet to c o m e 1. The prospective identification and purification o f neural stem cells. Prospective identification refers to the ability to reliably predict which cells are stem cells and which are other cell types in vivo or among freshly dissociated cells. based on marker expression. The importance of prospective identification is illustrated by the experience with hematopoiesis where our understanding of hematopoietic stem cells was greatly accelerated by' their prospective identification and flow-cytometric purification (Spangrude et al.. 1988). So far. our understanding of neural stem cells has been based mainly on retrospective analyses in vivo and on analyses of cultured multipotent progenitors. As a result, fundamental questions about neural stem cells remain unanswered. Do multipotent neural progenitors that are cultured from the adult CNS arise from cells with similar properties in vivo or do they arise from more restricted progenitors by dedifferentiation (Gage. 1998)? Do stem cells in the adult brain reside in the ependymal layer (Johansson e t al.. 1999) orin the subependymal layer (Doetsch et al., 1999). or both? Markers are finally being identified that will allow us to predict which cells are neural stem cells, so that they can be studied as they exist in vivo. Uncultured N C S C s were recently purified by flow-cytomet~, (Morrison et al., 1999). As a result of the ability to prospectively identify these cells it was demonstrated that surprisingly they persist into late gestation by selfrenewing in peripheral nerves. These approaches should soon enable the purification of uncultured CNS stem cells as well. As it becomes possible to purify different classes of neural stem cells, to study their properties in vivo. and to directly compare the properties of different classes of stem cells our understanding of their biology will quicken. 2. The potential o f neural stem cells. Is the nervous system formed by many different kinds of stem cells that are independently born in different regions of the nervous system and restricted in the types of neurons they can form? Do cell-intrinsic differences between stem cells from different regions of the nervous system interact with environmental differences to generate neural diversity? We need to deter-

mine what types of neurons can be f o r m e d by stem cells from different regions of the nervous system, and w h e t h e r there are cell-intrinsic differences between neural stem cells in terms of self-renewal potential or their r e s p o n s e to lineage determination factors. 3. The genetic regulation o f stem cell s e l f - r e n e w a l a n d differentiation. With the advent of microarray analysis it should be possible to conduct increasingly sophisticated screens for genes whose expression is associated with stem cell self-renewal or differentiation. In c o m b i n a t i o n with analyses of gene function in neural stem cells in vitro and in genetically modified mice in vivo, such screens may make it possible to decode the genetic programs that regulate stem cell function. 4. The response o f neural stem cells to disease. It will be important to test whether stem cells in the adult CNS respond to injury and disease, and whether their potential for regeneration can be therapeutically enhanced. The approach of trying to optimize a regenerative response by e n d o g e n o u s neural stem cells would be an important new approach to repairing the nervous system after injury, or disease.

References Altman. J. (1962). Are new neurons formed in the brains of adult mammals? Science 135, 1127- I 128. Altman, J. (1969). Autoradiographic and histological studies of postnatal neurogenesis. IV. Ceil proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. NeuroI. 137, 433-458. Altman. J.. and Das. G. D. (1966). Autoradiographic and histological studies of posmatal neurogenesis. J. Comp. Neurot. 126, 337-390. Anderson. D. J. (1989). The neural crest cell lineage problem: Neuropoiesis? Neuron 3, 1- 12. Anderson. D. J. (1997). Cellular and molecular biology of neural crest cell lineage determination. Trends Genet. 13, 276-280. Anderson. D. J.. and Axel. R. (1986). A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids. Cell (Cambridge. Mass.) 47, 1079- 1090. Baroffio. A.. Dupin. E.. and Le Douarin. N. M. (1988). Ctone-tbrming ability and differentiation potential of migrator" neural crest cells. Proc. Natl. Acad. Sci. [~S.A. 85, 5325-5329. Baroffio. A., Dupin. E.. and Le Douarin, N. M. (1991). Common precursors for neural and mesectodermal derivatives in the cephalic neural crest. Development (Cambridge, UK) 112, 301-305. Barres, B. A. (1999). A new role for glia: Generation of neurons! Cell (Cambridge. Mass.) 97, 667-670. Batchelder, C.. Dunn. M. A.. Choy, B., Suh, Y., Cassie, C., Shim, E. Y., Shin. T. H.. Mello, C., Seydoux. G., and Blackwell. T. K. (1999). Transcriptional repression by the Caenorhabditis elegans germ-line protein PIE- 1. Genes Dev. 13, 202-212. Bengzon, J., Kokaia. Z., Elmer, E., Nanobashvili, A., Kokaia, M.. and Lindvall, O. (1997). Apoptosis and proliferation of dentate gyrus neurons after single and intermittent Iimbic seizures. Proc. Natl. Acad. Sci. U.S.A. 94, 10432-10437. Bitgood, M. J.. and McMahon, A. R (1995). Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172, 126-138. BjorkIund. A., and Lindvall. O. (1999). Transplanted nerve cells survive and are functional for many years. Laekarridningen 96, 3407-3412.

12 Stem Cells of the Nervous System Bjornson. C. R. R.. Rietze. R. L., Reynolds. B. A.. Magli. M. C.. and Vescovi. A. L. (1999). Turning brain into blood: A hematopoietic fated adopted by adult neural stem cells in vivo. Science 283, 534-537. Brezun. J. M.. and Daszuta. A. (1999). Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats. Neuroscience 89, 999-1002. Bronner-Fraser. M.. and Fraser. S. (1989). Developmental potential of avian trunk neural crest cells in situ. Neuron 3, 755-766. Brown. E. S.. Rush. A. J., and McEwen. B. S. (I999). Hippocampal remodeling and damage by' corticosteroids: Implications for mood disorders. Neuropsychopharmacology 21,474 - 484. Cal. L.. Hayes. N. L.. and Nowakowski. R. S. (1997). Local homogeneity of cell cycle length in developing mouse cortex. J. Neurosci. 17, 20792087. Cameron. H. A.. and McKay. R. D. (t999). Restoring production of hippocampal neurons in old age. Nar Neurosci. 2, 894-897. Campbell. K.. Olsson. M.. and Bjorklund. A. (1995). Regional incorporation and site-specific differentiation of striatal precursors transplanted to the embryonic forebrain ventricle. Neuron 15, 1259 - t 273. Chai, Y.. Jiang. X.. Ito, Y.. Bringas. R, Han. J., Rowitch, D. H.. Soriano. R. McMahon. A. R. and Sucov. H. M. (2000). Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development (Cambridge. UK) 127, 1671-1679. Chen. Z.-F.. Paquette. A. J.. and Anderson. D. J. (t998). NRSF/REST is required in vivo for repression of multiple neuronal target genes during embwogenesis. Nar Gener 20, 136-142. Cheshier. S. H.. Morrison, S. J.. Liao. X., and Weissman, I. L. (I999). In vivo proliferation and cell cycle kinetics of long-term hematopoietic stem cells. Proc. Nail. Acad. Sci. U.S.A. 96, 3120-3125. Chong. J. A., Tapia-Ramirez. J., Kim. S., Toledo-Aral, J. J., Zheng. Y.. Boutros, M. C.. Altshuller. Y. M.. Frohman. M. A.. Kraner, S. D.. and Mandel. G. (1995). REST: A mammalian silencer protein that restricts sodium channel expression to neurons. Cell (Cambridge, Mass.) 80, 949-957. Clapp. D. W:. Freie. B.. Lee. W-H.. and Zhang. Y.-Y. (1995). Molecular evidence that in situ-transduced fetal liver hematopoietic stem/progenitor cells give rise to medullary hematopoiesis in adult rats. Blood 86, 2113-2122. Clarke, D. L.. Johansson. C. B.. Wilbertz. J.. Veress, B.. Nilsson, E.. Karlstrom. H.. Lendahl. U.. and Frisen. J. (2000). Generalized potential of adult neural stem cells. Science 288, 1660-1663. Corotto, E S.. Henegar. J. R., and Maruniak, J. A. (1994). Odor deprivation leads to reduced neurogenesis and reduced neuronal survival in the ol. facto W bulb of the adult mouse. Neuroscience 61,739-744. Cross, A. J. (1990). Serotonin in Alzheimer-type dementia and other dementing illness. Anna. N. E Acad. Sci. 61)0, 405-417. Dale. L., and Wardle. F. C. (1999). A gradient of BMP activity specifies dorsal-ventral fates in early Xenopus embQos. Semin. Celt Dev. Biol. 10,319-326. Date. I. (1996). Parkinson's disease, trophic factors, and adrenal medullary" chromaffin cell grafting: Basic and clinical studies. Brain Res. Bull. 40, 1-19. Date. I.. and Ohmoto. T. (1999). Neural transplantation for Parkinson's disease. Cell. MoI. Neurobiol. 19, 67-78. Davis. A., and Temple, S. (I 994). A self-renewing multipotential stem ceil in embry'onic rat cerebral cortex. Nature (London) 372, 263-266. deHaan, G.. Nijhof. W:, and VanZant. G. (1997). Mouse strain-dependent changes in frequency and proliferation of hematopoietic stem cells during aging: Correlation between lifespan and cycling activity. Blood 89, 1543-1550. Deville. F. S.-S.-C.. Ziller. C., and Le Douarin. N. (1992). Developmental potentialities of cells derived from the truncal neural crest in clonal cultures. Dev. Brain Res. 66, 1-10. Deville. F. S.-S.-C.. Ziller. C.. and Le Douarin. N. M. (1994). Developmental potentials of enteric neural crest-derived cells in clonal and mass cultures. Dev. Biol. 163, 141 - 151.

249 Doetsch. F.. Caille. I., Lim, D. A., Garcia-Verdugo, J. M., and AlvarezBuylla. A. (1999). Subventricutar zone astrocytes are neural stem cells in the adult mammalian brmn. Cell (Cambridge, Mass.) 97, 703-716. DonE. J.-MI. Smith. R. Hall, C., and Lira, L. (1995). Promoter region of the transcriptional unit for human c~1-chimaerin, a neuron-specific GTPaseactivating protein for p21rac. Eur J. Biochem. 227, 636-646. Donovan. R J. (I994). Growth factor regulation of mouse primordial germ cell development. Curr. Top. Dev. Biol. 29, 189-225. Duff. R. S.. Langtimm, C. J.. Richardson. M. K.. and Sieber-Blum, M. (I991). In vitro clonal analysis of progenitor cell patterns in dorsal root and sympathetic ganglia of the quail embryo. Dev. Biol. 147, 451-459. Duman. R. S.. Heninger. G. R., and Nestler. E. J. (t997). A molecular and cellular theop," of depression. Arch. Gen. Psychiatry 54, 597-606. Erickson. C. A.. and Goins. T. L. (t995). Avian neural crest cells can migrate in the dorsolateral path only if they are specified as melanocytes. Development (Cambridge, UK) 121, 915-924. Eriksson, R S.. Perfilieva, E., Bjork-Eriksson. T.. Alborn, A.-M.. Nordborg, C.. Peterson. D. A., and Gage. F. H. (i998). Neurogenesis in the adult human hippocampus. Nar Meg 4, 1313 - 1317. Ferrari, G.. Cusella-De Angelis. G.. Coletta. M., Paolucci. E., Stornaiuolo. A., Cossu. G.. and Mavilio. F. (i998). Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 1528-I530. Fishell. G. (1995). Striatal precursors adopt cortical identities in response to local cues. Development (Cambridge, UK) 121, 803-812. Fleischman. R. A., Custer. R. R. and Mintz. B. (1982). Totipotent hematopoietic stem cells: Normal self-renewal and differentiation after transplantation between mouse fetuses. Cell (Cambridge. Mass.) 30, 351359. Francis. N.. Farinas, I., Brennan. C., Rivas-Plata, K., Backus. C.. Reichardt. L.. and Landis. S. (1999). NT-3, like NGE is required for sur-vivaI of sympathetic neurons, but not their precursors. Dev. Biol. 210, 411-427. Frank. E., and Sanes. J. R. (199I). Lineage of neurons and glia in chick dorsal root ganglia: Analysis in vivo with a recombinant retrovirus. Development (Cambridge, UK) 111, 895-908. Fraser, S. E.. and Bronner-Fraser, M. E. ( 1991 ). Migrating neural crest cells in the trunk of the avian embryo are muttipotent. Developmenv (Cambridge, UK) 112, 913-920. Frazier-Cierpial, L., and Brunjes. R C. (1989). Early postnatal cellular proliferation and survival in the otfactow bulb and rostral rpdgratory stream of normal and unilaterally odor-deprived rats. J. Comp. Neurol. 289, 481-492. Fuji. Y.. Hongo, T., Nakagawa, Y., Nasuda, K., Mizuno, Y., Igarashi, Y., Naito, Y.. and Maeda, M. (I989). Cell culture of small round cell tumor originating in the thoracopulmonary region: Evidence for derivation from a primitive pluripotent cell. Cancer 64, 43-51. Gage. F. H. (I998). Stem cells of the central nervous system. Curr. Opin. Neurobiol. 8, 671-676. Galli, R.. Borello. U., Gritti, A., Minasi, M. G.. Bjornson, C.. Coletta, M., Mora. M., Cusetla De Angelis, M. G., Fiocco, R., Cossu, G.. and Vescovi, A. L. (2000). Skeletal myogenic potential of human and mouse neural stem cells. Nat. Neurosci. 3, 986-991. Gould, E.. and Tanapat, R (1997). Lesion-induced proliferation of neuronal progenitors in the dentate gyrus of the adult rat. Neuroscience 80, 427436. Gould, E., Tanapat, R, McEwen, B. S., Flugge, G., and Fuchs, E. (1998). Proliferation of ganule ceil precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc. Natl. Acad. Sci. USA 95, 3168317t. Gould, E., Beylin, A.. Tanapat, R. Reeves, A., and Shors, T. J. (1999a). Learning enhances adult neurogenesis in the hippocampal formation. r ?var Neurosci. 2, 260-265. Gould, E., Reeves, A. J., Graziano, M. S. A.. and Gross. C. G. (I999b). Neurogenesis in the neocortex of adult primates. Science 286, 548-552. Greenwood. A. L., Turner. E. E., and Anderson, D. J. (1999). Identification of dividing, determined senso W neuron precursors in the mammalian neural crest. Development (Cambridge, UK) 126, 3545-3559.

250 Gritti, A., Parati, E. A., Cova, L., Frolichsthat, R, Galli, R.. Wanke. E.. Faravelli, L., Morassutti, D. J., Roisen, F.. Nickel, D. D.. and Vescovi. A. L. (1996). Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J. Neurosci. 16, 1091-1 I00. Gross, R. E.. Mehler, M. F., Mabie. R C., Zang. Z., Santschi, L.. and Kessler. J. A. (1996). Bone Morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells. Neuron 17. 595-606. Guillemot, F., Lo, L.-C., Johnson, J. E., Auerbach. A., Anderson. D. J., and Joyner, A. L. (1993). Mammalian achaete-scute homolog-1 is required for the early development of olfactor)' and autonomic neurons. Cell (Cambridge. Mass. ) 75, 463-476. Gussoni, E.. Soneoka, Y.. Strickland. C. D.. Buznev. E. A.. Khan. M. K., Flint, A. F.. Kunkel, L. M.. and Mulligan. R. C. (1999). Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature ('London) 401, 390-394. Hagedorn, L., Suter, U., and Sommer. L. (1999). PO and PMFm,2 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-/3 family' members. Development (Cambridee. UK) 126, 3781-3794. Hardy, R. R.. and Hayakawa, K. (1994). CD5 B cells, a fetal B cell lineage. Adv. lmmunol. 55, 297-339. Harrison, D. E., and Lerner, C. R ( 1991 ). Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil. Blood 78, 1237-1240. Henion. R D.. and Weston, J. A. (1997). Timing and pattern of cell fate restrictions in the neural crest lineage. Development CCambridge. UK) 12,4o 4351-4359. Hentges, K., Thompson, K., and Peterson. A. (1999). The fiat-top gene is required for the expansion and regionalization of the telencephalic primordium. Development (Cambridge, UK) 126, 1601 - 1609. Hirsch, M. R., Tiveron, M. C., Guillemot, F., Brunet, J. F., and Goridis, C. (1998). Control of noradrenergic differentiation and Phox2a expression by' MASH1 in the central and peripheral nervous system. Development (Cambridge. UK) 125, 599-608. Ikuta, K,, Kina, T., MacNeil, I., Uchida, N., Peault, B., Chien, Y. H., and Weissman, I. L. (1990). A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell (Cambridge. Mass.) 62, 863-874. Jiang, X., Rowitch. D. H., Soriano. R, McMahon. A. R. and Sucov. H. M. (2000). Fate of the mammalian cardiac neural crest. Development (Cambridge. UK) 127, 1607-1616. Johansson, C. B., Momma, S., Clarke, D. L.. Risling, M., Lendahl, U., and Frisen, J. (1999). Identification of a neural stem cell in the adult mammalian central nervous system. Cell (Cambridge. Mass.) 96, 25-34. Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M.. and McKay, R. D. G. (1996). Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev. 10, 3129-3140. Johnson, J. E., Birren, S. J., and Anderson, D. J. (1990). Two rat homologues of Drosophila achaete-scute specifically expressed in neuronal precursors. Nature (London) 346, 858 - 861. Johnson, J. E., Birren, S. J., Saito, T., and Anderson, D. J. (1992). DNA binding and transcriptional regulatory, activity of mammalian achaetescute homologous (MASH) proteins revealed by interaction with a muscle-specific enhancer. Proc. Natl. Acad. Sci. U.S.A. 89, 3596-3600. Kalyani, A. J., Hobson, K.. and Rao, M. S. (1997). Neuroepithelial stem cells from the embr-yonic spinal cord: Isolation, characterization, and clonal analysis. Dev. Biol. 186, 202-223. Kalyani, A. J., Piper, D., Mujtaba. T.. Lucero, M. T., and Rao, M. S. (1998). Spinal cord neuronal precursors generate multiple neuronal phenotypes in culture. J. Neurosci. 18, 7856-7868. Kantor, A. B., Stall. A. M.. Adams, S., Herzenberg. L. A., and Herzenberg, L. A. (1992). Differential development of progenitor activity for three B-cell lineages. Proc. Natl. Acad. Sci. U;S.A. 89, 3320-3324.

Ii Lineage Specification and Differentiation Kaplan, M. S., and Hinds, J. Wi (1977). Neurogenesis in the adult rat: Electron microscopic analysis of light radioautographs. Science 197, I 0 9 2 1094. Kempermann. G., Kuhn, H. G.. and Gage. F. H. (I 997). More hippocampal neurons in adult mice living in an enriched environment. Nature (London ) 386, 493 - 495. Kempermann. G.. Brandon. E. R. and Gage. F. H. (19981). Environmental stimulation of 129/SvJ mice causes increased cell proliferation and neurogenesis in the adult dentate gyms. Curt. Biol. 8, 939-942. Kondo. M.. Weissman, I. L., and Akashi, K. (1997). Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell (Cambridge. Mass. ) 91, 661-672. Kondo. T., and Raft, M. (2000). Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 289, I 7 5 4 1757. Kopen. G. C.. Prockop, D. J., and Phinney, D. G. (1999). Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc. Natl. A cad. Sci. U.S.A. 96, 10711 - 10716. Lagasse, E.. Connors, H.. AI-Dhalimy, M., Reitsma, M.. Dohse, M., Osborne, L.. Wang. X.. Finegold. M.. Weissman, I. L.. and Grompe, M. (2000). Purified hematopoietic stem cells can differentiate to hepatocvtes in vivo. Nat. Med. 6, I229-1234. Le Douarin. N. M. (1982). "'The Neural Crest." Cambridge University Press. Cambridge. UK. Le Douarin. N. M. (1986). Cell line segregation during peripheral nervous system ontogeny. Science 231, 1515-1522. Le Douarin. N. M.. and Dupin. E. (1992). Cell lineage analysis in neural crest ontogeny. J. Neurobiol. 24, 146-16 i. Lemke. G. E., and Brockes. J. P. (I984). identification and purification of glial growth factor. 3'. Neurosci. 4, 75-83. Li, P.. Yang, X., Wasser. M.. Cai, Y.. and Chia, W. (1997). Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions. Cell (Cambridge, Mass.) 90, 437-447. Li. W:. Cogswell. C. A.. and LoTurco, J. J. (1998). Neuronal differentiation of precursors in the neocortical ventricular zone is triggered by BMR J. Neurosci. 18, 8853-8862. Lo, L.. and Anderson. D. (1995). Postmigrato~' neural crest cells expressing c-RET display restricted developmental and proliferative capacities. Neuron 15, 527-539. Lo. L., Sommer. L.. and Anderson. D. J. (1997). MASH1 maintains competence for BMP2-induced neuronal differentiation in post-migrator3, neural crest cells. Curr. Biol. 7, 440-450. Lo, L., Tiveron. M.-C., and Anderson, D. J. (1998). MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identiDr. Development (Cambridge. UK) 125, 609-620. Lois, C.. and Alvarez-Buylla. A. (1993). Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci. US.A. 90, 2074-2077. Lois, C., and Alvarez-Buylla, A. (1994). Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145-I 148. Lowenstein, D. H.. and Parent, J. M. (1999). Brain, heal thyself. Science 283, 1126-1127. Luskin, M. B., Pearlman. A. L., and Sanes. J. R. (1988). Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus. Neuron 1,635-647. Lyons, K. M.. Hogan, B. L. M., and Robertson. E. J. (1995), Colocalization of BMP 7 and BMP 2 I~NAs suggests that these factors cooperatively mediate tissue interactions during murine development. Mech. Dev. 50, 71-83. Mabie. P. C., Mehler, M. F., and Kessler, J. A. (1999). Multiple roles of bone morphogenetic protein signaling in the regulation of cortical cell number and phenovype. J. Neurosci. 19, 7077-7088. Marchionni, M. A., Goodearl, A. D. J., Chen, M. S., Bermingham-

12 Stem Cells of the Nervous System McDonogh. O.. Kirk. C.. Hendricks. M.. Danehy. E. Misumi. D.. Sudhalter. J.. Kobayashi. K.. Wroblewski. D.. Lynch. C.. Baldassare. M.. Hiles. I.. Davis. J. B.. Hsuan. J. J.. Totty. N. F.. Otsu. M.. McBurney. R. N.. Waterfield. M. D.. Stroobant. R. and Gwynne. D. (1993). Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous s_x'stem. Nature (London) 362, 312 -318. Marina. N. M.. Etcubanas, E.. Parham. D. M.. Bowman. L. C.. and Green. A. (1989). Peripheral primitive neuroectodermal tumor (peripheral neuroepithelioma)in children. Cancer 64, I952-1960. Matsui. Y.. Zsebo. K.. and Hogan. B. L. M. (1992). Derivation of pluripotential embD;onic stem cells from murine primordial germ cells in culture. Cell (Cambridge. Mass. ) 70, 841 - 847. Mayer-Proschel. M.. Kalyani. A. J.. Mujtaba. T.. and Rao. M. S. (t9971). Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron 19, 773-785. McKay. R. (1997). Stem cells in the central nervous system. Science 276, 66-7 I. Mehler. M. F., Mabie. R C.. Zhang. D.. and Kessler. J. A. (1997). Bone morphogenetic proteins in the nervous system. Trends Neurosci. 20, 309-317. Mello. C. C.. Schubert. C., Draper. B.. Zhang. '~r Lobel. R.. and Priess. J. R. (I996). The PIE-1 protein and germline specification in C. etegans embryos. Nature (London) 382, 710-712. Meyer. D., and Birchmeier. C. (1994). Distinct isoforms of neuregulin are expressed in mesenchymal and neuronal cells during mouse development. Proc. Natl. Acad. Sci. U.S.A. 91, 1064-1068. Meyer, R., and Birchmeier. C. (1995). Multiple essential functions of neuregulin in development. Nature (London) 378, 386-390. Morrison. S. J.. and Weissman. I. L. (1994). The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1, 661-673. Morrison. S. J.. Hemmati. H. D.. Wandycz. A. M.. and Weissman. I. L. (1995a). The purification and characterization of fetal liver hematopoietic stem cells. Proc. Natl. Acad. Sci. U S.A. 92, 10302-10306. Morrison. S. J.. Uchida. N.. and Weissman. I. L. (1995b). The biology of hematopoietic stem cells. Annu. Rev. Cell Dev. Biol. 11, 35-71. Morrison. S. J.. Wandycz. A. M., Akashi, K.. Globerson. A., and Weissman. I. L. (1996). The aging of hematopoietic stem cells. Nar Med. 2, 10111016. Morrison. S. J., Shah. N. M.. and Anderson, D. J. (1997a). Regulatory mechanisms in stem cell biology. Cell (Cambridge. Mass.) 88, 287298. Morrison. S. J.. Wandvcz. A. M., Hemmati. H. D.. Wright, D. E.. and Weissman. I. L. (1997b). Identification of a lineage of multipotent hematopoieric progenitors. Development (Cambridge. UK) 1924, I929-1939. Morrison, S. J., Wright. D.. and Weissman. I. L. (1997c). Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization. Proc. Natl. Acad. Sci. U.S.A. 94, 1908-1913. Morrison, S. J.. White, R M.. Zock. C.. and Anderson. D. J. (1999). Prospective identification, isolation by flow cytometr-y', and in vivo selfrenewal of multipotent mammalian neural crest stem cells. Cell (Cambridge. Mass.) 96, 737-749. Morrison, S. J., Csete, M.. Groves, A. K., Melega. W:. Wold. B., and Anderson, D. J. (2000). Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J. Neurosci. 20, 7370-7376. Morrissey. T. K.. Levi. A. D.. Nuijens. A., Sliwkowski. M. X., and Bunge. R. R (1995). Axon-induced mitogenesis of human Schwann cells involves heregulin and p185erbB2. Proc. ~%~tl.Acad. Sci. U.S.A. 92, 143 I 1435. Morshead. C. M.. Reynolds. B. A.. Craig. C. G.. McBurney, M. W.. Staines, W. A.. Morassutti, D., Weiss. S.. and ,,'an der Kooy, D. (1994). Neural stem cells in the adult mammalian forebrain: A relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071 - 1082. Morshead. C. M.. Craig. C. G.. and van der Kooy, D. (1998). In vivo clonal

251 analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain. Development (Cambridge, UK) 125, 2251-2261. Murphy. R, Topilko. R. Schneider-Maunour?, S., Seitanidou, T.. Baron van Evercooren, A.. and Charnay, R (1996). The regulation of Krox-20 expression reveals important steps in the control of peripheral giial cell development. Development (Cambridge. UK) 122, 2847-2857. Na. E., McCarthy. M.. Neyt, C.. Lai. E., and Fishell. G. (1998). Telencephalic progenitors maintain anteroposterior identities cell autonomously. Curt Biol. 8, 987-990. Olanow. C. W.. Freeman, T. B.. and Kordower. J. H. (1997). Neural transplantation as a therapy for Parkinson's disease. ,In "The Basal Ganglia and New Surgical Approaches for Parkinson's Disease" (J. A. Obeso, M. R. DeLong. C. Ohye. and C. D. Marsden. eds.), pp. 249-269. Lippincott-Raven. Philadelphia. Osawa. M.. Hanada. K.-I.. Hamada. H., and Nakauchi. H. (1996). Longterm lymphohematopoietic reconstitution by a single CD34-1ow/negative hematopoietic stem cell. Science 273, 242-245. Palmer. T. D.. Takahashi, J.. and Gage, F. H. (I997). The adult rat hippocampus contans primordial neural stem cells. Mot. Celt. Neurosci. 8, 389-404. Palmer. T. D.. Markakis. E. A.. Willhoite. A. R.. Safar, E. and Gage. F. H. (1999). Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J. Neurosci. 19, 8487- 8497. Parent. J. M.. Yu. T. W., Leibowitz, R. T.. Geschwind. D. HI.. Sloviter. R. S.. and Lowenstein. D. H. (1997). Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J. Neurosci. 17, 3727-3738. Parent. J. M., Janumpalli, S., McNamara, J. O., and Lowenstein. D. H. (1998). Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci. Letr 247, 9-12. Peterson. B. E.. Bowen. W. C., Patrene, K. D., Mars. W. M., Sullivan. A. K.. Murase. N.. Boggs. S. S.. Greenberger. J. S.. and Goff, J. R (1999). Bone marrow as a potential source of hepatic oval cells. Science 284, 11681170. Pham. T. D.. Gershon. M. D.. and Rothman. T. R (1991). Time of origin of neurons in the murine enteric nervous system: Sequence in relation to phenotype. J. Comp. Neurol. 314, 789-798. Rakic. R (1985). Limits of neurogenesis in primates. Science 227, 10541056. Ramdn y Cajlil, S.. and May. R. T. (I959). "'Degeneration and Regeneration of the Nervous System," Vol. 2. Hafner. New York. Rao, M. S., Noble. M., and Mayer-Proschel. M. (1998). A tripotential glial precursor cell is present in the developing spinal cord. Proc. Natl. Acad. Sci. U.S.A. 95, 3996-400I. Rebel. V. I.. Miller. C. L.. Eaves, C. J., and Lansdorp. P. M. (1996). The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their adult bone marrow counterparts. Blood 87, 35003507. Reddy, P. H., Williams. M., and Tagle, D. A. (1999). Recent advances in understanding the pathogenesis of Huntington's disease. Trends Neurosci. 22, 248-255. Reid, C. B., Liang. I.. and Walsh~ C. (I 995). Systematic widespread clonal organization in cerebral cortex. Neuron 15, 299-310. Reissman. E., Ernsberger, U.. Francis-West, P. H.. Rueger, D.. Brickell, P. D., and Rohrer, H. (1996). Involvement of bone morphogenetic protein-4 and bone morphogenetic protein-7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons. Development (Cambridge, UK) 122, 2079-2088. Reynolds, B. A., and Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707-1710. Richardson, M. K.. and Sieber-Blum. M. (1993). Pluripotent neural crest cells in the developing skin of the quail embryo. Dev. Biol. 157, 348358.


252 Riethmacher. D.. Sonnerberg-Riethmacher. E., Brinkmann. "v:, Yamaai, T.. Lewin, G. R.. and Birchmeier, C. (1997). Severe neuropathies in mice with targeted mutations in the erbB3 receptor. Nature (London) 389, 725-730. Rosselli-Austin. L.. and Williams. J. (1989). Enriched neonatal odor exposure leads to increased numbers of olfactory' bulb mitral and granule cells. Dev. Brain Res. 51, 135-t37. Rutkowski. J. L., Kirk. C. J.. Lerner. M. A.. and Tennekoon. G. I. (1995). Purification and expansion of human Sch,xann cells in vitro. Nature Med. 1, 80-83. Sauvageau. G.. Thorsteinsdottir, U.. Eaves, C. J.. Lawrence. H. J.. Largman. C.. Lansdorp. R M.. and Humphries, R. K. (I 995). Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev. 9. 1753-1765. Schneider. C.. Wicht. H.. Enderich. J.. Wegner. M.. and Rohrer. H. (1999). Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons. Neuron 2,4, 861-870. Schoenherr. C. J.. and Anderson. D. J. (1995). The Neuron-Restrictive Silencer Factor (NRSF): A coordinate repressor of multiple neuronspecific genes. Science 267, 1360-1363. Schoenherr. C. J., Paquette. A. J.. and Anderson. D. J. (1996). Identification of potential target genes for the neuron-restrictive silencer factor. Proc. Natl. Acad. Sci. U.S.A. 93, 9881-9886. Scott. B. W.. Wang. S.. Burnham. W. M.. Boni. U. D.. and Wojtowicz. J. M. (1998). Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci. Left. 248, 73-76. Sell, S., and Pierce, G. B. (1994). Maturation arrest of stem cell differentiation is a common pathway for the cellular origin of teratocarcinomas and epithelial cancers. Lab. Investig. 70, 6 - 2 2 . Serbedzija, G. N.. Bronner-Fraser, M., and Fraser, S. E. (1994). Developmental potential of trunk neural crest cells in the mouse. Development (Cambridge. UK) 120, 1709-1718. Seydoux, G.. Mello. C. C., Pettitt, J.. Wood. W. B.. Priess, J. R.. and Fire, A, (1996). Repression of gene expression in the embryonic germ lineage of C. elegans. Nature cLondon) 382, 713-716. Shah, N. M.. Marchionni. M. A.. Isaacs, I., Stroobant. R W.. and Anderson. D. J. (1994). Glial growth factor restricts mammalian neural crest stem cells to a glial fate. Cell (Cambridge. Mass.) 77, 349-360. Shah, N. M., Groves, A.. and Anderson. D. J. (1996). Alternative neural crest cell fates are instructively" promoted by TGF/3 superfamily members. Cell(Cambridge, Mass.) 85, 331-343. Sieber-Blum, M. (1989). Commitment of neural crest cells to the sensory,' neuron lineaoe Science 243, 1608-1610. Sieber-Blum, M., and Cohen, A. (1980). Clonal analysis of quail neural crest cells: They are pluripotent and differentiate in vitro in the absence of non-neural crest cells. Dev. Biol. 80, 96-106. Smith, W. C., and Harland, R. M. (1992). Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell (Cambridge. Mass. ) 70, 8 2 9 - 840. Sommer, L., Shah, N., Rao. M.. and Anderson, D. J. (1995). The cellular function of MASH I in autonomic neurogenesis. Neuron 15, 12451258. Spangrude, G. J., Heimfeld, S., and Weissman. I. L. (I988). Purification and characterization of mouse hematopoietic stem cells. Science 241, 58-62. Spangrude. G. J., Brooks, D. M., and Yumas. D. B. (1995). Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: In vivo expansion of stem cell phenoty, pe but not function. Blood 85, 1006 - 1016. Stemple. D. L.. and Anderson, D. J. (1992). Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell (Cambridge. Mass.) 71,973-985. -

Suhonen, J. A.. Peterson, D. A., Ray. J.. and Gage, F. H. (11996). Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature (London) 383, 624-627. Takahashi. M.. Palmer. T. D.. Takahashi. J.. and Gage. E H. (1998). Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Mol. Cell. ?@urosci. 12, 340-348. Temple, S., and Alvarez-Buylla~ A. (1999). Stem cells in the adult mammalian central nervous system. Curt. Opin. ?4eurobiol. 9, 135-141. Tenenhaus. C.. Schubert. C.. and Seydoux. G. (1998). Genetic requirements for PIE-1 localization and inhibition of gene expression in the embryonic germ lineage of Caenorhabditis elegans. Dev. Biol. 200, 212-224. Thiele. C. J. (1991). Biology' of pediatric peripheral neuroectodermal tumors. Cancer Metastasis Rev. 10, 311-3 i 9. Topilko, R. Levi. G., Merlo. G., Mantero. S., Desmarquet. C.. Mancardi, G., and Charnav. R (1997). Differential regulation of the zinc finger genes Krox-20 and Krox-24 (ERr-l) suggests antagonistic roles in Schwann Cells. J. Neurosci. Res. 50, 702-712. Uchida, N.. Aguila. H. L.. Fleming. W H.. Jerabek. L., and Weissman. I. L. (1994). Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1 loLin-Sca-1 + hematopoieric stem cells. Blood 83, 3758-3779. Vicario-Abej6n. C.. Johe. K. K.. Hazel, T. G.. Collazo. D.. and McKay. R. D. G. (1995). Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons. Neuron 15, 105114. Wakamatsu. Y.. Mochii, M.. Vogel. K. S.. and Weston. J. A. (t998). Avian neural crest-derived neurogenic precursors undergo apoptosis on the lateral migration pathway. Development (Cambridge. UK) 125, 42054213. Walsh. C., and Cepko, C. L. (1993). Clonal dispersion in proliferative layers of developing cerebral cortex. Nature (London) 362, 632-635. Weiss. S.. Dunne, C.. Hewson, J.. Wohl. C.. Wheatley, M.. Peterson. A. C., and Reynolds. B. A. (1996). Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J. Neurosci. 16, 7599-7609. White. P. A.. and Anderson, D. J. (1999). In vivo transplantation of mammalian neural crest cells into chick hosts reveals a new autonomic sublineage restriction. Development (Cambridge, UK) 126, 4351-4363. Williams. B. E. and Price, J. (1995). Evidence for multiple precursor cell types in the embQonic rat cerebral cortex..Neuron 14, 1I81-1188. Williams. B. E, Park. J. K., Alberta. J. A., Muhlebach, S. G., Hwang, G. Y., Roberts. T. M.. and Stiles. C. D. (I997). A PDGF-regulated immediate early response initiates neuronal differentiation in ventricutar zone progenitor cells. Neuron 18, 553-562. Wilson Berry. L.. Westlund. B.. and Schedl, T. (I997). Germ-line tumor formation caused by activation of glp-I, a Caenorhabditis elegans member of the Notch family of receptors. Development (Cambridge. UK) 124, 925-936. Winkler, J.. Thai. L. J.. Gage, E H.. and Fisher, L. J. (1998). Cholinergic strategies for Alzheimer's disease. J. Mol. Med. 76, 555-567. Xuan, S. H., Baptista. C. A., Balas, G.. Tao, W. F., Soares, V. C., and Lai, E. (1995). Winged helix transcription factor BF- 1 is essential for the development of the cerebral hemispheres..Neuron 14, 1141-1152. Yokoi, M., Mori, K., and Nakanishi, S. (I995). Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory, bulb. Proc. Natl. Acad. Sci. U.S.A. 92, 3371-3376. Zhu. A. J.. and Watt. F. M. (1999)./3-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development (Cambridge, UK) 126, 2285-2298. Zimmerman. L. B., Dejesus-Escobar, J. M.. and Harland, R. M. (1996). The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein-4. Cell (Cambridge, Mass.) 86, 599-606.

Cellular and Molecular Mechanisms Regulating Skeletal Muscle Development Atsushi Asakura a n d Michael A. Rudnicki Program in Molecular Genetics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada KIH 8L6

!. Introduction

ies that elucidated mechanisms regulating myogenesis. Consequently, the study of vertebrate myogenesis has provided a powerful biological system to investigate the molecular regulation of the developmental program that controls the genesis, ~owth, migration, and differentiation of an embryonic cell lineage. Our knowledge of the molecular mechanisms that regulate skeletal myogenesis was dramatically accelerated about a decade ago following the discovery of the MyoD family of transcription factors, termed the myogenic regulatory factors (MRFs). The MRFs comprise a group of skeletal-musclespecific bHLH (basic helix-loop-helix) transcription factors, consisting of MyoD, Myf5, myogenin, and MRF4. The MRFs activate transcription of muscle-specific genes through binding of DNA motifs called E-boxes. Forced expression of the MRFs in various cell types in vitro and in vivo induces muscle differentiation. The restricted expression of the MRFs together with their ability to dominantly induce differentiation led to the suggestion that the MyoD family members are master regulators of the muscle developmental program. Subsequently, gene targeting in mice allowed for genetic dissection of these regulatory pathways and clearly defined the roles played by the MyoD family of transcription factors in myogenesis.

!1. Embryonic Origin of Skeletal Muscle i!1, MyoD Family of Myogenic Regulatory Factors IV. Muscle-Specific Transcriptional Regulation V. Inductive Mechanisms of Myogenesis VI. Specification of Muscle Fiber Types Vii. Muscle Regeneration VIII, Conclusion References v

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I. I n t r o d u c t i o n The study of skeletal muscle differentiation has been intensive since the 1960s because of readily discerned criteria for differentiation (e.g., multinucleated syncytium) and the availability of biochemical markers such as actin and myosin. In addition, the early isolation of cell lines derived from skeletal muscle was essential for cellular and molecular studMouse Development

253

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254 During vertebrate development, the mesodermal progenitors of skeletal muscle originate from precursor cells that arise from the somites and prechordal mesoderm. The specification of muscle progenitor cells from the somites is regulated by signals from the neural tube. notochord, and other surrounding tissues that provide both positive and negative cues (see Section V). Indeed. the embQologicaI experiments currently under way to investigate the specification of skeletal muscle arguably represent the state of the art in vertebrate developmental biology. The purpose of this chapter is to provide an overview of the molecular mechanisms that regulate myogenesis in the mouse. Topics discussed include the embuonic origin of skeletal muscle, the MyoD family' of myogenic regulatou factors, muscle-specific transcriptional regulation, inductive mechanisms regulating embryonic myogenesis, fiber-type specification, and the regulation of adult muscle regeneration.

II. Embryonic Origin of Skeletal Muscle A. The Terminology of Myogenesis Defined terms are used for particular muscle cell types during myogenesis (Stockdale. 1992: Hauschka. 1996: Bischoff. 1996). During vertebrate development, muscle progenitor cells, which still possess developmental plasticity, derive from the mesodermal lineage. Muscle progenitor cells differentiate into myoblasts that express the MRFs. MyoD (Myodl) and MyfS. Proliferating myoblasts withdraw from the cell cycle to become terminally differentiated mvocvtes that express muscle-specific genes such as myogenin (Myog). MRF4/Herculin (M3f6), myosin heavy chain (MyHC), and muscle creatine kinase (MCK, Clonm) genes. These mononucleated myocytes fuse with each other to form multinucleated mvotubes that as mature muscle fibers initiate muscle contraction. Skeletal muscle differentiation occurs in several phases. First formed at about 13 days postcoitus (dpc) in the mouse are the primao" fibers, followed by the secondar 3, fibers beginning at about 16 dpc, which develop parallel to and within the same basal lamina of the primary fibers. Elaboration of muscle fiber subtypes begins late in development and extends into the neonatal period. Fasttwitch (t3pe H) glycolytic and slow-twitch (t3pe 1) oxidative muscle fibers differ in their functional properties and in the expression of distinct isoforms of contractile proteins. Last. satellite cells, the quiescent stem cell of adult muscle, are activated (initiate proliferation) in response to stress to produce proliferating myogenic precursor cells (mpc) that mediate the postnatal growth and regeneration of muscle. Muscle-derived stem cells represent the recently described pluripotent stem cells that reside in adult skeletal muscle.

B. Mesodermal Progenitor Cells Are the Origin of Skeletal Muscle Cells During vertebrate embryogenesis, the musculature of the trunk and limbs is derived from progenitors that originate from the somites, epithelial spheres of paraxial mesoderm that form in a rostrocaudal progression on either side of the neural tube (Christ and Ordahl, 1995). Craniofacial muscles, including muscle masses of the occulo motors and branchial arches, are derived from cephalic paraxial mesoderm (somitomeres), prechordal mesoderm, and occipital sornites (a few of the most rostral somites) (Noden, 1991 Trainor et al., 1994). The seven pairs of somitomeres derived from paraxial head mesoderm give rise to the voluntary craniofacial muscles and. together with prechordal mesoderm and the occipital somites, contribute progenitors to the branchial arches to form the muscles of the jaws and neck. Trunk and limb muscle development is relatively welt understood compared to craniofacial myogenesis. During early embryogenesis, trunk mesoderm is separated into four distinct regions: axial, paraxial, intermediate, and lateral mesoderm. The axial mesoderm gives rise to the notochord located beneath the neural plate. The lateral mesoderm becomes the lateral plate that gives rise to cardiac and smooth muscle. The presomitic mesoderm (or unsegmented mesoderm) derived from the paraxial mesoderm undergoes segmentation to form epithelial spheres or somites on either side of the neural tube in a rostrocaudaI direction beginning at 7.5 dpc in the mouse (see Chapter 7).

C. Trunk and Limb Muscles Originate from Somite Cell lineage experiments using chick-quail chimeras and transplantation of rotated somites reveal that cells in newly formed somites retain the potential for all somitic lineages. Therefore, specification of somitic lineages occurs following the exposure of somitic cells to patterning signals derived from the surrounding tissues (Figs. 1A and 1B and see Section V). The ventral part of the newly formed somites deepithelializes to form mesenchymal cells that give rise to the sclerotome. The cells in the sclerotome subsequently migrate around the neural tube as well as laterally to form the cartilage of the vertebral column and ribs (Fig. 1C). Between 8.0 dpc and 8.5 dpc in the mouse, the dorsal part of the most rostral somite (see Plate 15b-c, d in Kaufman, 1992), consisting in part of proliferating cells, maintains an epithelial structure and is called the dermomyotome. Cells in the dorsomedial region of dermomyotome (dorsomedial lip; DML), residing close to neural tube, gadually extend laterally beneath the entire width of the dermomyotome (Figs. 1A-D). By' 8.5 dpc in the mouse, DML-derived cells in the most

13 Regulation of Skeletal Muscle Development

F i g u r e ] Schematicrepresentation of mouse embryo at thoracic level somites. Somite (s) formation followed by dermomyotome(din) and myotome (mr) formations proceed in a rostral (a: anterior) to caudal (p: posterior) sequence. By 8.0 dpc. the ventral part of the most rostral somite deepit_helializesto form mesenchymalcells, which give rise to the sclerotome (st) (A-C). By 8.5 dpc in the mouse, the dorsal part of the rostraI somite maintains a proliferating epithelial structure called the dermomyotome(AD). Cells in the dorsomedial region of dermomyotome (dorsomedial lip: DML) withdraw from cell proliferation and longitudinallyelongatebeneath the dorsomedial part of dermomyotometo form the dorsal myotome(epaxial myotome; C-E). At 9.5 dpc. cells in the ventrolateraIregion of dermomyotome [ventrolateral lip (VLL) or hypaxial somite bud] withdraw from cell proliferation and longitudinally elongate beneath to ventral part of the dermomyotome to form the ventral myotome (hypaxial myotome: D-F). Eventually, both epaxial and hypaxial myotome extend continuously beneath the dermomyotome to form a planar myotome structure (F). By 11.0 dpc. the center of the dermomyotomehas dissociatedinto dermiswhile the epithelial DML and VLL still remain and continue to provide myotomal cells (G). At 10.0 dpc. epithelial cells in the VLL at the forelimb bud level delaminate and ventrally migrateout from the dermomyotomeinto the emb~'o (migrator, muscle progenitorcells: F and G). nt, neural tube: d, dorsal: v, ventral: e. ectodermallayer: nc. notochord.

rostral somite (see Plate 15b-c, d and Plate 18b-e, i in Kaufman. 1992), withdraw from cell proliferation and longitudinally elongate beneath the dorsomedial part of der-

255 momyotome to form the first terminally differentiated myocytes in a structure called the myotome (Fig. I D; Williams and OrdahI. 1997; Christ and Ordahl, 1995). The developmental processes of the cells in the ventrolateral region of dermomyotome [ventrolateral lip (VLL) or somite bud] are the mirror image of those of the dorsomedial part (Christ and Orda_hl. 1995; Brand-Saberi et ai., 1996a). By 9.75 dpc in mouse. VLL-derived cells in the interlimb somite (see Plate 2 0 c - d , e, f, g. h in Kaufman, 1992) begin to withdraw from the cell cycle and longitudinally elongate beneath to the ventrolateral part of the dermomyotome to form the ventral myotome (Figs. 1D and 1E). The compartment consisting of postmitotic cells beneath the dorsomedial portion of the dermomyotome is called the epaxial myotome and gives rise to the epaxial musculature or muscles of the deep back. The compartment residing beneath the ventrolaterat part of the dermomyotome is called the nonmigratory" hypaxial myotome and gives rise to lateral skeletal muscles in the trunk such as intercostal muscle and body-wall muscle (Figs. 1E and 1F). Eventually, both epaxial and hypaxial rnyotomes at the interlimb bud level extend continuously beneath the dermomyotome to form a planar myotomaI structure (Figs. 1F and 1G and see Plate 19b-i, Plate 19c-a, and Plate 2 0 c - e , f. g, h in Kaufman, 1992) that expresses muscle structural proteins at the center of myotome. By 12.0 dpc, the central portion of myotomes at trunk level form the first multinucleated myotubes (see Plate 26c and 26d in Kaufman, 1992). Experimental analysis of avian somitogenesis has revealed unexpected insights into the mechanism of myotome formation (Fig. 2, Denetclaw et aL, 1997, 2000; Kahane et aI., 1998a, b; Cinnamon et al., 1999: Kalcheim et al., 1999). The first differentiated myocyctes, termed muscle pioneers, are derived from mitotic cells in the DML that withdraw from the cell cycle as they migrate beneath the dermomyotome and translocate to the rostral edge (Figs. 2A and 2B). Subsequently, pioneer cells longitudinally elongate across the rostral to the caudal lip of the dermomyotome to form the primary myotome (Fig. 2C; first wave of myotome development) (Kahane et at.. 1998a). At this stage, the nuclei of pioneer myofibers become restricted to the center of the myotome (Fig. 2C). Next, mitotic cells in all four lips (DML, VLL, rostral lip, and caudal lip) of the dermomyotome cease proliferation, migrate beneath the dermomyotome, and translocate into the rostral and caudal edges (Fig. 2D). These migratory cells longitudinally elongate toward the rostral and caudal lips of the dermomyotome between pioneer muscle fibers to form the secondary myotome and constitute the second wave of myotome development (Figs. 2E and 2F; Ka~hane et al., 1998b). Subsequently, secondary myotome formation continues ventrolaterally to form the hypaxiaI myotome and medially to increase the thickness of the myotome (Fig. 1F). Following the disap-

ii Lineage Specification and Differentiation

256

Figure 2

Schematic model representing various stages of avian myotome development. Each view represents the myotomal plane from which the dermomyotome has been lifted apart (indicated by thick arrow in B). The first myotomal cells, termed muscle pioneers, are derived from cells (muscle progenitor cells) in the DML that withdraw from the cell cycle, migrate beneath the dermomyotome (A), and translocate to the rostral edge (indicated by thick arrow in B) of the somite. Subsequently, pioneer cells elongate across the distance spanning from the rostral to the caudal lip of the dermomyotome (indicated by dashed arrows in C) to form the primary, myotome (first wave of myotorne development). The second wave of myotome is formed by the migration of cells from all four edges of the dermomyotome to the perimeter of the pioneer myotome (D). These cells extend longitudinally in between the pioneer muscle fibers and form the secondarv mvotome (E, F) (Kahane et al.. 1998 a,b: Cinnamon et al.. 1999).

pearance of the dermomyotomal lips, a population of mitotically active precursors within the myotome continues to contribute to myotome formation in a process termed third wave of myotome formation. Much of the skeletal muscle of the body, however, is derived from migratory progenitors that arise as somitically derived multipotential cells. Epithelial cells in the VLL delaminate and ventrally migrate out from the dermomyotome into the embryo (Figs. 1F and 1G and see Fig. 5D in a later section). Such migratory muscle progenitor cells enter the ventral region of trunk and limb buds and continue proliferation. Migratory muscle progenitor cells coalesce as muscle anlagen prior to withdrawal from the cell cycle and terminal

differentiation. Migratory muscle progenitors give rise to the migratory hypaxial muscles" the pectoralis, abdominal, and diaphragm muscles of the trunk, and all limb muscles (Ordahl and Le Douarin, 1992; Ordahl and Williams, 1998).

!11. MyoD Family of Myogenic Regulatory Factors A. The MRFs Are bHLH Transcription Factors In a series of seminal experiments, Weintraub and his colleagues cloned a bHLH transcription factor called MyoD

13 Regulation of Skeletal Muscle Development that appeared to function as a master regulator of muscle differentiation (Davis et aI., 1987). In vertebrates, four highly related genes, MyoD, M)fS, myogenin, and MRF4, belong to this class of genes also known as the myogenic regulatory factors (Fig. 3). Forced expression of the M R F s in various cell lines in vitro and in different tissue types in vivo dominantly induces skeletal muscle differentiation (Weintraub et al., 1991a). The bHLH region of the M R F s is about 70 amino acid residues and is highly conserved with more than 80% identity in amino acid sequence level. The basic region contains many positively charged amino acid residues and is responsible for DNA binding (Davis e t al., 1990). The HLH region is responsible for dimerization with a distinct bHLH class of

257 E proteins, E I 2 / E 4 7 (Tcfe2a), ITF-2 (Tcf4), and M E 1 / H E B (Tcfl2) (Murre et al., 1989b, Lassar et aL, 1991). In general, bHLH protein dimers bind the D N A motif called an E-box with the core consensus sequence C A N N T G (Murre et aL, 1989a). However, heterodimers of M R F s / E proteins preferentially bind to the sequence C A ( G / C ) ( G / C ) T G (Blackwell and Weintraub, 1990). The M R F s efficiently dimerize with E proteins and these heterodimers activate muscle-specific transcription by binding E-boxes present in the regulatory regions of most muscle-specific genes (Hauschka, I996; Molkentin and Olson, 1996). Whereas the bHLH region is highly conserved, the conservation of regions outside the bHLH is relatively low among the MRFs, except for some regionally conserved

Figure 3 Two phylogenetic groups of the MRFs. (A) Vertebrate MRF genes were derived from a common ancestor by gene duplication. Invertebrates, Caenorhabditis elegans. Drosophila, sea urchin, and ascidians have but one MRF gene. Phylogenetic analysis suggests that the duplications occurred before or early during the radiation of the vertebrate. Amino acid sequences of Myf5 and MyoD are more similar to one another (53%) than either is to myogenin (40% and 38%, respectively) orto MtLF4 (40% and 43%, respectively). Similarly, myogenin and MRF4 are more related to one another (53%) than to either Myf5 or MyoD. (B)Myf5 and MRF4 arose from ancestral MRF gene by gene duplication at the same locus: myogenin and ~yoD arose from MRF4 and .~f5, respectively, after a gene duplication event to a second c~'-omosome, and finally, myogenin and MyoD loci were separated. Therefore. sequence homology, phylogeny, and chromosomal location supports the notion that the MRF family falls into two sub~oups that arose through evolution by successive gene duplication events (Atchley. 1994).

258

!! Lineage Specification and Differentiation

islands. Experiments using chimeric proteins suggest that the bHLH regions are functionally equivalent between the MRFs (Asakura et aI., 1993). Several conserved regions are found outside the bHLH domain including a cystein and histidinerich (C/H) region just N terminal of the basic domain, as well as box l and box2 located on the C-terminal side of the HLH domain (Tapscott et al., 1988: Weintraub et at.. 1991b). Of these regions only box2 exhibits weak conservation with C. elegans and Drosophila MyoD. and Drosophila acheate-scute complex, neurogenic bHLH genes (Fujisawa-Sehara et al., 1990).

B. Expression of MRF Genes during Development Analysis of MRF expression in mouse embryos by in situ hybridization (Lyons and Buckingham. 1992) indicates that M3f5 is the first MRF expressed in the dorsomedial region of most rostral somites on embryonic 8.0 dpc (Ott et al.. 1991 also see Plate 1 0 - o in Kaufman. 1992). Expression of M3f5 is maintained in the dorsomedial lip of the dermomyotomes and in the newly formed epaxial myotome (Figs. 4A. 4B, and 4I. and see Plate 15b-b. c. d in Kaufman. 1992). The initial localization of ~ v f 5 mRNA is significant since the dorsomedial lip is a center for supplying muscle progenitor cells to form the differentiated epaxial myotome (Fig. 4I). Subsequently. expression spreads in a rostral to caudal direction until expression is detected in the whole myotome (Fig. 4J). Thereafter. Myf5 expression is downregulated in mature myotomes (Fig. 4C) although expression is maintained throughout fetal muscle development. Myogenin mRNA first appears in the most rostral myotome at 8.5 dpc (Fig. 4E and see Plate 15b-b. and 16b-h. i in Kaufman. 1992) and thereafter spreads caudally (Fig. 4F, Sassoon et al., 1989). Myogenin expression is maintained in adult muscle where its expression is localized to motor endplates. MRF4 mRNA appears transiently in myotome in a rostral to caudal manner from 9.0 dpc to 11.5 dpc (Fig. 4G) and is reexpressed in muscles at 16.0 dpc to become the most abundant MRF expressed after birth (Bober et al., 1991 Hinterberger et al., 1991 Hannon et al., 1992). MyoD mRNA appears in the dorsomedial edge of myotome at the cervical to thoracic level and the ventrolateral edge of myotome at the interlimb bud level at 9.75 dpc (Figs. 4H and 4I; Sassoon et al.. 1989: Faerman et al., 1995: Tajbakhsh et al. 1997: also see Plate 20c-e, f, ~, h in Kaufman, 1992). Whereas the onset of MyoD expression in the dorsomedial myotomes follows that of MyfS. the onset of MyoD expression in the ventrolateral myotome is prior to or at the same time as that of M3f5 (Figs. 4J and 4K). The initial localization of MvoD mRNA in the ventrolateral myotome supports the notion that MyoD is important in hypaxial myotome formation (Faerman et al., 1995: Cossu et aI., 1996: Kablar et al., 1997 Asakura and Tapscott, 1998). By

11.0 dpc, MyoD expression spread throughout the myotome and is maintained thereafter throughout development. Limb and abdominal muscles are derived from muscle progenitor cells migrating from the VLL/somite bud of the dermomyotome (Figs. 2E. 21:. 6: Franz et al., 1993; Bober et al., 1994: Williams and OrdahI, 1994). Cells in the VLL and migrating muscle progenitor cells derived from the VLL do not express any MRF until after they arrive at sites of myogenic differentiation. In the limbs, muscle progenitor cells begin to express MvoD and M3f5 at about the same time (10.5 dpc in forelimb and 11.0 dpc in hindlimb in mouse) (Lyons and Buckingham, 1992). Myogenin expression is detected at 11.0 dpc in the forelimb and at 11.5 dpc in the hindlimb. The complex pattern of MRF expression evident during embryogenesis likely reflects overlapping phases of myogenic determination and differentiation. Consideration of MRF expression in cultured cells supports a division of the MRFs into two groups. Proliferating myoblasts express MyoD and/or Myf5 but do not express myogenin or MRF4 or other markers of differentiation. By contrast, differentiated myocytes express myogenin and MRF4 and downregulate expression of Myf5 and MyoD (Weintraub, 1993). Hence. on this basis the MRF family appears to consist of two groups that likely reflect distinct functional specialization in either proliferating myoblasts (Myf5 and MyoD) or in terminally differentiated myocytes (myogenin and MRF4) (Table I and Fig. 6).

C. Two Functional Groups of MRFs Gene targeting has allowed a genetic dissection of the roles played by the MRF genes in the determination and differentiation of skeletal muscle during embryonic development. The introduction of null mutations in M3f5, MyoD, myogenin, and MRF4 into the germline of mice has revealed the hierarchical relationships existing among the MRFs, and established that functional redundancy is a feature of the MRF regulatory network (Weintraub, 1993" Megeney and Rudnicki, 1995; Olson et at., 1996). Newborn mice lacking a functional MyoD gene display no overt abnormalities in muscle but express about fourfold higher levels of M3f5 (Rudnicki et aI., 1992). Newborn Myf5-deficient animals also exhibit apparently normal muscle, but die after birth due to malformation of the ribs (Braun et al., 1992; Grass et al., 1996). Muscle development in the trunk of embryos lacking M ) f 5 is delayed until the onset of MyoD expression, which occurs with somewhat delayed kinetics (Braun et al., 1992; Tajbakhsh et al., 1997; Kablar et al., 1997). Importantly, newborn mice deficient in both M3f5 and MyoD are totally devoid of myoblasts and myofibers. Thus. Msf5 and MyoD appear to be required for the determination of muscle progenitor cells and act upstream of myogenin and MRF4 (Rudnicki et al., 1993). The

Figure 4 Temporal and spatial expression pattern of mRNAs for MRFs, Pax3, and MyHC. M3f5 mRNA is the first MRF expressed in the dorsomedial region of rostral somites and, subsequently, in the dorsomedial region of dermomyotome and myotome (arrowheads in A, 8.75 dpc; B, 9.5 dpc). At 9.75 dpc, M~f5 expression appears in the ventral region beneath the dermomyotome (arrow in K). Thereafter (11.0 dpc), M~f5 expression is downregulated in the central region of mature myotomes (C) where expression of MyHC initiates (L). Myogenin mRNA first appears in the most rostrat myotome at 8.5 dpc (arrowheads in D) and thereafter spreads caudally (E. at 9.5 dpc). Note that both sides of myotomes are displayed in D (arrowheads). By 11.0 dpc, the whole myotomes express rnyogenin (F). MR1=4mRNA appears transiently in myotome (arrowheads in G, at 9.5 dpc). Last, MyoD mRNA appears in the ventrolateral edge of myotome at the interlimb bud level. By I 0.5 dpc (H) ~voD is expressed in the dorsomediaI edge of myotome at the cervical to thoracic level (arrowhead in H) and the ventrolateral edge of myotome at the intertimb bud level (arrow in H). By tl.5 dpc, the whole myotomes express MyoD (I). Pax3 is detected in the dermomyotomes in trunk level as well as in the dorsal neural tube at 10.5 dpc. At limb bud level, ventral regions of the dermomyotome (VLL) delaminate (arrow in J), and the migratopy muscle progenitor cells migrate out from the VLL into the limb buds (arrowheads in J). ft, forelimb bud: hl, hindlimb bud.


260

Table i Targeted Gerrnline Mutations in Mice Affecting Myogenesis Myogenic phenotype

Targeted mutation Myogenic Regulatory Factors M y o D -~- (1-5) M s f 5 -/- (3, 4, 6 - 8 ) myogenin-"- (9-11 ) M R F 4 -~- (12) M y o D - ~ - : M 3 f 5 -~- (13.14) M y o D - J - : m y o g e n i n -/- (15) M v o D - / - : M R F 4 - J - (16) M.~f5 .....:myogenin-"- (15) myogenin-~-: M R F 4 -~'- (16) M y f S - J - . ' M R F .... (16) MyoD-~-:myogenin-~'-:MRF4 -i- (17) M.~f5 -~-: S p l o t c h (Pax3 -~-) ( 8 )

Delayed migrator" and nonmigrator?"hypaxial muscle formation: impaired adult muscle regeneration and adult mvoblast differentiation: upregulation of Myf5 Delayed MyoD expression in mvotome, epaxial muscle, some nonmigrato~ hypaxial muscle formation and esophagus transdifferentiation Absence of secondar? muscle fiber formation: increased myoblast number Upregulation myogenin (mildest allele) Complete absence of myogenesis Phenocopy of m y o g e n i n -~Phenocopy of m y o g e n i n - Phenocopy of m y o g e n i n -~Phenocopy of m y o g e n i n .... Phenocopy of .'v/)fS-Presence of mvoblasts: absence of muscle fibers Absence of trunk and limb muscle

Other Transcription Factors Ibx] -~- (18-20) M o x 2 (21 ) c-ski -~'- (22) p a r a x i s -/- (23,24) p a r a x i s - / - : M s f 5 -'~- (24) Pax7-"- (25)

Absence of lateral migrator?' hypaxial muscle formation Impaired subset of limb muscle formation: decreased Myf5 expression in limb bud Reduced diameter of muscle fibers: reduction of muscle mass Impaired epaxial and nonmigrato~' hypaxial muscle formation Absence of epaxial muscle formation Absence of myogenic satellite cells

Signaling Molecules Shh -~- (26,27) Writ1 -~'-: Wnt3a-1- (28) c - m e t .... (29,30) S F / H G F - ; - (30) G D F 8 -~- (31 ) Noggin-~- (32)

Absence of formation and Myf5 expression in epaxial myotome Impaired formation and reduced Myf5 expression in epaxial dermomyotome Absence of migrator hypaxial muscle Absence of migratory hypaxial muscle Adult muscle hypertrophy Reduced epaxial myotome formation

References cited: 1. Rudnicki et al.. 1992.2. Megeney e t al.. 1996, 3. Kablar et al., 1997, 4. Kablar et al., 1998, 5. Sabourin et at., 1999.6, Braun e t a l . , 1992, 7. Tajbakhsh e t al., 1997, 8. Kablar et al., 2000, 9. Hasty e t al.. 1993. 10. Nabeshima et al., 1993, 11. Venuti, 1995. 12. Zhang e t aI., 1995, 13. Rudnicki et al.. 1993. 14. Kablar et al.. 1999, 15. Rawls et al., 1995, 16. Rawls et ai.. I998, 17. Valdez et aL, 2000, 18. Schafer and Braun, 1999, 19. Gross e t al.. 2000, 20. Brohmann et al.. 2000, 21. Mankoo et al.. 1999, 22. Berk et aI., I997. 23. Burgess e t al., 1996, 24. Wilson-RaMs et al., 1999, 25. Seale e t al.. 2000, 26. Chiang e t al., 1996, 27. BoQ'c "ki et at.. 1999, 28. Ikeya and Takada, 1998, 29. Bladt et al., I995.30. Dietrich et al., 1999a, 31. McPherron etaI.. 1997.32. McMahon etal.. 1998

assertion that Myf5 and MyoD are determination factors is supported by the observation that putative muscle progenitor cells remain multipotential and contribute to nonmuscle tissues in the trunk and limbs of M~f5-/-:MyoD -/- embryos (Kablar et al., 1998). Mice lacking myogenin are immobile and die perinatally due to deficits in myoblast differentiation as evidenced by an almost complete absence of myofibers (Hasty et al., 1993; Nabeshima et al., 1993). However, normal numbers of MyoD-expressing myoblasts are present and these are organized in groups similar to wild-~pe muscle. Myogenindeficient embwos form primary myofibers normally, but appear unable to form secondary myofibers (Venuti et al., 1995). Moreover, the observation that mice lacking both myogenin and Msf5, or both myogenin and MyoD, are identical to mice lacking only myogenin has confirmed that Msf5 and MyoD act upstream of myogenin (Rawls et al., 1995). Therefore, myogenin plays an essential in vivo role in the terminal differentiation of myoblasts (Fig. 5).

Mice carrying different targeted MRF4 mutations display a range of phenotypes consistent with a late role for MRF4 in the myogenic pathway. Zhang and coworkers observed that mice lacking MRF4 are viable with seemingly normal muscle and display a fourfold increase in myogenin expression (Rawls et al., 1995; Zhang et al., 1995). By contrast, Braun and Arnold (1995) reported a targeted MRF4 mutation, in which Myf5 expression (located about 6 kb away) is completely ablated in cis (Olson et al., 1996). A third targeted MRF4 mutation displays an intermediate phenotype due to partial ablation of M3f5 expression in cis (Patapoutian et al., 1995: Yoon et al., 1997; Olson et al., 1996). Interestingly, mice lacking both MyoD and MRF4 display a phenotype similar to the myogenin-null phenotype (Rawls et al., 1998). Therefore, MRF4 function may be substituted by the presence of myogenin, but only in the presence of MyoD (Fig. 5). Taken together, these data support the hypothesis that the MRFs function as couplets, M)f5 and MRF4 to regulate epaxial muscle development,

13 Regulation of Skeletal Muscle Development

261

Figure 5 Spatial expression pattern of mRNAs for,~f5, MvoD. ,~'cHC Pax3, and Pax. At 10.0 dpc M)f5 expression appears in the dorsomedial region (dml) of caudal somite (s) at the tail bud region (A). At trunk level, the expression spreads in the whole myotomes (B). MyoD mRNA appears in the ventrolateraI edge (vll) of myotome but not in the dml at the interlimb bud level (C). Pax3 mRNA is detected in the dermomyotome and in the dorsal neural tube at forelimb bud level (D). Ventral regions of the dermomyotome (vii) delaminate and the migratory' muscle progenitor cells migrate out from the vll into the forelimb bud (asterisk). MyHC expression is detected in the mature myotome at the central region but not in the dml or vii region. Paxl is expressed in the sclerotome (st) (F). Shh (Sonic hedgehog) expression is detected in the midline structures, notochord (arrow), and floor plate (arrow head), nt, neural tube. and MyoD and myogenin to regulate hypaxial m u s c l e development.

o f M R F s in m y o b l a s t s . Alternatively, ~dvf5 m a y p o s s e s s a limited function i n d u c i n g terminal differentiation o f m u s c l e .

T r i p l e mutant m i c e lacking MyoD, myogenin, and MRF4 fail to f o r m differentiated m u s c l e fibers but do contain a normal n u m b e r o f m y o b l a s t s (Valdez et al., 2000). This study

In conclusion, o b s e r v a t i o n s f r o m g e n e - t a r g e t i n g e x p e r i m e n t s have divided the M R F f a m i l y into t w o g r o u p s . T h e

suggests that M ) f 5 alone is insufficient to induce terminal differentiation o f muscle, p e r h a p s due to the insufficient levels

primary' M R F s , MyoD and M~f5, are r e q u i r e d for m y o g e n i c d e t e r m i n a t i o n . T h e s e c o n d a r y M R F s , m)'ogenin and MRF4, act later in the p r o g r a m as differentiation factors (Fig. 6).

Figure 6 The MyoD family defines discrete myogenic lineages in the myotome and migratoD' muscle development. Dermomyotomal cells differentiate into myoblasts by the expression of primary MRFs, M.~f5 or MyoD, in dorsal subdomain (epaxial myotome) or ventral subdomain (hypaxial myotome), respectively. Thereafter. both MyoD and ~ f 5 are coexpressed in the same muscle cells. Proliferating myoblasts withdraw from the cell cycle and initiate to express seconda_W MRFs. myogenin and MRF4 to form mulfinucleated myotubes. The two subdomains of the myotome give rise to back musculature and intercostal and body musculature, respectively. The rnigato~, hypaxial muscles also give rise to diaphragm and limb musculature (Kablar et aL, 1997, 1999: Ordahl and Williams. 1998).


262

D. Myf5 and lVlyoD Regulate the

Development of Distinct Myogenic Lineages The temporal-spatial patterns of myogenesis in M3fSand MvoD-deficient embryos provides compelling evidence for unique roles for M3f5 and MyoD in the development of epaxial and hypaxial musculature (Kablar et al., 1997). Embryos lacking MyoD display normal development of paraspinal and intercostal muscles in the body proper, whereas muscle development in limb buds and branchial arches is delayed by about 2.5 days. By contrast, embryos lacking Myf5 display normal muscle development in limb buds and branchial arches and a marked delay in development of paraspinal and intercostal muscles. Transdifferentiation of esophageal smooth muscle into skeletal muscle is also dependent on the presence of Msf5 (Kablar et aI., 2000). Although MyoD mutant embryos exhibit a delay in the development of limb musculature, the migration of Pa_r3 (a paired type homeobox gene) expressing cells into the limb buds and subsequent induction of Msf5 in muscle progenitor cells occur normally. Therefore. the phenotypes of M3f5 and MyoD mutant mice strongly support the hypothesis that M ) f 5 and MyoD have unique roles in the regulation of the developmental programs of the myogenic lineages giving rise to epaxial versus hypaxial musculature. Moreover. the observation that mice lacking both MyoD and MRF4 display a phenotype similar to the myogenin-null phenotype (Rawls et at., 1998) lends genetic support to the hypothesis that MyoD and myogenin are together required for the appropriate development of hypaxial musculature (Fig. 6).

IV. Muscle-Specific Transcriptional Regulation A. Positive Regulators for Muscle-Specific Transcriptions Analysis of several genes such as MCK, MLCI/3 (My/f), cz-actin (Actal), and myogenin has revealed the existence of additional cis-acting elements involved in muscle-specific transcriptional regulation (Hauschka, 1996: Molkentin and Olson, 1996). The identification of DNA motifs such as the MEF2 (CTA(A/T)4TAG), CArG box (CC(AT)6GG), ATrich (T(A/T)ATAAT(A/T)A), MCAT (CATTA), and MEF3 (TCAGGT) sites as required elements in muscle-specific regulatory sequences led to the identification of diverse transcription factors that play important roles in regulating transcription in muscle. Mesodermal cell-specific homeobox protein (mHOX, Prrxl) binds the AT-rich site located in the MCK enhancer region and activates MCK gene transcription (Cserjesi et al., 1992). Serum response factor (Srf) and factors belonging to the MADS family (MCM 1, Agamous, Deficiens, Serum re-

sponse factor) bind the CArG box as well as SRE (serum responsive element) (Mohun et al., 1991). TEF- 1 (Teadl), a tissue nonspecific transcription factor, binds to the MCAT site (Farrance et al., 1992; Stewart et aL, 1994). Sixl (Sixl), a vertebrate homolog of Drosophila sine oculis (so) homeobox factor, binds to the MEF3 site and upregulates myogenin and fast muscle specific aldolase (Atdol) promoters (Hidaka et aL. 1993 Spitz et aI., 1997, 1998). MEF2 sites are bound by members of the MEF2 family of transcription factors (Yu et al., 1992). In vertebrates, the MEF2 family of genes is composed of MEF2a (Mef2a), MEF2b (Mef2b), MEF2c (Mef2c), and MEF2d (Mea~d). MEF2 factors contain a MADS domain, which is essential for dimer formation and DNA binding (Olson et al., 1995" Molkentin and Olson, 1996). MEF2 genes are expressed in heart muscle, internal smooth muscle, and the neural tube as well as in skeletal muscle during early embwogenesis (Edmondson et at., 1994; Subramanian and Nadal-Ginard, 1996). In vitro experiments suggest that MEF2 factors function in differentiation rather during skeletal myogenesis as MEF2 genes are upregulated by expression of MRFs.

V, Inductive Mechanisms of Myogenesis The molecular mechanisms leading to the de novo induction of Msf5 and MyoD transcription in uncommitted progenitor cells during development have remained a central problem in myogenesis. However, several upstream signaling pathways have been implicated as positively or negatively affecting the de novo induction of M3f5 and MyoD transcription. For example, Sonic hedgehog (Shh)expressed in the floor plate and the notochord and Wnt family members expressed in the dorsal neural tube combinatorially activate myogenesis in the somite (see Fig. 9 in a later section; Johnson et al., 1994; Munsterberg et al., 1995: Stern etat., 1995). Shh also activates Noggin expression in the dorsal somite, and Noggin (Nog) inhibits the negative activity of lateralplate-derived BMP4 (bone morphogenetic protein) on myogenesis (Hirsinger et al., 1997; Marcelle et aI., 1997; Reshef et al., 1998). Notch signaling pathways also appear to exert negative effects on myogenesis (Kopan et aL, 1994; Shawber et al., 1996: Delfini et al., 2000). Although these pathways have been demonstrated to exert effects on the timing and patterning of myogenesis, the molecular mechanisms that activate the de novo transcription of Myf5 and MyoD are completely unknown.

A. Neural Tube, Notochord, and Dorsal Ectoderm Induce Myotome Formation Experiments in which somites were rotated suggest that cells in the newly formed somite are developmentally equiv-

13 Regulation of Skeletal Muscle Development alent and maintain multipotentiality independent of position (Aoyama and Asamoto. 1988: Ordahl and Le Douarin, 1992). However. dermomyotome, myotome, and sclerotome formation obviously proceed in a position-dependent manner presumably mediated by signals from the surrounding tissues. Indeed. in vitro experiments reveal that the dorsal neural tube secretes factor(s) capable of inducing myogenic commitment and maintaining myotome differentiation in cocultured somites. The proximity of the dorsal neural tube to the DML of the dermomyotome suggests that signals from the dorsal neural tube induce the formation of the DML and the formation of the myotome from the DML (Bober et al., 1994: Buffinger and Stockdale, 1994: Stern and Hauschka, 1995: Munsterberg and Lassar, 1995: Cossu et aI., 1996; Xue and Xue. 1996: Dietrich et al., 1997: Marcelle et al., 1997: Hirsinger et al.. 1997). In organ culture experiments with cocultured neural tube and somites, young neural tube mainly induces M y f 5 expression, whereas mature neural tube mainly induces MyoD expression (Tajbakhsh et al., 1998). The notochord alone induces Paxl/Pax9, paired type homeobox genes, and sclerotome (Brand-Saberi et aI., 1993, 1996a; Pourqui~ et al., 1993: Balling et al., 1996). However, inductive (Buffinger and Stockdale, 1994; Bober et al., 1994; Munsterberg and Lassar, 1995: Stern and Hauschka, 1995: Pownall et al., 1996; Xue and Xue. 1996) and suppressive effects (BrandSaberi et al., 1993" Pourqui6 et al., 1993" Goulding et al., 1994: Xue and Xue, 1996) of notochord on myogenesis have been observed, and these differences may be generated as a function of the distance between somite and notochord, or they may reflect the developmental stage of notochord. In addition, signals from notochord cooperating with signals from neural tube induce skeletal muscles in somites (Munsterberg and Lassar, 1995" Stern and Hauschka. 1995). In mouse, this cooperation mainly induces M ) f 5 gene expression in the somites (Cossu et al., 1996). These results are consistent with the notion that M x f 5 expression is initiated in the DML of the somite and dermomyotome, which are topologically closed to the dorsal part of neural tube. Furthermore, signals from dorsal ectodermal layer above the somites induce dermomyotome and myotome mediated through M y o D induction in the mouse somite (Fig. 9; Fan and TessierLavigne, 1994; Cossu et al., 1996, Tajbakhsh et al., 1998). In mouse, dorsal ectoderm for the most part induces M y o D gene expression in the somite (Cossu et al., 1996). These results are consistent with the notion that M y o D expression at the interlimb level is initiated in the ventrolateral part of myotome (T. H. Smith et aI., 1994; Faerman et al., 1995" Cossu et al., 1996). Lateral plate promotes Iateralization of dermomyotome, opposing the dorsal neural tube effect, which promotes medialization of dermomyotome (Pourqui6 et al., 1995. 1996). Subsequently, muscle differentiation in the dermomyotome is suppressed by lateral plate. This inhibitow effect for muscle differentiation by lat-

263 eral plate is important since the VLLs of the dermomyotome generate undifferentiated migratory muscle progenitor cells.

B. Cell-Cell Interactions and the Community Effect Regulate Muscle Differentiation In vitro culture experiments using Xenopus and chicken embwos revealed that dissociated blastomeres (Xenopus) and epiblasts (chicken) induce M y o D expression and subsequently differentiate into skeletal myocytes (Holtzer et al., 1990: Godsave and Slack, 1991; George-Weinstein et al., 1996). Intact blastomeres and epiblasts do not exhibit any M y o D expression or muscle differentiation. Similarly, a subset of cells in mouse embryonic brain expressing M>f5 rnRNA but not protein does not differentiate into muscle unless the cells are dissociated (Tajbakhsh et al., 1994; Daubas et al., 2000). Dissociated presomitic mesodermal cells cocultured with both notochord and neural tube unde~o myogenic differentiation. However, a critical mass of mesodermal cells is required in what has been termed the community effect. For example, at least 3 0 - 4 0 dissociated cells derived from mouse presomitic mesoderm (Cossu et al., 1995) and more than 100 dissociated cells from Xenopus gastrula are required for the muscle conversion (Gurdon, 1988; Kato and Gurdon, 1993; Gurdon et al., 1993a,b). Taken together, these observations suggest several common features of the mechanisms that regulate skeletal myogenesis. First, dissociated undetermined cells in vitro possess a capacity to readily express M y o D and to undergo myogenic commitment. Second, myogenic differentiation requires interactions between a certain critical mass of cells. Therefore. transcriptional regulation of MRF genes is likely regulated by a variety of environmental cues including cellcell interactions. The myogenic differentiation of Notch-expressing ceils is suppressed following interaction with Delta (DIll)/Jagged (Jagl)-expressing cells (lateral inhibition), mediated by direct :interaction between Notch and Delta/Jagged that belong to transmembrane proteins (Kopan and Cagan, 1997; Ar-tavanis-Tsakonas et al., 1999). The cytoplasmic domain of Notch is released by the interaction with Delta/Jagged and is translocated into the nucleus. Indeed, expression of the cytoplasmic domain of Notch 1 is sufficient to suppress differentiation of myoblasts in vitro (Kopan et al., 1994). Ectopic Delta 1 expression inhibits myogenic differentiation in developing limb muscle (Delfini et aL, 2000). Muscle differentiation is upregulated by cell-cell interactions as mediated by adhesion molecules such as the cadherins in which cell adhesion is achieved by homophilic binding. For example, myoblasts derived from chicken epiblasts express N-cadherin (Cdh2), and blocking N-cadherin function with antibodies suppresses muscle differentiation


264 (George-Weinstein et aI., 1997). In addition, expression of a dominant-negative cadherin in Xenopus embryos suppresses skeletal muscle differentiation (Holt et al.. 1994). Moreover, incubation of antagonistic M-cadherin ( C d h l 5 ) peptides or antisense RNA inhibits both myoblast fusion and cell cycle withdrawal in conditions that normally promote differentiation (Zeschnigk et al., 1995).

C. Positive (Shh and Wnt Proteins) and Negative (TGF-fi family) Factors Regulate Myotome Formation Recent experiments have revealed the molecular aspects for the positive and negative signals that regulate myotome formation (Currie and Ingham, 1998). Wnt proteins possess the ability' to induce muscle differentiation in somites (Munsterberg et al.. 1995 Stern et al.. 1995). In addition, notochord provides Shh protein, which can induce both myotomal cells and sclerotomal cells in the somite (Johnson et al., 1994: Fan and Tessier-Lavigne, 1994" Munsterberg et al.. 1995). Shh secreted from notochord also induces floor plate in the most ventral region of neural tube and the floor plate also provides Shh (Ingham, 1994). Shh has been suggested to act in diverse roles in somitogenesis, for example as an inductive factor, a proliferation factor, and a cell survival factor (Borycki et aI., 1999: Duprez et aI., 1998: Teillet et al., 1998). The different roles played by Shh may be due to combinations of different signals and the differences in stage of cell differentiation. In mouse, cells of the dorsal ectodermal secrete Wnt7a protein, whereas the dorsal neural tube secretes Wnt 1. Cultured presegmental mesoderm upregulates M y o D expression when exposed to Wnt7a and upregulates M y f 5 when exposed to Wntl (Tajbakhsh et aI., 1998). These observations are consistent with the relationship between the topologically distinct regions of Wnt expression and the induction of ,V13f5 in the DML and M y o D in VLL (see Fig. 9 in a later section). Moreover, Wnt4. Wnt5a. and Wnt6 all induce ex-

Table il

pression of both M y o D and M y f 5 in the somite. Interestingly, expression of M y f 5 mRNA in the brain is observed close to domains where Wntl and Shh are expressed (Daubas et al., 2000). BMP4. belonging to the transforming growth factor/3 (TGF-fl) family, was identified as a negative regulator of myotome and sclerotome formation. B M P 4 is expressed in the dorsal neural tube, dorsal ectoderm, and lateral plate mesoderm (Pourqui6 et al., 1996: Marcelle et at., 1997; Maroto et al.. 1997 Hirsinger et al., 1997; Reshef et al., 1998; Curfie and Ingham. 1998). Therefore, BMP4 appears to maintain and expand the number of precursor cells in the dermomyotome (see Fig. 9 in a later section). However, the DML of the dermomyotome expresses Wntl 1 and BMP4 antagonists Follistatin (Fst) and Noggin (Nog). Therefore, BMP4 protein in the DML is inactivated, allowing induction of myotome formation in the dorsomedial region (Amthor et aL, 1996: Hirsinger et al.. 1997: Marcelle et al.. 11997; Reshef et al., 1998). The notochord expresses Noggin, which also blocks B MP4 function in the DML (Hirsinger et al., 1997" McMahon et al., 1998). Myostatin (Gdf8), belonging to the TGF-fl family, is expressed in the myotome as well as adult skeletal muscle where it acts to negatively control muscle mass. Myostatin is mutated in double-muscled cattle (Belgian Blue) in which muscle mass is markedly increased (Grobet et al., 1997; McPherron and Lee, 1997). Similarly, mice lacking the myostatin gene display similar muscle hypertrophy (McPherron et al., 1997; Lee and McPherron, 1999).

D. Genetic Analysis of Myogenic Induction Several gene knock-out experiments have revealed the important roles played by various signaling molecules in myotome formation (Tables I and II). Mice lacking Wntl or Wnt3a do not display any defect during somitogenesis. By contrast, mice lacking both Wnt l and Wnt3a genes display reduced myotome formation (Ikeya and Takada, 1998). In addition, because Wntl and Wnt3a are expressed in the dor-

S p o n t a n e o u s M u t a t i o n s in Mice and Their Effect on Myogenesis

Spontaneous mutation

Gene

Splotch (Sp) (1-4)

Pax3

Open brain (opb) (5) Danforth "s short rail (Sd) (6,7)

Unknown (Ch 1) Unknown (Ch 2)

Brachyuo" curtailed (To)(7) Pintail (Pt) (7) Truncate (tc) (7)

Brachyur~" T

Unknown (Ch 4) Unknown (Ch 6)

Homozygous myogenic phenotype Partially impaired myotome formation, epaxial and nonmigratory.' hypaxial muscle formation: absence of migTatory hypaxial formation Absence of Myf5 expression in epaxial myotome; impaired its formation Absence of Myf5 expression in epaxial myotome: increased apoptosis in epaxial myotome: absence of epaxial muscle Absence of epaxial myotome (heterozygotes) Absence of epaxial myotome Absence of epaxial myotome

References cited: 1. Franz et al., 1993.2. Bober et al.. 1994. 3. Goulding et al., 1994.4. Williams and Ordahl, 1994. 5. Sporle et al., 1996. 6. Asakura and Tapscott. 1998.7. Dietrich et al., 1999b

13 Regulation of Skeletal Muscle Development sal neural tube close to the dorsomedial dermomyotome, loss of both genes induced lateralization of the entire dermomyotome. In the mutant dermomyotome, expression of the lateral dermomyotome marker Siml, a transcription factor homolog of Drosophila single minded (sire), is medially expanded to the dorsomedial edge, and expression of the medial dermomyotome markers such as Enl, a mouse homolog of Drosophila engrailed (en). and dorsomedial markers such as noggin and Wntl I are not detected. Mice lacking Shh gene display substantial defects in somitogenesis. Initially. a low level of the sclerotomal marker Pax1 is detected that completely disappears at later times. Therefore, Shh may act as a survival factor or a proliferation factor rather than as an inducer of sclerotome formation (Chiang et al., 1996). During myotome formation. M)S5 expression is not initiated in the DML of the dermomyotome, but is detected in the hypaxial myotome. MyoD expression in the hypaxial myotome is not affected (Chiang et al., 1996; Bo~'cki et al., 1999). These results are consistent with the hypothesis that Shh acts as an inducer of dorsomedial myotome formation, mediated by M)f5 gene activation by cooperating with Wnt proteins. Phenotypic characterization of various spontaneous mouse mutants reveals useful insights into the important roles played by the surrounding tissues in myotome formation. In the neural tube mutant mouse, open brain (opb), M ) f 5 gene expression is severely reduced, whereas MyoD expression remains normal (Sporle et aI., 1996). This result indicates that the dorsal neural tube provides signals (likely Wnts) necessary for the induction of M)f5 expression in the DML of the dermomyotome, whereas MyoD induction in the hypaxial myotome occurs independently of the neural tube. Danforth's short-tail (Sd) mouse is a semidominant mutation that prevents completion of notochord development. In homozygous mutant mice, the notochord completely degenerates by 9.5 dpc, whereas the neural tube and somites continue to form, permitting analysis of somite development in the absence of inductive signals from the midline structure, the notochord, and floor plate. In the somites formed after notochord degeneration, initial Msf5 expression is significantly reduced (Fig. 7" Asakura and Tapscott, 1998). Paxl expression in the sclerotome is not detected. Muscle gene expression including MyoD and M ) f 5 is normally detected in the hypaxial myotome and there is normal development of both migratory and nonmigratory hypaxial muscles. By contrast, muscle gene expression including Msf5 is not detected in the epaxial myotome and a high level of apoptosis is detected with significantly decreased formation of epaxial muscles. However, the initial Pax3 expression in the dermomyotome is relatively normal. Later, the hypaxial myotome is ventrally shifted into the place where the sclerotome normally forms (Asakura and Tapscott, 1998" Dietrich et al., 1999b). These results are similar to the somitic phenotype of Shh -/- mice (Chiang et al., 1996: Borycki et at., 1999) as

265

Figure 7 Notochord requires the epaxial myotome formation. Wildtype emb~o (A) expresses Shh in the notochord (arrow) and floor plate (arrowhead). In Sd/Sd embryo (B). Shh is not detected in the notochord or floor plate at the caudal level. In wild-type emb~o, M~f5 expression is observed in the entire myotome (A). In contrast, in Sd/Sd emb~'o, the expression is normal in the ventral (hypaxial) myotome but reduced in the dorsal (epaxiaI) myotome (B) at the caudal level, suggesting an important role of the notochord in the epaxial myotomeformation (Asakura and Tapscott. 1998). well as other distinct mutations affecting notochord development, for example, Truncate (tc), Pintail (Pt), and Brachyu 9 curtailed (Tc) (Dietrich et at., 1999b). Taken together, these results suggest that Shh is the main factor secreted from the notochord with a role in somitogenesis. The notochord also provides Noggin, which promotes myotome formation, mediated by antagonizing BMP4 in the DML. Noggin -/- mice display a severe reduction in the epaxiaI myotome (McMahon et aL, 1998). Both Patched (Ptch) gene, a Shh receptor, and Glil (Gti), a vertebrate homolog of Drosophila Cubitus interruptus (ci), which is a zinc finger type transcription factor, are known to be upregulated by Shh induction. Indeed, these genes are detected in the dermomyotome, sclerotome, and myotome, consistent with a direct role for Shh in sclerotome and myotorne induction (Marigo et al., 1996a; B o ~ c k i et al., 1997, 2000; Marcelle et al., 1997). In zebrafish, Shh secreted from the ventral axial structures induces the formation of the slow adaxial muscles. In the notochord mutants, bozozok (boz) and no tail (ntI), the adaxial muscle pioneer cells, are absent and slow muscle does not form. However. the formation of fast muscle is unaffected (Devoto et aL, 1996; Blagden et aL, 1997; Norris et al., 2000; Currie and Ingham, 1998). This observation contrasts with the mouse where, in the absence of the notochord, myotome formation of the DML of the dermomyotome is severely affected. However, slow-type MyHC is detected (Asakura and Tapscott, 1998, in discussion). Therefore, the notochord or Shh appears necessa,~' for a subset of muscle cells, the adaxial muscle pioneer cells in zebrafish, and muscle pioneer cells in the mouse.

E. Migratory Muscle Progenitor Cells All myotomal cells and migratory muscle progenitor cells in the trunk are derived from the dermomyotome. As

266 described above, signals such as Shh. Wnts. and BMP4 secreted from the surrounding tissues regulate the development of the dermomyotome and myotome (Amthor et al., 1998: Duprez et aI., 1998. 1999). Importantly, several transcription factors that potentially represent the effectors of these signaling molecules have been implicated in regulating the development of the migratory muscle progenitor cells derived from the VLL of the dermomyotome. Expression of Pccr3. a paired type homeobox gene. in the dermomyotome (Fig. 5D) is activated by BMP4 secreted from the dorsal ectoderm (Pourqui6 et al., I996. Amthor et al.. 1998. 1999). Furthermore. expression of Pax3 in proliferating muscle progenitor cells arriving in the limb bud is maintained by B M P 2 / 4 / 7 expressed by the surrounding limb cells. Pear3 expression suppresses both MRF expression and muscle differentiation. Therefore. BMP proteins positively stimulate Pax3 expression in muscle progenitor cells in both the dermomyotome and the limb bud. In the spontaneous mouse mutant Splotch (Pccr3 sp) carv i n g a defective Pax3 gene. homozygous mutants die during embryogenesis. Homozygous Splotch mice display malformation of neural tissues and neural crest-derived tissues, decreased elongation of dermomyotome and myotome, and absence of delamination at the DML of the dermomyotome (Table II: Franz et al.. 1993 Goulding et al.. 1994: Bober et al.. 1994: Williams and Ordahl. 1994). Subsequently. epaxial muscles of the deep back and nonmigratoo" hypaxial muscles, such as intercostal and abdominal muscles, are reduced and migratory, hypaxial muscles at trunk level such as limb. pectoralis muscles, and diaphragm are completely absent (Tremblay et al., 1998). Because Pax3 is expressed in both dermomyotome and migrating muscle progenitor cells. Pax3 may regulate both short-range migration, such as that occurring during myotome formation, as well as long-range migration of muscle progenitor cells into the limbs (Tremblay et al.. 1998). Pax7, a Pax3 related gene, is also expressed in the similar region of Pax3 expression, suggesting roles for Pccr7 in development of migratory cells during somitogenesis. Interestingly, the ascidian has a single Pax3~7 related gene, which is expressed in the neural tube. like vertebrates, but not in the somite (Wada et al., 1997). Therefore, because migratory muscle lineages as well as migratory, neural crest cells emerged during early vertebrate evolution, an additional expression domain of Pax3~7 in the vertebrate somite may have resulted in subsequent emergence of novel migratory muscle lineages, which form craniofacial and limb bud/ paired fin muscles (Gee. 1994: Neyt et al., 2000). Many ceil migration processes have been shown to require specific receptor-ligand interactions. Indeed, the migration of muscle progenitor cells requires a functional c-metSF/HGF cascade (Brand-Saberi et al., 1996b Heymann et al., 1996). Mice lacking c-met, a receptor tyrosine kinase (Met), or lacking its ligand, scatter factor/hepatocyte

!1 Lineage Specification and Differentiation growth factor (SF/Ho~), display similar muscle phenotypes to Splotch mice; that is. an absence of migratory hypaxial muscle formation as well as defective delamination at the VLL of their dermomyotome. Therefore. c-met or SF/Hgf plays an essential role in the delamination and subsequent migration of muscle progenitor cells (Table I, Bladt et ai., 1995: Maina et al., 1996; Dietrich et al., 1999a). Because c-met is expressed in the dermomyotome and m i ~ t o r y muscle progenitor cells and S F / H o J is expressed in the limb buds. loss of Pax3 function may disturb the c-met-SF/Hgf cascade. However, in Splotch mice. reduced c-met expression is still detected in the dermomyotome and a subset of migratory cells is detected in limb bud. suggesting that Pax3 does not entirely regulate c-met expressions in the dermomyotome (Yang et al., 1996: Scaal et al., 1999). In addition, in Splotch mice. the presence of migratory cells that might be derived from the dermomyotome and which do not have myogenic potential suggests the existence of a novel Pax3independent migratory population (Mennerich et al., 1998). Interestingly, a population migrating from somite has been reported to differentiate into the angioblast lineage, which forms the endothelial cells of the blood vessels (Wilting et al.. 1995). However. cell lineage analysis of the novel Pax3-:c-met- population remains to be conducted. Recently. a new' marker for the migratory muscle progenitor cells has been identified. L b x l ( L b x l h ) , a vertebrate homolog of Drosophila homeobox protein ladybird (Ib), is expressed in the ventrolateral region of the dermomyotome as well as in migrator?" muscle progenitor cells. In Splotch mice. L b x l gene expression is not detected in the dermomyotome at trunk level. However, expression of L b x l is detected in the occipital somites but mi~ation of tongue muscle progenitors is delayed (Mennerich et al., 1998). These results suggest a possible role for Pax3 as an upstream regulator of L b x l gene in the dermomyotome at trunk level. Mice lacking L b x l gene display a complete absence of lateral limb (extensor) muscle formation (Table I, Schafer and Braun. 1999 Gross et al., 2000; Brohmann et al., 2000). However, in Lbxl-deficient mice formation of ventral limb (flexor) muscle, tongue muscle, and diaphragm is normal, suggesting that L b x l is required for the migration of a subset of muscle progenitor cells.

F. Transcription Factors Regulating Myotome Formation As described above, signaling molecules such as Shh, Wnts, and BMPs act to regulate the induction of M~f5 and MyoD, leading to subsequent myogenic determination. However. the specific transcription factors that directly activate M y o D or M ~ f 5 transcription remain unidentified. Nevertheless. the transcriptional regulatory, elements of MRF genes have been analyzed using in vivo transgenic mice to address this question.

267

13 Regulation of Skeletal Muscle Development For example, analysis of transgenic mice carrying MyoD gene regulatory regions driving bacterial ~-galactosidase (lacZ) genes, demonstrated that two different regions, a 258-bp core enhancer and a 700-bp distal regulatory, region (DRR), located at - 2 0 and - 5 kb upstream of the transcriptional initiation site, respectively, are involved in mouse MyoD gene activation (Fig. 8). Transgenic mice carrying a lacZ gene driven by 6-kb upstream MyoD gene containing the DRR (MyoD6.0-lacZ) revealed that 6 kb upstream of mouse MyoD gene contains elements required for MyoD gene expression in a subset of myotomal cells, which appear to represent differentiated myocytes. (Figs. 8A and 8B: Asakura et al., 1995). By contrast, transgenic mice carrying the core enhancer and - 2 . 5 - k b promoter region of MyoD gene driving lacZ gene (MyoDcore/2.5-lacZ) showed that the core enhancer is sufficient for initiation of MyoD gene transcription in transgenic mice during early myotome formation (10-11 dpc) (Goldhamer et al., 1992, 1995). In addition, lacZ-expressing cells in trunk and limb were detected in MyoD-/-:?d~f5 -/- mutant embryos, suggesting that the core enhancer contains elements that are required for the initial activation of MyoD transcription in muscle progenitor cells (Kablar et al., I998). From I 1 to 12 dpc, the expressions of lacZ in myotomes of MyoD6.0-lacZ (Fig. 8B) and MvoDcore/2.5-1acZ (Fig. 8C) appear mutually exclusive, suggesting that both the core and DRR enhancers together cooperatively regulate mouse MyoD gene expression during myotome formation (Kablar et aI., 1999). However, transcription factors involved in MyoD gene expression mediated through those enhancers remain to be elucidated. Nevertheless, several transcription factors have been suggested to exert positive or negative effects on MRF transcription. Avian and mouse Msxl is expressed in the ventrolateral region of dermomyotome and limb bud and appears to negatively regulate MyoD transcription and muscle differentia-

tion (Bendall et at., 1999" Houzelstein et aI., 1999). Suppression of MyoD transcription is mediated by directly binding to the core enhancer of the MyoD gene (Woloshin et al., 1995). Although Msx 1 is a negative regulator of MyoD transcription, it represents the only factor known that directly regulates MyoD transcription during embryogenesis. The mouse homeobox gene Mox2 (Meox2) is expressed in the dermomyotome as well as in migratory muscle progenitor cells. Mice lacking Mox2 showed downregulated Msf5 expression in limb buds and a subsequent loss of a subset of limb muscles (Table I; Mankoo et at., 1999). How'ever, MyoD expression is relatively normal. Because mice lacking M3f5 display normal limb muscle development (Kablar et al., 1997), Mox2 does not appear to act as a simple upstream regulatory factor regulating Myf5 transcription. The M-twist (Twist) related bHLH gene Para_xis (Tcfl5) is expressed in the dermomyotome during mouse embryogenesis (Burgess et al., 1995). Mice lacking Paraxis display abnormal somite formation. However, muscle differentiation occurs within the disorganized myotome (Burgess et al., 1996). The migration of epaxial myotomal cells is disorganized and nonrnigratory hypaxial myotome formation is delayed by several days, due to a delay in MyoD expression (Wilson-Rawls et aI., 1999). The formation of migrator; muscles is also delayed. Compound mutant mice lacking both Para_ris and M3)e5 display delayed MyoD expression in the myotome and an absence of epaxial muscle formation, such as back muscle, and reduced formation of a subset of hypaxial muscles, such as the proximal region of intercostal muscles and appendicular muscles (Wilson-RaMs et al., 1999). These results suggest that Paraxis is an important regulator of a subset of the muscle progenitor cells derived from the dermomyotome (Table I). Mice lacking both Myf5 and Pax3 (Splotch) display a complete absence of MyoD expression and an absence of

Figure 8 Transcriptional analyses of MyoD gene using transgenic mice carrying lacZ reporter genes. Two elements, a core enhancer (258 bp) located at -20 kb and a DRR (700 bp) located at -5 kb of the initiation site of MyoD gene, are required for mouse MvoD gene expression. (A and B) Double staining with X-gaI for lacZexpression (blue) and in sire hybridization for MyoD expression (dark brawn) in embu'os (11.5 dpc)from transgenic mice carrying 6 kb upstream containing the DRR driving lacZ gene (MyoD6.0lacZ). (C) Whole-mount X-gal staining for lacZ expression (blue) in emb~,o (11.5 dpc) from transgenic mice carrying 258-bp core enhancer driving lacZ gene (MyoDcore/2.5-IacZ). MyoD mRNA is expressed in the entire myotomesand limb buds (Panels A and B and see Fig. 4I). Expression of MyoD6.0-lacZ is detected in the medial region of myotome but not in the limb bud (A. B). In contrast. MvoDcore/2.5-lacZ is expressed in the dorsal and ventral regions of myotome, and limb bud (C) (Asakura et al., 1995: Kablar et aL, 1999).

268 myogenesis in the trunk. This result led to the suggestion that both Myf5 and Pax3 are upstream regulators of MyoD (Table I Tajbakhsh et al., 1997). Indeed, ectopic expression of Pax3 induces skeletal myogenesis in nonmuscle tissues in avian embry, os (Maroto et al.. 1997). However. ectopic expression of Pax3 in C2C 12 myoblasts efficiently inhibits muscle differentiation (Epstein et at.. 1995). In addition, coexpression of MyoD and Pax3 is not observed during mouse myotome formation (Williams and Ordahl. 1994). Mutations in human Pax3 have been identified and are responsible for Waardenburg syndrome (WS) in which tissues derived from neural crest cells are markedly affected. Importantly, fusions between Pax3 or Pax7 and FKHR (ia forkhead related transcription factor) are frequently identified in human alveola rhabdomyosarcoma (RMS). Pax3-FKHR or Pax7-FKHR acts as a potent activated form of Pax3 or Pax7. Therefore. activated Pax3/7 appear to promote RMS cell proliferation or suppress terminal differentiation (Merlino and Helman. 1999). Therefore, Pax3 may act as a indirect upstream factor that induces migration or other cellular changes to facilitate a subsequent induction of MyoD transcription. Recent experiments in chicken embryos have demonstrated that novel transcriptional regulators. Six l. Eya. and Dach, are involved in myotome formation and may act as upstream regulators of the MRFs. Six l is the vertebrate homolog of Drosophila homeobox protein sine oculis (so). Eya is the vertebrate homolog of Drosophila eyes absent (eya). Dach is the vertebrate homolog of Drosophila dachshund (dac), which exhibits partial homology to the ski/sno protein, which is known to regulate adult muscle mass (Table I, Colmenares and Stavnezer. 1989: Berk et al.. 1997). Six l protein is a transcription factor that binds the MEF3 site originally identified as a regulatory element in the fast muscle-specific aldolase (Hidaka et al., 1993 Salminen et al., 1996: Spitz et al., 1997) and rnyogenin genes (Spitz et al., 1998). Recent experiments, using the avian system demonstrated that Dach2 and Eya act as transcriptional cofactors that physically interact with either Six l or Dach proteins (Heanue et al., 1999: Relaix and Buckingham, 1999). Dach2, Eya2, and Six l are expressed in the developing somite, the DML of the dermomyotome, the myotome, and the migratory muscle progenitor cells (Relaix and Buckingham, 1999). Ectopic expression experiments have demonstrated that Dach2 and Pax3 can induce each other's transcription in the somite, suggesting the presence of a positive feedback loop among the genes (Fig. 9). Dach2/Eya2 or Six l/ Eya2 complexes can induce ectopic myogenesis, including M R F and MyHC expression within the somite (Heanue et al.. 1999). Because ectopic expression of Pax3 can induce ectopic myogenesis in nonmuscle tissue, the downstream targets of Pax3 may include DachP/Eva2/Sixl_ .. complexes, which then initiate the muscle differentiation program (Fig. 9). However, it remains to be shown whether the de novo induction of MyoD or Myf5 ~ene is directly

il Lineage Specification and Differentiation

Figure 9 Secreted factors and transcription factors regulate the myotome formations. Secreted factors from surrounding tissues, the neural tube (NT). notochord (NC). dorsal ectoderm (DE). and lateral plate (LP) act to pattern the dermomyotome (IDM) and myotome (MT). Positive factors for mvotome formation are underlined. activated by these complexes and whether MyoD or MU5 regulatory regions contain MEF3 sites that are bound by Six l protein. Interestingly, ski (Ski) and sno (Skir) proteins, components of a histone deacetylase complex, suppress the TGF-/3/BMP signaling cascade mediated by blocking Smad2 (Madh2), Smad3 (Madh3), or Smad4 (Madh4) proteins (Luo et al., 1999). In addition, ectopic expression of both ski and sno induces myogenesis or muscle hypertrophy (Colmenares and Stavnezer, 1989; Berk et al., 1997). Therefore, Dach2/ Eya2 complex may initiate myogenesis by blocking the BMP signaling cascade that normally suppresses myotome formation. In the future, targeted mutations of Dach, Eya, and S/x genes and in vitro transcriptional activation assays will reveal the genetic hierarchy that regulates myotome formation and MRF gene regulation.

VI. Specification of Muscle Fiber Types In adult skeletal muscle, each muscle contains different proportions of distinct muscle fiber types. In the routine hindlimb for example, the lateral gastrocnemius mainly consists of fast-twitch fibers and the soleus mainly consists of slow-twitch fibers. The fiber type is judged by the physiological aspect and a distinct MyHC isoform profile. While the slow-twitch fibers consist of oxidative fibers (type I fiber) that express slow" MyHC, MyHC-/3/MyHC type I (MHCb), the fast-twitch fibers consist of glycolytic fibers (type II fiber) that express fast MyHC including type IIA (Myh2), type IIB (Myh4), and type IIX (Myhl) MyHC (Stockdale, 1992; Hughes and Salinas, 1999: Talmadge, 2000).

13 Regulation of Skeletal Muscle Development During myotome formation, emb~onic and slow isoforms of MyHC, MyHC-ernb (,~vh3), and MyHC-~, respectively, are detected in the early differentiating myotome by 9.5 dpc in mouse, and subsequently the perinatal isoform of MyHC. MyHC-pn (Myh8) is detected at 10.5 dpc (Lyons et al., 1990). However, the distribution of these isoforms is uniform in the myotome, suggesting fiber type is not determined in the myotome. By contrast, during zebrafish myotome formation, adaxial muscle that is proximal to the notochord initiates slow-type MyHC expression and these cells migrate laterally within the myotome (Devoto et at., 1996; Blagden et al., 1997). Shh secreted from the notochord and floor plate induces slow muscle fiber type. Subsequently, myotomal cells expressing fast-type MyHC reside in the medial part of myotome. Therefore, in zebrafish development of distinct muscle fiber types is initiated at an early stage of myotome formation (Currie and Ingham, 1998). During fetal myogenesis, MyHC-ernb and MyHC-pn genes are expressed at high levels. By contrast, fast type MyHC, MyHC-IIB, MyHC-IIA, and MyHC-IIX mP,NA is only detectable at quite low levels from 14.5 dpc to birth. MyHC-/3 is expressed throughout fetal myogenesis at medium level. After birth the relative proportions of MyHC isoforms are changed dramatically. Both expressions of MyHC-emb and MvHC-pn are downregulated in leg muscles, although a high level of MyHC-pn expression is maintained during neonatal stage. Instead, expression of slow-type (MyHC-fl) or fast-type (M>,HC-IIA, liB, or IIX) isoform is upregulated in each muscle fiber during postnatal development to constitute slow'-, fast-, or mixed-fiber-type muscle (Lu et al., 1999). During avian or rodent limb muscle development, primary muscle fibers are uniformly formed, and then rapidly acquire slow or fast features depending on their location within the limb. Subsequently, secondary fibers form in association within the primary fiber scaffold (Stockdale, 1992; Hughes and Salinas, 1999). The determination of the fiber types is dependent on both cell lineage and local signals. For example, embryonic myoblasts differentiate into small myotubes that express slow-type MyHC and form primary fibers. By contrast, fetal myoblasts mainly express fast-type MyHC and form secondary fibers. In addition, clonal culture experiments reveal the presence of cell populations that form distinct fiber types (Miller and Stockdale, 1986a,b; Hughes and Blau, 1992; DiMario et al., 1993). However, implantation experiments using distinct cell populations suggest that both cell lineage and local signals affect muscle fiber types (Robson and Hughes, 1996, 1999). Interestingly, two distinct muscle populations, first slow lineage and second fast lineage, may enter the limb buds migrating from dermomyotome (Seed and Hauschka, 1984; Van Swearingen and Lance-Jones, 1995). During fetal myogenesis, motorneurons innervate muscle fibers and the resulting electric activity also affects fiber type specification. The signal from nerve is strong enough to override the cell lineage or local cues,

269 which determines fiber types, to induce slow/fast MyHC changes (Gundersen, 1998" Hughes and Salinas, 1999: Talmadge, 2000). Recent work demonstrated that a signaling pathway of calcineurin (Ppp3ca), a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase may regulate manifestation of the slow muscle fiber type (Chin et aL, I998; Wu et al., 2000; Olson and Williams, 2000; Talmadge, 2000). The activated calcineurin dephosphorylates the phosphorylated NFATc (Nfatcl), allowing NFATc to enter the nucleus and cooperate with MEF2 to activate slow-fiber specific genes. Whereas MyoD is mainly detected in fast-type fibers, myogenin is mainly detected in slow-type fibers in mouse adult muscle (Hughes et al., 1993). MyoD -/- mice display subtle shifts in fiber type of fast muscles toward a slower character and a shift of slow muscles toward a faster phenotype (Hughes et al., I997). Ectopic expression of myogenin in adult skeletal muscles induces an increase in slow fiber composition. These results suggest that MyoD and myogenin are somehow involved in development or maintenance of muscle fiber types but likely via an indirect mechanism (Hughes et al., 1999).

VII. Muscle Regeneration A. Satellite Cells in Adult Muscle Muscle satellite cells are a distinct lineage of myogenic cells responsible for mediating the postnatal growth and repair of adult skeletal muscle. Satellite cells reside beneath the basal lamina of adult skeletal muscle, closely juxtaposed against skeletal muscle fibers. Satellite cells are normally mitotically quiescent, but are activated (initiate proliferation) in response to stress induced by weight bearing or other trauma such as injury, to mediate the postnatal growth and regeneration of muscle. The progeny of activated satellite cells, termed myogenic precursor cells (MPCs), undergo multiple rounds of cell division prior to their terminal differentiation to form new muscle fibers (Fig. 10B). Satellite cells account for 2 - 5 % of sublaminal nuclei in adult muscle (2 months in mice). The number of quiescent satellite cells in adult muscle remains relatively constant over multiple cycles of degeneration and regeneration, indicating an inherent capacity for self-renewal (Grounds et al., 1992; Bischoff, 1996; Seale and Rudnicki, 2000).

B. MRFs in Satellite Cell Activation and Differentiation The MRF expression pro~am during satellite cell activation, proliferation, and differentiation is analogous to the p r o g a m manifested during the embryonic development of skeletal muscle. Quiescent satellite cells express no detect-

270


Figure 1 0 Satellite cells and pluripotential muscle-derived stem cells. (A) Pluripotent muscle-derived stem cells, also called side population (SP) cells, were identified in adult skeletal muscle tissue by fluorescence activated cell sorting (FACS) on the basis of Hoechst dye exclusion. Following intravenous injection, both muscle-derived and marrow-derived stem SP cells give rise to skeletal muscle cells and hematopoietic cells (Gussoni et al., 1999" Jackson et al., 1999). (B) Mitotically quiescent satellite cells are activated (initiated the cell division) and initiate expressions of MvoD and/or Myf5 during muscle regeneration. The self-renewal activated satellite cells give rise to the muscle precursor cells (MPCs) or go back to the quiescent satellite cells as a stem cell compartment. The MPCs cease the cell proliferation to initiate expression of myogenin, MRF4, and several muscle structural proteins then fuse each other to form the multinucleated myombes. Satellite cells also possess a potential to give rise to adipocytes and osteocytes. Muscle-derived stem cells possess a potential to differentiate into both skeletal muscle cells and hematopoietic cells. P a x 7 gene knock-out experiments suggest that myogenic specification of pluripotent muscle-derived stem cells requires P a x 7 gene (Seale et aI., 200O).

13 Regulation of Skeletal Muscle Development able MRFs but do express c-met and M-cadherin (Irintchev et al., 1994 Cornelison and Wold, 1997). During in vitro single muscle fiber culture and in vivo muscle regeneration,

in which satellite cells initiate their activation and entrance into the cell cycle, either M y o D or M?,f5 is rapidly upregulated concomitant with entrance of the cell cycle, followed soon after by coexpression of M y o D and M3'f5. Following proliferation, m y o g e n i n and M R F 4 are expressed in cells beginning their differentiation program (C. K. Smith et al., 1994; Yablonka-Reuveni and Rivera. 1994; Cornelison and Wold. 1997: Cornelison et al., 2000). Therefore. MyoD and Myf5 appear to play an early role in the satellite cell developmental program (Fig. 10B). Mice lacking M y o D gene display marked deficits in satellite cell function (Table I). M y o D mutant mice interbred with mdr mice, which are a model for Duchenne's and Becker's muscular dystrophies, exhibit increased penetrance of the mdv phenotype characterized by muscle atrophy and increased myopathy leading to premature death (Megeney et al., 1996). In spite of the presence of morphologically normal satellite cells in M y o D -7- muscles, muscle from M y o D -Imice displays a strikingly reduced capacity for regeneration following injury. Whereas growing M y o D -/- myoblasts express normal levels of c-met, expression of M-cadherin and desmin is notably reduced. By contrast, Myf5 expression is markedly upregulated in these cells. Under conditions that normally induce differentiation of wild-type myoblasts, M y o D -/- myoblasts continue to proliferate and exhibit delayed differentiation. Supporting this, M y o D -/- myoblasts show delayed induction of myogenin and MRF4. Therefore, M y o D -/- myoblasts display characters that are more primitive than wild-type myoblasts and may represent an intermediate stage between a satellite cell and a myogenic precursor cell (Sabourin et al., 1999: Cornelison et al., 2000; Seale and Rudnicki, 2000).

C. Pluripotent Muscle-Derived Stem Cells Tissue-specific stem cells have been found in various adult organs where they participate in replacement of differentiated cells for physiological turnover or injury. Recent experiments have demonstrated that tissue-specific stem cells appear to have a wider differentiation capability than previously thought. Therefore, many or all tissues may contain a population of pluripotent stem cells that differentiates in an appropriate manner in response to growth factors and signals provided by host tissues (Fuchs and Segr& 2000; Seale and Rudnicki, 2000; Cossu and Mavilio, 2000; Orkin, 2000). The pluripotent nature of adult stem cells isolated from diverse tissues raises the possibility of stem cell therapy for a variety of degenerative diseases including muscular dystrophy. Ferrari and colleagues were the first to demonstrate that marrow-derived cells could participate in muscle regeneration (Ferrari et al., 1998). Subsequently, Gussoni et al.

271 (1999) demonstrated that highly purified hematopoietic stem cells recruited to skeletal muscle of max mice resulted in a partial restoration of dystrophin expression (Gussoni et al., 1999). Gussoni et al. (1999) and Jackson et aI. (1999) further identified the presence of side population (SP) cells, enriched in pluripotent muscle-derived stern cells, isolated in adult muscle tissue by fluorescence activated cell sorting (FACS) on the basis of Hoechst dye exclusion (Fig. 10A: Gussoni et al., 1999; Jackson et al., 1999). Following intravenous injection, muscle-derived SP cells efficiently participate in regeneration of skeletal muscle, appear to give rise to myogenic satellite cells, and reconstitute the complete re D ertoire of the hematopoietic systems (Gussoni et al., 1999; Seale and Rudnicki, 2000). Cell culture experiments have demonstrated that continuous myoblast lines derived from satellite cells can transdifferentiate into adipoblasts and osteoblasts t~llowing treatment with adipogenetic inducers such as thiazolidinedione (Teboul et at., 1995) or by blocking the Wnt signaling pathway (Ross et aI., 2000), and BMP2 (Katagiri et aI., 1994), respectively. Several in vivo observations have postulated the existence of pluripotential stem cells within skeletal muscle. For example, expansion of adipose tissue within skeletal muscles occurs in response to denervation (Dulor et aI., 1998) and in some muscle diseases, including muscular dystrophy (Lin et aI., 1969) and mitochondrial myopathy (DiMauro et al., 1980). Moreover, transplantation of B MP into adult skeletal muscles can induce ectopic osteogenesis within muscle (Urist and Strates, 1971). Similarly, B M P 2 / 4 have been implicated in ectopic ossification of muscles in a human disease, fibrodysplasia ossificans progressiva (FOP) (Shafritz et al., 1996). However, it remains unknown whether satellite cells are the origin of adipocytes and osteocytes in vivo (Fig. 10). Satellite cells first appear in the limbs of 17.5 dpc mouse embryos and appear to constitute a myogenic cell lineage distinct from the embryonic muscle lineages derived from somites and prechordal mesoderm. Resent work has revealed that clonal skeletal myogenic cells that closely resemble satellite cell-derived myogenic precursors are readily isolated from the embryonic dorsal aorta of mouse embryos in vitro (De Angelis et al., 1999; Ordahl, 1999; Cossu and Mavilio, 2000). Aorta-derived myogenic cells express MyoD and/or Myf5 and several endothelial markers that are also expressed in activated satellite cells. Myoid cells in thymus, which express MyoD and myogenin are also a candidate source of myogenic stem cells (Grounds et aI., 1992; Wong et al., 1999; Wakkach et aL, 1999). However, the presence of myogenic cells in dorsal aorta and thymus does not preclude the possibility of a somitic origin for satellite cell progenitors. Recently, we found that P a x 7 is expressed in both quiescent and proliferating satellite cells. Strikingly, mutant mice lacking P a x 7 display a complete absence of satellite cells. Nevertheless, muscle-derived SP cells are present in

272


normal proportions (Table I Seale et aI., 2000). These results strongly; suggest that muscle-derived stem cells are a distinct stem cell population from satellite cells which yet possess mesenchymal differentiation plasticity. Additional experiments have revealed that isolated satellite cells readily give rise to colonies composed of myocytes, adipocytes, or osteocytes, whereas muscle-derived SP cells efficiently formed colonies of hematopoietic cells in vitro (A. Asakura and M. A. Rudnicki unpublished data). Taken together, these data support the hypothesis that pluripotent muscle-derived stem cells are the progenitors of myogenic satellite cells and suggest that the mechanism that induces Peer7 expression in pluripotent stem cells is under strict control (Fig. 10B).

VIII. C o n c l u s i o n The MRFs play key regulatory roles in the development of skeletal muscle during embryogenesis and in postnatal growth and repair of muscle. Recent experiments have revealed several key regulatory molecules including both transcription factors such as Pax3 and Six and signaling molecules such as Shh. Wnts, and B MPs. which positively and negatively regulate muscle development. In addition, experiments using the avian system have exposed revealing insights into the dynamic mechanisms regulating myotome formation. In the near future, the molecular mechanisms regulating myotome formation will certainly be elucidated by combining information from different animal systems, including the continued genetic analysis afforded by gene targeting in mice. Recent studies have demonstrated that a novel population of muscle-derived stem cells gives rise to hematopoietic lineages and also participates in muscle regeneration following intravenous transplantation. The molecular mechanisms that control the specification of adult pluripotent stem cells into differentiated cells types must involve induction of developmental c o n t r o l g e n e s s u c h as P a x 7 in the s a t e l l i t e cell myogenic

lineage. However, how these mechanisms

func-

tion to e f f e c t this i n d u c t i o n r e m a i n s e n t i r e l y u n c l e a r . N e v e r t h e l e s s , the r a p i d p r o g r e s s in u n d e r s t a n d i n g t h e m e c h a n i s m s r e g u l a t i n g m y o g e n i c s t e m cell s p e c i f i c a t i o n a n d f u n c t i o n in a d u l t m u s c l e will c l e a r l y l e a d to new" i n s i g h t s in t h e a n a l o gous mechanisms regulating embryonic myogenesis.

Acknowledgments We thank Boris Kablar and Robert Perry for critical comments on the manuscript. This work was supported by grants to M.A.R. from the Muscular Dystrophy Association. the National Institutes of Health. and the Canadian Institutes of Health Research. A.A. is supported by a development grant from the Muscular Dystrophy Association. M.A.R. is a research scientist of the Canadian Institutes of Health Research and is a member of the Canadian Genetic Disease Network.

References Amthor. H.. Connolly. D.. Patel. K.. Brand-Saberi. B.. Wilkinson. D. G.. Cooke. J.. and Christ. B. (1996). The expression and regulation of follistatin and a follistatin-like gene during avian somite compartmentalization and myogenesis. Dev. Biol. 178, 343-362. Amthor. H.. Christ. B.. WeiI. M.. and Patel. K. (1998). The importance of timing differentiation during limb muscle development. Curt. Biol. 8, 642-652. Amthor. H.. Christ. B.. and Patel. K. (11999). A molecular mechanism enabling continuous embryonic muscle g r o w t h w a balance between proliferation and differentiation. Development (Canvbridge. UK) 126, 10411053. Aoyama. H.. and Asamoto. K. (1988). Determination of somite cells: Independence of cell differentiation and morphogenesis. Development (Cambridge. UK) 104, 15-28. Artavanis-Tsakonas. S.. Rand. M. D.. and Lake. R. J. (1999). Notch signaling: Cell fate control and signal integration in development. Science 284, 770 -776. Asakura. A.. and Tapscott. S. J. (11998). Apoptosis of epaxial myotome in Danforth's short-tail (Sd) mice in somites that form following notochord degeneration. Dev. Biol. 203, 276-289. Asakura. A.. Fujisawa-Sehara. A.. Komiya. T.. and Nabeshima, Y. (1993). MvoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors. Mol. Cell. Biol. 13, 7153-7162. Asakura. A.. Lyons. G. E.. and Tapscott. S. J. (1995). The regulation of MvoD gene expression: Conserved elements mediate expression in emb~,onic axial muscle. Dev. Biol. 171, 386-398. Atchley. W R.. Fitch. \x,: M., and Bronner-Fraser. M. (I994). Molecular evolution of the MyoD family of transcription factors. Proc. Natl. Acad. Sci. US.A. 91, 11522-11526. Bailing. R.. Helwig. U.. Nadeau. J.. Neubuser. A.. Schmahl. W, and Imai. K. (1996). Pax genes and skeletal development. Ann. N. K Acad. Sci. 785, 27-33. Bendall. A. J., Ding. J.. Hu. G.. Shen. M. M., and Abate-Shen. C. (1999). Msxl antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors. Development (Cambridge, UK) 126, 4965-4976. Berk. M.. Desai. S. Y.. Heyman. H. C., and Colmenares, C. (1997). Mice lacking the ski proto-oncogene have defects in neurulation, craniofacial, patterning, and skeletal muscle development. Genes Dev. 11, 20292039. Bischoff. R. (11996). The satellite cell and muscle regeneration. In "'Myology'" (A. G. Engel and A. Franzini-Armstrong. eds.), pp. 97-1 I8. McGraw-Hill. Blackwell. T. K.. and Weintraub. H. (I990). Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science 250, 1104-1110. Bladt. F., Riethmacher. D.. Isenmann. S.. Aguzzi. A.. and Birchmeier, C. (1995). Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature (London) 376, 768771. Blagden. C. S.. Currie. R D.. Ingham, R W:. and Hughes, S. M. (1997). Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog. Genes Dev. 11, 2163-2175. Bober, E., Lyons, G. E., Braun, T,. Cossu, G., Buckingham. M., and Arnold, H. H. ( 1991 ). The muscle regulato~ gene, Myf-6, has a biphasic pattern of expression during early mouse development. J. Cell Biol. 113, 12551265. Bober. E., Franz, T., Arnold, H. H., Gruss. P., and Tremblay, P. (1994), Pax-3 is required for the development of limb muscles: A possible role for the migration of dermomyotomal muscle progenitor cells. Development (Cambridge. UK) 120, 603- 612. Borvcki. A. G.. Strunk. K. E.. Savar . R.. and Emerson. C. R. Jr. (1997). Distinct signal/response mechanisms regulate pax l and QmyoD activa-

13 Regulation of Skeletal Muscle Development tion in sclerotomal and myotomal lineages of quail somites. Dev. Biol. 185, 185 -200. Bo~'cki, A. G.. Brunk. B.. Tajbakhsh. S., Buckingham. M.. Chiang. C.. and Emerson, C. R. Jr. (1999). Sonic hedgehog controls epaxial muscle determination through Myf5 activation. Development (Cambridge. UK) 126, 405 3 - 4063. Bo~'cki. A.. Brown. A. M.. and Emerson. C. R, Jr. (2000). Shh and Wnt signaling pathways converge to control Gli gene activation in avian somites. Development (Cambridge. UK) I27, 2075-2087. Brand-Saberi. B.. Ebensperger. C.. Wilting. J.. Bailing, R.. and Christ. B. (1993). The ventralizing effect of the notochord on somite differentiation in chick embrvos. Ana~. Embra'ot. 188, 239-245. Brand-Saberi. B.. Wilting. J., Ebensperger. C., and Christ. B. (1996a). The formation of somite compartments in the avian embryo. Int. J Dev. Biol. 40, 411-420. Brand-Saberi. B.. Muller. T. S.. Wilting. J.. Christ. B., and Birchmeier, C. (1996b). Scatter factor/hepatocyte growth factor (SF/HGF) induces emigration of myogenic cells at interlimb level in vivo. Dev. Biol. 179, 303308. Braun. T. and Arnold, H. H. (1995). Inactivation of Myf-6 and Myf-5 genes in mice leads to alterations in skeletal muscle development. EMBO J. 14, 1176-1186. Braun. T.. Rudnicki. M. A.. Arnold. H. H.. and Jaenisch. R. (1992). Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell (Cambridge. Mass.) 71, 369-382. Brohmann. H.. Jagla. K.. and Birchmeier. C. (2000). The role of Lbxl in migration of muscle precursor cells. Development (Cambridge. UK) 127, 437-445. Buffinger. N.. and Stockdale. F. E. (1994). Myogenic specification in somites: Induction by axial structures. Development (Cambridge. UK) 120, 1443-1452. Burgess. R., Cserjesi. R. Ligon. K. L.. and Olson. E. N. (I995). Paraxis: A basic helix-loop-helix protein expressed in paraxiaI mesoderm and developing somites. Dev. Biol. 168, 296-306. Burgess. R.. Rawls. A.. Brown, D., Bradley. A., and Olson. E. N. (1996). Requirement of the paraxis gene for somite formation and musculoskeletal patterning. Nature (Londonj 384, 570-573. Chiang. C.. Litingtung. Y.. Lee. E.. Young, K. E., Corden, J. L.. W~stphat, H., and Beachy. R A. (I996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature (London) 383, 407413. Chin. E. R.. Olson, E. N.. Richardson. J. A.. Yang. Q., Humphries. C.. Shelton. J. M.. Wu. H.. Zhu. W.. Bassel-Duby, R.. and Williams. R. S. (1998). A catcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes Dev. 12, 2499-2509. Christ, B., and Ordahl. C. R (1995). Early stages of chick somite development. Anat. Embo'ol. 191, 381-396. Cinnamon. Y.. Kahane. N.. and Kalcheim. C. (1999). Characterization of the early" development of specific hypaxial muscles from the ventrolateral myotome. Development (Cambridge. UK) 126, 4305-43 I5. Colmenares. C.. and Stavnezer. E, (1989). The ski oncogene induces muscle differentiation in quail embryo cells. Ceil (Cambridge, Mass.) 59, 293303. Cornelison. D. D., and Wold. B. J. (1997). Single-cell analysis of regulator' gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev. Biol. 191,270-283. Cornelison. D. D.. Olwin. B. B.. Rudnicki, M. A.. and Wold. B. J. (2000). MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient. Dev. Biol. 224, t22-137. Cossu, G., and Mavilio. F. (2000). Myogenic stem cells for the therapy of primary' myopathies" Wishful thinking or therapeutic perspective? J. Clin. Invest. 105, 1669-1674. Cossu. G., Kelly', R., Di Donna. S.. Vivarelli, E., and Buckingham, M. (1995). Myoblast differentiation during mammalian somitogenesis is

273 dependent upon a community effect. Proc. Natl. Acad. Sci. US.A. 92, 2254-2258. Cossu, G., Kelly, R., Tajbakhsh, S., Di Donna, S., Vivareili, E., and Buckingham. M. (1996). Activation of different myogenic pathways: Myf-5 is induced by the neural tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm. Development (Cambridge. UK) 122, 429-437. Cserjesi. R, Lilly, B.. Bryson. L., Wang, Y.. Sass~x)n, D. A., and Olson. E. N. (1992). MHox" A mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer. Development (Cambridge. UK) 115, 1087-I I01. Cuttle. E D.. and Ingham, R ~ : (1998). The generation and interpretation of positional information within the vertebrate myotome. Mech. De',,. 73, 3 - 2 ! . Daubas, R, Tajbakhsh, S., Hadchouel, J., Primig, M.. and Buckingham, M. (2000). Myf5 is a novel early axonal marker in the mouse brain and is subjected to post-transcriptional regulation in neurons. Development (Cambridge. UK) 127, 319-33t. Davis. R. L.. V~:eintraub. H., and Lassar. A. B. ( t 987). Expression of a single transfected cDNA converts fibroblasts to m,voblasts. Cell (Cambridge, Mass.) 51,987-10('o0. Davis. R. L.. Cheng, R F.. Lassar. A. B., and Weintraub. H. (1990). The MyoD DNA binding domain contains a recognition code for musclespecific gene activation. Cell (Cambri@e. Mass.) 60, 733-746. De Angelis, L., Berghella, L.. Coletta, M., Lattanzi, L., Zanchi. M.. CuselIaDe Angelis, M. G.. Ponzetto. C., and Cossu. G. (11999). Skeletal myogenic progenitors originating from embQ'onic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration. J. Ceil Biol. 147, 869-878. Delfini. M.. Hirsinger, E.. Pourqui6, O.. and Duprez, D. (2000). Deltalactivated Notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis. Development (Cambridge. UK) 127, 5213-5224. Denetclaw, W. F.. and Orda~hi. C. R (2000). The growth of the dermomyotome and formation of early myotome lineages in thoracolumbar somites of chicken emb~os. Development (Cambridge. UK) 127, 893-905. Denetclaw. W: F., Jr.. Christ. B., and Ordahl. C. R (1997). Location and growth of epaxial myotome precursor cells. Development (Cambridge. UK) 124, I601-1610. Devoto. S. H.. Melancon. E.. Eisen, J. S.. and Westerfield. M. (1996). Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development (Cambrid,oe. UK)122, 3371-3380. Dietrich, S.. Schubert. F. R., and Lumsden. A. (1997). Control of dorsoventral pattern in the chick paraxial mesoderm. Development (Cambridge, UK) 124, 3895-3908. Dietrich. S.. Abou-Rebyeh. F.. Brohmann. H., Bladt, F.~ SonnenbergRiethmacher. E., Yamaai, T.. Lumsden. A.. Brand-Saberi. B.. and Birchmeier. C. (1999a). The role of SF/HGF and c-Met in the development of skeletal muscle. Development (Cambridge, UK) 126, 1621-I629. Dietrich. S., Schubert. E R.. Gruss, R. and Lumsden. A. (I999b). The role of the notochord for epaxial myotome formation in the mouse. Cell. Mol. Biol. (Noisy-te-grand) 45, 601 - 616. DiMario. J. X., Fernyak. S. E., and Stockdale. F. E. (1993). Myoblasts transferred to the limbs of embryos are committed to specific fibre fates. Nature (London.) 362, 165-167. DiMauro. S.. Mendell, J. R.. Sahenk. Z.. Bachman. D., Scarpa, A., ScofieId, R. M., and Reiner, C. (1980). Fatal infantile mitochondriaI myopathy and renal dysfunction due to cytex:hrome-c-oxidase deficiency. Neurology 30, 795-804. Dulor. J. R. Cambon, B., Vigneron. R. Reyne, Y., Nougues, J., Casteilla, L.. and Bacou. E (1998). Expression of specific white adipose tissue genes in denervation- induced skeletal muscle fatty degeneration. FEBS Lett. 439, 89-92. Duprez, D.. Fournier-Thibault, C,, and Le Douarin, N. (I998). Sonic Hedgehog induces proliferation of committed skeletal muscle cells in the chick limb. Development (Cambridge. UK) 125, 495-505.

274 Duprez, D.. Lapointe, F., Edom-Vovard. F.. Kostakopoulou, K., and Robson, L. (1999). Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud. Mech. Dev. 82, 151163. Edmondson, D. G., Lyons, G. E., Martin, J. F., and Olson, E. N. (1994). Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embwogenesis. Development (Cambridge. UK) 120, 125 t-1263. Epstein, J. A.. Lam, E. Jepeal, L., Maas. R. L.. and Shapiro. D. N. (1995). Pax3 inhibits myogenic differentiation of cultured myoblast cells. J. Biol. Chem. 270, 11719-11 "Y~ Faerman, A., Goldhamer, D. J.. Puzis. R.. Emerson. C. E. Jr.. and Shani. M. (1995). The distal human myoD enhancer sequences direct unique muscle-specific patterns of lacZ expression during mouse development. Dev. Biol. 171, 27-38. Fan. C. M., and Tessier-Lavigne. M. (1994). Patterning of mammalian somites by surface ectoderm and notochord: Evidence for sclerotome induction by a hedgehog homolog. Cell (Cambridge. Mass.) 79, 11751186. Farrance, I. K., Mar. J. H., and Ordahl. C. E (1992). M-CAT binding factor is related to the SV40 enhancer binding factor. TEF-1. J. Biol. Chem. 267, 17234-17240. Ferrari, G., Cusella-De Angelis. G.. Coletta. M.. Paolucci. E.. Stornaiuolo, A.. Cossu. G.. and Mavilio. F. (1998). Muscle regeneration by' bone marrow-derived myogenic progenitors. Science 279, 1528-1530. Franz, T.. Kothary'. R., Surani. M. A., Halata, Z.. and Grim, M. (1993). The Splotch mutation interferes with muscle development in the limbs. Anat. Embo'ol. 18"7, 153-160. Fuchs, E., and Segr4, J. A. (2000). Stem cells: A new lease on life. Cell (Cambridge. :'class.) I(R}, 143 - 155. Fujisawa-Sehara, A., Nabeshima, Y., Hosoda. Y., and Obinata, T. (1990). Myogenin contains two domains conserved among myogenic factors. J. Biol. Chem, 265, 15219-15223. Gee, H. (1994). Vertebrate morphology'. Return of the amphioxus. Nature (London) 370, 504-505. George-Weinstein. M., Gerhart. J.. Reed, R.. Flynn, J.. Callihan. B.. Mattiacci. M.. Miehle, C., Foti, G.. Lash, J. W:. and Weintraub. H. (1996). Skeletal myogenesis: The preferred pathway of chick embryo epiblast cells in vitro. Dev. Biol. 173, 279-291. George-Weinstein. M.. Gerhart, J.. Blitz. J.. Simak, E., and Knudsen. K. A. (1997). N-cadherin promotes the commitment and differentiation of skeletal muscle precursor cells. Dev. Biol. 185, 14-24. Godsave, S. F., and Slack, J. M. (1991). Single cell analysis of mesoderm formation in the Xenopus embr?o. Development (Cambridge. UK) 111, 523-530. Goldhamer, D. J., Faerman, A., Shani, M., and Emerson, C. P., Jr. (1992). RegulatoD' elements that control the lineage-specific expression of myoD. Science 256, 538-542. Goldhamer, D. J., Brunk, B. E. Faerman. A.. King, A., Shani. M.. and Emerson. C. P., Jr. (1995). Embryonic activation of the myoD gene is regulated by' a highly" conserved distal control element. Development (Cambridge. UK) 121, 637-649. Goulding, M.. Lumsden, A., and Paquette, A. J. (1994). Regulation of Pax3 expression in the dermomyotome and its role in muscle development. Development (Cambridge, UK) 120, 957-971. Grass, S., Arnold, H. H., and Braun, T. (1996). Alterations in somite patterning of Myf-5-deficient mice: A possible role for FGF-4 and FGF-6. Development (Cambridge. UK) 122, 141 - 150. Grobet, L., Martin, L. J., Poncelet, D., Pirottin, D., Brouwers, B., Riquet, J., Schoeberlein, A.. Dunner, S.. Menissier. F., Massabanda, J.. Fries, R.. Hanset, R., and Georges, M. (1997). A deletion in the bovine myostatin gene causes the double-muscled phenoty,pe in cattle. Nat. Genet. 17, 71-74. Gross, M. K., Moran-Rivard, L., Velasquez, T., Nakatsu, M. N., Jagla, K., and Goulding, M. (2000). Lbxl is required for muscle precursor migra-

!1 Lineage Specification and Differentiation tion along a lateral pathway into the limb. Development (Cambridge, UK) 127, 4 1 3 - 424. Grounds, M. D., Garrett. K. L., and BeilharT., M. W. (1992). The transcription of MyoD1 and myogenin genes in thymic cells in vivo. Exp. Cell Res. 198, 357-361. Gundersen, K. (1998). Determination of :muscle contractile properties: The importance of the nerve. Acta Physiol. Scand. 162, 333-34 I. Gurdon. J. B. (1988). A community effect in animal development. ?v~ture (London2 336, 772-774. Gurdon. J. B., Lemaire. E, and Kato. K. (1993a). Community effects and related phenomena in development. Cell (Cambridge, Mass. ) 75, 831-834. Gurdon, J. B., Kato, K.. and Lemaire. E (1993b). The community effect, dorsalization and mesoderm induction. Curr. Opin. Genet. Dev. 3, 6 6 2 667. Gussoni, E., Soneoka, Y.. Strickland, C. D.. Buzney. E. A.. Khan. M. K., Flint, A. F., Kunkel. L. M., and Mulligan, R. C. (1999). Dystrophin expression in the mdx mouse restored by' stem cell transplantation. Nature (London) 401,390-394. Harmon. K.. Smith. C. K. D.. Bales, K. R.. and Santerre, R. E (1992). Temporal and quantitative analysis of myogenic regulatow and growth factor gene expression in the developing mouse embr?'o. Dev. Biol. 151, 137-144. Hasty. E. Bradley. A.. Morris. J. H.. Edmondson. D. G., Venuti. J. M., Olson. E. N.. and Klein. W. H. (1993). Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature (London) 364, 501-506. Hauschka, S. D. (1996). The embDonic origin of muscle. In "Myology" (A. G. Engel and A. Franzini-.~a-mstrong, eds.), pp. 3-73. McGraw-Hill. Heanue. T. A.. Reshef. R.. Davis, R. J., Mardon. G.. Oliver, G.. Tomarev, S., Lassar. A. B.. and Tabin, C. J. (1999). Synergistic regulation of vertebrate muscle development by Dach2. Eya2, and Six i. homologs of genes required for Drosophila eye formation. Genes Dev. 13, 3231-3243. Heymann, S., Koudrova, M., ,Arnold. H., Koster, M., and Braun, T. (1996). Regulation and function of SFIHGF during migration of limb muscle precursor cells in chicken. Dev. Biol. 180, 566-578. Hidaka. K.. Yamarnoto, I., Arai, Y., and Mukai, T. (1993). The MEF-3 motif is required for MEF-2-mediated skeletal muscle-specific induction of the rat aldolase A gene. Mol. Cell. Biol. 13, 6469-6478. Hinterberger, T. J., Sassoon. D. A., Rhodes, S. J.. and Konieczny, S. F. ( 1991 ). Expression of the muscle regulatow factor MRF4 during somite and skeletal myofiber development. Dev. Biol. 147, 144-156. Hirsinger, E.. Duprez, D., Jouve, C., Malapert. P., Cooke, J., and Pourqui& O. (1997). Noggin acts downstream of Wnt and Sonic Hedgehog to antagonize B MP4 in avian somite patterning. Development (Cambridge. UK) 124, 4605-4614. Holt, C. E., Lemaire, E, and Gurdon. J. B. (1994). Cadherin-mediated cell interactions are necessary for the activation of MyoD in Xenopus mesoderm. Proc. Natl. Acad. Sci. US.A. 91, 10844-10848. Holtzer, H.. Schultheiss, T.. Dilullo, C., Choi, J., Costa, M., Lu. M., and Holtzer. S. (1990). Autonomous expression of the differentiation programs of cells in the cardiac and skeletal myogenic lineages. Ann. N.Y. Acad. Sci. 599, 158-169. Houzelstein, D., Auda-Boucher, G., Cheraud, Y., Rouaud. T., Blanc, I., Tajbakhsh, S., Buckingham, M. E., Fontaine-Perus, J., and Robert, B. (1999). The homeobox gene Msxl is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimb. Development (Cambridge, UK) 126, 2689-2701. Hughes. S. M., and Blau, H. M. (1992). Muscle fiber pattern is independent of cell lineage in postnatal rodent development. Cell (Cambridge. Mass.) 68, 659-671. Hughes, S. M., and Salinas, E C. (1999a). Control of muscle fibre and motoneuron diversifcation. Curr. Opin. Neurobiol. 9, 54-64. Hughes. S. M., Taylor, J. M., Tapscott. S. J., Gurley, C. M., Carter, W: J., and Peterson. C. A. (1993). Selective accumulation of MyoD and myo-

13 Regulation of Skeletal Muscle Development genin mRNAs in fast and slow adult skeletal muscle is controlled bv inne~'ation and hormones. Development (Cambridge. UK) 118, 11371147. Hughes. S. M.. Koishi. K. Rudnicki, M.. and Maggs. A. M. (1997). MyoD protein is differentially, accumulated in fast and slow skeletal muscle fibres and required for normal fibre type balance in rodents. Mech. Dev. 61, 151-i63. Hughes. S. M.. Chi. M. M.. LowQ ~. O. H.. and Gundersen, K. (1999b). Myogenin induces a shift of enzyme activity' from glycolytic to oxidative metabolism in muscles of transgenic mice. J. Cell Biol. 145, 633-642. Ikeya. M.. and Takada. S. (1998). Wnt signaling from the dorsal neural tube is required for the formation of the medial dermomyotome. Development (Cambridge. UK) 125, 4969-4976. Ingham. R W. (1994). Pattern formation. Hedgehog points the way. Curt'. Biol. 4, 347-350. Irintchev, A.. Zeschnigk. M.. Starzinski-Powi~. A.. and Wernig. A. (1994). Expression pattern of M-cadherin in normal, denervated, and regenerating mouse muscles. Dev. Dvn. 199, 326-337. Jackson. K. A.. Mi. T.. and Goodell. M. A. (1999). Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 96, 14482-1'4486. Johnson. R. L.. Laufer. E.. Riddle. R. D.. and Tabin. C. (!994). Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites. Cell (Cambridge. Mass.) 79, 1165-i 173. Kablar. B.. Krastel. K.. Ying. C.. Asakura. A., Tapscott. S. J.. and Rudnicki. M. A. (1997). MyoD and Myf-5 differentially regulate the development of limb versus trunk skeletal muscle. Development (Cambridge, UK) 124, 4729-4738. Kablar. B.. Asakura. A.. Krastel. K., Ying. C.. May. L. L.. Goldhamer. D. J., and Rudnicki. M. A. (1998). MyoD and Myf-5 define the specification of musculature of distinct embQ'onic origin. Biochem. Cell. Biol. 76, 1079-1091. Kablar. B.. Krastel, K.. Ying. C.. Tapscott. S. J.. Goldhamer. D. J.. and Rudnicki. M. A. (1999). Myogenic determination occurs independently in somites and limb buds. Dev. Biol. 21)6, 219-231. Kablar. B.. Tajbakhsh. S.. and RudnickS. M. A. (2000). Transdifferentiation of esophageal smooth to skeletal muscle is myogenic bHLH factordependent. Development (Cambridge. UK) 127, I627-1639. Kahane. N.. Cinnamon. Y.. and Kalcheim. C. (1998a). The origin and fate of pioneer myotomal cells in the avian emb~,o. Mech. Dev. 74, 59-73. Kahane. N.. Cinnamon. Y.. and Kalcheim, C. (1998b). The cellular mechanism by which the dermomyotome contributes to the second wave of myotome development. Development (Cambridge, UK) 125, 42594271. Kalcheim. C.. Cinnamon. Y.. and Kahane. N. (19991). Myotome formation: A multistage process. Cell Tissue Res. 296, 161 - 173. Katagiri. T.. Yamaguchi, A.. Komaki. M.. Abe. E.. Takahashi, N.. Ikeda. T., Rosen. "v:, Wozney, J. M.. Fujisawa-Sehara. A., and Suda. T. (1994). Bone morphogenetic protein-2 converts the differentiation pathway of C2C 12 myoblasts into the osteoblast lineage. J. Cell Biol. 127, 17551766: erratum: Ibid. Kato. K.. and Gurdon. J. B. (1993). Single-cell transplantation determines the time when Xenopus muscle precursor cells acquire a capacity for autonomous differentiation. Proc. Natl. Acad. Sci. L~S.A. 90, 13101314. Kaufman. M. H. (1992). "'The Atlas of Mouse Development." Academic Press. San Diego. Kopan. R., and Cagan. R. (1997). Notch on the cutting edge. Trends Genet: 13, 465 -467. Kopan. R.. Nye. J. S.. and Weintraub. H. (1994). The intracellular domain of mouse Notch: A constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development (Cambridge. UK) 120, 2385-2396. Lassar. A. B.. Davis. R. L.. Wright, ~: E., Kadesch, T.. Murre, C.. Voron-

275 ova, A., Baltimore. D., and Weintraub, H. (1991). Functional activity of myogenic HLH proteins requires hetero- oligomerization with E12/E47like proteins in vivo. Ceil (Cambridge. Mass.) 66, 305-315. Lee, S. J., and McPherron. A. C. (1999). Myostatin and the control of skeletaI muscle mass. Curt. Opin. Genet. Dev. 9, 604-607. Lin. C. H.. Hudson, A. J., and Strickland, K. R (1969). Fatty acid metabolism in dystrophic muscle in vitro. Life Sci. 8, 21-26. / Lu. B. D., Allen, D. L., Leinwand, L. A., and Lyons, G. E. (1999). Spatial and temporal changes in myosin heavy chain gene expression in skeletal muscle development. Dev. Biol. 216, 312-326. Luo. K.. Stroschein, S. L., Wang, W.. Chen, D., Martens. E., Zhou, S., and Zhou. Q. (1999). The Ski oncoprotein interacts with the Smad proteins to repress TGFbeta signaling. Genes Dev. 13, 2196-2206. Lyons, G. E.. and Buckingham. M. E. (1992). Developmental regulation of myogenesis in mouse. Semin. Dev. Biol. 3, 243-253. Lyons, G. E.. Ontell, M., Cox, R., Sassoon, D., and Buckingham, M. (1990). The expression of myosin genes in developing skeletal muscle in the mouse emb~'o. J. Cell BioL 111, 1465-1476. Maina, E, Casagranda. E, Audero, E., Simeone, A., Comoglio, R M., Klein. R.. and Ponzetto, C. (1996). Uncoupling of Grb2 from the Met receptor in vivo reveals complex roles in muscle development. Cell (Cambridge, Mass.) 87, 53!-542. Mankoo, B. S., Collins. N. S., Ashby, R, Grigorieva, E., Pevny, L. H., Candia. A., Wright, C. V., Rigby, R W., and Pachnis, V. (1999). Mox2 is a component of the genetic hierarchy controlling limb muscle development. Nature (London)4i)i), 69-73. Marcetie. C.. Stark. M. R., and Bronner-Fraser, M. (1997). Coordinate actions of BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite. Development (Cambridge, UK) 124, 3955-3963. Marigo, V., Johnson, R. L., Vortkamp, A., and Tabin, C. J. (1996a). Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev. Biol. 180, 273-283. Maroto. M., Reshef, R., Munsterbe~. A. E., Koester, S., Goulding, M., and Lassar. A. B. (1997). Ectopic Pax-3 activates MyoD and Myf-5 expression in emb~'onic mesoderm and neural tissue. Cell (Cambridge, Mass.) 89, I39-148. McMahon, J. A., Takada, S., Zimmerman, L. B., Fan, C. M., Harland, R. M.. and McMa~hon, A. R (1998). Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes Dev. 12, 1438-1452. McPherron, A. C.. and Lee, S. J. (1997). Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. US.A. 94, 12457-12461. McPherron. A. C.. Lawler, A. M., and Lee, S. J. (1997). Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature (London) 387, 83-90. Megeney, L. A.. and Rudnicki. M. A. (1995). Determination versus differentiation and the MyoD family of transcription factors. Biochem. Cell. Biol. 73, 723-732. Megeney. L. A.. Kablar. B., Garrett, K., Anderson, J. E., and Rudnicki, M. A. (1996). MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev. 10, 1173-1 t 83. Mennerich, D.. Schafer. K.. and Braun, T. (1998). Pax-3 is necessary but not sufficient for lbxl expression in myogenic precursor cells of the limb. Mech. Dev. 73, 147-158. Merlino. G.. and Helman, L. J. (1999). Rhabdomyosarcoma--working out the pathways. Oncogene 18, 5340-5348. Miller, J. B.. and Stockdale, E E. (1986a). Developmental regulation of the multiple myogenic cell lineages of the avian emb~o. J. Cell Biol. 103, 2197-2208. Miller. J. B., and Stockdale, E E. (1986b). Developmental origins of skeletaI muscle fibers: Clonal analysis of myogenic cell lineages based on expression of fast and slow myosin heavy chains. Proc. Natl. Acad. Sci. US.A. 83, 3860-3864.

276 Mohun, T. J., Chambers, A. E., Tov,ers, N., and Taylor, M. V. (1991). Expression of genes encoding the transcription factor SRF during early' development of Xenopus Iaevis: Identification of a CArG box-binding activity as SRF. EMBOJ. 10, 933-940. Molkentin. J. D.. and Olson. E. N. (1996). Defining the regulatory' networks for muscle development. Curt. Opm. Genet. Dev. 6, 445-453. Munsterberg, A. E., and Lassar, A. B. (1995). Combinatorial signals from the neural tube. floor plate and notochord induce myogenic bHLH gene expression in the somite. Development (Cambridge. UK) 121, 651-660. Munsterberg, A. E., Kitajewski, J., Bumcrot, D. A., McMahon, A. R, and Lassar, A. B. (1995). Combinatorial signaling by Sonic hedgehog and Wnt family' members induces myogenic bHLH gene expression in the somite. Genes Dev. 9, 291 i-_"9 "~'~.~.. Murre, C., ,X.IcCaxv, R S.. and Baltimore. D. (1989a). A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins, Cell (Cambridge. Mass. ~ 56, 777-783. Murre, C., McCaxv, R S.. \:aessin. H., Caudv, M., Jan. L. Y., Jan. Y. N., Cabrera, C. V.. Buskin. J. N., Hauschka. S. D.. Lassar, A. B. et al. (1989b). Interactions between heterotogous helix-loop-helix proteins generate complexes that bind specifically' to a common DNA sequence. Cell (Cambridge. Mass. j58. 537-544. Nabeshima. Y.. Hanaoka. K.. Havasaka. M.. Esumi. E.. Li. S.. and Nonaka. I. (!993). Myogenin gene disruption results in perinatal lethalitx because of severe muscle detect. Nature (Londonj 364, 53 ,"~- 3 -'~_3. Neyt. C.. Jagla. K.. Thisse. C., Thisse. B.. Haines. I_,., and Currie. P. D. (2000). Evolutionary, origins of vertebrate appendicular muscle. Nature (London) 408, 82 - 86. Noden. D. M. ( 1991 ~,. Ve~ebrate craniofacial development: The relation between ontogenetic process and morphological outcome. Brain. Behav. Evol. 38. 190-225. Norris, W.. Neyt. C.. In(ham. P. W.. and Currie, R D. (2000). Slow muscle induction bx Hedgehog sio_nailino_~ ~ in vitro. J. Cell Sci. 113, 2695-_70_~." " Olson, E. N., and \Vitliams, R. S. i2000). Remodeling muscles with calcineurin. BioEssavs 22, 510-519. Olson, E. N., Pen3, M., and Schulz, R. A. (1995). Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors. Dev. Biol. 172, 2 - 14. Olson, E. N., Arnold, H. H.. Rigby, R W., and Wold, B. J. (1996). Know your neighbors: Three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell (Cambridge. Mass.) 85, 1-4. Ordahl. C. P. (1999'!. Myogenic shape-shifters. J. Cell Biol. 147, 6 9 5 - 698. Ordahl. C. P.. and Le Douarin. N. M. (1992). Two myogenic lineages within the developing somite. Development (Cambridge. UK) 114, 339-353. Ordahl, C. R, and W'illiams. B. A. (1998). Knowing chops from chuck: Roasting myoD redundancy. BioEssavs 20, 357-362. Orkin. S. H. (2000). Stem cell alchemy. ,\~t..~Ied. 6, 1212-1213. Ott. M. O.. Bober. E.. Lyons. G...Arnold. H., and Buckingham. M. (1991). Early expression of the myogenic regulatory gene. myf-5, in precursor cells of skeletal muscle in the mouse embryo. Development (Cambridge. UK) 111, 1097-1107. Patapoutian, A., Yoon, J. K., Miner, J. H., Wang. S., Stark. K., and Wold. B. (1995). Disruption of the mouse MRF4 gene identifies multiple waves of myogenesis in the myotome. Development (Cambridge. UK) 121, 3347-3358. Pourquir. O., Coltey, M.. Teillet. M. A., Ordahl. C.. and Le Douarin. N. M. (1993). Control of dorsoventral patterning of somitic derivatives bv notochord and floor plate. Proc. ,~tl. Acad. Sci. 6'~S..4. 90, 5242-5246. Pourquir. O., Coltey, M.. Brrant. C., and Le Douarin, N. M. (1995). Control of somite patterning by signals from the lateral plate. Proc. :Vatl. Acad. Sci. (iS..4.92, 3 "~19 "~'~" Pourqui6. O.. Fan. C. M.. Coltev. M.. Hirsinger. E.. Watanabe. Y.. Brdant. C.. Francis-West. R. Brickell. R. Tessier-Lavigne. M.. and Le Douarin. N. M. (1996). Lateral and axial signals involved in avian somite patterning: A role for BMP4. Ceil (Cambridge. Mass.) 84, 461-471. Pownall. M. E.. Strunk. K. E., and Emerson. C. R. Jr. (1996). Notochord

!1 Lineage Specification and Differentiation signals control the transcriptional cascade of myogenic bHLH genes in somites of quail embryos. Development (Cambridge, UK) 122, 1475I488. Rawls. A.. Morris. J. H.. Rudnicki. M., Braun. T . . . ~ n o l d . H. H.. Klein. \V. H.. and Olson. E. N. (1995). Myogenin's functions do not overlap with those of MyoD or Myf-5 during mouse embD, ogenesis. Dev. Biol. 172, 37-50. Rawls. A.. Valdez. M. R.. Zhang. \~,:. Richardson. J.. Klein. W. H., and Olson, E. N. (19981). Overlapping functions of the myogenic bHLH genes MRF4 and MyoD revealed in double mutant mice. Development (Cambridge. UK) 125, 2349-2358. Retaix. E, and Buckingham. M. (1999). From insect eye to vertebrate muscle: Redeployment of a regulator' network. Genes Dev. 13, 31713178. Reshef. R., Maroto, M.. and Lassar. A. B. (1998). Regulation of dorsal somitic cell fates: BMPs and Noggin control the timing and pattern of myogenic regulator expression. Genes Dev. 12, 290-303. Robson. L. G., and Hughes. S. M. (1996). The distal limb environment regulates MyoD accumulation and muscle differentiation in mousechick chimaeric limbs. Development (Cambridge. UK) 122, 3899-3910. Robson. L. G.. and Hughes. S. M. (1999). Local signals in the chick limb bud can override myoblast lineage commitment: Induction of slow myosin heavy chain in fast mvoblasts. Mech. Dev. 85, 59-71. Ross. S. E.. Hemati. N.. Longo. K. A.. Bennett. C. N.. Lucas, R C.. Erickson. R. L.. and MacDougald. O. A. (2000). Inhibition of adipogenesis by Wnt signaling. Science 289, 950-953. Rudnicki. M. A.. Braun. T.. Hinuma. S.. and Jaenisch. R. (1992). Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell (Cambridge. Mass.) 71,383-390. Rudnicki. M. A.. Schnegetsberg, R N.. Stead, R. H.. Braun, T., :Mnold, H. H.. and Jaenisch. R. (1993). MyoD or Myf-5 is required for the formation of skeletal muscle. Cell (Cambridge. Mass.) 75, 135 t - 1359. Sabourin. L. A.. Girgis-Gabardo. A.. Seale. R, Asakura. A.. and Rudnicki, M. A. (1999). Reduced differentiation potential of primary M y o D - / my'ogenic cells derived from adult skeletal muscle. J. Cell Biol. 144, 631 - 643. Salminen. M.. Lopez, S., Maire. R. Kahn. A.. and Daegelen. D. (1996). Fast-muscle-specific DNA-protein interactions occurring in vivo at the human aldolase A M promoter are necessary for correct promoter activity in transgenic mice. Mol. Cell. Biol. 16, 76-85. Sassoon. D., Lyons. G.. Wright, W. E., Lin. V.. Lassar, A., Weintraub, H., and Buckingham, M. (1989). Expression of two myogenic regulatory,, factors myogenin and MyoD1 during mouse embryogenesis. Nature (London) 341,303-307. Scaal. M.. Bonafede. A.. Dathe, V.. Sachs. M.. Cann. G., Christ. B., and Brand-Saberi. B. (1999). SF/HGF is a mediator between limb patterning and muscle development. Development (Cambridge. UK)126, 48854893. Schafer, K., and Braun, T. (1999). Early specification of limb muscle precursor cells by the homeobox gene Lbxlh. Nat. Genet. 23, 213-216. Seale, P., and Rudnicki, M. A. (2000a). A new look at the origin, function. and "'Stem-Cell'" status of muscle satellite cells. Dev. BioL 218, 115-124. Seale. P.. Sabourin, L. A.. Girgis-Gabardo, A., Mansouri. A., Gruss, P., and Rudnicki, M. A. (2000). Pax7 is required for the specification of myogenic satellite cells. Cell (Cambridge. Mass.) 102, 777-786. Seed. J.. and Hauschka. S. D. (19841), Temporal separation of the migration of distinct myogenic precursor populations into the developing chick wing bud. Dev. Biol. 106, 389-393. Shafritz, A. B., Shore, E. M., Gannon, F. H., Zasloff, M. A., Taub, R,, Muenke, M., and Kaplan, E S. (1996). Overexpression of an osteogenic morphogen in fibrodysplasia ossificans progressiva. N. EngI. J. Med. 335, 555-561. Shawber, C,, Nofziger, D., Hsieh, J. J., Lindsell, C., Bogler, O., Hayward, D,,

13 Regulation of Skeletal Muscle Development and V~'einmaster. G. (1996). Notch signaling inhibits muscle cell differentiation through a CBFl-independent pathway. Development (Cambridge. UK) 122, 3765-3773. Smith. C. K.. 2nd. Janney, M. J.. and Allen. R. E. (1994). Temporal expression of myogenic regulatory. genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells. A Cell. PhvsioL 159, 379-385. Smith. T. H.. Kachinsky. A. M.. and Miller. J. B. (1994). Somite subdomains, muscle cell origins, and the tour muscle regulatory factor proteins. J. Cell Biol. 127, 95-t05. Spitz. F.. Salminen, M., Demignon. J.. Kahn. A.. Daegelen. D.. and Maire. R (1997). A combination of MEF3 and NFI proteins activates transcription in a subset of fast-twitch muscles. MoI. Cell. Biol. 17, 656-666. Spitz. F.. Demignon. J.. Porteu. A.. Kahn, A., Concordet. J. R, Daegeten. D., and Maire, R (1998). Expression of myogenin during embry'ogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site. Proc. Natl. Acad. Sci. 6:S.A. 95, 14220-14225. Sporle. R.. Gunther. T.. Struwe. M.. and Schughart. K. (1996). Severe defects in the formation of epaxial musculature in open brain (opb) mutant mouse embryos. Development (Cambridge, UK) 122, 79-86. Stern. H. M.. and Hauschka. S. D. (1995). Neural tube and notochord promote in vitro myogenesis in single somite explants. Dev. Biol. 167, 87-103. Stern. H. M.. Brown. A. M.. and Hauschka. S. D. (1995). Myogenesis in paraxial mesoderm: Preferential induction by dorsal neural tube and by cells expressing Wnt-1. Development (Cambridge, UK) 121, 36753686. Stewart. A. F.. Larkin. S. B.. Farrance. I. K. Mar. J. H.. Hall. D. E.. and Ordahl. C. R (1994). Muscle-enriched TEF-I isoforms bind M-CAT elements from muscle-specific promoters and differentially activate transcription. J. Biol. Chem. 269, 3 I47-3 t50. Stockdale. F. E. (1992). Myogenic cell lineages. Dev. Biol. 154, 284-298. Subramanian, S. "v:. and Nadal-Ginard, B. (1996). Early expression of the different isotbrms of the m,vocyte enhancer factor-2 (MEF2) protein in myogenic as well as nonmyogenic cell lineages during mouse embryogenesis. Mech. Dev. 57, 103 - I 12. Tajbakhsh, S., Vivarelli, E.. Cusella-De Angelis. G.. Rocancourt. D.. Buckingham. M., and Cossu. G. (I 994). A population of myogenic cells derived from the mouse neural tube. Neuron 13, 8 t 3 - 8 2 I . Tajbakhsh. S.. Rocancourt. D.. Cossu. G.. and Buckingham. M. (1997). Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Celt (Cambridge. Mass.) 89, 127138. Tajbakhsh. S., Borello. U.. Vivarelli. E.. Kelly'. R.. Papkoff. J.. Duprez. D., Buckingham. M.. and Cossu, G. (1998). Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development (Cambridge. UK) 125, 4155-4162. Talmadge, R. J. (2000). Myosin heavy chain isoform expression following reduced neuromuscular activity: Potential regulatow mechanisms. Muscle Ner't'e 23, 661-679. Tapscott. S. J.. Davis, R. L.. Thayer, M. J., Cheng, R R. Weintraub. H.. and Lassar, A. B. (1988). MyoDl: A nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. Science 242, 405-411. Teboul, L., Gaillard, D.. Staccini. L.. Inadera. H., Amri. E. Z.. and Grimaldi. P. A. (1995). Thiazolidinediones and fatty acids convert myogenic cells into adipose- like cells. J. Biol. Chem. 270, 28183-28187. Teillet. M.. Watanabe. Y.. Jeffs, P.. Duprez, D.. Lapointe. E. and Le Douarin. N. M. (1998). Sonic hedgehog is required for survival of both myogenic and chondrogenic somitic lineages. Development (Cambridge, UK) 125, 2019-2030. Trainor. P. A.. Tan, S. S., and Tam. P. P. (1994). Cranial paraxial mesoderm: Regionalisation of cell fate and impact on craniofacial development in mouse embr'yos. Development (Cambridge, UK) 120, 2397-2408.

277 Tremblay, R. Dietrich. S.. Mericskay. M., Schubert. R R., Li. Z.. and Paulin. D. (1998). A crucial role for Pax3 in the development of the hypaxial musculature and the tong-range migration of muscle precursors. Dev. Biol. 203, 4 9 - 6 !. Urist M. R., and Strates. B. S. (197t). Bone morphogenetic protein. J. Dent. Res. 5,0, 1392-1406. Valdez. M. R.. Richardson. J. A.. Klein. "~,s H., and Olson. E. N. (2000). Failure of Myf5 to support myogenic differentiation without myogenin. MyoD. and MRF4. Dev. Biol. 219, 287-298. "Van Swearingen. J., and Lance-Jones. C. (1995). Slow and fast muscle fibers are preferentially derived from myoblasts migrating into the chick limb bud at different developmental times. Dev. Biol. 170, 32t-337. Venuti. J. M.. Morris. J. H.. Vivian. J. L., Olson, E. N., and Klein. W. H. (1995). Myogenin is required tor late but not early aspects of myogenesis during mouse development. J. Cell Biol. 128, 563-576. Wada. H.. Holland. R "~,'n. 216, 411-419. Whitfield. L. S.. Lovetl-Badge. R.. and Goodfellow. R N. (1993). Rapid sequence evolution of the mammalian sex-determining gene SRY. Nature (London) 364, 713-715. Whitworth. D. J.. Licht R, Racey. R A.. and Glickman. S. E. (1999). Testislike steroidogenesis in the ovotestis of the European mole, Talpa europaea. Biol. Reprod. 60, 413-418. Wilkins, A. S. (1995). Moving up the hierarchy: A hypothesis on the evoIution of a genetic sex determination pathway. BioEssays 17, 71-77. Wunderle, V. M., Critcher. R., Hastie, N.. Goodfellow. R N.. and Schedl, A. (1998). Deletion of long-range regulatory, elements upstream of SOX9 causes campomelic dysplasia. Proc. ,~;atl. Acad. Sci. U.S.A. 95, 1064910654. Yi, "v'v:.and Zarkower, D. (I999). Similarity of DNA binding and transcriptional regulation by Caenorhabditis elegans MAB-3 and Drosoz)hila melanogaster DSX suggests conser~'ation of sex determining mechanisms. Development (Cambridge, UK) 126, 873-88I. Yu. B. D., Hess. J. L., Homing. S. E., Brown, G. A., and Korsmeyen S. J. (1995). Altered Hox expression and segmental identity in Mll-mutant mice. Nature (London) 378, 505-508. Yu, R. N.. Ito. M.. Saunders. T. L.. Camper, S. A.. and Jameson, J. L. (1998). Role of Ahch in gonadal development and gametogenesis. Nat. Genet. 20, 353-357. Zanaria, E., Muscatelli. F., Bardoni. B., Strom, T. M., Guioli, S., Guo, W:, Lalli, E.. Moser. C., Walker, A. R, McCabe, E. R. et aL (1994). An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature (London) 372, 635641. Zazopoutos, E., Lalli, E., Stocco, D. M., and Sassone-Corsi. R (I 997). DNA binding and transcriptional repression by DAX- 1 blocks steroidogenesis. Nature (London) 390, 311 - 315. Zhu, L., Belo. J. A., De Robertis, E. M.. and Stern, C. D. (1999). Goosecoid regulates the neural inducing strength of the mouse node. Dev. Biol. 216, 276-281. Zimmermann, S.. Steding, G., Emmem J. M., Brinkmann. A. O., Nayernia, K.. Holstein, A. 1=., Engel, W. and Adham, I. M. (1999). Targeted disruption of the Inst3 gene causes bilateral cwptorchidism. Mol. EndocrinoL 13, 681-69 I.

Development of the Excretory S},stem G r e g o r y R, Dressier

Department of Pathology, Universi~"of Michigan, Ann Arbor, Michigan 48109

I. Introduction II. Patterning of the Intermediate Mesoderm !11. G r o w t h of the Nephric Duct a n d Ureteric Bud Diverticulum IV. Inductive Interactions

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V. Mesenchyme-to-Epithelial Conversion VI. Glomerular D e v e l o p m e n t and Vascularization Vii. D e v e l o p m e n t a l Basis of H u m a n Renal Disease ViIi. Future Perspectives References 9

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i, I n t r o d u c t i o n

Perhaps more so than any other tissue, the development of the urogenital system in higher vertebrates illustrates quite clearly how "ontogeny follows phylogeny." In mammals, the embryonic kidney follows a developmental progession that mimics its evolutionary origin, proceeding through a series of transition structures before generating the Mouse Development

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adult kidney. Although detailed embryology and anatomy can be gleaned from a variety of excellent sources (Carlson, 1988; Kaufman, 1992), this chapter provides a brief introduction to the basic design of the mouse urogenital system and the relationships between the parts. In all mammals, the excretory system begins as a single epithelial duct formed from the intermediate mesoderm, a region between the paraxial, or sorrfitic, mesoderm and the lateral plate mesoderm (Fig. 1). As this nephric duct extends caudally, it induces a linear array of epithelial tubules, which extend medioventrally and are thought to derive from periductal mesenchyme. In mammals, a graded evolution of renal tubule development occurs with the most anterior, or pronephric tubules, being very rudimentary, and the more posterior, mesonephric tubules becoming well developed. In contrast, the pronephros of the zebrafish larvae is a fully de",'eloped, functional filtration unit with a single m_idline glomerulus (Drummond et al., 1998). Amphibian embryos such as Xenopus laevis have bilateral pronephric glomeruli and tubules that are functional until replaced by a mesonephric kidney in the tadpole (Vize et aI., 1997). In fact, it is not altogether obvious in mammals where to draw the distinction between pronephric tubules and mesonephric tubules. Although mature mesonephric tubules are characterized by a vascularized glomerulus at the proximal end of the tubule that empties into the nephric duct, the most anterior and Copyright 9 2002 Academic Press All rights of reproduction in any form reserved.

396

ill Organogenesis

Figure 1 The three stages of kidney development in the mouse. Between E9 and El3. the three embry'onic kidneys are formed in a sequential manner along the anterior-posterior axis..-ks the primary' nephric duct extends caudally, pro- and mesonephric tubules are induced along the periductal mesenchyme. By' El0, the nephric duct has reached the cloaca, the mesonephros is present as a linear array of tubules, and the metanephric mesenchyme remains uninduced at the posterior end. Bv El0.5, ureteric bud growth becomes evident and invasion into the mesenchyme is complete by El 1.5, In response to inductive signals, the metanephric mesenchyme aggregates around the ureteric bud tips. In turn, the ureteric bud undergoes repeated dichotomous branching morphogenesis. By E13, the M/,illerian duct is observed running parallel to the posterior nephric duct. The male mesonephric tubules, adjacent to the testis, will form the epididymis and vas deferens, as the Miillerian duct degenerates. The female MiJllerian duct will form the oviduct and uterus, while the nephric duct and mesonephric tubules have completely degenerated. posterior mesonephric tubules are more rudimentary, with the posterior tubules not connected to the duct at all. The adult kidney, or metanephros, is formed at the caudal end of the nephric duct when an outgrowth, called the ureteric bud or metanephric diverticulum, extends into the surrounding metanephric mesenchyme. This process begins at E 10.5 and requires signals emanating from the mesenchyme for bud initiation. Inductive signals from the bud begin the process of converting the metanephric mesenchyme to epithelium. Mesenchymal cells ag~egate around the tips of the bud. form a primitive polarized epithelial vesicle, and undergo a well-defined morphological series of events leading to the mature nephron (Fig. 2). Reciprocal inductive signals, derived from the mesenchyme, promote growth and branching of the ureteric bud. Branching is dichotomous and resuits in new aggregates forming at the tips of the branches and further induction of nephrons. This repeated branching and induction results in the formation of nephrons along the radial axis of the kidney, with the oldest nephrons being more medullary, and the younger nephrons located toward the periphew. It was generally believed that most of the epithelium of the nephron was derived from the metanephric mesenchyme, whereas the ureteric bud epithelium generates the collecting ducts and the most distal tubules. As will be discussed in following sections, more precise cell lineage studies have challenged this hypothesis. However, not all the mesenchyme becomes induced and converted to epithelium, with some cells contributing to the interstitial mesenchyme, or

stroma, and others generating endothelial cells of the renal vasculature. Mesenchyme-derived epithelial cells become highly specialized and express markers specific for glomerular podocytes, proximal tubules, cells of the ascending and descending limbs of Henle's loop, and distal tubules. How cells are instructed to differentiate along individual pathways remains obscure. The analysis of gene expression patterns and the generation of many mouse mutants, with distinct renal phenotypes, have provided a wealth of data with respect to early inductive events, mesenchyme-to-epithelium conversion, and branching morphogenesis. Much of the available data are accessible on-line through the Kidney Development Database (http://www.ana.ed.ac.uk/anatomy/data base/kidbase/kidhome.html) (Davies, 1999), a systematic compilation that is searchable and frequently updated.

II. Patterning of the Intermediate Mesoderm The complex pattern of gene expression during kidney development begins with the specification of the intermediate mesoderm, which already expresses a number of unique markers prior to the initiation of urogenital development. Molecular genetic analysis has confirmed the essential functions of a variety of early patterning genes in the intermediate mesoderm. Yet, the genetic network that positions the urogenital tract, restricts the cell lineages of the developing

18 Development of the Excretory System

397

Figure 2 The conversion of metaneph_ricmesenchyme to epithelium. The sequential stages in the formation of the nephron are outlined. Induction by the ureteric bud results in mesenchymal aggregates forming at the bud tips. The aggregates become polarized and form a primitive epithelial renal vesicle. That part of the renal vesicle adjacent to the ureteric bud tip fuses to the collecting duct primordium to form a continuous lumen at the S-shaped body stage. The ureteric bud has grown outward to induce the next generation of nephrons along the radial axis of the kidney. Furthest from the collecting duct, endothelial precursor cells invade the glomerular cleft. The developing nephron is fully elaborated as glomerular vascularization and proximal tubule extension begins. kidney, and remodels the epithelium to generate the precise three-dimensional architecture of the nephron still remains obscure.

A. Early Regionalization Among the two earliest genes expressed as makers of nephric progenitors are liml and Pax2. The lira1 gene encodes a homeobox containing protein that is expressed in the developing head and the intermediates mesoderm, extending along the anterior-posterior axis. At E8.5, liml expression is found in both the somatopleure and the splanchnopleure of the lateral plate mesoderm but becomes restricted to the nephrogenic cord by E9.5 (Tsang et al., 2000). Based on studies using a LacZ-tagged lirnl null allele (Tsang et at., 2000), the gene appears essential for the aggregation of these early mesodermal cells into the nephric cord. Furthermore,

lim l seems to restrict the fate of the mesodermal precursors to preferentially generate intermediate and lateral plate mesoderm. In the absence of liml activity, formation of the nephric duct is very rudimentary and expression of the Pax2 gene is suppressed. The Pax2 gene is also essential for early urogenital development. Pax2 expression is first detected in the intermediate mesoderm, prior to formation of the nephric duct, beginning at approximately E8.5 (Fig. 3). In the chick, transplantation experiments suggest that Pax2 expression and specification of the nephric cord is dependent on signals emanating from the trunk paraxial mesoderm (Mauch et al., 2000). Despite its early expression pattern, Pax2 is not required for initiation and extension of the nephric duct. Mouse mutants lacking Pax2 show an epithelial duct extending caudally toward the cloaca, from E9.5 to E 11.5. However, by E 11.5 the nephric duct starts to degenerate. Pax2 mutants show no evidence

398

!11Organogenesis

Figure 3 Earl 3' expression pattern of Pax2 as marked by Pa.~:2/lacZ transgene. Approximately 4 kb of upstream regulatou' sequences from the mouse Pax2 gene were fused to the Escherichia coli 8" galactosidase (lacZ) coding sequences. The expression of lacZ mimics the early pattern of Pax2 expression, as shown by whole-mount staining for lacZ activity. At E8, approximately 0 - 4 somites, Pax2 expression is prominent in the midbrain-hindbrain (MH) junction. By E8.5 ( 4 - 8 somites), Pax2 expression is detectable in the intermediate mesoderm (IM) prior to nephric duct formation, in the midbrain-hindbrain junction and in and around the optic placode (OP). By E9.5, Pax2 expression in the nephric duct (ND) is evident.

of mesonephric tubule formation, completely lack the metanephros, and are deficient in the sex-specific epithelial components derived from the intermediate mesoderm (Tortes et al., 1995). Thus. the periductal mesenchyme is unable to generate mesonephric tubules or cannot respond to signals derived from the nephric duct. Metanephric mesenchyme isolated from Pax2 mutants is also unable to respond to inductive signals from heterologous tissues in the organ culture assay (G. R. Dressier. unpublished observation). Thus it appears that Pax2 is necessary for specifying the region of intermediate mesoderm destined to undergo mesenchyme-toepithelium conversion. Furthermore. Pax2 mutant nephric duct does not respond to GDNF and shows no evidence of ureteric bud outgowth despite expression of at least some markers of normal ductal epithelium. In the nephric duct epithelium. Pax2 is also required for maintenance, or responsiveness perhaps through regulation of the glial-derived neurotrophic factor (GDNF) receptor, RET. In the developing metanephros, Pax2 expressing cells are closely associated with the ureteric bud tips. This observation led to the proposition that Pax2 expression was activated by inductive signals emanating from the bud (Dressier et al., 1990; Dressier and Douglass, 1992). Further support for the dependence of Pax2 expression on inductive signals came from studies with the Danforth ~ short tail (Sd) mouse, in which Pax2 expression was detected in the nephric duct and ureteric bud but not in the mesenchyme, presumably due to lack of ureteric bud invasion (Phelps and Dressier, 1993). Yet, the primary defect in the Sd mouse is posterior degeneration of the notocord. Thus, gene expression in the intermediate mesoderm could be affected by loss of notocord-derived patterning signals. Until recently, it has proved difficult to separate Pax2 expression in the mesenchyme from ureteric bud invasion, since it is not easy to define the mesenchyme morphologically prior to E11. However, re-

cently we observed Pax2 expression in the metanephric mesenchyme of E11.5 RET homozygous mutants, which have no ureteric bud but essentially wild-type mesenchyme (Fig. 4). Thus. Pax2 expression in the metanephric mesenchyme predates ureteric bud invasion and marks the posterior intermediate mesoderm as the metanephric anlagen. The Pax gene family encodes transcription factors essential for the development of the eye, the vertebral column, certain derivatives of the neural crest, B lymphocytes, the thyroid, and various neural structures (Dahl et al., 1997; Noll. 1993: Stuart et al., 1993). In mice and humans, Pax genes are haploinsufficient, indicating a strict requirement for Pax gene dosage during normal development. The phenotype of heterozygous Pax mutant individuals is variable and less severe than that found among homozygotes. Human mutations in the PAX2 gene have been described. These heterozygous individuals have a variety of renal defects, including small, poorly differentiated kidneys and reflux of urine from the bladder (Sanyanusin et aI., 1995" Schimmenti et at., 1997). Within the mesonephric and metanephric mesenchymal cells along much of the nephric duct, expression of the mouse Eyal gene is prevalent. Eyal is a mammalian homolog of the Drosophila eyes absent gene and is a member of a small gene family. In humans, mutations in the E y a l gene are associated with branchio-oto-renal syndrome, a complex multifaceted phenotype (Abdelhak et al., 1997). In mice homozygous for an Eyal mutation, kidney development is arrested at E 11 because ureteric bud growth is inhibited and the mesenchyme remains uninduced, though Pax2 and WT1 expression appears normal (Xu et al., 1999). However, two other markers of the metanephric mesenchyme, Six2 and G D N F expression, are lost in the Eyal mutants. The loss of G D N F expression most probably underlies the failure of ureteric bud growth (see Section III). However, it is not clear if


399

F i g u r e 4 Pax2 marks the metanephric mesenchymeeven in the absence of inductive signals. The figure shows whole-mount antibody staining for Pax2 (red-orange) and cytokeratins (green) in El i.5 metanephroi. The region in and around the metanephric me~nchyme was dissected from wild-type and RET homozygous null mutants. Note the growth and branching of the ureteric bud. which is absent in the RET mutants. Pax2 protein is evident even in the absence of the ureteric bud. as indicatedby nuclear staining of the metanephric mesenchymeof RET mutants.

the mesenchyme is competent to respond to inductive signals if a wild-type inducer were to be used in vitro. The Eyes Absent gene family is part of a conserved network that underlies cell specification in several other developing tissues. Eya proteins share a conserved domain but lack DNA-binding activity. The Eya proteins interact directly with the Six family of DNA-binding proteins. Mammalian Six genes are homologs of the Drosophila sina oculis homeobox gene. This cooperative interaction between Six and Eya proteins is necessary for nuclear translocation and transcriptional activation of Six target genes (Ohto et al., 1999) (see Fig. 5). Eya proteins in turn can also bind to the dachshund family of proteins. In Drosophila eye development (Pignoni et al., 1997) and during muscle cell specification in the chick embryo (Heanue et al., 1999), the regulatory network involving Pax. Eya, Six. and dachshund genes is well conserved. Based on conservation of expression patterns and mutant phenotypes, a similar regulatory network has been proposed in the developing mouse kidney (Xu et al., 1999), with Pax2 being upstream of Six2 and Eyal, although a dachshund family homolog has not yet been described in the kidney.

B. Posterior Specification of the

Metanephric Mesenchyme

As the mesonephric tubules develop along the anteriorposterior axis of the nephric cord and the nephric duct ex-

tends to the cloaca, the most posterior part of the duct is not associated with mesonephric tubules at all, rather it runs adjacent to an aggregate of cells called the metanephric mesenchyme (Fig. I ). This metanephric mesenchyme is morphologically identifiable by El0.5-11. Even in Pax2 mutants, the metanephric mesenchyme appears as a distinct aggregate of cells apart from the surrounding mesoderm. Several genes are now known to mark this mesenchyme and to play critical roles in the survival and response of this mesenchyme to inductive signals. The Wilms' tumor suppressor gene, WTI, is one of the early markers of the metanephric mesenchyme and is essential for its survival. Wilms' tumor is an embryonic kidney neoplasia that consists of undifferentiamd mesenchymaI cells, poorly organized epithelium, and surrounding stromal cells. Because the neoplastic cells of the tumor are able to differentiate into a wide variety of cell types, the genes responsible for Wilms' tumor were thought to be important regulators of early kidney development (van Heyningen and Hastie, 1992). Expression of WT1 is regulated spatially and temporally in a variety of tissues and is further complicated by the presence of at least four isoforms, generated by alternative splicing. In the developing kidney, WT 1 can be found in the uninduced metanephric mesenchyme and in differentiating epithelium after induction (Armstrong et al., 1992: Pritchard-Jones et al., 1990). Early expression of V~q'l may be mediated by PAX2 (Dehbi et aI., 1996). Initial expression levels are low in the metanephric mesenchyme, but become upregulated at the S-shaped body stage in the precursor cells

400

Iii Organogenesis

Figure

5 Interactions among the Pax, Eya. Six. and dachshund gene families. (A) In the E11 metanephros. Pax2 expression is present in the ureteric bud and the mesenchyme, whereas Six2 and Eyal are present only' in the mesenchyme. (B) Current model of genetic interactions based on work in the fly'. chick, and mouse embr)os. Pax genes are required for the activation of Six and dachsund genes. Eya. which may also be regulated by' Pax. functions to translocate Six to the nucleus to cooperatively bind a target sequence and activate transcription. Eya can also interact with dachshund to activate other sets of target genes.

of the glomerular epithelium, the podocytes (Fig. 6). High WT1 levels persists in the podocytes of the glomerulus into adulthood. The impact of WT1 on kidney development is still under intense investigation, though some conclusions can be drawn from the phenotypes of both mouse and human mutations. The homozygous null WT1 mouse has complete renal agenesis (Kreidberg et al., 1993), because the ureteric bud fails to grow out of the nephric duct and the metanephric mesenchyme undergoes apoptosis. The arrest of ureteric bud growth appears to be noncell autonomous and is most probably due to lack of signaling by the WT1 mutant mesenchyme. Yet. the mesenchyme is unable to respond to inductive signals even if a wild-type inducer is utilized in the organ culture assay. Taken together, it appears that WT1 is required early in the mesenchyme to promote cell survival, such that cells can respond to inductive signals and express ureteric bud growth promoting factors. At later stages of nephron development, increased WT1 expression levels in the podocyte precursor cells correlate with repression of the Pear2 gene (Ryan et al., 1995). WT1 binds to the promoters of a variety of genes, including Pax2, IGF2. and the IGF2 receptor, by direct contact of the zinc finger DNA binding domain to a conserved GC rich nanomer. Much of the data

indicate that the amino terminal region of WT1 acts as a transcription repression domain (Madden et al., 1991), consistent with the increased levels of growth factor expression observed in Wilms" tumors. Yet in other cases, WT1 is able to function as a transcription activator (Lee et al., 1999). Furthermore. specific isoforms of WT1 proteins have been colocalized to the mRNA splicing machinery suggesting a post-transcriptional role (Davies et al., 1998). Thus, many questions still remain to be addressed. The expression levels of WT1 are precisely regulated during "kidney development and may indicate different functions depending on the threshold level of protein present and the ratio of individual isoforms.

III. Growth of the Nephric Duct and Ureteric Bud Diverticulum A. Initiation of the Nephric Duct The first epithelial structure formed within the intermediate mesoderm is the pronephric duct. Because this duct extends caudally it is also known as the mesonephric duct or the Wolffian duct. All three of these terms describe essen-


Figure 6 Expression of WTt and Pax2 in nephron development. Serial sections are shown from a kidney at E l 4.5. Antibody staining (A) for Pax2 (orange) and E-cadherin (green) and (B) for WT1 (orange) and E-cadherin (green). All stages of nephron development are shown, from undifferentiated mesenchyme along the periphery to maturing glomeruli, tubules, and collecting ducts located more centrally. The ureteric bud has branched repeatedly at this point and the major collecting ducts (CD) are now evident. WTI expression is low in the mesenchyme, but increases at the S-shaped body stage (S) in the cells of the glomerular cleft. These are the podocyte precursors, which surround the developing glomerulus (G) before migrating into the glomerular tuft. Pax2 expression is high in the condensing mesenthyme (CM) and its early derivatives but becomes more restricted ~ the nephron matures. In the developing podocytes. Pax2 levels decrease as WTI goes up. Also note the lack of Pat Z

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changes are noted when occipital somites are replaced with more caudal trunk somites, indicating the extrinsic nature of the migration cues (Mackenzie et at., t 998). This migration route is also taken by the hypoglossat nerve and circumpharyngeal NC. though ablations of these populations have no effect on muscle ceil migration.

C. Ectoderm Cranial ectoderm is a critical component of the craniogenesis machinery, contributing both directly and indirectly to the development of craniofaciat structures. It has been suggested that the ectodermal field can be segregated into ectomeres, though the functional significance to these divisions remains to be determined. The cranial surface ectoderm has been shown to play a vital role in the differentiation of CNC into skeletal tissue. In amphibian and chick studies, isolated CNC does not appear to self-differentiate in ~'itro to form primary cartilage, dentine or intramembranous bone (Thorogood. 1993" Hall. 1999). and there is compelling extirpation and tissue recombination evidence that focal (localized)epitheliomesenchymal tissue interactions have a fundamental role in leading ectomesenchyme to differentiate into these hard tissues (reviewed by Hall. 1987. I999: Lumsden, 1988: Thorogood, 1993). For example, extirpation of surface or neural ectoderm leads to an absence of associated dermal bones (Schowing. 1968a, b). Tissue recombination studies conducted by Hall and colleagues suggest that the basal lamina of a mitotically active ectoderm is necessarsj for CNC osteogenesis; the ectoderm need not be cephalic because limb bud. dorsal trunk. and periscleral epithelium can induce osteogenesis in mandibular ectomesenchyme (Tyler and Hall. 1977: Bradamante and Hall. 1980: Hall et aI., 1983: Tyler. 1983). Thorogood and colleagues have extensively studied the chondrogenic promoting capacities of the collagen II-rich ECM secreted by the neurepithelium at the condensation sites of the chondrocranium (reviewed by Thorogood, 1993). Though a capacity as a direct inductor has been ruled out. this collagen II-rich ECM may play a role as part of a three-dimensional reposito~' for ECM-sequestered chondrogenic factors (Bissell and Barcellos-Hoff. 1987; Thorogood. 1993; Hall, I999). The potential to form cartilage, but apparently not intramembranous bone, may be informed while the CNC is still associated with the NT or soon thereafter (Hall and Tremaine, 1979; Bee and Thorogood, t 980: Hall. I980a, b, i 999). It has further been demonstrated that the skeletogenic factors associated with the craniofacial ectoderm can be either matrix mediated or diffusible (Bee and Thorogood. 1980" Thorogood and Smith. 1984). These studies further address an acknowledged but perhaps underappreciated aspect of craniofacial development: the temporal delay between the advent of the chondrocranium (the initial, cartilaginous emb~'onic cranial skeletal

structures) and the dermatocranium (the perinatal skull that arises with the advent of the nascent dentition and the intramembranously ossified elements of the skull that develop around the chondrocranium). This is likely to be under the control of the ectoderm (Hatl, 1987). It may be that the one is necessary for the development of the other, as is suggested by the work of Corsin (1966. i975, 1977) showing the need of amphibian BA dermatocranium for the presence of the viscerocranium. The embryonic skull has an unique set of functional demands for which the cartilaginous skull is best fit to fulfill, and likewise for the perinatal dermatocranium built around the chondrocranium (Hanken and Thorogood, 1993; Prestey, I993). Clearly it is advantageous to temporally regulate the onset of each (Presley, I993). How this is achieved is not yet clear. Thus. the sketetogenic promoting factors of the embryonic skull are not uniform with regard to mechanism, and the diversity in cellular fate is due to the influences of other cells and the extracellular environment. Significantly, regulation of these events in B A t M w h e r e cartilage, membrane bone. and teeth all form must be highly regulated spatially and temporally. For example, teeth develop on the rostral (oral) surface of the developing mandibular primordium, whereas bone and cartilage develop more caudally (aboralty). The different positional fates of these CNC cells must be determined early in the formation of the primordium. Expression of the closely related LIM domain homeobox genes Llcr6 and L h x 7 is restricted to oral ectomesenchyme of the mandibular and maxitla~)' processes and complements that of Gsc, which is expressed in aboral ectomesenchyme (Tucker et at., t999). The ectoderm appears to be involved in inducing both oral and aboral mesenchymal gene expression. The ectoderm expresses a wide range of signaling molecules, including FGFs, BMPs, WNTs. and HHs. and it is the restriction of F g f 8 expression to the oral (and pericleftal) ectoderm that appears to set up the anterior-posterior (AP) axis of BAt (Grigoriou et al., 1998" Trumpp et al.. 1999). The restriction of Gsc expression to aboral mesenchyme involves repression by L h x 6 / 7 expressing cells, although the mechanism that restrictsLhx6/ 7 expression to oral mesenchyme is independent of Gsc and is more probably related to the distance from the source of FGF8. Targeted mutations in Lhx6 or Lhx7, however, do not result in dental defects; defects may only be revealed when these mutations are combined (Zhao et at., 1999; ~v: Pachnis, personal communication). Mutations in Gsc do, however. lead to mandibular bone defects (see below) but the teeth develop normally (Rivera-P6rez et at., 1995; Yamada et al., 1995). EndotheIin-1 expression in the entire mandibular epithelium appears to act as a maintenance factor for Gsc expression (Tucker et al., 1999) and it is regulated in part by FGF8 (Trumpp et al., 1999); as with Gsc, both Endothelin-1 and Endothetin receptor A knock-outs have mandibular defects where bone is affected but dental development essentially is not (Kurihara et al,, 1994; Cloukhier et al., t998).

433

t 9 Craniofacial Development More specific roles for the ectoderm in dental development are discussed later in this chapter. The expression of signaling molecules in the BA ectoderm has been shown to be independent of the NC, such that when the NC is ablated the expression of signaling molecules still comes on in a defined spatially restricted pattern tVeitch et al., t999). Thus, it would appear that although the skeletal identity of an arch might be determined by the NC cells, the AP polarity within an arch is determined by the ectoderm. This perhaps explains why, when Rt and R2 (which both produce crest destined for the BA I) are rotated, no change in first arch AP pattern is seen (Noden, t983b; unpublished observations). This may also explain why, when mesencephatic CNC is grafted caudally (Noden, 1983b), or in the Hoxa2 knock-out (Rijli et al., t 993; Gendron Maguire et aI., 1993), the duplicated first arch elements have a mirror image symmetry. In addition to expressing signaling molecules involved in local epithelial-mesenchymat interactions, B A ectoderm also expresses a range of transcription factors, including Hox genes. Hoxa2, for example is expressed in the ectoderm of BA2 as well as its CNC. Such ectodermal expression comes on after the NC has migrated and was initially thought to be induced by the crest (Hunt et at., t 991 a). However, the ectoderm's Hox code has now been shown to be independent of the NC" When non-Hox-expressing crest replaces Hox-expressing crest, the ectoderm still turns on its normal Hox code (Couly et aL, 1998). Arch identity may therefore come from a combination of patterning information from the CNC and patterning information in the tissues into which it migrates, possibly explaining why Hoxexpressing NC appears unable to form cartilage elements when placed in a normally Hox-devoid setting (Couly et al., 1998: Grammatopoulos et al., 2000). Hence, the ectoderm may impart positional information and the CNC its interpretation, necessitating rigorous investigation of the proximate regulators of ectodermal gene expression.

D. Endoderm Pharyngeal endoderm lines the internal surface of the BA, and the pouches between the BA yield a variety of structures. In the first pha.D'ngeaI pouch (between BA1 and BA2). the endoderm forms the pharyngeotympanic tube and tympanic cavity, which meets up with the ectodermal, first phar?'ngeal cleft-derived external auditow meatus. Where these two epithelial structures join, the ear drum forms. Between the second and third arch, the endoderm invaginates to form the palatine tonsil. Between the third and fourth arch the endoderm invagination forms the inferior parathyroid gland and thymus, while at the base of B A4 the superior parathyroid gland and ultimobranchial body form. The thyroid descends in front of the pharynx until it reaches its final position in front of the trachea. It remains connected to the tongue by a narrow canal, the thyroglossat duct, which even-

tually regresses. The origin of the migration route is marked by a persistent blind o ~ n i n g at the back of the tongue, the foramen caecum. The endoderm of the floor of the pharynx proliferates to help generate with contributions from each of the arches--the tongue. Other than these derivatives, the endoderm has a limited contribution itself to craniofaciat tissues but has an important roIe as an inducer, for example, through its induction of the epibranchiat ptacodes (see below). The epibranchial ptacodes contain the gustatory sensory neurons that link up with the taste buds in the tongue (Northcutt and Bartow, 1998). Therefore, in this case, by inducing the sensor' neurons, the endoderm appears to be inducing its own afferent innervation. Endodermal markers, such as B m p Z are also remined after CNC cell ablation, indicating that, like the ectoderm surrounding the crest, its patterning information is independent (Veitch et at., 1999).

III. Organ Development A. Mouth Development Mouth development begins with the formation of the stomodeum (mouth pit), an ectodermal depression around which the facial primordia grow and extend to create the oral cavity. An early regional landmark is an area of cellular apposition (here an epithelial-epithelial contact) that develops just caudal to the forming cardiac tissue between the prechordal/foregut mesendoderm and the stomodeal ectoderm at the cranial end of what will be the foregut (Schwind, 1928; "Waterman, 1977). The division between ectoderm and endoderm is not readily distinguishable until after this cellular apposition~the buccopharyngeal m e m b r a n e ~ f o r m s (---E8.25). The buccopharyngeal membrane breaks down by E8.75 through a process of cell rearrangement and death, at which point once again there is no morphological or histological distinction between ectoderm and endoderm. With the breakdown of the buccopharyngeal membrane an obvious mouth is distinguishable, consisting exclusively of the oropharynx. Rupture of the membrane is critical to organisreal survival and may be a developmentally important early bamer to mesenchymal migration across the ventral midline (Waterman and Schoenwolf, 1980). At E8.75, the development of the first structure from the stomodeal ectoderm, the pituitary, begins when a diverticulum (Rathke's pouch) grows cranially from the oropharynx toward the infundibulum growing toward it from the floor of the third ventricle of the brain. The first characterized molecular marker of the stomodeal ectoderm is the homeobox gene Pitx2, which is initially expressed throughout the ectoderm from E8.5 and gradually becomes refined to presumptive dental ectodermal development (Gage et at., 1999" Lin et aL, 1999; Lu et aI., t 999a). Mutations in the human PI7X2

434

ili Organogenesis

gene result in Reiger syndrome which affects several craniofacial tissues, including teeth. Mouse knock-outs of Pitx2 surprisingly show tooth development arrested at the early bud stage, indicating that dental ectoderm does form in the absence of Pitx2.

B. Secondary Palate Development The primmT palate develops from the medial frontonasal processes and is a transient structure from which the upper lip and the prema.xillary palate (anterior to the secondary palate) form. The secondarT (or definitive) palate, which forms the roof of the mouth, develops from the maxillary processes of BA1. Bilateral secondary' palatal shelves initially arise from the maxillary processes at E t 2. They begin as vertical (dorsoventral) projections down the sides of the developing tongue whose growth is driven by mechanisms that probably involve controlled cellular proliferation. At a particular time in development (E 13.5), the shelves rapidly elevate to a horizontal position above the tongue. The medial edges approach each other toward the midline where thev contact and fuse. The epithelial edges of the apposing shelves fuse to form a seam. rapidly degenerating by processes that may involve apoptosis, epithelial-to-mesenchymal transitions, retractions of epithelial cells to the dorsal and ventral aspects of the shelves, or possibly any combination of these. Epithelial seam degeneration allows the underlying mesenchymal cells to contact along the horizontal plane thus forming a continuous structure. As the seam epithelial cells degenerate, the epithelia on the nasal aspect of the palate differentiate into pseudostratified ciliated columnar cells, while those on the oral aspect of the palate, become stratified squamous cells. Osteogenesis begins at sites in the anterior mesenchyme of the palate, forming the maxillary and palatine palatal shelves of the hard palate. Myogenesis occurs in the posterior third of the palate giving rise to the soft palate. Because cleft palatemwith or without cleft l i p - - i s the most common human birth defect, the biology of palatal development has attracted much attention (Ferguson, 1988). Cleft palate can result from disturbances at any phase of development up to epithelial seam degradation. In humans cleft palate has long been recognized as a multifactoriaI disorder and despite major efforts little progress has been made via the human genetics route toward understanding palatal development. The multifactorial nature of cleft palate has been confirmed by the many mouse gene knock-outs that give rise to cleft palates: even so. in only a few cases has a direct primary association been established. Because palatal development is a very dynamic process, any disturbance in the development of surrounding tissues can affect shelf elevation and/or fusion resulting in a cleft. Indeed. even minor disturbances affecting the timing of elevation, for example, can ultimately result in a cleft. Thus, the outcome of many disturbances in facial development can lead to the same phe-

notype, namely, a cleft, but via what can be very different mechanisms. One molecule identified as having an essential primary role in palatal shelf development is transforming growth factor t93 (7,@3). Tgjb3 is expressed in medial edge palatal shelf epithelium at the time of fusion and degradation, and Tgfb3 knock-out mice develop cleft palate and die within 24 hr of birth (Kaartinen et al., t 995; Proetzet et aL, 1995). Unlike knock-outs in many other genes that produce cleft palate, Tgfb3 -j- mice have no other obvious craniofacial defects. Palatal shelves isolated from Tgfb3 -/- mutant embryos are unable to fuse in vitro when cultured but are able to fuse when placed next to wild-type or heterozygous shelves (Taya et al., 1999). Addition of exogenous TGF-/33 or other TGF-/3s to T g ~ 3 -/- palatal shelves in culture enables the shelves to fuse. These in vitro experiments confirm a primary role for TGF-/33 in palatal shelf fusion although the biochemical details of this action remained to be determined. As described above, palatal shelves can be cultured as isolated explants using methods similar to those used for teeth. An advantage of this culture system is that shelves from mouse mutants with cleft palates can be placed alongside those from wild-type emb~,os to see if they are capable of fusing. In addition, rescue of a fusion defect by the specific addition of factors into these cultures provides a powerful method of extending genetic analysis and developing possible nonsurgical correction approaches. These types of in vitro experiments are important for determining whether any cleft palate phenotype results from failure of elevation or fusion and if the phenotype is primary or secondary.

C. Neurogenic Placodes Another unique feature of craniofacial development relates to the development of sensor" neurons. In the trunk all sensory neurons develop from NC, while in the head the earliest differentiating neurons in the sensor" ganglia are derived from specialized ectodermal fields, the dorsolateral and epibranchiat placodes (collectively known as the neurogenic or ganglionic ptacodes) (H6rstadius, 1950; D'Amico-MarteI and Noden. 1983; Begbie et aL, t999). The dorsolateral placodes (the vestibular and trigeminal) develop alongside the central nervous system, while the epibranchial placodes (the geniculate, pertrosal, and nodose) develop atthe base of each BA at the top of the branchial clefts. The significance of this dual origin of sensory ganglia is unkanown, but they have been found to respond differently to neurotropic factors such as NFG (Lindsay et al., 1985). The placodaI neurons differentiate early and establish both peripheral and central projections before the NC-derived neurons initiate axonogenesis (Webb and Noden, i993). They play an important role in establishing the peripheral projections of the NCderived ganglia, because in their absence these are abnormal

19 Craniofacial Development (Moody and Heaton, 1983). The epibranchial placodes are induced to form by BMP7 signaling from the pharyngeal endoderm (Begbie et al., 1999). Trunk ectoderm, unlike cranial ectoderm, is unable to respond to dais inductive signal in vitro, although in vivo studies have shown that trunk ectoderm is able to contribute neurons to the distal Xth ganglion when grafted to the position of the nodose ptacode in vivo (Vogel and Davies, i993). The dorsolateral placodes, however. appear to have a different system of induction. The trigeminal, for example, does not form in proximity to a source of endoderm. Instead. induction by the NC appears more likely (Stark et al., t997). The tact that the two neurogenic placode types use distinct developmental pathways is emphasized by the fact that the dorsolateral placodes are lost in neurogenin t knock-out mice, while the epibranchial placodes are lost in neurogenin 2 knock-outs (Fode et al., t 998; Ma et at., 1998).

D. Olfactory development The olfactory; placodes (OfP) develop bilaterally in the rostroventral surface ectoderm over the prosencephalon. A focal thickening of the ectoderm is the initial histologic indication of the onset of placodogenesis (Jacobson, t963; Verwoerd and van Oostrom, t979). Fate mapping using chick-quail chimeras has demonstrated that the placoda! epithelium descends from the ectoderm of the ANR of the rostraI neural fold (Couly and Le Douarin, I985, i 987, 1990). However, that the Ot~ is formed from the fringes of the neural plate, and not from the adjacent non-neural ectoderm, has recently been shown in zebrafish (Whitlock and Westerfietd,

2OOO). Transplantation and recombination experiments suggest that placodogenic inductive processes involve the presumptive nasal epithelium, chordomesoderm, and endoderm during gastrulation, and the nasal epithelium and the telencephalic primordia later (Webb and Noden, I993). After induction, the placode invaginates to form a pit. The ectoderm of this invagination will yield the respiratow, olfactory, and vomeronasal (Jacobson's) organ (VNO) epithelia. The neuroblasts of the olfactory nerves (CN I) do not delarrdnate, but develop in situ within this pit sending axonal projections to the FB. The new'us terminalis (CN 0) develops out of the medial placodaI epithelium (Webb mad Noden, 1993). Cells immunocytochemically identified as producing gonadotropin releasing hormone/leutenizing hormone releasing hormone (GnRH/LHRH) delaminate from the epithelium and migate into the FB (Schwanzet-Fukuda and Pfaff, 1989" Wray et al., 1989). The path of the nervus terminalis is marked by these LHRH-positive cells. Couly and Le Douarin (1985) provide evidence that CN I myelin-producing cells also arise from the placode. Olfactory pit invagination is a morphogenic process coupled with the formation of a ring of tissue surrounding

435 the pit, delineating a medial frontonasal prominence (MFP) and a lateral frontonasal prominence (LFP). The basal lamina of the Of R and later the olfactow and respiratow epithelium, contains collagen It, foreshadowing the sites of chondrogenesis of the nasal capsules and basal plate formation (Croucher and Tickle, 1989; Thorogood, 1993). Extirpation experiments provide evidence that the epithelium of the invaginated olfactory pit, moreover, induces chondrogenesis in the CNC mesenchyme to form the nasal capsule (Corsin, t 971). The MNP wilt support the induction of CNC (posterior prosencephalic and anterior mesencephalic) to yield medial capsular structures (e.g., rostraI nasal septum, paraseptal cartilages etc. see below); the LFP supports the induction of the lateral capsular structures (e.g., paries nasi, turbinals, etc.) (Osumi-Yamashita et at., t 997a,b). The trabecular basal plate to which the nasal capsules are ultimately connected (see below) develops as a rostrad extension from initially paired struts (trabecula cranii) developing adjacent to the prechordaI plate anterior to the notochord. A mechanism is in place that directs the meeting and fusion at the midline of these paired trabecula cranii. This mechanism is likely to involve signals from the prechordal plate and developing FB. The premaxitlae (forming the primary" palate) and upper incisors develop out of the more superficial CNC in this region. A transient constriction of epithelium, the nasolacrimat groove, separates the LFP from the expanding maxillary branch of the first branchial arch. Analysis of olfactow development, perhaps lagging behind that of optic and otic development, has generally addressed the developmental relationships of the olfactow bulbs, olfactow neurons, VNO, and LHRH cells, their neurogenesis and targeting, as well as (to a lesser extent) the role played by the intervening ectomesenchyme (Keverne, t 999; Lin and Ngai, 1999; Mombaerts, t999; Mori et aI., I999). Mutations of a number of genes affect the development of olfactow structures, including of BF-] (forgl), DlxS, Gsc, HesxI, Otx2, RAR, and Pax6 (see below). Some of these genes are ectodermally expressed, such as BF-1, Dtx5, and Pax6, while others are primarily mesenchymally expressed, such as Gsc and RAR. As with the BA, epithelial-mesenchymal interactions appear vital to proper nasal development. For example, the nasal capsular defects in Gsc -/- mice are, in effect, a subset of the defects seen in the Dtx5 -/- mice; not surprisingly, ectodermal loss of Dtx5 expression leads to loss of mesenchymal G s c expression, further suggesting that ectodermal regulation of the mesenchyme is necessary for proper capsulogenesis (Depew et al., !999; see below). Similarly, a toss of placodal Pax6 leads to a loss of M s x I expression and retinoic acid signaling in the underlying frontonasal mesenchyme (Grindley et at., 1995; Anchan et at., 1997; Enwright and Grainger, 2000). In general, however, analysis of early nasal development has been obscured by the tight developmental relationship of the rostral neuraxis and the Of P.

436

111Organogenesis

E. SubmandibularGland Development A number of important glands develop within the craniofacial tissues, the best studied of which is perhaps the submandibul~ gland (SMG). Among other things, study of these glands has provided a wealth of information of the nature of branching morphogenesis (Hieda and Nakanishi. t 9 9 7 Jaskoll and Melnick, 1999). SMG development begins around E11.5 as an in-growth of the oral ectoderm into the mandibular mesenchyme in an initial "bud stage." The invaginating epithelium proliferates to form an elongate, solid epithelial stalk terminating in a bulb. With continued proliferation and end-bud branching, along with selective growth inhibition and cell death, a network of large and small ducts is built, eventually resulting in a mature gland.

F. Tooth Development Teeth are remarkable structures. They contain the hardest substance in the human body. enamel: their preservation in the fossil record provides the main source material for palaeontologists and anthropologists: and their unique function in feeding means tooth shape and organization are powerful driving forces in evolution. The origins of teeth have been traced back to the earliest vertebrates where skin denticles of extinct fishes are thought to have "moved" into the oral cavity as jaws evolved (reviewed by Butler. 1995). Early jawed fish (e.g., acanthodians) possessed denticles in a variety of shapes attached to the endoskeleton of the jaw. The similarity of these denticles to modern teeth suggests that they could have been precursors of modern teeth. It has been suggested that teeth have evolved from a specific group of denticles similar to those in the oropharyngeal region of agnathan (jawless fish) vertebrates (Smith and Coates. 1998). One classic theory, suggests that the evolution of dermal dentictes into teeth coincided with the evolution of the mandibutar arch into a jaw: contrary, to this it has been suggested that oropharyngeal denticles were specialized feeding structures prior to the evolution of jaws in early fishes and thus teeth may have evolved before jaws (Smith and Coates, 2O0O). Historically. dental research has focused on the teeth themselves and how to repair and preserve them in adults. More recently, the embryonic development of teeth has attracted increasing interest not only for an understanding of how these important organs develop but also because tooth development offers a powerful experimental system to address general questions in organogenesis. Mice have thecodont dentition (teeth occupying bony sockets) which exhibits heterodonty: that is, they have teeth of different shapes: in this case. two incisors (which continuously grow throughout life) and six molars in each jaw. Mice do not develop canine or premolar teeth; instead, the incisors and molars are separated by a region, the diastema, devoid of teeth. Mice only

develop one set of teeth (monophyodont dentition), unlike human dentition, which is diphyodont. In considering the differences between the human (and many other vertebrates) and murine dentition, and the fact that mice lack canines and premolars, the extrapolation of data from mice to humans and other mammals should be approached with caution. However. the early development of teeth in most species, at least histologically, is fundamentally the same. Teeth develop on the oral surfaces of the facial processes (mandibular. maxillary, and frontonasal) from interactions between the oral epithelium and underlying CNC. The early epithelial-mesenchymat interactions led to the formation of epithelial tooth buds surrounded by condensed mesenchyme cells. The formation of epithelial buds is a common feature of the development of several organs such as hair. lung, kidney, and sweat glands and not surprisingly many of the same signaling pathways are involved. In mammals, the position at which an epithelial tooth bud invaginates into the ectomesenchyme specifies the type of tooth that develops. Tooth buds forming in distal regions of the facial processes will develop as monocuspids (incisors and canines), whereas tooth buds in more proximal regions will develop with multiple cusps (premolars and molars). Tooth type is thus intrinsically linked to tooth position to produce the characteristic pattern of tooth types, the dentition. 1. Origins of Tooth Cells Teeth have several unique cell types that are found nowhere else in the body. These include amelobtasLs, odontoblasts, and cementoblasts. The differentiated ceil types that are responsible for secreting and organizing the specific hard tissues of teeth develop early in embryogenesis. Ametoblasts secrete enamel matrix and are derived from oral ectodermal cells. Odontoblasts produce dentine and develop from CNC cells, as do all other supporting dental cells. Ameioblasts are the only cells remaining in teeth at birth that are derived from the ectoderm. The stages of tooth development are outlined in Appendix I. Tooth development is initiated by local interactions between oral (stomodeal) ectoderm cells and the underlying CNC. These first interactions are specific and unique to these two tissues. Recombination experiments using explant cultures and anterior eye transfers have identified a requirement for these two tissues in tooth development. Thus, recombinations of stomodeal ectoderm with any CNC cell population can support tooth development. Recombinations between stomodeal ectoderm and non-NC mesenchyme cannot support tooth development. Recombinations between nonstomodeal ectoderm and CNC cells cannot support tooth development. Similar experiments (see below) also show that premigratow CNC cells can support tooth development. Thus. teeth can only form when stomodeat ectoderm is in contact with ectomesenchyme and the only site of such contact in mouse embwos is the developing oral cavity.

19 Craniofaciat Development Lineage tracing using DiI in rodent embryos has identified the axial origins of the CNC cells that contribute to tooth formation (Nichols, 1981; Tan and Morriss-Kay, I986; Fukiishi and Morass-Kay, 1992: Osumi-Yamashita er aI., 1994, I997a: Imai et al., 1996). These studies have defined the caudal MB and rostral HB as the source of odontogenic NC cells for mandibular incisors and molars and maxilla~' molars. Premaxillary incisors develop on the frontonasaI process, and, though the origins of these odontogenic cells have not been directly mapped, studies in avian embryos implicate the rostraI MB and FB as the source of these cells. 2. Patterning of Dentition

Patterning of the dentition leads to the development of different shapes (types) of teeth in their correct positions in the jaws. In mice this pattern consists of incisors distally and molars proximally. A theory for dental patterning based on a field model was proposed by Butler (reviewed by ten Care, t995). This model predicts that diffusible morphogens determine aeas within the jaw in which incisors, canines, and molars will develop. Alternatively, Osborn (1978, cited by ten Care, 1995) proposed a clonal model in which isolated clones of ectomesenchymal cells specie; each different type of tooth. The first model to be proposed based on experimental evidence was the homeobox code model, which proposes that the restricted domains of homeobox gene products are responsible for specifying the pattern of the dentition. The homeobox code model was based on observations of the spatially restricted expression of several homeobox genes in ectomesenchymal cells prior to El t (Sharpe, 1995: Thomas and Sharpe, t998). The early expression of M s x t and M s x 2 prior to the initiation of tooth germs is restricted to distal, midline ectomesenchyme in regions where incisor but not molar teeth will develop, while D l x t and Dla~ are expressed in ectomesenchyme cells where molars but not incisors will develop (MacKenzie et al., 199t" Qiu et al., t995, 1997). These expression domains are broad and do not exactly correspond to presumptive molar and incisor odontogenic cells. Rather, they are considered to define broad territories. Expression of B a r x t overlaps with D l x l / 2 and corresponds closely to ectomesenchymat cells that will develop into molars. The homeobox code model proposes that the overlapping domains of these (and other genes) provide the positional information for tooth type morphogenesis. The first support for this model came from the dental phenotype of D l x l / 2 -/- compound mutant mice where development of maxillar?~" molar teeth is arrested at the epithelial thickening stage (Qiu et at., 1997: Thomas et al., 1997). As predicted by the code model incisor development was normal in these mice; normal development of mandibular molars (not predicted by the code) was assumed to result from the functional redundancy with other Dtx genes such as Dtx5 and D l x 6 that are expressed in ectomesenchyme in the mandibular primordium. Maxillao, molar teeth are re-

437 placed by ectopic cartilage in D L r ! / 2 -/- mice, suggesting that. in the absence of these genes, ectomesenchymal cells become reprogrammed from an odontogenic to a chondrogenic phenotype. The phenotype of M s x ] - / - ; 2 -/- compound mutant embryos shows development of all teeth arrested at the epithelial thickening stage, suggesting a functionally redundant rote for these genes in tooth bud Ibrmation (Satokata et at., 2000). This phenotype has incorrectly been interpreted as indicating that M s x and Dlx functions are mechanistically the same in tooth development. This conclusion ignores the fact that the D l x l / 2 -/- mutant phenotype only affects maxillary molars, whereas in MsxI-/-~;2 -/ ..... mutants all teeth are affected. Significantly, the lack of expression of D l x genes in presumptive incisor mesenchyme prior to initiation is consistent with D t x genes having a specific role in molar tooth development. The reported presence of ectopic bone in molar regions of newborn M s x l - / - mice (Satokata and Maas, 1994) has also been suggested to indicate a common mechanism with DLr genes since in D l x t / 2 -/- mutants, maxilta~, molar teeth are replaced by ectopic cartilage. However, whereas it is very clear that chondrogenesis replaces odontogenesis in D l x ] / 2 -/- mutant maxillary molar mesenchyme, the cartilage does not mineralize and the occasional formation of ectopic bone in M s x ! - / - mutants is most likely to be a result of effects on alveolar bone development. Thus taking into account all the available evidence it is likely that Msx and DLv genes function through different mechanisms to regulate tooth formation. D l x genes have a clearly established rote in patterning molar tooth development, M s x genes are clearly required for the bud-to-cap transition and may, also have a rote in bud formation and possibly incisor patterning based on the odontogenic homeobox code. This early role for M s x I specifically in incisor development is supported by the observation that whereas ectopic BMP4 can rescue molar tooth development in M s x t mutants, it does not rescue incisor development (Zhao et al., 2000). Thus the requirement for M s x l in incisor development is different from that in molar development and is not mediated by BMPs. This is consistent with the early, incisor region expression pattern of M s x l , which is induced by BMP4 but which does not correlate with dow'nstream activation of B m p 4 in the mesenchyme. Strong functional support for the code model came from misexpression of B a r x I in distal ectomesenchyme cells, which resulted in incisor tooth germs developing as molars (Tucker et al., I998a). B a r x t expression was found to be localized to proximal ectomesenchyme (molar) by a combination of positive and negative signals from the oral ectoderm. FGF8 localized in proximal ectoderm induces B a r x l expression while BMP4 in the distal ectoderm represses B a r x l expression. B a r x l expression was experimentally induced in distal (presumptive incisor) ectomesenchyme by inhibition of BMP signaling following implantation of Noggin (a BMP antagonist) beads. This also had the effect of re-

1110rganogenesis

438 pressing Msx gene expression, which is induced in distal ectomesenchyme by BMP4 and thus it is not yet established whether the transformation of incisors into molars requires misexpression of BarxI alone or whether it also needs accompanied toss of Msx gene expression. However, the fact that tooth type could be transformed in this way provides powerful support for the model.

3. Instructive Signals for Patterning Recombinations between oral ectoderm and ectomesenchyme have been carried out to determine the origin of the instructive information for dental patterning. Do,burgh (t967) published an abstract outlining preliminary work in which he recombined incisor and molar epithelium and mesenchyme from E l 0 - 1 4 emb~'os. It was found that when molar epithelium was recombined with incisor mesenchyme, a molar tooth formed; and when incisor epithelium was recombined with molar mesenchyme, an incisor formed. This led to the conclusion that the epithelium was responsible for determining the type/shape of a tooth. Further experiments by Miller (1969), in which recombinations were cultured on the chick chorioallantois, supported Dryburgh's work. Similar recombinations revealed that at E 11 - 12. the ectoderm determined the tooth type. The results of a series of classic experiments by Kollar and Baird (1969, 1970a,b) disagreed with the previously reported findings that epithelium was responsible for patterning. The work of Miller (1.969) and D~'burgh (1967) was repeated at E 13-16, and it was shown that molar epithelium recombined with incisor mesenchyme resulted in incisor teeth and incisor epithelium recombined with molar mesenchyme resulted in molar teeth (Kollar and Baird, 1969). KolIar and Baird performed recombinations between dental tissues and nondental tissues and after an initial in vitro culture period, transferred the tissue in oculo. In the first set of experiments, lip furrow epithelium (El5 and E 16) was recombined with either molar or incisor mesenchyme of the same stage (Kollar and Baird, 1970a). The shape of the teeth was determined by the origin of the mesenchyme. In addition, when E16 cervical loop epithelium from an incisor was recombined with incisor mesenchyme, a fully formed incisor tooth formed: and when recombined with molar mesenchyme, a fully formed molar tooth formed. Further experiments used tissue from the hairless (plantar) surface of the foot in combination with dental tissues (Kollar and Baird, 1970b). At either El4, or E I5, dental epithelium, when recombined with foot mesenchyme, showed no tooth development; however, when plantar epithelium was combined with dental mesenchyme, tooth development occurred (Kollar and Baird, I970b). The apparent conflict of whether the ectoderm or ectomesenchyme provides the instructive information for patterning has now been resolved by studying the temporal regulation of homeobox gene expression in ectomesenchyme by ecto-

dermal signals. Removal of the ectoderm from E t0 mandibular arch explants resulted in loss of expression of ectomesenchymaI homeobox gene expression within 6 hr. indicating that expression requires signals produced by the ectoderm. Expression could be restored by implantation of beads soaked in FGF8, a factor expressed in oral ectoderm at this time (see below). Expression of DLvl/2, Msx, and Barxl was seen around the implanted beads regardless of their position in the explant, indicating that all ectomesenchymal cells at this time are competent to respond to FGF8 and implying that the CNC cells are not entirely prepatterned (Ferguson et al., 2000). When this experiment was repeated at El0.5 ectomesenchymal gene expression was again lost following removal of ectoderm but this time implantation of FGF8 beads only restored expression in the original domains. Thus at E10.5 ectomesenchymal ceil competence to express homeobox genes in response to FGF8 has become restricted to those cells that expressed the gene at El0. By E 11 removal of ectoderm had no effect of ectomesenchymal gene expression showing that by this stage expression is independent of ectodermal signals. These results provide a molecular understanding of the control of dentat patterning and an explanation for the conflicting recombination results. The distoproximal (incisor-molar) spatial domains of homeobox gene expression (homeobox code) are produced in response to spatially restricted ectodermal signals acting on pluricompetent ectomesenchymal cells. Recombinations carried out before El0.5 will therefore show the instructive influence of ectoderm on tooth shape, whereas those carried out after El0.5 will show an instructive influence of ectomesenchyme since by this stage expression is independent of ectodermal signals.

4. Initiation Well in advance of the first morphological signs of tooth development, processes are occm-ring that will determine where a tooth will develop and which cells will contribute to the developing tooth germs. It has previously been suggested that nerves may play a role in determining the sites of tooth development (reviewed by Kottar and Lumsden, 1979). This suggestion was based mainly on descriptive data which showed that the trigeminal nerve arose at E9 and fibers branched from this nerve by E9.5 into presumptive tooth forming regions (Lumsden, I982). Neuronal induction of odontogenesis was disproved when dennervated E9 mad E l 0 mandibular processes developed teeth in the absence of innervation (Lumsden and Buchanan, 1986). More recently it has been suggested that the foregut endoderm may be involved in establishing the sites of tooth development. Imai and coworkers (1996)performed lineage labeling experiments in which they infected rat foregut endoderm with adenovirus-inserted lacZ. After 9 days of culture (3 days of whole embryo rolling culture and 6 days of mandibular organ culture), foregut endoderm cells were

19 Craniofacial Development shown to be immediately adjacent to developing tooth germs. The conclusion was that dental epithelium originates from the oral ectoderm in a region adjacent to foregut endoderm and that this endoderm does not directly contribute to the developing tooth. However, no direct experimental evidence was presented to suggest that dental epithelium could not arise in oral ectoderm in areas not adjacent to foregut endoderm. Recombination experiments have been the main tool with which the capacity to initiate tooth development has been studied. Wagner (1955, cited in ten Cate, 1995) worked on anuran (whose "teeth" axe merely keratinous appendages and are not true teeth) and urodele larvae (which develop true teeth) (ten Care, 1995). He transplanted anuran NC cells under the newt oral epithelium and found that teeth developed where the dental papilla (NC-derived) was of frog origin and the dental organ (ectodermal) was from the urodele (Wagner, 1955). When the reciprocal experiment was performed (i.e., newt NC cells transplanted under anuran oral epithelium), teeth did not develop. This suggested that although anuran ectomesenchyme was capable of forroSng teeth, anurans lacked a signal from the oral epithelium that would normally initiate tooth development. Lumsden (1988) performed informative experiments in which he either (1) recombined oral epithelium with CNC cells, TNC cells, or limb mesenchyme, or (2) recombined odontogenic mesenchyme with limb epithelium. These recombinations were performed with E9 or El0 tissue, then transferred in vivo into the anterior chamber of the mouse eye. Lumsden did not observe any signs of tooth development in the grafts of odontogenic mesenchyme with limb epithelium, limb epithelium with cranial neural crest cells, or, perhaps surprisingly, oral epithelium with limb mesenchyme. Lumsden did observe tooth development when oral epithelium was recombined with CNC cells or TNC cells. This work suggests that oral ectoderm can signal to mesenchyme and initiate tooth development as long as the mesenchyme is of neural crest origin; however, mandibular ectomesenchyme can only participate in tooth development if it is in combination with oral epithelium. Experiments carried out by Mina and Kollar (1987) supported the view that the oral epithelium was the instructive component with regard to initiation. Mina and Kollar performed recombination experiments with mouse tissue between the ages of E9.0 and E 13.0. They recombined oral epithelium with second branchial arch (nonodontogenic) neural crest-derived mesenchyme and found that when the recombinations were grafted in oculo, normal tooth development occurred with ~afts between E9 and E 11; the incidence of tooth development decreased in the El2 recombinations and no teeth were formed from the E t 3.0 experiments. Conversely, mandibular odontogenic mesenchyme recombined with second arch epithelium did not give rise to tooth rudiments at any age of the recombinations.

439 BMP4 was the first secreted signaling molecule to be identified that had a potential role in tooth initiation (Vainio et aL, 1993). Bmp4 shows a dynamic expression pattern during early tooth development. It is first detectable at E 1 0 10.5 in distal midline ectoderm where it is involved in controlling the spatial domains of ectomesenchyme expression of homeobox genes such as Msxl and Barxl (see above, below). At E11 this expression resolves into patches of ectodermal expression that correspond to the sites of tooth formation, and by E 11.5 expression in the ectoderm is downregulated and transferred to the underlying ectomesenchyme. Throughout these changes in expression, Bmp4 is intimately linked with expression of Msxl (Tucker et aL, 1998b). Thus, at El0.5 BMP4 induces Msxl expression in the underlying mesenchyme and at E t 1 is responsible for maintaining M s x t expression specifically in the ectomesenchyme at the sites of tooth formation. The transfer of Bmp4 expression to the ectomesenchyrne at E11.5 is dependent on Msxl. Bmp4 expression is ~us linked with tooth initiation but it is not known whether it is required for this process. Bmp2 is coexpressed with Bmp4 at E i I in dental epithelium, but unlike BmtM, expression does not shift to dental mesenchyme but remains in the epithelium at the bud and cap stages (Vanio et al., 1993; Va~tok,axi et al., 1996a; Dassule and McMahon, 1998). Unfortunately, both Bmp2 -/- and Bmp4 -/- mutant mice are early embu'onic lethal and loss-of-function analysis is therefore, limited GVinnier et aL, 1995; Zhang and Bradley, 1996). Expression of Shh is localized to the presumptive dental ectoderm at E11, and is thus another good signaling candidate for tooth initiation. Shh -/- mice have little development of the facial processes mad thus any role in tooth initiation cannot be identified from these (see Appendix 3; Chiang et al., 1996). Mutations in GIi genes do suggest a role in early tooth development since Gli2-/-;3 -/- double mutant embryos do not produce any recognizable tooth buds (Hardcastle et aI., 1998). Addition of SHH-soaked beads to oral ectoderm can induce local epithelial cell proliferation to produce invaginations that are reminiscent of tooth buds but which do not develop into teeth (Hardcastle et aL, 1998). Shh thus appears to have a role in stimulating epithelial cell proliferation and its local expression at the sites of tooth development implicate SHH signaling in tooth initiation, it is unclear to what extent the BMP and SHH pathways interact during initiation since all are coexpressed in epithelial cells and it is not until EI2.5 that Bmp4 expression shifts to the mesenchyme. BMPs and SHH are not capable of reciprocal induction of expression (Dassule and McMahon, 1998). However there are clear interactions between BM~P and SHH signaling pathways at the bud stage (see below). Lefl is a member of the HMG family of nuclear proteins that includes the TCF proteins, "known to be nuclear mediators of Wnt signaling. Left is first expressed in dental epithelial thickenings and on bud formation shifts to being ex-

440 pressed in the condensing mesenchyme. In Left knock-out mice. all dental development is arrested at the bud stage; recombination assays, however, have identified the requirement for Lefl in the dental epithelium as occurring earlier. prior to bud initiation (van Genderen et al., 1994 Kratochwil et al., 1996). Ectopic expression of Lefl in oral epithelium has also been shown to result in ectopic tooth formation, albeit in a single transgenic mouse (Zhou et al., 1995). Lefl expression does not appear to be linked to BMP signaling, and tooth arrest at the bud stage may be rescued by a source of fibroblast growth factor (FGF) signaling (K. Kratochwil, unpublished, referenced in Bei et at.. 2000). Lefl is coexpressd with ~l,%tlOb in tooth epithelial thickenings and thus may lie downstrem of signaling by this ligand. Wnt 10b has been shown to be capable of inducing Lefl expression in tooth mesenchyme (Dassule and McMahon. 1998). Expression of several genes in ectomesenchyme mark the sites of tooth germ initiation. These include Pax9 and activin-A both of which are expressed beginning around E 11 in small localized groups of cells corresponding to where tooth epithelium will invaginate to form buds. In the case of Pax9 it has been shown that antagonistic interactions between FGF8 and BMP4 from oral ectoderm may act to localize Pccr9 to these sites and thus this was proposed to be a mechanism specifying the sites of tooth formation (Neubiiser et at., 1997). Significantly, however tooth buds form in Pax9 mutant embryos. Activin-A expression is not regulated by the same mechanism suggesting that such FGF8/BMP4 interactions may not have a direct role in tooth initiation (Ferguson et al., 1998).

5. Regionalization of Oral and Dental Ectoderm Because regionally restricted expression of signaling protein genes in oral ectoderm controls dental initiation and patterning, it follows that the mechanisms that control the regional restriction of ectodermal signals need to be understood. At present there is no experimental evidence to explain how the ectodermal domains of Fgf8 and Bmp4 are generated. Some limited pro~ess has been made toward understanding how Shh expression is restricted to dental ectoderm. During Drosophila segmentation, interactions between Hh and wingless signaling are involved in ectodermal celt boundary specification. Several Wnt genes are expressed during tooth development and one. WntTb. has a reciprocal expression pattern to Shh in oral ectoderm (Sarkar and Sharpe, i999; Sarkar et al., 2000). WntTb is expressed throughout the orat ectoderm except for presumptive dental ectoderm where Shh is expressed. The possible interactions between these two pathways was investigated by misexpressing Wnt7b in presumptive dental ectoderm using a mufine retrovirus. Expression of Wnt7b in the Shh domain of ectoderm resulted in loss of Shh and Ptc expression and failure of tooth bud formation. This repression of Shh expression by Wnt7b appeared to be specific since expression of

II! Organogenesis genes regulated by other ectodermal signals was not affected. More significantly, tooth development could be rescued by addition of exogenous SHH to I~nt7b-infected exptants. This suggests that Wnt7b acts to repress Shh expression in oral ectoderm and thus the boundaries between oral and dental ectoderm are maintained by an interaction between Wnt and Shh signaling similar to ectodermal boundary maintenance in segmentation in Drosophila.

6. Bud-to-cap Transition The transition from bud to cap marks the onset of morphological differences between tooth germs that wilt give rise to different types of teeth. Because several gene knockouts have resulted in tooth development being arrested at the bud stage, much attention has focused on this transition. Msx] is expressed with Bmp4 in the mesenchymal ceils that condense around tooth buds. Msx] -/- embr3jos have tooth development arrested at the bud stage, and Bmp4 expression is lost from the mesenchyme suggesting that Msx] is required for Bmp4 expression (Y. Chen et aI., 1996). BMP4 is able to maintain Msxl expression in wild-type tooth bud mesenchyme indicating a positive feedback loop between the two where B MP4 induces its own expression via Msx 1. Tooth development can be rescued in M s x l - / - embryos by addition of exogenous BMP4 (Y. Chen et at., 1996, Bei et al.. 2000). BMP4 is also required at the bud stage to induce expression of an early EK marker gene, p2], suggesting that BMP4 is a key mesenchymal-to-epithelial signal that is required for buds to progess into caps. BMP4 is also capable of inducing Dtx2 and Lefl expression in bud mesenchyme but these actions are independent of Msxl (Bei and Maas, t 998). FGF3 also probably plays a role at the bud stage because Fgf3 expression in mesenchyme is reduced in M s x l - / - embryos and FGF3 is able to maintain Msxl expression in wild-type e m b ~ o s (Bei and Maas, 1998). The lack of a dental phenotypes in FGF3 -/mutant emb~'os does not support a role in tooth development. though functional redundancy with other FGFs, such as FGF7, may be important. Bmp4 expressed in the bud mesenchyme is required to maintain Bmp2 and Shh expression in the epithelium. Loss of Bmp4 expression in Msxl mutants is accompanied by loss of Shh expression at E12.5, which can be restored by exogenous BMP4 (Zhang et al., 2000; Zhao et al., 2000). Blocking Shh function with neutralizing antibodies also results in toss of Bmp2 expression, suggesting that Shh and Bmp2 may be in the same pathway and that downregulation of Bmp2 in Msxl mutants may be downstream of the loss of Shh (Zhang et aI., 2000). Paradoxically, overexpression of Bmp4 in wild-type tooth buds leads to a repression of Shh expression that has been suggetsed to imply that the levels of Shh expression are tuned by BMP4 concentration (Zhang et al., 2000). The requirement of Shh for normal tooth development has been contentious largely because facial development is

44t

19 Craniofacial Development too disrupted in Shh mutant embryos to draw' any conclusions about tooth development. The loss of Shh signaling at different stages of tooth development, either by addition of neutralizing agents in vitro or by Cre-mediated excision in vivo, has identifed distinct time-dependent requirements for Shh. Blocking Shh signaling using neutralizing antobodies or forskolin shows that at Et 1-12 Shh is required for dentat epithelium proliferation to form tooth buds whereas blocking at E t 3 affects tooth bud morphology but these buds can still form teeth (Cobourne et al .... 2001). Genetic disruption of Shh signaling from E12.5 by Cre-mediated excision of targeted Shh null alleles results :in a disruption of molar tooth morphology but cytodifferentiation appears normal, suggesting that Shh has a major role at the cap stage of development (Dassule et aL, 2000). Another homeobox gene with a rote in the bud-to-cap transition is Pax9. Pa_~:9 is expressed in bud stage mesenchyme and also earlier in similar domains to activin-~A and Msxl in patches of mesenchyme that mark the sites of tooth formation. Pax9 -/- mutant embryos have all teeth an'ested at the bud stage. Despite being coexpressed, early activin-~A expression is not affected in Pax9 -/- emb~'os and Pax9 expression is not affected in activin-13A -/- embryos (Matzuk et al., 1995b). These two genes that are essential for tooth development to progess beyond the bud stage thus appear to function independently. There are, however, changes in expression of other genes such as Bmp4, Msxl and Lefl in Pax9 -/- tooth bud mesenchyme (Peters et al., t 998). 7. The Enamel Knot

The enamel knot (EK) is made up of transient clusters of epithelial cells visible in sections of molar cap stage tooth germs (see Fig. 5 later in this chapter). The EK was believed to have some ill-defined physical role in cusp formation but it was not until the expression of Msx2 was shown to be localized to the EK at the cap stage that interest in this structure was renewed (MacKenzie et aI., 1992). The significance of the EK has now been established due largely to the work of Irma Thesleff in identifying it as a signaling center with many similar characteristics to other signaling centers such as the ZPA, notochord, and floor plate (Vamhtokari et al., 1996b). EK precursor cells can first be detected at the tip of the tooth buds by expression of p2I, followed shortly after by Shh (Jernvall et al., 1998). By the cap stage, when it is visible histologically, the EK expresses genes for many signaling molecules including Bmp2, Bmp4, Bmp 7. Fgf9, WntlOb, and Shh (Thesleff and Sharpe, t997) Three-dimensional reconstructions of the expression of these genes have revealed highly dynamic spatial and temporal nested patterns in the rodlike structure of the EK. On the whole, receptors for the EK signals are localized in the epithelial cells surrounding the EK, consistent with a highly active signaling center. Moreover analyses of cell proliferation and apoptosis reveal a low rate of proliferation of EK ceils that rapidly disappear

toward the end of the cap stage via apoptosis (Vaahtokari et at., t994). The exact physical rote of the EK is not yet established but changes in its morphology in tooth germs of spontaneous mouse mutants with abnormal molar cusp formation such as Tabby (Eda) and downless (dl) have started to reveal some detail of this remarkable structure. Tabby mice have abnormal molar tooth cusps (in addition to other abnormalities most particularly in hair and sweat gland formation), and the mutated gene has been identified as a protein that has homology with (he Tnfligand family and is a transmembrane protein that probably trimerizes through an internal collagen-like domain. The Tabby gene (Ta) is analogous to the human gene EDt, which is responsible for X-linked hypohidrotic (anhydrotic) ectodermal dysptasia (XLI4AED) (ChristSiemens-Touraine syndrome) (Kere et al., 1996). The Tabby phenotype is indistinguishable from that seen for mutations in downtess (Philips, 1960; Gr;Sneberg, 1965). The downless gene has recently been identified as encoding a novel tumor necrosis factor (TNF) receptor (Headon and Overbeek, 1999). Mutations in the human homolog of mouse dt cause ARHED (autosomal recessive HED) and ADHED (autosomal d o ~ n a n t HED), which are clinically indistinguishable from the more common XLHED (MonreaI et al., 1999). Ta is expressed in the outer enamel epithelium but not the inner enamel epithelium, whereas dI is expressed in the EK. This suggests that if these proteins are going to interact directly at least one of them must be secreted. In Tabby mice the EKs are smaller than in wild type but have a normal shape (Pispa et ai., 1999). In downIess mace, however, the EKs are of normal size but the structure of the EK is different with the cells being arranged into an elongated "ropelike" structure (Tucker et al., 2000). Thus despite the apparently identical tooth phenotypes and the interactive structure of the proteins, the molecular biology indicates a more complex mechanism of action. The fact that the abnormal EKs are evident in both Tabby and downless provides evidence that this structure is involved in cusp formation. Incisor tooth germs also have EKs distinguishable by expression of the signaling genes but not evident morphologically. Significantly the difference between monocuspid incisors and multicuspid molars is the formation of secondary" EKs at the tips of the forming cusps in bell stage molars that are not present in incisors.

G. Skeletal Development The vertebrate skull has been a focal point for the investigation of vertebrate development and evolution for more than a centu~'. Note that most vertebrate adaptive transitions involve a role for the skull (Hanken and Thorogood, 1993), a fact that, along with its relative preservation in the fossil record and the ability to reconstruct developing skulls (either with wax models made from sections or differential staining

442 of bone and cartilage), continues to make it a vital structure for investigation of development and evolution. Studies of cranial development provide perhaps the best means of a continuum of investigation and thought from the early masters of evolution and development to the modern world of molecular genetics and cellular biology. The use of Alizarin red and Alcian blue for differential staining of bone and cartilage (a system that allows for the clear three-dimensional identification of both normal and abnormal cranial skeletal development) is a nearly ubiquitous feature of any knockout paper. Unfortunately, there is often little appreciation or understanding of the intricacies and nuances of vertebrate cranial development (or of the classic comparative, evolutiona_ry, and genetic studies) in many discussions of knockouts. In large part this is due to the lack of a clear text describing the developing murine skull and the scattered and abstruse nature of the classic descriptions of cranial development. In Appendix 2 we attempt to address this situation with a brief description of the embu'onic and perinatal cranial skeletons (further details of which can be found in Flower. 1885: Jenkinson. 1911" Fawcett. 1917. 1922: Kesteven. 1926: Thomas. 1926; Broom. I930: Johnson. 1933" Parrington and Westoll. 1940: Greene. 1955" Goodrich, i958" Gr:dneberg, 1963; Youssef, 1966, 1969: Barghusen and Hopson, 1979; Sher. 1971" Presley and Steel. 1976, 1978" Moore. 1981" de Beer. 1985" Frick. 1986" Rugh, 1968" Kuhn and Zeller, 1987 Zeller. 1987" Allin and Hopson. 1992: Novacek, 1993 Kuratani. 1989: Kuratani et al., 1997. and references therein).

1. Regulation of Skeletogenesis Well over 150 heritable defects in murine skeleto-odontogenesis have been recorded (Kalter, 1980: Li and Olsen. 1997) and craniofacial malformations occur in one third of all human congenital maladies (Gorlin et aL, 1990; Thorogood, 1997)mattesting to the complexities of craniofacial skeletal and dental formation, growth, and homeostasis (Mundlos and Olsen. 1997a.b). Such deficiencies may be either osteochondrodysplasias, affecting elements (and teeth) in a generalized fashion, or dysosteochondroses, only affecting particular skeletal elements (or teeth) while leaving all others unchanged (reviewed by Mundlos and Olsen, 1997a,b). Moreover, skeletal ontogenesis is lifelong, with shifts in balance of ~owth and resorption, modeling and remodeling (Frost, 1969; Glimcher, 1989). Each step in skeletogenesis is (in large part) characterized by differential cellular behaviors, whether, for example, that means proliferation, death, transcription, translation and secretion, division, and/or mi~ation (Hall and Miyake, 1992, 2000). As agents of change, both morphogenesis (detected as change in an organism over a lifetime) and evolution (detected as change in a population over generations) share regulation of these behaviors in part by controlling the timing (Thompson, 1942; Gould, t977; Richtsmeier, 1992;

I!10rganogenesis Hall, 1999). Local alterations in the timing (heterochrony) of this control, say, either in induction or response, can result in new morphology and perhaps organisms. Thus, understanding craniofacial morphogenesis or evolution is an exercise in understanding the local regulation of cellular behaviors and the resulting development of structures. Although mechanisms of skeletogenesis are described in detail in Chapter t4, it should be axiomatic that misregutation at any step in these processes can have consequences for craniofacial development (Gr/ineberg, 1963). This is well exemplified by the range of cranial defects present when misreguIation of endochondral ossification occurs leading to malformations. Such malformations can occur, for example, through (1) deficiencies in the formation and maturation of the ECM, as seen with collagen XI a l in cho mice (Li et aL, 1995), various collagen H a1 mutations, including the D m m mice (Brown et aI., 1981" Li et al., 1995" Pace et aL, 1997; Rintala et al., 1997), and aggrecan in cmd mice (Rittenhouse et al., 1978: Watanabe et aL, 1994); or (2) deficiencies in cellular maturation in endochondral ossification, as seen with PTHrP -/- and P T H / P T H r P - R e c e p t o r -/- mutant Irdce, which exhibit diminished chondrocyte proliferation and accelerated hypertrophy (Karaplis et aL, 1994; Lanske et aL, 1996), and Ihh -/- mutant mice (St. Jacques et aL, 1999; Karp et aL, 2000). Skeletal defects in the skull are obvious in mice with either a block in osteoblast maturation [Cbfal, or Cliedocranio dysostosis (Ccd) mice: Komori et aL, I997; Otto et al., 1997] or in bone-resorbing osteoclasts (e.g., c-fos, Johnson et aL, 1992, Wang et al., 1992; c-src, Sofia,no et al., 1991" M-CSF/op, Marks and Lane, 1976; Yoshida et aL, 1990; NF-KB, Franzoso et aL, 1997). Table II lists selected genes implicated as regulators of skeletal ontogeny whose mutations affect craniofacial development.

2. Molecular Evidence of Regulation of Craniofacial Development An exponentially increasing number of genes is being implicated in the regulation of pattern and development of craniofacial structures. Above we have related the essential requirement for epithelial-mesenchymal cross-talk for the realization of craniofacial development. How this requirement is elaborated into discrete, morphologically stereotypic structures is a central focus in craniofacial research. The embr3'onic complexity of the cranial region, as evidenced by the number of cellular origins, potentially instructive and responsive tissues, and craniofacial primordia involved, has complicated the search for spatiotemporal manifestations of coordinating tissues. Because the skull is a composite structure. this elaboration of signal and response into discrete, morphologically stereo~'pic elements may be considered with respect to a number of interrelated tasks (Fig. 2a), including the establishment of (1) inter-BA identity such that each BA within the series is unique; (2) intra-BA identity such that each element within a given BA has an unique

19 Craniofacial Development Table Ii

443

Selected Regulators of Skeletogenesis Revealed through Genetic Analysis

Gene

Principal defects

Citation

Aggrecan

Crnd mice. with depleted ECM and disorganized arrangement of chondrocytes

c~-globin

c-fos

Hydrop fatalis, due to loss of haemaglobin Sulfate transport and metabolism affecting proteoglycans in the ECM in diastrophic dysplasia and brn mice Bpa (bare patches) mice. chondrodysplasia Ccd mace, wormian bones, expanded fontanelles, delayed ossification of calvarial elements; diminished clavicles Osteopetrosis due to a failure of resorption and remodeling

Collagen t

Osteogenesis imperfecta

Collagen H a ]

Dmm/Dmm mice. with a paucity of collagen fibrils in the ECM and retention of procollagen II in the endoplasmic reticulum Older Dell mice

brn Bpa

Cbfal

Collagen H a] Collagen IX al Collagen X a 1 Collagen XI al

cho/cho mice, with misassemblage of collagen II fibrils, disorganized chondrocytes in

C-SIC

GHRH-Receptor

~owth centers and deceased numbers of HTC Osteopetrosis due to a failure of resorption and remodeling Growth hormone has no receptor in the pituitary and dwarfism ensues

Ihh Csfl/op PTH PTH/PTHrP-R Mo

Osteomalacia and altered magnesium metabolism in X-linked hypophosphatemic mice Inappropriate timing of skeletal maturation Osteopetrosis due to a failure of resorption and remodeling Inappropriate timing of skeletal maturation Inappropriate timing of skeletal maturation Copper transport

identity; (3) the neurocranium-parachordaI, prechordal/trabecular, and s e n s o ~ capsular; and (4) the calvarium. a. E s t a b l i s h i n g I n t e r - B A I d e n t i o : It has generally been thought that the B A are segmental, metameric (branchiomerle) strucvares (Barghusen and Hopson, 1979; Langille and Hall, 1989). Following the paradigm established for the vertebrae (and, in effect, the limb as well), the generation of BA identity has been thought to be regulated by H o x gene expression such that the meristic, overlapping pattern of gene expression of this gene family correlates with the identity of the branchiomeres (Fig. 2b). In effect, distinct populations of uniquely H o x - e x p r e s s i n g CNC (perhaps with the complementation of H o x - p o s i t i v e ectoderm) are hypothesized to endow inter-BA identity. The most compelling arguments for such a " ' H o x code" have come from the knock-out studies of H o x a 2 (see Appendix 3 and above; Rijli et aL, t993; Gendron-Maquire et al., 1993). Although H o x a 2 is expressed in R2 (transiently) and more caudal rhombomeres, BA1 CNC is H o x a 2 negative. BA2, in contrast, expresses H o x a 2 in both the CNC and ectoderm. H o x a 2 - / - mutants

Rittenhouse et aL (1978); Watanabe er aL (1994) P~iszD"et at. (t995) Mundlos and Olsen (I997a, b); Pennypacker et aL (I 98 I) Happle et at. (1983) Sitlence er aL (1987); Komori et al. (t997): Otto et al. (1997) Grigoriadis et at. (1994); Wang et aL (1992) Mundlos and Olsen (I997a,b); Stacey et al. (i988); Bonadio et aL (1990) Brown er aL (1981); Pace et aL (1997) Rintala et aL (I 997) Nakata et aL (t993); F~ster et aL (1994) Mundtos and Olsen (1997a,b): Jacenko er aL (t993) Seegmiller and Fraser (1977); Li et al. (1995) Sorim'~oet aL (1991 ) Godfrey et aL (1993); Wajnmjch et aL (I996) Meyer er at. (1979) St. Jacques er al. (I999) Kaku et al. (1999) Karaptis et aL (1994) Lanske et aL (1996) Camakaris et aL (1979) (cit: Sillence et al. (1987)

exhibit mirror image homeotic-like transformations of the BA2-derived structures into proximal B A l - l i k e structures (Fig. 2i). These B A t - l i k e transformations are limited, however, to proximal, peficleftal structures and not the entire arch. Targeted disruption of other rostral H o x genes, H o x a l , for example, disrupts BA2-derived structures (though defects are generally dysmorpholoNes and not A P arch transformations), as well as the supraoccipital arch and associated structures (Chisaka and Capecchi, 1991" Chisaka et at., 1992; LufkAn et at., 199!, 1992; Mark e t aL, 1993). Compound H o x a t - / - ; 2 - / - mutants have exacerbated defects in relevant structures (Barrow and Capecchi, 1999; Fig. 1 l j). Further, indirect evidence for H o x regulation of inter-BA identity comes from analysis of P b x l - / - mutants (see Appendix 3; Fig. 1 lr; Selleri e t al., 2001). P b x l is the vertebrate homolog of the D r o s o p h i l a gene e x t r a d e n t i c I e (exd), which functions genetically in parallel with H o x genes to alter the morphological consequences of Hox activities in flies (Peifer and Weischaus, 1990; Rauskolb and Weischaus, 1994). Nuclear PBX binds D N A with Hox parmers thus formAng complexes on appropriate D N A sites (Beruhelsen e t al.,

444

1!10rganogenesis

Figure 2 Schema of selected mutants with craniofaciaI defects revealed through genetic analysis of murine development. The schema represent the authors' interpretations of published descriptions and figures. Furflaer description and details can be found within the text. Follow the color key for interpretation of defects. As a rule. where representation of elements would obscure that of other elements, unilateral representations of skeletal alterations are depicted.

19 Craniofaciai Development

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":

19 Craniofacial Development

1998a.b; Jacobs et aI., 1999; Ferretti et aL, 2000). As with the Hoxa2 -/- mutants, splanchnocranial structures derived from B A2 and the first pha~'ngeal groove display striking morphological alterations in Pbxl -/- mutants (which die ---El6.0; Selleri et aL, 2001). Morphologically; the lesser horns of the hyoid are transformed into elongated cartilages whose structures are reminiscent of MeckeI's cartilage or of the suspensorial BA2 derivatives of certain nonmammatian vertebrates. Although both Hoxa2 -/- and PbxI -/- mutants display transformations of BA2 structures into BAt-like structures, they are distinct in nature: Hoxa2 -/- mutants have transformations that are more proximal-BA1 in nature, and include a distinct mirror image duplication around the first cleft, while P b x t -/- mutan~ have transformations of a more distal-BA1 nature. Homeotic transformations of other BA, for example, BA3 to BA2. have yet, however, to be demonstrated for any knock-out (Hox or otherwise), and while a Hox code may regulate inter-BA identity other, non-Hoxassociated factors are likely involved. Moreover, chick-quail grafting experiments (see above), complemented by work suggesting that Fgf8 expression at the isthmus restricts the rostral extent of Hox gene expression in the rhombencephalon and possibly the CNC, suggest that BAt is under a distinct regulation from the other BA, and is thus unique w'ithin the BA series to begin with (Fig. 2b; Noden, 1983b; Hunt et al., 1998b; Irving and Mason, 2000). Finalb; fuller appreciation of what sets inter-BA identity may only come with an understanding of the nature of the final arbiter setting the number of BAs which develop.

449 b. Establishing Intra,BA Identio: The generation of intra-BA identity can be viewed as the culmination of processes forming polarities [PD, AP (orat-aboral), and ML] within each BA, processes perhaps most complex and well studied within BAI. Paleontological and comparative emb~ological studies have suggested that the prototypical B A may have been comprised of five PD-oriented chondrocranial and associated dermal elements that would have been repeated in each BA (Goodrich, 1958; Barghusen and Hopson, 1979; Moore, 198t; de Beer, 1985; Hildebrand, 1988; Langitle and Hall, 1989). This fundamental bauplan has tended toward modification in all major vertebrate ~oups, where in mammals, for example, the B A1 chondrocranium is represented by only two components: the maxillary and mandibular derivatives of the palatoquadrate and Meckel's cartilage, respectively. Moreover, within BA 1 at least, an AP axis must be established in order that the teeth appear on the appropriate, functional surface; and, because, for example, tlhe palatal shelves need to meet at the midline, the ML axis must be regulated as welt. While it is not entirely clear how the establishment of each axis is determined, or iffhow each developing axis informs the establishment of the other axes, it seems clear that intra-BA identity is informed by multiple epithelial signals (BMP, FGK SHH, ET-1, and WNT driven) that either induce and/or maintain discrete populations of B A ectomesenchyme (as assessed by unique profiles of gene expression). The epithelium coveting the BA, including both the ectodermal and endode~mat linings, is characterized by distinct spatiotemporal fields of gene expression (see above). These genes generally encode secreted factors (e.g., Bmps, ET-1, Fgfs, Shh) but not exclusively so (e.g., Pitx). Each BA receives signals from at least two sources centered around the pharyngeal clefts: one along the anterior (rostral) and another along the posterior (caudal) surface (Fig. 2c). For BA1, the anterior source emanates from the oral ectoderm. Patteming information in the BA comes from a balance of these sources. The targeted loss of Hoxa2, leading to a mirror image duplication of proximal mandibular structures in BA2, provides evidence that the cleft is at least a point source of patterning information read by the mesenchyme; Hoxa2 -+mutants, however, do not show a full PD transformation of BA2 (see Appendix 3 and Fig. t li; Rijli et aL, i993; Gendron-Maquire et aL, 1993). A conditional loss of function of F g f 8 in BA1 ectoderm (Fgf8 :~c'~-/- mice; see Appendix 3; Fig. 11b; Trumpp et aI., 1999) fm--ther supports this idea of multiple signals. Fgf8 is normally expressed in the epithelium of the oral ectoderm as well as the peri-cleftai ectoderm and endoderm (Crossley and Martin, 1995; Trumpp et al,, 1999). In F g f 8 u~c~e-/- mutants (acNeved by driving expression of Cre under a Nestin promoter to flox FgfS), F g f 8 is lost within the BA1 ectoderm with the exception of a transient posterior (aboral) pericleftat patch of expression. This results m a near complete loss of all BA 1 skeletal struc-

450 tures. The exceptions are informative: one. a malleus (with a body, manubrium, and processus brevis) develops in the mesenchyme underlying this posterior patch: and two, the distal mandibular midline develops with a rostral process of Meckel's cartilage, incisors, and associated dermal bone as welt as maxillary bone associated with the nasal capsules. Mutation of ET-1, another epithelial factor, also leads to drastic loss of maxillary and proximal mandibular structures while the distal mandibular structures form (though they may be aberrantly fused; see Appendix 3 and Fig. 1 l a: Kurihara et aI.. 1994). ET-t is also expressed within the mesodermat core of the BA. though discrimination of the respective roles of its expression within ectodermal and mesodermal cell populations remains to be achieved. ET-1 expression is lost in BA1 ectoderm of the Fgf8 mutants m except within the transient posterior patch of Fgf8 expression (Trumpp et al., 1999). Thus. it appears that multiple (e.~.,,, both anterior and posterior) sources of FGF8 inform intra-BA identity, though not of the entire PD axis because the distal midline is clearly under a distinct regulatory regime. Principal candidates for this role. at least within B A1, come from studies of Shh, Bmps, and associated factors. For example, although loss of Bmp4, which marks the midline ectoderm, results in embryonic lethality, BMP-soaked beads can induce the expression of Msx within mandibular mesenchymal explants (see above). M s x l and Msx2 are expressed in this midline tissue, and their potential induction by BMP signals may be significant because mutation of Msxl leads to midline defects including incisors (though proximal teeth are also affected; see Appendix 3" Satokata and Maas, 1994). Such defects appear to be more severe in compound M s x l - / - 2 -/mutants (Satokata et al., 2000). Moreover, when the BMP antagonists Noggin and Chordin are both mutated a general, catastrophic loss of midline structures results (Bachiller et al., 2000). Within the oral ectoderm, furthermore, experimental evidence suggests that these epithelial factors regulate the dental fields that establish molar (proximal) versus incisor (distal) fields: .Antagonism of BMP signaling along the mid!ine results in ectopic expression of Barxl, loss of Msxl, and the transformation of tooth type (see above; Tucker et al., 1998a). Unfortunately, as with FgfS, the proximate regulation of Bmp epithelial expression has yet to be clarified. The distinction of PD domains of epithelial gene expression is reflected by similar, discrete populations within the underlying ectomesenchyme. One discrete ectomesenchymal population is defined by the expression of the D/x genes (Fig. 10d; see Appendix 3). This gene family has been implicated in the elaboration of element morphology along the PD axis (Qiu et al., 1995, 1997: Depew et al.. 1999). In mace, six DIx genes have been described: Dlxl, Dlx2, Dlx3, DtxS, Dlx6, and DIx7 (Doll6 et at., 1992: Robinson and Mahon, 1994; Simeone et at., 1994: Qiu et al., 1995, 1997; Stock et al., 1996). Studies of genomic organization indicate that

!il Organogenesis the Dlx genes are arranged as tightly linked, convergently transcribed (tail-to-tail) pairs (DlxI and DIx2, Dtx5 mad Dtx6, and Dlx3 and Dlx7) located adjacent to Hox gene clusters. Analysis of DNA sequence similarity and chromosomal location indicates that the Dtx genes can be placed in two paralogous groups: Dtxt, Dtx6, and Dlx7 in one, and Dlx2, Dlx5, and Dlx3 in the other. Moreover, tightly linked Dlx pairs appear to share regulator), regions and are expressed in similar patterns within the BA, placodes, and embryo as a whole (Dolt6 et at., t992; Akimenko et al., t994; Simeone et at., 1994; Robinson and Ma.hon, 1994; Qiu et at., 1995, 1997: Anderson et al., 1997a,b; Yang et aI., 1998; Depew et al.. 1999; Eisenstat et al., 1999: Zerucha et al., 2000). Importantly for B A development, paralogous DIx genes share nested expression patterns within the developing BA: D l x l / 2 are expressed throughout most of the BA ectomesenchyme (i.e., both proximally and distally within the BA), while DlxS/ 6 and Dlx3/7 share progessively restricted domains distally. These genes are not mesenchymally expressed, however, along the distal midline of the arches. The correlation of the nested Dlx gene family expression with a PD skeletal series led to the hypothesis that a combinatorial D/x code regulates the establishment of the distinct skeletal elements within a particular BA unit. This idea has been genetically examined in DLvl, DL~2, Dlxl/2, and Dlx5 mutant mice (see Appendix 3 and Figs. 10g,h; Qiu et aI., 1995, 1997; Acampora et al., 1999; Depew et al., 1999) where D l x l and Dtx2 have been shown to regulate proximal (e.g., maxillary) BA development and Dlx5 to regalate (among other things) proximal mandibular (distal BA) structures. That Dlxl -/-, Dlx2 -/-, and D l x l / 2 -/- mutants evince no change of morphology where other Dtx genes are expressed, suggest that these other Dlx genes, in particular DIx5 and Dlx6, compensate for the loss. In line with this, a genetic interaction between Dlx2 and Dlx5 is seen in compound Dlx2-/-;5 -zmutant mice in which the mandibular (i.e., distal BA) sta-'uctures~except the distal-most midline, as represented by the incisor fields~are drastically altered (M. J. Depew and J. L. R. Rubenstein, unpublished observations). A further prediction of this combinatorial model is that loss of Dtx5 and DIx6, resulting in duplicated regions whereupon only DlxI and Dlx2 are expressed, should proximaiize a segment of the skeletal series. This prediction is currently being tested in Dlx5/6 -/- double mutants (M. J. Depew, T. Lufkin, and J. L. R. Rubenstein, unpublished data). Regulation of Dlx expression remains an open question. While FGF8soaked beads are capable of inducing Dtx2 and DLv5 in mandibular mesenchyme, mutants in which Fgf8 h ~ been floxed in BA 1 epithelium maintain early (E9.5) Dlx expression patterns. B MP-soaked beads are also capable of inducing D/x expression, but Bmp expression domains in the BA are generally not in register with mesenchymat Dtx expression patterns and assessment of D/x expression in Bmp mutants has not been genera!Iy available (Fig. 2d).

19 Craniofaciai Development PD polarity within the arches patentIy extends beyond the expression domain of the Dlx genes to the distal rnidline (as well as to the olfactory ptacode in B A t). A number of other genes are mesenchymally expressed along the distal midline, often members of a family of genes likewise distally expressed [e.g., M s x l and Msx2; Prxt and (to a much lesser extent) Prx2; Alx3, AIx4, and CartI; d H A N D and eHAND]. Distal midline skeletal defects often accompany mutations in such genes, including in the P r x l -~-, P r x t - / - ; 2 -/-, M s x l - / - , M s x t - / - ; 2 - / - , Alx4 -/-, Cartl -/-, Otx2 ~/-, and Oto mutants (see Appendix 3 and Figs. i 0o,p; Satokata and Maas, t994; Acampora et al., 1995; Martin et at., 1995" Matsuo et al., 1995" Ang et at., 1996; Qu et at., 1998, 1999; ten Berge et at., 1998b; Lu et aI., 1999b; Zoltewicz et at., 1999; Satokata et al., 2000). Defects are usually associated with hypoplasia of the midline though (perhaps unexpectedly) proximal portions of the BA may be nearly normal despite the absence of the midline. Mutations of the distal midline factor dHAND, however, die early (E t I) but have hypoplastic B A (Srivastava et at.. 1995. 1997; Thomas et al., 1998). W~hile most patterns of gene expression assayed in these mutants appear normal, M s x l expression is decreased (Thomas et al., 1998). Additional genes are expressed in regions within D/xpositive domains but may not extend throughout the proximal most or distalmost mesenchyme, including Lhx6 and Lhx7, Barxl, Pax9, and Gsc. Expression of some of these are affected in Dlx mutants (e.g., Gsc in Dlx5 -/- and Barxl in D l x I / 2 -/- mutants; Depew et at., t999; Thomas et al., 1997) while others less so. At the other end of the PD axis in BA1 lies the olfactory apparatus. Numerous genes are distinctly expressed either within the epithelium of the placode and pit (e.g., Dlx, Fgf8, Pax, Pitx, Shh, a~rld Bmp) or the underlying mesenchyme (e.g., Msx, Gsc, AIx, Pax, RAR, and Prx), where the general correlation of gene expression within the distal mSdline and the olfactor-y-maxillaw conjunction is striking and may reflect a mechanism to maintain dental occlusion/alignment of the maxillae (BAi derived), premaxillae (frontonasal prominence derived), and the mandibutae (see above and Appendix 3 for further details). While many genes have been identified as required for some aspect of intra-BA elaboration (Fig. 2e), their developmental interrelationships are only rudimentarily understood. c. Forming the Neurocraniurn. Proper generation of the neurocranium involves forming the parachordal and trabecuIar basal plates, the paired sensor' capsules, and their eventual inte~ation to form a structural unit (see Appendix 2). Signals from the notochord (e.g., SHH; see Appendix 3) and a competent adjacent mesoderm (e.g., expressing the appropriate GIi, Pax, and Nkx genes) appear essential for parachordal development (see Appendix 3; Chiang et aI., 1996; Mo et al., t997; Lettice et al., 1999; Peters et al., I999; Tribioti and Luftdn, 1999). The development of the neurocranium anterior to the notochord is rather complex as competent

451 chordomesoderrnaI, AVE. facial ectodermal, olfactow placodaI, neurepithetiat, and CNC tissues are each essential for trabecular basal plate and/or nasal capsular formation (optic and otic capsular development are touched on elsewhere). This region (essentially the rostral head) subsequently is sensitive to perturbation. Many types of disruption occur in the neurocranium anterior to the hypophysis in mutant mice, including toss of the nasal capsules without full loss of associated dermal bone or midline (trabecular) structures (e.g., Pax6 -i-" Roberts, 1967" Hogan et at., 1986; Hill et at., 1991" Matsuo et at., 1993; Grindley et al., t995; Kaufman et al., 1995; Osumi et al., 1997" Osumi-Yamashita et aI., 1997b; Quinn et at., 1997; Sander et al., 1997; Sosa-Pineda et al., t997); asyrmnetfic loss of the nasal capsular structures ( H e s x I - / - ; Dattani et aL, 1998) with associated trabecular defects (e.g., D t x 5 - / - ; Depew et at., 1999); loss of medial nasal capsular structures without particular trabe.cular defects (e,,.=., Pax7 -/-, Pax9-/-; Mmnsouri et al., t996; Peters et at., 1998); toss or, or collapse about, the midline trabecula_r structures resulting in holoprosencephaly and cyclopsia (e.g., Oto, Shh -/-, Noggin -/-. Chordin -/-" Chiang et at., t996; Zoltewicz et at., 1999; Bachitler et aL, 2000); separation but not toss of trabeculae without loss of capsules (e.g., P D G F - R a -/-, A t x 4 - / - ; Cartt -/-, GIi2-/-); separation with loss of both trabecular and capsular structures (e.g., Otx2"/-, , ~ 4 R a - / - ; y - / - ; Lohnes er aI., 1994; Acampora et at., I995; Mark et al., 1995" Matsuo et at., I995; Ang et al., t996); disruption of both trabecular and capsular structures without specific midline separation (e.g., Wnt5a -/-, Gti3-/-; Mo et al., !997; Yamaguchi et al., 1999); and total toss of both trabecutar and capsular structures (e.g., L i m - / - ; Otx2~/-; Acampora et al., 1995" Matsuo et al., 1995; Shawlot and Behfinger, 1995" A n g e t al., 1996). Moreover, the integration of the CNC-derived trabecular plate and taenia marginalis (see Appendix 2) with the mesoderm-derived parachordal plate may variously be affected (e.g., B m p 7 - / - ; D l x t / 2 - / - ; M s x t - / - ; O t x t - / - ; Satokata and Maas, 1994; Dudley et at., t995; Luo et al., t995; Suda et at., i996; Qiu et aI., t997). Overall, anterior neurocranial development follows a delicate balance of factors tending to expand the midline and those tending to collapse, or constrict, it (i.e., a balance of anterior DV and ML patterning mechanisms; Fig. 2e). Untangling the relative patterning contributions of the chordomesodermal, AVE, facial ectodermal, olfactory placodaI, foregut endodermal, neural plate/neurepithelial, and CNC tissues (and genes expressed therein) to the patterning and development of the skull anterior to the hypophysis remains a principal challenge in craniofaciat research. d. Forming the Catvarium: Cranial Articulations and Suturogenesis. The growth of the dermatocranial catvarial

bones (see Appendix 2) begins as mesenchymal precursors condense at specific sites on the dorsal surface of the head and produce an osseous matrix. These condensations extend out-

452 ward, eventually meeting at the presumptive sutures. Growth then becomes localized to the periphery of the extending bone, at the lateral margins of the sutures known as the osteogenie front (Johansen and Hall. t982). The location of cells within the osteogenic front appears to be correlated with their state of differentiation, with the lateralmost cells expressing terminal differentiation markers such as bone sialoprotein. Although an increasing number of genes are being implicated in calvariat development (see Table III), to date our understanding is limited of the cellular and molecular regulation of the individual calvarial elements, why they form where they do. and what establishes their boundaries, growth. and sutural articulations. Sutural closure involves a transition from synchondroses (bones connected via cartilage) or syndesmoses (bones connected through dense fibrous connective tissue) to synostoses (Zimmerman et at., 1998). Early in development the calvarial elements are principally syndesmotic, being "'open" to allow for growth. The normal development of the calvarium requires mechanisms that ensure that the rate of growth and morphology of the elements is coordinated with that of the brain and jaws (Iseki et al.. 1999), and involves the tissues between them. including the meninges. The dura mater is integral to the maintenance of a suture because without it the suture fails to remain open and will synostose (Opperman et at.. 1993, 1995. 1998: Kim et al., 1998). Light and electron microscopic examinations of sutural maturation reveal a transition from a bone forming front to a bone-tendon junction accompanied by the replacement of osteoblasts (in part through apoptosis) and mineralizing osteoid to fibroblasts and nonmineralizing, straight collagen bundles (Zimmerman et al., I998). The development, form and time of sutural closure have traditionally been thought to result from an interplay been hereditar?: determination and functional adaptation to brain growth (Young. 1959: Moss et al.. 1972: Oudhof. 1982: Herring. 1993: Jabs et al.. 1993; Bellus et at., 1996a: Zimmerman et al., 1998). For example, calvarial elemental morphology and sutural sequence and maturation were influenced by cranial contents in studies of experimentally induced macrocephaly (hydrocephaly) and microcephaty (Young, 1959). Each suture delineates a growth center (Iseki et al., t 999). Different sutures share some signaling mechanisms thoueh developmental specificity is reflected by the uniqueness of the affected suture in craniosynostotic syndromes (conditions of premature sutural closure: see Table III). Clues to the molecular and cellular events involved in sutural growth and closure have come from genetic and experimental analysis. Msx2 is expressed in some of the cells of the osteogenic front and dura mater. Boston type craniosynostosis has been shown to be due to a single amino acid substitution in MSX2, which produces a dominant positive (Jabs et al.. 1993). Consistent with this. overexpression of Msx2 causes bone cells to invade the sutures (Dodig et at., I999). This appears to be

ill Organogenesis due to a transient retardation of osteogenic cell differentiation within the suture, leading to an increased pool of proliferative osteogenic cells and ultimately to an increase in bone growth (Y. H. Liu et al., 1999). Msx2 has been shown to be regulated by BMP signaling during suture development (Kim et al., 1998). Bmp2 and Bmp4 are expressed in the osteogenic front and sutural mesenchyme, arid it has been proposed that they regulate the balance between the undifferentiated and differentiated states of osteogenic ceils via regulation of Msx. B MP4 beads cause an increase in tissue volume when placed on the osteogenic front or midsuture area. but do not affect sutural closure (Kim et at., 1998). Activated mutations in the FGF Receptors 1 - 3 lead to craniosynostosis, including Apert, Jackson-Weiss, and Crouzon and Pfeifer syndromes (reviewed by Webster and Donoghue, 1997 9V~;11kle. "" 1997" Burke et al., t998). FGF4 beads are able to induce Msxl, but not Msx2, in sutural tissue, and lead to accelerated sutural closure when placed on the osteogenic fronts (Kim et al., 1998). FGF2 beads have been shown to upregulate Fgfr], downregulate Fgfr2, and inhibit cell proliferation (Iseki et al., 1999). Fgf2 is abundant in skeletal membranes and is upregulated within sutures as they fuse. while Fgf9 is expressed throughout the calvarial mesenchyme (Iseki et al.. t999: Mehrara et at., t998; Kim et at., 1998). Fgfr2 is intensively expressed in the osteogenic fronts in proliferating osteoprogenitor cells. The onset of proliferation is preceded by Fgfr2 downregulation with a corresponding upregulation of Fgfr]. As the differentiation marker osteopontin turns on, Fgfrl is downregulated. Fgfr3 is expressed in cranial cartilages, including the plate of cartilage underlying the coronal suture, as w'ell as in osteogenic cells, suggesting a dual role (Iseki et aI., 1999). The phenotype of the FGFR3 mutation is similar to that of Saethre-Chotzen syndrome. This syndrome is caused by a loss-of-function mutation in TWISZ a gene encoding a basic helix-loop-helix (bHLH) protein. The Drosophila omholog of TWIST regulates transcription of heartless, a homolog of vertebrate Fgfrs: Saethre-Chotzen syndrome is thus likely to have a defect in FGF signaling (El Ghouzzi et at., 1997). Overall, experimental assessment of coronal and sagittal sutural development has suggested a mechanism involving the balanced coupling of the recruitment of cells into osteogenic differentiation (involving Fgfrl and ligands) and the maintenance of osteoprogenitor cells in a proliferative state associated with the outer osteogenic front (involving F~oSr2, its ligands, and possibly BMP. Msx, Twist, and Idl; Kim et at., 1998" Iseki et aI., 1999; Rice et at., 2000). Other signaling molecules have also been shown to play a rote in suture development. Shh and its receptor gene, patched, are expressed in a mosaic pattern along osteogenic fronts at the end of emb~,'onic development, by which time the expression of Msx2, Bmp2, and Bmp4 are reducing (Kim et al., 1998). T g f - ~ l - 3 also exhibit distinct patterns of expression during sutural formation (Opperman et al., 1997).

453

19 Craniofacial Development Table III Gene Alx4 ALr4 /Cart]

compound Bapxl BMP-I Cbfa- I (Ccd) Crti l Csfl/osteopetrosis Dtx2 DlxI/2 Dlx5 FbnI FGF2 FGF3/4 (Bulgy.eye) Fgf8 Fgfrl Fgfr2

Selected Regulators of Skeletogenesis Revealed through Genetic Analysis Principal defects

Undermineratized, diminished parieuds Undermineratized diminished frontals, parietals, supraoccipital, and squamosals Agenic supraoccipitat Deficiency of frontal, parietal, interparietM, and expanded associated fontaneIles Arrested development of osteogenic tissues: cleidocranio dysostosis Domed skull: misshapen parietal and frontal Misregulated remodeling with stenosis and synostosis of the sagittal suture in maturing mice Split and duplicated squamosal, loss of lamina obturans Split and duplicated squa.rno~t, loss of lamina obturans Undermineralized parietal and interparietals Craniosynostoses: Maffanoid, Shprintzen, Goldberg Enlarged occipital, chondrodysplasia Coronal craniosynostoses: Crouzon Loss of lamina obturans, dysmorphic squamosal Craniosynostoses: Pfeiffer Coronal Craniosynostoses: Crouzom Pfeiffer, Jackson-Weiss, Apert and Beare-Stevenson

Fgd'r3

Muenke craniosynostosis, achondroplasia and macrocephaly

Fidget Gbx2

Fusion of pm--ietosquamosat suture and presphenoid and baxsisphenoid Agenic interparietaI, diminished supraoccipital

Gti2 Gli3/Xt IKK1/IKKa ( Chuk)

Deficiency and delayed ossification of frontal parietal, interparietN Failure of skull vault formation Domed skull with ectopic infraparietaI, dysmorphic zygomatic

Lmx l a/dreher

Dysmorphic interparietal with fenestra, dysmorphic tambdoidat and parieto-interparietal sutures Split supraoccipital Hydrocephalus, with no bony calvarium coveting the bulging cerebral hemispheres and ectopic midline frontal disorganized neurocranium

M 3 3 / b m i l (Pc) i ~ f l / F o x c l cong. hydro-cephalus M sx l /,Vlsx2 compound Msx2 PlO7/p130 Pax6 (Small eye) Prx] (Mhox) Pugnose Querkopf K 4 R a /~, Ryk T gf-~2 Twist

Craniosynostosis: Boston type Enlarged parietal foramen, enlarged parietosagittat fontanelle Diminished supraoccipital/interparietal Dysmorphic frontal, parietal, and squamosai Agenic supraoccipital (including tectum) Domed skull: misshapen, shortened parietal and frontal Dysmorphic frontal, parietal, occipital, and associated sutures (not cmniosynostotic) Minor dysrnorphogenesis of vault elements Reduced and dysmorphic (undermineralized?) frontal, parietal, interparietal, squamosaI and occipital Coronal craniosynostosis: Saethre-Chotzen syndrome

Wnt5a

Indicates knock-out, conditional knock-out, mutagenesis, or classical mutant stock. ~"Indicates human studies. Indicates overexpression. Indicates a dominant negative allele. "Indicates haploinsufficiency.

Citation Qu et aL (1997) ~ Qu et aL (t 999)~ Lettice et aL (1999Y; Yribioli and Lufkin (I999)" Suzuki et aL (1996)"~ Sillence etaL (t987): Komori et aL (1997)": Otto et aL (1997)" Watanabe and Yamada ( 1999y Kaku et aL ( 1999)~' Qu et aL (1995) ~ Qu et aL (1997) '-' Acampora et aL ( 1999)'~; Depew er aL ( 1 9 9 9 y Sood et aL (1996) ~ Coffin et aL ( 1995)" Carlton et aL (1998) Trumpp et aL (I999) ~' Muenke et al. ( t994)~': Betlus et aL (1996a,b) ~' Jabs et aI. (1994)#: Reardon et aL (t994)~; Lajeunie et aI. (1995)9; Neilson and Fricset (1995)b: Rutland et aL ( 1995)~; Wilkie et aL (1995)b: Bellus et aL (1996a,b)b: Gatvin et al. (1996) ~ Rousseau et aL ( t994)b: Shimng et aL ( 1994)e: Naski et aL (1996)b; Deng et aL (i996) ~' Truslove (t 956) Wasserman et aL (I997); M. J. Depew (personal observation)) Mo et aL (1997) '~ Johnson (I 967)~; Mo et aL (1997) '~ McLaughlin et aL (1997)~: Hu et aL (1999)'~: Li et aL ( 1999)'~; Takeda et aL ( 1999)'~ Bierwotf (1958y; Manzaneres et aL, 2000y; MilIonig et aL. 20C~))~ Be! et aL ( 1998Y Gr/ineberg (t953); Gridneberg and McKramaratne (1974); Kume et aL (1998)

Jabs et aL (t993. I994)~~";Y. H. Liu et aL (1999): Wilkie et aL (2000)~.~; Satokata et aL (2000y Cobrinik and Knechel (1996y Kaufman et al. (I996); Osumi-Yamashita et aL (1997b) KddweI1 et aL ( 1961)~ Thomas er aL (2000)~ Lohnes et aL (1994) Halford et aL (2000) Sanford et aL (1997) ~ E1 Ghouzzi et al. (1997y.b; Howard et aL (t 997)'~'; Bourgeois et aL (! 998)~ Yamaguchi et aL ( 1999)~

111Organogenesis

454 IV. Conclusion In this chapter, we have focused on the organization, development, and pattern of craniofacial tissues. We have seen that in order to generate a functional skull, an e m b u o must specie' its body axes, along with its CNC. mesoderm, ectoderm, and endoderm. CNC must "migrate" correctly in concert with the ectoderm: CNC must stop at appropriate locations; cells must differentiate and condense" cells must be competent to recognize change of status at each junction and be aware of their position" cells must "know how to respond appropriately to apoptotic signals and cells must behave differentially in order to generate morphology. How this occurs is regulated by both genetically intrinsic factors (e.g., gene expression eventually regulating the capacity to signal, competence to receive signals, etc.) and extrinsic factors (e.g., when and where cells get signals, how loudly the signals come/go, etc.). Though we can now recognize the need to fulfill each of the above functions, the particulars of each step are not always clear, and answers to many of the basic questions that have been asked for a centu~' regarding craniofacial development remain incomplete: What dictates where each element will form and how many blastamata it will incorporate? What sets its boundaries and eventual shape? What is the nature of the fundamental organization of the skull? What is the nature and origin of the cells forming cranial structures? What is the precise nature of the interplay of genetic and epigenetic regulators of craniofacial patterning? While strides have been made in answering the question "Vvqaat is the molecular evidence for the generation of pattern (organization) within the developing cranium, especially within the ectoderm, neural tube. and CNC.. the ever proximate establishment (induction) of regional gene expression in these tissues remains a goal. For example. what is it that induces the expression of Fgf8 in the facial ectoderm and by what mechanism(s) is the nested pattern of D& gene expression in the B A established? The realization of the vast contribution of the CNC to the skull revitalized studies of craniogenesis: yet, we still seek to understand the mechanisms that are involved in the generation of the CNC from the ectoderm, and how they possibly impart positional information, cell fate. and regional pattern. Studies of the CNC still address basic questions: What are the relative contfibutions of CNC and ectoderm to patterning ~ Do separate patterning mechanisms exist for CNC rostral to R3 relative to that caudal to R2? What is the significance of the uniqueness of BA1 relative to the other BA? What causes the CNC in the maxillar?" B A 1 to proliferate toward the eye and nose? What cell-cell and cell-ECM interactions are critical for the elaboration of craniofaciat development, and do the epithelial-mesenchymal interactions necessary for dentoskeletal development unmask pattern, generate pattern, or both? What establishes the precise, idiosyncratic and stereotypical patterns of cell migration? What regulates the subset

of cells that enters the mesodermal core of the BA? Investigation of the neuraxiaI patterning has further revealed pertinent questions: What roles do the iskhmic organizer, anterior visceral endoderm, anterior neural ridge, and chordomesoderm play' in patterning craniofaciat hard tissues? Clearly, the combination of an array' of critical questions and the accessibility of cranial hard tissues to examination will make the next century of investigation on craniogenesis as productive as the last.

Acknowledgments R T. S. would like to acknowledge the support of the MRC. Weltcome Trust. and BBSRC. M. J. D. would like to thank John L. R. Rubenstein and Darrell Evans for their time and input, and the support of the March of Dimes, NqDR gant T32 DE07204 and ARCS. A. S. T would like to acknowledge the WellcomeTrust for its support

V. Appendix 1" Descriptive Dental Development

A. Epithelial Thickening Stage CNC cells migrating to BA1 complete their journey by E9 (Lumsden and Buchanan, 1986). The first morphological signs of tooth development can be seen at approximately E 11.0 (E 1 t.5 for the molars) as localized thickenings of the oral epithelium: hence, this stage of tooth development is known as the epithelial thickening stage. Hay" (1961) reported that the mouse mandibular tooth germs arose from three separate in-gowths of the oral epithelium into the mesenchyme. She observed that the molars developed from the two proximal in-gow,"ths on each side of the jaw and that both incisors formed from one anterior, distal-midline thickening. This thickened epithelium is comprised of approximately five layers of cuboidal cells at its thickest part; by comparison, the rest of the oral epithelium is composed of one or two layers of cuboidal cells (Cohn, 1957). The nuclei of the cuboidat cells in the thickening are oriented perpendicularly to the basement membrane (Fig. 3) (Peterkova et al., 1993). The epithelial thickening yields the dental lamina on the lingual aspect and the vestibular lamina on the buccal aspect. The vestibular lamina forms a sulcus between the cheek and the teeth. The dental lamina gives rise to the teeth. As the dental larrfina thickens, the deep cells of the lamina remain cuboidal in shape with their nuclei oriented perpendicular to the basement membrane. However, the epithelial cells on the oral surface of the dental lamina form a layer of flattened squarnous cells whose tong axes of their nuclei are parallel to the oral surface (Peterkova et aL, 1993). Three-dimensional reconstructions of the premaxilla.~ incisor region reveal multiple dental laminae, which are perhaps remnants of incisors lost by mice during their evolution (Peterkova et aL, 1993). These dental laminae (six in total) are


455

Figure 3 Tooth development. The diagrams represent frontal sections of developing tooth germs. (A) The epithelial thickening stage of tooth development. The oral epithelium has thickened in presumptivetooth-forming areas. (B) The bud stage of tooth development. The dental lamina ha,; invaginated into the underlying ectomesenchymeand the mesenchymatcells have begun ~o group together to form the dental papilla. (C) The early cap stage of tooth development. Morphogenesis of the tooth germs has begun, the ectomesenchymehas condensed to form the dental papilla. Internal dental epithelial cells can be distinguished from external dental epithelial cells: see text. The transient, putative signaling center, the enamel knot, is visible at this stage and is associated with another transient swacture, the enamel septum. (D) The late cap stage of tooth development. Epithelial cells of Lhedenta! organ have formed the stellate reticulum. Internal dental epithelial cells become more columnar in shape, whereas the external dental epithelial cells are cuboidaI. (E) The late bell stage of tooth development. Internal dental epithelial cells have differentiated into ametoblasts that secrete enamel matrix. Dental papiIla ceils have differentiated into odontoblasts that give rise to predentine. all found to contribute to the further development of the premaxillary incisor. A further seven transito W tooth primordia were found between the premaxiltary incisors and maxillary molars in the diastemal regions (Peterkova et al., t995, 1996; Lesot et al., 1998). However, these all regressed (probably by apoptosis). It has been suggested that these transitory primordia might be anlagen of ancestral canines and premolars. Three-dimensional reconstruction of mandibular diastemal and molar regions showed that, unlike in the upper jaw diastema, only one shallow lamina forms (Lesot et at., 1998).

and molars at E13.5-14.0). The epithelial invagination is now termed the dental organ. The peripheral cells of the organ remain cuboidal, whereas the ceils on the oral surface are squamous and those internal to the peripheral epithelial cells are densely packed and round in shape (Fig. 3B). The formation of the epithelial bud is associated with a localized condensation of the underlying ectomesenchyme. Most of the mesenchyme condenses at the base of the dental organ forming the dental papilla.

B. Bud Stage

The next stage of tooth development is the "cap" stage; incisors reach an earIy cap stage by E t4.0 and molars by E 14.5. The dental organ changes in shape, looking less budlike and more conical with an indentation on its deep surface (Fig. 3C). Peripheral epithelial cells are now different in appearance. External dental epithelial cells are cuboidat in

After the dental lamina has formed, further invagination of the epithelium into the underlying ectomesenchyme resuits in an epithelial structure that resembles a bud shape; hence, this is the "bud" stage (reached for incisors at E13

C. Cap Stage

!110rganogenesis

456 shape, having a very large nucleus and little cytoplasm, whereas internal dental epithelial cells are slightly more coIumnar in shape with a central nucleus. The region where the internal epithelial cells meet the external epithelial cells is called the cervical loop. The dental organ sits on the dental papilla, now a "balI" of densely packed ectomesenchyme. Both the papilla and the dental organ are encased in the dental follicle, which consists of condensed mesenchyme cells, oriented in a radial pattern. At this stage of tooth development, the enamel knot (EK) first becomes evident as a transient localized thickening of internal dental epithelial cells that has been proposed to act as a signaling center with a putative role in cusp formation (Butler. 1956: Yhahtokari et aI., 1996a). The enamel septum is another transient structure of the dental organ present at the early cap stage of molar tooth development. The septum is a strand of epithelial cells that runs from the EK to the external dental epithelial cells, dividing the dental organ into two parts. The function of the enamel septurn is as yet unknown. At this early cap stage the incisor tooth germs begin to differ in shape slightly from the molars. In the incisors the labial projection of the cap stage tooth grows more posteriorly (more anteriorly in the maxillary- incisors) than the lingual projection of the dental organ. By E 15. the central cells of the dental organ become recognizable as the stellate reticulum (Fig. 3D). The stellate reticulum cells are star shaped with large intercellular spaces potentially playing a role in supplying enamel-secreting ameloblasts with nutrients. By El6.0 in the incisors and El7.0 in the molars, the stratum intermedium begins to be recognizable from the internal dental epithelial cells as a layer of flattened epithelial cells between the stellate reticuIum and the internal dental epithelium. The internal dental epithelial cells increase in height and become preameloblasts.

D. Bell Stage Incisors reach the bell stage by EI7.0 and molars by Et7.5-18.0. The dental papilla cells differentiate into odontoblasts, beginning in the most anterior mesenchymal cells (Fig. 3E). The external dental epithelial cells decrease in thickness and become a one or two cuboidal cell layer. The preameloblasts almost double in height and differentiate into ameloblasts, their nuclei peripherally placed: this differentiation occurs in the most anterior regions first. There are areas of internal dental epithelial cells in both the incisors and molars that do not differentiate into enamel-secreting ameloblasts. The lingual side of the incisors does not become coated with enamel because internal dental epithelial cells do not differentiate into amelobIasts on this side of the incisor tooth. In addition, in the molars the tips of the cusps do not become coated with enamel" again, this is as a result of internal dental epithelial cells tailing to differentiate into ame-

tobtasts. At El7.0 these nondifferentiating internal dental epithelial cells are at their greatest height. In subsequent stages of development, they diminish and become cuboidal in shape, then flattened, and eventually merge with adjacent connective tissue cells. By E I 8 in the incisors and El9 in molars, the odontoblasts begin to secrete predentine. The dentine laid down on the labial side of the incisors forms a much thicker layer than that on the lingual side. After a further 24 hr of development, predentine becomes calcified and enamel matrix is secreted by, the amelobtasts. Enamel matrix is only laid down in areas where amelobIast differentiation has occurred, therefore, in the incisors, no enamel is seen on the most anterior labial side. the incisal edge, or the lingual side of the tooth. Calcification of the enamel matrix occurs postnatally and the incisors and first molars erupt by 20 days after birth (P20). When mineralization of dentine and enamel occurs the shape of the tooth becomes fixed.

E. Second and Third Molar Development The second and third molars develop when the jaw of the mouse has elongated enough to accommodate their large size. The first signs of the second molar, the dental lamina, can be seen at E15.5 forming as an outgrowth of the first molar tooth germ epithelium. By E18.5 the second molar is at the cap stage of tooth development and these teeth erupt approximately 25 days after birth (P25). The lamina of the third set of molars is not apparent until the fourth day' o f postnatal development; they reach the cap stage by P 7 - 9 and the bell stage by PI0, the third set of molars have erupted by the .~.~th day postnatal. A percentage of wild-type mice may fail to develop third molars (Griineberg, 1963). More than 200 genes have been identified as having been expressed during tooth development, and their expression patterns are available on a database (http://www.honeybee .helsinki.fi/toothexp). Molecular markers exist for all cell types and stages of tooth development.

VI. Appendix 2" Morphological Organization of the Murine Skull The murine skull is a complex, composite, modular assemblage of skeletal elements with diverse developmental origins that encases the brain, its associated primary sensory organs, and the oral and respiratory openings. Ontogenetically it can be viewed as three separate skulls: embD'onic, perinatal, and adult (Moore, 1981; Zeller. t987; Presley, 1993). The initial, embryonic cranial skeletal structures to appear are the cartilaginous (chondrocraniaI) elements, whose development reflects the bauplan of the vertebrate skull (Fawcett, 1927; Goodrich, t 958; Bm~husen and Hop-

19 Craniofacial Development son, i979" Moore. t981-de Beer. 1985" Kuhn and ZelIer, 1987" Zeller, t987: Northcutt. 1990: Gans, 1993; Hanken and Thorogood, 1993" Novacek, I993: Kuratani et al., I997). The perinatal skull arises with the advent of the nascent dentition and the intramembranously ossified elements of the skull (the dermatocranium) which develop around the chondrocranium. The adult skull, or syncranium, develops with the refined modeling and remodeling of the cranial elements to fit the functional demands of the adult.

A. General Description: Units and Divisions Historically a number of terms have been used to describe divisions and units within the skull based on embryonic origin, function, or mode of ossification. The chondrocranium (endocranium) is composed of those structures, which develop as cartilaginous units. These units have numerous possible fates: endochondral ossification, direct investment by dermal bone, degeneration, or transdifferentiation. Two chondrocranial components are distinguished: the neurocranium and the splanchnocranium (viscerocranium). The neurocranium is based on a/;anctional division and includes those structures that support the central nervous system (CNS), and the primary sensory; organs. The neurocranium can be further subdivided into parachordal, trabecular (prechordal or achordaI), and sensory capsular divisions. The viscerocranium is composed of those elements derived from the branchial (visceral or pharyngeal) 'arches. which give rise to the masticatory, pharyngeal, and laryngeal apparatuses. In mice, as in eutherians in general, splanchnocranial elements also form portions of the cranial side walt (see below). The dermatocranium (exocranium) is composed of those intramembranous elements that surround and develop in coordination with the chondrocranium and dorsal CNS.

B. Overview of the Embryonic Chondrocranium The early' (---El3) embryonic neurocranium develops out of the coalescence of a number of chondrogenic centers (Fig. 4). The floor is subdivided rostrocaudally at the hypophysis into the parachordaI basal plate (pars parachordalis) and the trabecular basal plate (pars trabecutaris, trabecula cranii or prechordal plate). This division is fundamental in nature, dividing the NC-derived from the mesoderm-derived structures. As the name suggests, the pa_rachordat cartilage develops adjacent to, and appears to be induced by', the notochord (Pourqui6 et al., 1993; Martaugh et at., 1999). The rostratmost end forms from the paired parachordaI cartilages proper, while the caudal end forms from the somite-derived occipital cartilages. Initially, the chondrogenic centers of the paired parachordals are separate, but they coalesce medially; the rostral gap between them, the basicraniat fenestra, may

457 perinatalIy persist as a small hole. The occipital cartilages develop in greater proximity to the notochord and no gap is seen. Caudally, lateral alae wii1 extend to meet at the dorsaI midline. The caudal basisphenoid and basioccipitat will form within the parachordat basal plate. The trabecular basal plate has a complex evolutionary genesis and is formed by a number of chondrogenic centers. The caudatmost end is formed by the acrochordat and polar cartilages which eventually form as a plate (hypophyseat lamina) across the midline just anterior to the rostralmost end of the parachordat plate (clearly seen ---EI4.0). Rostrat to the acrochordals are the associated, paired trabecular cm'tilages. In the mouse, the trabecutar cartilages are closely apposed at the midline so as to usually appear as a single midtine cartilaginous rod. The trabecular and acrochordal cartilages quickly come into continuity, yielding the bodies of the basisphenoid (caudad) and presphenoid (rostrad). These structures wilt further conjoin with the parachordaI cartilage culminating in a continuous basal plate (central stem). At the rostratmost tip of the notochord, within the basisphenoid where the pituitary forms from Rathke's pouch, a hypophyseal fenestra may be seen early and a hypophyseal fossa later. Hence, the mature basisphenoid, on which lies the hypophysis, is both a mesodermat and an ectomesenchymat element. The basisphenoid extends bilateral basitrabecular processes (processus alaris), which conjoin with the alicochlear commissures and alae temporatis (see below). The trabecular rod extends anteriorly to contribute to the midIine nasal septum (septum nasi) to which the nasal cartilages join, forming the mesethmoid under the olfactory bulbs. The trabecular plate thus provides the support for CNS structures from the hypophysis to the olfactory bulbs. To either side of the basal plate ~ e the paired nasal, orbital, and otic sensory" capsules. The nasal and most of the orbital cartilages appear to be NC derived whiIe the otic is principally mesodermal in origin. Ventral and lateral to the neurocranium the elements of the splanchnocranium develop, the most prominent of these being Meckel's cartilage (MC)--the cartilaginous core of the mandibular branch of the first branchial arch (BAt). [Terminology for the brancNaI/visceral/phary'ngeal arches, and for the skull in general, grew out of a number of distinct anatomic and palaeontologic traditions and suffers from a lack of cohesion. For example, %r some the first "branchial arch" develops caudal to the "mandibular" (BAt) and "byold'" (BA2) arches and is equivalent to the third branchial arch of the terminology employed here.]

C. Regional Morphology 1. Nasoethmoidal (Frontonasal) Region (Regio Ethrmoidalis): Chondrocranial The nasoethmoidat (frontonasaI) region is comprised of those structures which support the olfactory apparatus, in-

458

II! Organogenesis

Figure 4 Generalized late embr?'onic murine chondrocranium seen from (A) norma basalis, (B) norma verticalis, and (C) norma !ateralis. Dark blue represents spIanchnocranial elements: turquoi~ represents the cribriform plate. See list of abbreviations for structural identification. cluding both respiratory and olfacto~ epithelium, the VNO, the olfacto~, bulbs, and rostrat palate. The regio ethmoidalis of the chondrocranium is represented therefore by the nasal capsules and portions of the trabecula cranii (Fig. 4). The nasal capsules themselves a.re paired cm-tilaginous sacs

formed around the invaginating olfactow pits. They fuse to the trabecular nasal septum at the midline, thereafter forming a continuous dorsal roof (tectum nasi), a fenestrated ventral floor (solum nasi), and side walls (paries nasi). The anterorostral ends of the capsules yield the cupula nasi anterior,

459

19 Craniofacial Development and the anterodorsal tectum is lined with a median sulcus dorsalis nasi which forms a shallow rostrocaudal groove. Paired projections at the rostralmost end, the processus alaris superior, form the posterosuperior margins of the narial openings (fenestra narina). On each capsule, a processus alaris inferior is separated from the superior by the fenestra superior nasi. The caudal end of the rectum is perforated at the foramina epiphaniale, which transmit the lateral branches of the ethmoidal (nasal) nerve of the ophthalmic (profundaI) branch of the trigeminal (V1). These mn from within to without the nasal cavity under cover of the nasal bones. The caudal border of the rectum, the crista cribroethmoidalis, meets the obliquely oriented paired laminae cribrosae (cribriform plates), which underlie the o!facto~' bulbs. These are mammalian neomorphs. Numerous olfactory foramina (foramina olfactoria) perforate this cartil~inous plate. At the dorsal margin of the plate, just ventral to the crista cfibroethmoidale, are the cribroethmoidal foramina, through which pass into the nasal cavity a few fila olfactoria (CN I) and the parent trunks of the ethmoidal nerves (Fig. 5). Where the posterior end of the paries nasi meets the caudomedial end of the lamina cribrosa (the lamina infracribrosa) the planum antorbitale (cartilago antorbitatis, or lamina of Ntonasalis) forms. This meets the trabecutar plate (here, the interorbitaI septum) medially, the lamina infracribrosa superiorly and the lamina transversalis posterior inferiorty (see below) thus forming the cupuIa posterior. The paries nasi are divided into an anterior wail (pars anterior nasi, the interior of which is the pars maxillonasoturbinalis), an intermediate wall (pars intermedia, the interior of which is the pars !ateralis) and a

posterior wail (pars posterior nasi, or pars ethmoidale, the interior of which is the pars ethmomrbinalis). The pars anterior is perforated by the fenestra dorsalis. The pars intermediale contains a laterally expanded prominentia Iateralis, in correspondence to the internal recessus frontomrbinalis (wherein lies the crista semicirutaris) and is separated from the pars anterior by the sulcus antefio!ateratis. The external surface of the caudolateral prominentia extends a smatI spur. The anterior nasal floor is formed by the paired lamina transversal is anterior, wNch meet at the midline to form the zona anutaris. Projecting from the lateral margins of the lamina transversatis anterior are the processus Naris inferior (see above). Caudal to this, on either side of the nasal sepmm (septum nasi), are the cuffed paraseptal cartilages, which house the VNO and represent the junction of the medial nasal ca~sute and the septum. Further caudad the floor takes contribution from the paired lamina transversalis posterior, extensions from the ptanum antorbitale representing the posteromedial nasal capsules. Between the medial septum nasi and paraseptat cartilages and the lateral paries nasi is an anteroposterior vacuiff the fenestra basatis. Within the interior of the nasal capsules a number of turbinal (turNhate) cartilages form. These are thin, convoluted shelves that greatly increase the surface area covered by the nasal epithelium. The anteriormost turbinats, the atriomrbinale (where the paries nasi meets the lamina transversalis anterior) and marginotufoinale (caudad to the atrioturbinate), develop from the nasal floor. These are followed caudad by the (inferior) maxiltoturbinale and (superior) nasoturbinale which project from the pars intermediale. A cartilaginous crest, the crista semicircularis, projects inward from the prominentia lateralis. From the paries nasi and lamina cribrosa arise the frontomrbinale (superiorly) and several ethmoturbinale. The turbinals are separated by various recesses. The mesethmoid is formed by the elements of the cribriform plate, nasal septum, and associated turbinals.

2. Nasoethmoidal (Frontonasal) Region (Regio Ethmoidalis): Dermatocranial

F i g u r e 5 Exit points of the cranial nerves (green) retative to the late emb~'onic murinechondrocranium(purple).

A number of dermatocraniaI elements are associated with the nasoethmoidaI region (Figs. 6 and 7). These include the nasal, frontal, lacrimal, premaxillary, vomer, and maxillary bones. The nasal bones develop as paired elements over most of the tectum nasi. Their rostral borders are free and just caudal to the processus alaris superior, while their caudal borders suture with the frontals. Medially they contact the maxillae and premaxillae. The frontals are expansive, paired bones overlyin~ the rostrodorsat telencephalon as well as the hindmost nasal capsule, mesethmoid (and the olfactory bulbs) medially, and the ala orbitalis (see below) laterally. They articulate rostrad with the nasals, caudad with the parietals (the coronal suture), and mediad with each other (metopic fontanelle, thereafter suture). Interfrontal bones can variably be seen within the rostral metopic suture in certain backgrounds. Ventrolateratly the frontals form the interior

460

111Organogenesis

Figure 6 Generalizedlate emb~'onic murine dermatocranium relative to the underlying chondrocranium seen from (A) norma basMis and (B) norma iateraiis. Red depicts the ossi~ing elements of the dermatocranium, blue depicts the chondrocranium, and yellow depicts the future position of late ossi~ing elements. See list of abbreviations for structural identification. orbital wall dorsal and anterior to the optic foramen of the orbitosphenoids (see below). Within the orbit, they articulate with the lacrimals, orbito- and alisphenoids, maxillae, palatines, and squamosals. Ventrolateral to the nasals, the premaxillae develop along the pars anterior of the capsular side wall. Each supports three main divisions" the body and alveolus, the palatine (palatal) process, and the nasofrontal process. Each body and alveolus houses an upper incisor and extends caudad to a maxilla. The palatine process represents the primary" palate and is extended mediad toward its contralateral partner, with a rostrocaudal expansion at the midline. and caudad to the vomer. Perinatally, the palatal process underlies the paraseptal cartilages and the VNO. The lacrimal. a small laminar bone, forms along the rostrodorsal orbit adjacent to the posterior of the prominentia lateralis of the pars intermedia. It houses the lacrimal glands and represents the caudodorsal end of the nasolacrimal duct. It articulates with the frontal processes of the maxillae and the frontals. The maxillae develop out of the maxillary branch of B A1 but their morphogenesis is intimately tied to that of the frontonasal prominences, which give rise to the nasal capsule. It is the largest bone of the upper face and takes part in forming the oral. nasal, and orbital cavities. It consists of a body and a number of processes alveolar, frontal (ascending). palatine, spheno-orbital, and zygomatic. The body will form the lateral osseous wall of the maxillary recess (undeveloped early on). The alveolar process extends caudad and houses the upper molar dentition. The zygomatic process lies lateral

to the alveolar and is a crest of bone that articulates with the jugal to contribute to the inferorostral lateral skeletal orbit. From the root of the zygomatic process and the body arises the dorsal frontal process, which eventually articulates with the frontal. This process develops around the prominentia lateralis. An expansive fenestration, the infraorbital tbramen, dominates the inferior aspect of the frontal process; the maxillar?' division of the trigeminal (CN V2) and the nasolacrimal duct pass through. The palatine process runs toward the midline from the caudal end of the maxilla. At the midtine the process runs rostrad toward the palatine process of the premaxillae. The fenestrations at either side of this meeting yield the incisive foramen. The spheno-orbital process is the flat sagittaly oriented process caudal to the frontal process which contacts the sphenoid and inferoposterior frontal. The vomers are paired, midtine dermal bones that (usually) will eventually fuse across the midline. In shape they resemble a cup under the lower margins of the septum nasi. Rostrad, they extend to the posterior extremities of the palatine processes of the premaxillae between the paraseptal cartilages. Caudad, the vomer stretch laterally to the lamina transversatis posterior and the nasopharyngeal openings between the rostral vertical lamellae of the palatine. 3. Orbitotemporal Region (Regio

Orbitotemporalis): Chondroeranial The orbitotemporal region is represented by those structures which develop in support of the optic apparatuses and


Figure 7 Neonatal routine cranial hard tissue seen from (A) norma basatis, (B) norma verticaiis, and (C) norma Iateralis. Dark red represents the branchial arch dermatocranial, elements: Ii~n.t red represents the remaining ossifying centers of the dermatocranium and chondrocranium. Orange depicts the dental tissues. See list of abbreviation.s for structural identification.

46t

462 structures within the cavum epiptericum, effectively forming the neurocranial side walls and bridging the nasoethmoidat and otic regions (Figs. 4. 6 and 7). A principal cartilaginous structure is a dorsal, rostrocaudally oriented strut running from the nasal capsule to the occipital arch and connected to the basal plate by mediolateral struts. The dorsal strut is divided into a rostrai ala orbitalis and a caudal taenia marginalis (processus marginalis, or orbitoparietaI commissure). From the ala orbitalis to the trabecular basal plate at the presphenoid run two pillars (radix): an anterior pila preoptica (preoptic root, or pillar) and a posterior pila postoptica (postoptic root/pillar, or pila metoptica). The dorsorostral border of the ala orbitalis connects with the nasal capsule via the sphenethmoidal commissure (commissura orbitonasalis). The ala orbitalis and its pillars comprise the orbitosphenoid. The orbitosphenoid and the presphenoid (within the trabecular basal plate) thus delimit the optic foramen for the second cranial nerves (CN II; Fig. 5). The orbitonasal fissure (fissura orbitonasalis), through which the ethmoidal branch runs dorsad (reentering the cavum cranii extradurally) along the cribriform plate to eventually exit through the cribroethmoidal foramen, separates the nasal capsule from the orbitosphenoid. In mice the mature pila postoptica is formed by an extension from the ala orbitalis (the posterior radix) which meets a separate cartilaginous body. the ala hypochiasmata. which extends to the basal plate. The posterior of the taenia marginalis forms an expansive parietal plate (lamina parietalis) dorsal to the otic capsule. Adding to the chondrocranial side wall of eutherians are the ala temporalis (see below), which is derived from the maxillary branch of BA1, and the mammalian neomorphic tegmen tympani of the otic capsule (see below). The cellular origins of the tegmen tympani. unique to therian mammals and intimately connected to the middle ear bones and geniculate ganglion, are uncharacterized and may well prove to be CNC derived. Expansion of the mammalian brain was accommodated in part by modification of the cavum epiptericum and cavum supracochleare (which is to the geniculate ganglion as the ala temporalis is to the trigeminal). This was achieved through the loss of portions of the original, primary side wall (including the pila antotica, which normally would run from the posterior taenia marginalis of the basitrabecutar process at the basisphenoid) and the incorporation of the sptanchnocranial ala temporalis and the tegmen tympani.

4. Orbitotemporal Region (Redo Orbitotemporalis): Dermatocranial The intramembranous bones associated with the orbital region include those described in association with the nasal capsule (lacrimal. maxillae, and frontal) and the BA-associated jugal, squamosal, palatine, and alisphenoid (Figs. 6 and 7). The jugal (zygomatic or malar) are small rostrocaudatty elongate dermal bones connecting the maxillary zygomatic process (overlying it) and the squamosal zygomatic process

!II Organogenesis (underlying it). The three together form the zygomatic arch, the lateral orbital skeleton. The squamosats are key integrating elements contributing to the orbit, the temporal side wall and the primary jaw articulation (but secondary craniomandibular joint). They possess a central body from which a number of processes and lamina extend. The zygomatic process runs rostrolateratly to articulate with the jugal. The squamosal lamina extends anteriorly to meet the lamina obturans (LO, or anterior lamina) of the alisphenoid and the frontal. A retrotympanic process runs caudad to overlie the tegmen tymapani and the middle ear elements. The sphenotic lamina extends ventrad toward the cupula cochlea and the sphenoid. The caudal process extends dorsocaudad between the squamosal lamina and the retrotympanic processes. The body forms at the confluence of these, forming the ventrally oriented glenoid cavity for the articulation with the condylar process of the dentary (mandible). The palatines make minor contributions to the caudoventral orbit. They are composed of horizontal and vertical lametlae. The horizontal lamellae form the posterior hard palate, the floor of the nasopharyngeal duct, and the anterior margin of the internal naries. The vertical lamellae extend up around the nasopharyngeal duct, where they eventually become closely related to the lamina transversatis posterior and the vomers. The sphenoid is a composite structure formed from trabecular, parachordal, orbital, and splanchnocranial cartilage, as well as the mammalian neomorphic LO. The LO, an appositional, investing, dermal bone. named after the homologous structure in monotreme mammals, spreads through the spheno-obturatory membrane and into the ala temporatis. The ala temporalis and the lamina obturans together form the alisphenoid. Its anterior border is initially separated from the pila postoptica by the large sphenoparietal foramen, which later persists as the sphenoidal fissure (sphenorotundal, superior orbital fissure, or foramen lacemm anterior). Cranial nerves III, IV. V 1, V2, and VI all pass through (Fig. 5).

5. Otic Region (Regio Otica) The otic region is extremely complex morphologically and is principally represented by capsular structures that fuse to support the auditory and vestibular apparatuses. The otic capsule (capsuta auditiva) itself develops around the otic vesicle and has two main components: the pars canalicularis, which supports the vestibular semicircular canals and utriculus, and the pars cochtearis, which supports the cochlear duct and sacculus. The pars canalicuiaris is dorsal, lateral, and caudal to the medioventrally oriented pars cochtearis. The intracranial surface of the otic capsule is dominated by the rostromedial interior acoustic meatus (for the exit of CN VIII: Fig. 5), the caudolateral prominence of the endolymphatic duct (which passes through the foramen endolymphaticum), and the caudolateral fossa subarcuata (which houses the dura around the cerebellar paraflocculus). The internal acoustic meatus is split by the crista falciformis into a fora-

19 Craniofacial Development men acousticum superior (for the vestibular division of CN VIII) and the foramen acousticum inferior (for the cochlear division). A cartilaginous commissure, the commissura suprafacialis (praefacia!is), forms a cartilaginous bridge on the dorsorostraI border of the capsule beneath which passes the facial nerve (CN VII: Fig. 5). At the copula cochlea (apex cochlea), the rostromedial pars cochlearis connects to the ala temporalis via the alicochtear commissure, tt is further connected to the parachordat basal plate via the anterior sphenocapsular commissure and the posterior chordocapsuIar commissure (connecting the medial edge of the processus recessus and the lateral edge of the parachordal plate). Between these commissures runs the basicapsular fissure. The alicochlear and sphenocapsutar cornmissures delimit the carotid foramen. The extracraniaI surface is dominated by two fenestrations. The first is the fenestra ovalis (vestibuli, or orale), into which the stapes is lodged, on the lateral surface at the junction of the pars canalicularis and pars cochlea,ris. The second, on the ventral, lateral and caudal surface of the capsule, is the fenestra rotunda (cochlea, or tympani) for the secondary tympanic membrane and housing the scala tympani and aquaductus cochleae (for the communication of the perilymph and the cerebrospinal fluid in the subarachnoid space). It is covered in part by a shelf, the processus recessus, and less directly by a portion of the splanchnocranially derived styloid process. The aquaductus cochlea and processus recessus as such are considered mammalian neomorphs. The fenestra and aquaductus are contiguous with the jugular foramen (posterior lacerate foramen), together forming the metotic fissure. The jugular vein and glossopharyngeal, vagal, and spino-accessory cranial nerves (CN IX, X and XI) pass through here (Fig. 5). The stytoid process is fused to the otic capsule at the crista parotica, and the eustachian tube passes forward in the interval between the styloid and the pars cochlearis. The crista parotica is related to the prominence of the lateral semicircular canal at the junction of the pars canalicutaris and pars cochlearis, which overlies the elements of the middle ear. The rostral extension of this prominence forms the tegmen tympani, a mammalian neomorph, which overhangs the incus and malleus. The tegmen is the laterat wall of the cavum supracochleare, which lodges the geniculate ganglion of the facial nerve. The caudal end of the prominence forms the mastoid process, which is weakly developed in mice. The incus and malleus are housed in the fossa incudis and epitympanic recess, cavitations created by the overhanging prominences. The remainder of the pars canalicularis is dominated by the prominences of the anterior and posterior semicircular canals and the subarcuate prominence. The otic capsule fuses eventually near the pa_racondylar process of the exoccipital cartilage (see below) at the exoccipitocapsular commissure and to the tectum posterior of the supraoccipitM arch via the supraoccipitocapsular commissure. These commissures help delimit two eutherian neomorphic fissurae, a dorsal superior occipitocapsular fissure

463 and a ventral inferior exoccipitocapsular fissure. Each capsule generally begins to ossi~:y at three or four periotic centers to form the uniform petrosal: (1) from the prootic, anterior to the fenestra ovalis, over the suprafacial commissure and covering the ampulla of the superior semicircular canal; (2) from the opisthotic, enveloping the ventral cochlea and around the fenestra rotundra; (3) from the epiotic, in the posterior capsule around the mastoid process and posterior semicircular canal; and (4) from the pterotic, when forming as a separate center, in the lateral surface of the capsule juxtaposed to the tympanic.

6. Occipital Region (Redo Occipitalis) The occipital region forms the caudal end of the chondrocranium. Within the caudal parachordal basal plate forms the basioccipital. Posterolaterally oriented occipital pillars (pilae occipitalis) lead to the dorsotaterally located exoccipita~ bones. Betw'een the exoccipitaI and basioccipital the hypoglossal foramen can be found (for CN XII). Cartilaginous protrusions on the rostral and caudal borders of the exoccipital form the paracondyta_r (parocciptiaI process) and occipital condyle cartilages, respectively. Continuous dorsally, the occipital cartilages form a complete ring around the foramen magnum. The dorsal aspect of this ring is the rectum synoticum (tectum posterior) within which the supraoccipital wilt form. An anterior extension of tectum, the anterior supraoccipitaI process, runs to form a continuous cartilaginous sheet with the parietal plate.

7. Splanchnocranimm The sptanchnocraniaI elements arise from the branchial arches and principally give rise to the masticatory, pharyngeal, and laryngeal apparatuses (Fig. 4). These elements have been under extensive evolutionary selective pressures and have subsequently undergone complex morphotogic modification. The first branchial arch of mice is the largest and has two major components: the maxillary and mandibular branch derivatives of the palatoquadrate cartilage (PQ) and Meckel's cartilage (MC), respectively. In mice, the PQ derivatives are the ala temporalis and the incus (but see Zelter, 1987, regarding other possible remnants), which are united at the blastemal stage but condense into separate cartilaginous elements. As a cranial structure, the ala temporatis has a distinguished, complex evolutionary history. It is attached to the basitrabecular process of the cranial base at the basisphenoid. The ala temporalis is comprised of a number of structures: a horizontal lamina, a pterygoid process, an ascending process (lamina ascendens or anteromediat process) and an anterolateraI process. At the rostromedial end of its fusion to the basitrabecular process forms the pterygold process. The horizontal lamina underlies the body of the trigeminal ganglion. The ascending process extends dorsoIaterally and is pierced by the alisphenoid foramen (for the stapediat, infraorbital artery). Between the ascending and the

tli Organogenesis

464 anterolateral processes is the incissura ovale, a cleft through which passes the mandibular branch of the trigeminM (CN V3). With the eventual investment of the lamina obturans, this will become an ossified tbramen ovale. The ophthalmic and maxillary branches (CN V I and V2) run rostromedial to the ascending process and exit the skull through the sphenoidat fissure (see above: Fig. 4). The incus (quadrate homolog) forms two primary synoviaI joints The body extends to the malleus and the crus longus to the stapes. A crus brevis is lodged into the fossa incudis. The mandibular branch of BAt is represented by the large rostrocaudally oriented MC. Though continuous. MC has three principal parts: the anterodistat, midline rostral process (Meckelian symphasis), the extended intermediate body. and the caudoproximaI malleus (articular homolog). Principal characters of the malleus include a neck, head. manubrium, and processus brevis. The head is in continuity with the body of MC and articulates with the incus (the primary craniomandibular joint). The neck (or synapsid retroarticular process) is angled ventrad relative to MC and accepts the inserts of the tensor tympani muscles. The end of the neck yields the bulbous processus brevis (muscular process) and the manubrium (a neomorph), which runs down and forward following the line of the spiral sulcus of the cochlea. The body of MC degenerates and the surrounding, sheathing tissue forms the sphenomandibular ligament and malleal anterior ligament. The second branchial arch (BA2) splanchnocranium is represented by four structures the stapes, the styloid process, the lesser horns of the hyoid, and the dorsoproximal hyoid body. The proximal end of BA2 at blastemal stages has two connected processes, a medial and a lateral a medial one pierced by the stapediaI artery and yielding the stapes (hyomandibular homolog), and a lateral one. Reichert's cartilage (RC. hyoid cornu), fused to the otic capsule at the crista parotica. The head of the stapes articulates with the incus, the neck receives the stapedial muscle insertions. the two crurae encircle either side of the stapedial foramen. and the base. or foot plate, is ligamentously attached to the margins of the fenestra ovalis. The proximal end of RC is formed by the tympanohyal (laterohyal, or suaropodial extrastapes), which is fused to the otic capsule at the crista parotica, and the proximodistally elongated stylohyal. Together, these form the styloid process. The styloid process is united to the lesser horns (cornu) of the hyoid (ceratohyaI homolog) via the stylohyoid ligament. The lesser horns meet the lateral ends of hyoid body. The body is a composite of BA2 (dorsoproximal body: certato and hypohyal) and BA3 (ventrodistal body: basihyal or basibranchial) element. The greater horns (cornu) of the hyoid, which extend caudad from the lateral margins of the body, are also BA3 derived. The fourth and more caudal BA gives rise to the lar?'ngeal cartilages. The thyroid cartilage (BA4) is a concave element composed of two tamellae (alae) that meet at the midline. From the posterior upper margin project (toward the greater horns of the

hyoid) the superior cornu, while the inferior cornu project from the posterior lower margin. Within the concavity of the thyroid, in the back of the larynx, are the paired awtenoid cartilages (BA4-6). Articulating with the inferior ~ t e n o i d borders is the cricoid cartilage (BA4-6). 8. Branchial Arch Dermatocrardum

The first branchial arch yields a number of dermal ossifications, many of which have been presented in relation to the nasoethmoidal (maxillae) and orbitotemporat regions (jugal, squamosat, palatine and atisphenoid). In addition to these. BA1 generates the pterygoid, dentary, goniaI, and ectotympanic bones (Figs. 2. 6 and 7). The pte~,goids develop in close association with the lateral basisphenoid and ala temporalis on the ventral neurocranium. They form Lshaped structures caudal to the palatines and ventral to the basitrabecular processes, occupying the lateral margins of the nasopharyngeal openings. In the body of murine pterygolds, as in many mammals, a pterygoid cartilage, composed of secondary cartilage, transiently forms around El6. The dentary is the largest dermal bone and forms the lower jaw (mandible), developing around most of the lengt_h of MC. As the name implies it bears the teeth of the Iower jaw: an elongate incisor, surrounded by an incisive alveolus, and fhree molars housed in a molar alveolus. An extended diastema separates the two. and they converge about a central body where the mental foramen exits buccatly. Lingually, there is a mandibular foramen. The proximal end is dominated by three processes: the anteriormost coronoid, the condylar, and the posteriormost angular. The coronoid forms a caudodorsat strut toward the zygomatic arch. The condyle runs back toward the squamosal (the secondary" craniomandibularjoint), and contains at its proximal heart a large secondary cartilage mass. The angle, likevdse maintaining a secondary cartilage proximally, runs caudally. The goniat (prearticular) is an investing bone that lies on the dorsodistal surface of the malleus. It will invade the malleus and become the processus folii (anterior, or gracilis) of the mature malleus at the point where the malleus separates from MC; hence (like the maxillary alisphenoid) the mature mandibular malleus is a composite endochondral (articular) and dermal (prearticular) bone. Underlying the same position on the malleus is the anterior process of the ectotympanic (angular). The ventral posterior (reflected lamina) process curves back caudad as a ring toward the styloid process, thus delimiting the tympanic membrane. 9. Calvarial Dermatocranium

By osteological tradition, the catvarium is the cranium minus the face (splanchnocranium) while the catotte is the calvarium minus the neurocranial base. Thus, the calvariat dermatocranium includes the frontals, parietats, interparietal. lamina obturans of the atisphenoids, and squamosats. The parietals are large, rectangular bones caudal to the fron-

465

19 Craniofaciai Development tals and dorsal to the squamosais. They come together mediad at the sagittal suture, and form the coronal suture with the frontals and the lambdoidal suture with the interparietat. T,~e interparietal lies dorsal to the otic capsule and just rostral to the endochondrat supraoccipital. The calvarium develops from the mesenchyme encompassing the brain. This mesenchyme has two layers: an inner endomenix and an outer ectomenix. The endomenix includes the deep pia mater, which covers the brain, and the arachnoid, through which the cerebrospinal fluid and blood supply interface. The ectomenix likewise has two closely apposed layers: a deeper dura mater and a more superficial mesenchyme in which the skeletal elements form. The dura acts as the endosoteum, or endocranial periosteum, of the catvarium. The ectomenix has both chondrogenic and osteogenic capacity. The dura mater has three principal septa subdividing the brain: the falx cerebri, the falx cerebelli, and the tentorium cerebelli. These structures have organized fiber bundles related and attached to the developing sutures. The eventual adult cranial form is shaped in part by directed forces due to growth of the brain and constraints of these fibers. Secondary cartilage, which is derived from the application of biomechanical forces to particular ectomesenchyme in the head, can often be seen perinatally in the sagittaI and lambdoidaI sutures.

VII, Appendix 3" Molecular Regulators of Craniofacial Pattern and Development A. Genes Encoding Secreted Signaling and Related Factors 1. Endothelin-1 (ET-1), ET-A, ECE-1 ET-t, initially reported as a potent vasoconstrictor produced by endothelial cells, is one of several closely related peptide tigands that bind to the G-protein-coupled receptor ET-A (Kurihara et at., 1994: Ctouthier et aL, 1998). Biosynthesis of active ET-1 occurs in a two-step process: First, a furin-like protease cleaves preproendothetin, 1: second, a cleavage by a metalloprotease, endothetin converting enzyme 1 (ECE-t), generates active ET-1 (Yanagisawa et at., 1998). ET-1 mRNA is detected in the ectoderm and endoderm of BA1-3 (Ku_rihara et al., i995; Clouthier et aI., 1998). It is also detected in the paraxial mesodermal core of BA1 and BA2 and the aortic arch artery and cardiac tissues. Conversely. the ET-I receptor gene, ET-A, is expressed in migrating and postmigratory rhombencephalic CNC of the first three arches. ECE-I is found expressed in both paraxial and CNC BA mesenchyme, neurepithelium, and both ectodermal and endodermat BA epithelia (Yanagisawa et aI., 1998). Targeted disruption of ET-t, E T A and ECE-I each yields mice virtually identical in craniofacial defects (Kurihara et ai., 1994; Clouthier et aI., 1998: Yanagisawa et aL,

I998) (Fig. 2a). Neonates are micrognathic, with variable midline fusion of the lower jaw, and the pinnae and ventral neck are hypoplastic. The proximal dentary is agenic, and the body of MC. malleus, goniaI, ectotympanic, incus, stapes (usually), and (presumably) the styloid process are absent. The external auditor' meatus is missing. The rostral process of MC, distal dentary, and teeth (which are abnormally embedded in loose mesenchyme) are, how'ever, present. The dentary does not extend to the squamosal at the otic capsule, instead articulating with a hypoptastic jugal. The dermal bone of the medial dentary fuses with the basal plate at the basisphenoid. The hyoid is severely mal~brmed, forming a ringlike structure fused to the finyroid and the basisphenoid near the ala temporatis and pterygoids. The atisphenoid, palatine, pterygoid, and squamosal bones are underdeveloped. Muscles of the tongue are severely diminished, and submandibular and sublingual glands are lacking. In ET-t-~embryos, distal midline expression of Hand2 and Handt is decreased but present (Thomas et at., t998). The sucker (suc) gene encodes the zebrafish homolog of ET-1; suc mutants fail to induce or maintain expression of dtx2, dLr3, msxE. gsc, EphA3, or dHAND in ventral (distal) BAI and BA2 tissues (Miller et ai., 2000). The micrognathia of ETA_/_ embryos is not due to a failure of CNC to migrate to the B A based on AP2, DIx2 and CrabpI expression, though Dix2 (and Barxl) expression is downreguIated in the BA2 (only) by El0.5 (Clouthier et aI., 2000). The BA are, however, smaller in these mutant embryos, and by Et 1.5 fewer BrdU positive cells and more apoptotic cells are detected in the B A. Neither Gsc nor Dtx3 (both ectodermal and ectomesenchymaI) expression is detected in the BA of ET-A -/- embryos at E t0.5 (Clouthier et aL, t998, 2000). A delay and downregulation of dHAND and eHAND expression, but not the associated gene ~fdl, are also seen. Dlxl and Prxl mRNA patterns, however~ appear similar to wild-type embryos.

2. Epidermal Growth Factor (Egf) and Transforming Growth Factor a (TGF-a) EGF. one of the first growth factors identified, was isolated from murine submaxillary glands (Cohen, 1962), while TGF-c~ was isolated from routine-sarcoma-virus transformed fibrobtast cultures (De Larco and Todaro, 1978); both appear to bind the same receptor (Egfr). In vitro, EGF acts as an inhibitor of chondrogenesis of mandibular arch mesenchyme, including BMP-stimutated chondrogenesis (CoffinCollins and Hail, 1989; Nonaka et al., t 999). Both Tgf-ce -/and Egfr -/- mice exhibit craniofaciaI defects, including cleft palate and defects in MC (Miettinen et at., 1999).

3. Fibroblast Growth Factors (Fg.f), F g f r I - 3 and Sprout)"

There are at least 14 mammalian fibrobtast growth factor (Fgf) genes based on the presence of a core sequence encod-

111Organogenesis

466 ing receptor-binding and heparin-binding domains (Lewandowski et aL, 1997). Most gene products, but not all (e.g., FGF1/aFGF and FGF2/bFGF) are secreted and avidly bind molecules at the cell surface, in particular heparan sulfate proteoglycans. FGF signaling is known to regulate craniofacial development: for example. Kg)3 -/~ mutants have dysmorphic otic capsules (Represa et aL. 1991" Mansour er aL. t993: McKay et aL. I996) and disruptions of the three known receptor tyrosine kinase (RTK) FGF Receptors lead to craniosynostosis or achondroplasia (reviewed by' Vv~bster and Donoghue. 1997: Wilkie. 1997: Burke et at., 1998: see Table III). Functional analysis of Fgf8 provides perhaps the clearest demonstration of FGF involvement in craniofacial development. FGF8 was originally identified as a secreted androgeninduced growth factor (Tanaka et aL. 1992). It has a dynamic spatiotemporal pattern of expression in a number of known signaling centers which regulate pattern and morphogenesis. including the primitive streak, the apical ectodermal ridge of the limb bud. the midbrain-hindbrain isthmus, and the intraembryonic coelom (to form the heart), olfactory placodes, phar?ngeal clefts (both endoderm and ectoderm) and oral ectoderm of B A1 (Heikinheimo et al.. 1994" Crossley and Martin. 1995" Trumpp er aL. 1999). To circumvent the embryonic death at gastmlation seen in Fgf8 -~- mutants (Sun et aL, 1999), Trumpp et aL. (1999) employed a Cre/loxP strategy to inactivate Fgf8 within the ectoderm of B A1 by E9.0. Significantly, gene inactivation in the caudal, pericleftal BA1. however, occurred later than in the rest of B A1 (Trumpp et al., 1999). This strategy resulted in an agnathic mouse (Fgf8 ''-'c~'-'-) with a dramatic loss of most of the B A 1-derived skeletal elements (Fig. 2b). The entire maxillary (incus and ala temporalis) and most of the mandibular splanchnocranium failed to form. Of Meckel's cartilage. only the rostral process (with associated dermal alveolar bone and diminutive incisors) and most of the malleus (including a well-formed manubrium and processus brevis) developed. The elongate body of MC did not. Most of the B A1 dermatocranial elements were agenic (e.g.. palatine. pterygoid, lamina obturans, jugal) or severely hypoplastic (e.g., maxilla, dentary, ectotympanic-goniale). By E9.5. B A 1 is clearly hypoplastic in these mutants. Based on Cad6, CRABPI, and AP2.2 expression, CNC migrate to BA1 and, in an assay of proliferation, the ratio of BrdU-labeled cells to unlabeled cells was similar to wild-type embwos. However, extensive BA 1 cell death was detected in Fgf8 "~-~ o~.-/- mice. suggesting that FGF8 acts as a survival factor in BA1. Evidence for disruption of patterning in both the ectoderm and mesenchyme in Fgf8 ~'e-'c~'-/- mice revealed that BA1 ectoderm maintained oral expression of Pitxl but lost ET-1 (Trumpp et al., 1999). Moreover. FGF8 dependent and independent regions of gene expression were found: The distal midline, which wilt eventually yield the rostrat process,

incisors, and associated dermal bone. maintained expression of both Pezv9 and Bmp4, while the ectoderm of the molar field associated with the body of MC and much of the dentaa2r' lost Pa_x9 expression. Expression of M~xl, a BMP4inducible gene, was maintained in the underlying midline mesenchyme as was Msx2, eHAND and dHAND. Loss of Msxl leads to a loss of those mandibular midline structures that persist in the Fgf8 ''~'-'cr~'-/ mice--namely, the rostral process and incisors (Satokata and Maas, t 994). At E9.5, the FGF8-inducible genes Dtx2 and Dlx5 were appropriately expressed in the proximodistal axis of BA1. further suggesting that B A1 retained some appropriate spatial information. Whether FGF8 is necessary for later maintenance of the DLr genes in the BA is uncertain. Mesenchymal expression of L/~rZ another FGF8-inducible gene, was maintained though that of the related gene, Lhx6, was not. Moreover, with a pertinent exception, mesenchymal expression of Gsc and Barxl was lost. This exception is in mesenchyme beneath the above-mentioned patch of caudal, peri-cleftat B A I Fgf8 expression. The ectoderm over this patch also maintained ET-1 expression. Those B A1 elements that do form in F0. o f 8 "~'~ mice are noteworthy. The portions of the maxillae that form in these mutants normally develop in close association with the nasal capsules and frontonasal processes an Fgf8-positive region--which may have influenced their growth. The malleus and rudimentary ossification in the region of the juxtaposition of the ectotympanic, gonial, and malleal processus folii developed out of a region of BA1 in which the gene inactivation in the caudal, pericteftat BA 1 occurred Iater than the rest of BA1. Expression of ET-1, Barxl, and Gsc in this spot remained: hence, the cells contributing to these structures may have been exposed to an elaborated FGF8 signal at the critical point of their ontogeny. The persistence of Brnp and Msx expression in the mandibular midline suggests the presence of an Fgf8-independent, Bmp/Msx-dependent field at the midline (Trumpp et al., 1999). Last, data from the F g f 8 \'~crc-/- mice support the idea that. as outgrowths, the BA receive patterning signals from both the rostral and caudal associated epithelium, as well as possibly from the olfactory field. Members of the SprouO~ (Sp~') gene family encode RTK inhibitors, including Fgfr RTKs (Hacohen et aI., t 998; Casci et al., 1999: Kramer et aL, 1999). Moreover. Spr3,2 has been identified as a regulatory" target of FGF8 signaling in BA1 (Minowada et al., 1999; Trumpp et at., 1999). Although overexpression of Spo,2 appears to lead to chondrodysptasia. craniofacial defects due a loss of function have yet to be presented (Minowada et aL, 1999).

4. Pdgf, Pdgf-R~ and Ph Platelet-derived growth factors (PDGFs) are disulfidebonded homo- or heterodimers of "A'" and "B" polypeptide chains. There are two RTKs for these tigands, PDGF-

19 Craniofacial Development Ra and-R/3, which likewise can homo- or heterodimerize upon ligand binding (Seifert et al., 1989). Homozygous mice carwing targeted deletions of Pdgf-Ra die during embryonic development with neural tube defects and subepidermat blebs and bleeding, particularly within the head (Soriano, t997). Both dorsal and ventral skull structures are unfused along the rostraI midline (Fig. 2c). Nasal capsules, with relatively intact paries nasi, develop and fuse to trabecula cranii~which, however, fail to fuse and are separated by a large gap. The face is thus cleft, as are the frontals and parietals. Other ventral neurocranial defects may also occur. Though largely undetailed, it seems likely that BA and otic structures are also extensively affected. Increased ceil death in the cephalic region and branchial arches was detected in regions of migrating CNC. This supports a model of PDGF action as a survival factor, although toss of Pdgja or Pdgfb does not appear to lead to skeletal defects of the nature seen in Pa~fra -/- mice (kev6en et al., 1994: Bostr6m et al., 1996; Soriano. 1997). Neuronal CNC derivatives in Pdgfra -/~ mice appear to migrate properly based on neurofilament assays. The Patch (Ph) mouse mutant, which presents a cleft face in particular backgrounds, is thought to have a deletion encompassing the Pdgfra gene (Orr-Urtreger et al., 1992).

5. Transforming Growth Factor ~ (Tgf-~)lBone Morphogenetic Protein (Bmp) Superfamily, Inhibitor,s, and Effectors of Signaling The Tgf-/3 superfamily is an extensive gene family encoding secreted proteins that regulate cellular behavior, including migration, proliferation, differentiation. ECM production, apoptosis and regional patterning (reviewed by Kingsley, 1994" Hogan, t996). This superfamily includes Tgf-~s, Bmps, Gdfs, activins, inhibins, nodal, and Mullerian-inhibiting substance. TGF- proteins were initially named for their ability to promote anchorage-independent growth of fibroblasts in culture. and they are expressed in the craniofacial mesenchyme (Roberts et al., 1981" Millan et aI., t 99 I" Pelton et al., 1991 ). Targeted disruption of Tojbi results in embryonic lethality by E 10.5 (Dickson et at., 1995). TgCbI mRNA is detected in the preimptantation blastocyst and persists in multiple tissues through adulthood (Sanford et at., t997). Among the sites of expression are chondrocytes and pefichondria, osteocytes of the head and optic and otic sensory epithelia. Tg~2 null neonates die with multiple craniofacial detects (Sanford et at., 1997). The frontals, squamosals, parietats, and interparietaI are dysgenic with reduced ossification and expanded fontanelles (Fig. 2d). The supraoccipital has no ossification and the exoccipitaI and basioccipital are connected. A malformed ala temporalis is present, extending two small ossicles taterad: the dermal lamina obturans is absent. A percentage have a cleft secondary palate. The mandible is reduced, in particular proximally where the angle is

46 7 absent and the condyle and coronoid much diminished. Defects are observed in the pars cochlearis. With the loss of f:unctionaI Tgfb3, the medial edge epithelia of the palatal shelves tail to adhere and eliminate as a seam, resulting in a clefting of the palate (Kaartinen et aI., 1995; Proetzel et aI., I995). Bone morphogenetic proteins (BMPs) were identified as components of bone extracts with the capacity to induce ectopic bone and cartilage when implanted in rodents (Sampath and Reddi, 1981 Wozney et al., 1988). Bmpl, the homolog of the Drosophila gene totloid (rid), encodes a secreted procottagen C-proteinase with this inductive capacity ('#~zney et at., 1988; Suzuki et al., 1996). Bmpl ~/- mice have malformed frontals, parietals, and interparietals along their sagittal sutures. Bmp2 is expressed in the ectomesenchyme and Bmp4 in discrete regions of the overlying ectoderm (in particular along the oral midiine) of the early craniofacial primordia, as well as dorsal midline of the neural tube (Francis-West et al., 1994; Bennett et at., t995). The effect of genetic inactivation of either Bn~p2 or Bmp4, however, is embry'onic death (Winnier et aI., 1995" Zhang and Bradley, 1996). Ectopic application of BMP2 or BMP4 in mandibular explants has been shown to induce the expression of Msxl and M~x2 (see above; Barlow and FrancisWest, 1997; Tucker et aI., I998b). Bmp5 is the gene mutated in the short ear mouse (Kingsley et al., 1992). As the name implies, toss of functional Bmp5 leads to shortened BAderived auditory pinnae. Functional loss of Bmp6 appears to only affect the sternum (Solloway et aL, 1998). Bmp7 is expressed in many cranial tissues, including in the stomodeal and surface ectoderm covering B AI, over and between the frontonasal processes, in the otic vesicle, the commissural plate between the tetencephalic vesicles, and the NT at the cephalic flexure (Lyons et at., 1995). Bmp7 is required for the development of structures around the interface of the mesodermat and CNC skeleton. Bmp7 -/- mutant mice have an extensive fenestra in the caudal basisphenoid at the hypophysis (Dudley et al., 1995; Luo et at., 1995). The ata temporalis is dysmorphic, apparently lacking an anterolateral process and supporting an aberrant alicochlear commissure with an ectopic strut, which runs to the middle ear and fuses to the otic capsule. The lamina obturans is malformed, as is the pte~:goid. Any defects in the otic or nasal capsule have not been reported. Bmp5-/-;7 -/- compound mutants die at E I0.5 with striking defects in tissues where both are expressed, including the rostral NT, the BA, and CNC (SolIoway and Robertson, t 999). GdfS, along with Brnp5, has been implicated in joint development (Storm and Kingley, 1996). Activin t3A is expressed in the mesenchyme of the facial primordia (Feijen et aL, 1994; Roberts and Barth, t 994) and its knock-out yields mice with cleft palates and without mandibular incisors (Matzuk et ai., 1995a,c). Nodal mRNA is detected early, marking the cells of the primitive streak. Nodal signaling is required for cephalic de-

468 velopment, being necessary for the formation of the primitive streak and most mesoderm and anterior neural tissues (Conlon et al., 1994: "varlet et at., 1997: reviewed by Schier and Shen. 2000). z V o ~ i n , chordin, and follistatin encode for proteins that appear to act: as specific antagonists of TGF-/3 family signaling. N o g g i n is expressed early in the anterior primitive streak, node and mesendoderm, and later in condensing and developing cartilaginous tissues (Brunet et al.. 1998: Bachiller et al.. 2000). Targeted disruption, theoretically leading to enhanced BMP signaling, leads to excessive chondrogenesis and disrupted skeletal morpho~enesis (Zimmerman et aI., 1996: Brunet er al., 1998). The skull and cervical vertebrae of mutants are nearly normal at birth (Brunet et al., I998), though defects in MC and other chondrocranial elements may be evident with further characterization. The remainder of the axial and the appendicular skeleton is malformed with multiple failures of joint formation. C h o r d i n is likewise expressed early in the primitive streak, node. and mesendoderm (Bachiller et al., 2000). C h o r d i n .... mutant mice display inner and outer ear defects as well as abnormalities of pharyngeal and cardiovascular organization (Bachiller et al., 2000). Compound N o g g i n -/- C h o r d i n - ' .... mutants (similar to Shh -~- mutants, see below) have cyclopsia with agnathia and a single nasal pit (Bachiller et al.. 2000). Although neither N o g g i n nor Chordin are expressed in the AVE. expression of H e s x l in the AVE fails to be maintained in the compound mutants. Using a knock-in approach. Kanzler et al. (2000) utilized an enhancer of the H o x a 2 gene to drive expression of X n o g g i n ( X e n o p u s noggin) in the BA to attempt to address the potential roles of B MP2 and B MP4 in regional development. This particular enhancer is active in the premigratory and migratory CNC of the BA2 and caudal arches (Nonchev et al.. t996). Affected embryos have hypoplastic B A 2 . BA3, and BA4, apparently not due to increased apoptosis but to a loss of migrating cells. The associated skeletal elements are malformed. Follistatin -/- mutants have. among other defects, cleft palates and lack lower incisors (Matzuk et al., t 995b). Smad proteins are cytoplasmic mediators of TGF-/3 superfamily signaling through receptor serine/threonine kinases (reviewed by Heldin et al., 1997). S m a d 4 associates with S m a d l and S m a d 5 in response to BMP signaling, while Smad4-Smad2 and Smad3 associations occur in response to TGF- and activin signaling: these complexes translocate to the nucleus where complexes may act as either transcriptional activators or repressors (Heldin et al., 1997 Gripp et aI., 2000). S m a d 4 -/- mutants have undifferentiated visceral endoderm, lack mesoderm, and have anterior patterning problems (Sirard et al., 1998). Inactivation of Srnad2 resuits in embryonic lethality (by El0.5) with defects in the visceral endoderm, epibtast, extraembryonic ectoderm, and an absence of embryonic mesoderm (Nomura and Li, 1998; Waldrip et at., 1998), suggestive of a role in the organization

111Organogenesis of the primitive germ layers prior to gastrutation. A percentage of S m a d 2 heterozygotes are rpScrognathic (or agnathic) and may be eyeless. These phenotvpes (severe cyclopsia and truncation of the rostral head) are compounded with transheterozygote S m a d 2 " / - ; N o d a t -/- mutants, indicating a genetic interaction if not a signaling relationship (Nomura and Li, 1998). A c t i v i n R e c e p t o r I L 4 - / - ; I I B -/- mutants, moreover, appear tike Srnad2 " / - ; N o d a l +/- compound mutants (Matzuk et aI., 1995c; Nomura and Li, 1998). Nuclear Smads interact with a number of cofactors, including TGIF and ski, thereby acting as transcriptional repressors (Gripp et at., 2000). Mutations in the human T G I F gene, which is expressed in craniofacial and forebrain tissues, result in hypotelorism and holoprosencephaly (Grippet al., 2000). Ski knock-out mutants exhibit variable upper facial clefting, neural tube closure, neurocranial base, and denta_ry defects (Berk et al.. 1997).

6. Retinoic AcM, RAR, RXR Vitamin A (retinoI) is crucial for normal pre- and postnatal gro,,~h and survival, acting as a regulator of gene expression and morphogenesis (reviewed by Morriss-Kay, t993: Sporn et at., I994: Brickell and Thorogood, 1997" Morriss-Kay and Ward. t999). Retinoic acid (RA)is generated as an active derivative of vitamin A. RA acts as a ]ipidsoluble ligand for two families of nuclear receptors, RARs and RXRs. which act as transcriptional regulators and are expressed in many embryonic tissues including the CNC (Mangelsdorf and Evans, I995). RA balance in the developing embryo is critical, because either a deficiency or an excess leads to developmental defects. The effects of vitamin A deficiency (VAD) syndrome include microophthalmia and cleft lip, palate, and/or face--defects that can be prevented with RA administration (Mark et aI., 1995; Morriss-Kay arid Skolova. i996). Ectopic exposure to RA leads to distinct, complex craniofacial malformations, including facial clefting and branchial arch alterations, in a manner that depends on dosage, location, and gestationat time of exposure (Webster et at., 1986; Wedden et al., 1988: Morriss-Kay; I993; Grant et at., 1997" Mallo, 1997). Excess RA administered early (presomite stages)leads to rostral shifts in the position of the otic vesicle and BA. partial fusion of trigeminal and facial ganglia and the first and second B A, and a diminished preotic hindbrain. Such treatments appear to alter HB segmentation, as assayed by rhombencephalic markers (Morriss-Kay et al., 1991; Holder and Hill, 199t" Conlon and Rossant. I992: Marshall et ai., t 992). Of particular interest is the range (from shortened to lost.) of MC morphology seen with the concomitant appearance of an ectopic MC-tike structure in the maxillary' BA1 (Morriss-Kay, 1993). The molecular nature of this shifting mo~hotogy is unclear but may be accompanied by changes in D t x gene family expression (see below).

19 Craniofacial Development Both of the RA receptors, RAR and RXR, exist in three forms c~,/3, and y ~ w i t h isoforms of each. For RAR, this includes c~l and c~2,/3I-/34, and y l and y2. RARa transcripts are apparently nearly ubiquitous developmentally, RAR/3 more restricted, and R A R T in specific mesenchymaI populations where there are precartitaginous condensations, including the frontonasat and branchial arch ectomesenchyme and the mesodermal limb buds (but apparently not i.n the FB. MB, or HB regions of presumptive CNC origin) (Dol16 et at., 1989. 1990: Ruberte et aI., 1990, 199t" Leroy et al.. I99t" Mendetsohn et at., t991, 1994a). 1,7 Hvo functional studies of these genes have been addressed via targeted mutation, where the range of defects seen in the various mutations recapitulates nearly all of the defects associated with VAD syndrome (Ghysetinck et al., 1997). Mice deficient for individual receptor isoforms, such as RARoel (Li et al., 1993" Lufidn et al., 1993), R A R f l 2 (Mendelsohn et al., 1994b), or RART2 (Lohnes et at.. 1993), appear normal" however, when all isoforms of ,~4Rce, R A R f i or R A R T are targeted, postpartum lethality and growth disturbances ensue (Lohnes et al., I993, 1994). The greatest malformations occur with compound mutants, especially with R A R c e - / - ; y -/- doubte mutant mice. suggesting some redundancy in RAR transduction of RA signaling. RARc~-/-;y-z- double mutant mice are clearly discernible from their littermates by their external features: diminished eyes, shortened snout and median facial cleft, occasional exencephaly, and agenic auditory pinnae (Lohnes et aI., 1994; Mark et al., 1995)(Fig. 2e). Massive cell death is seen in the ffontonasat CNC at El0.5, and atthough an olfactory pit forms the ffontonasal processes are fused to the ipsitateral maxillary process and never at the midline. Thus, RA signaling is required for proper coalescence of the facial pfimordia. Consequently, nasoethmoidaI development is severely deficient: The trabecular basal plate is widely split and the nasal capsules and mesethmoid are represented by rods of cartilage (laterocaudaI rudiments) without any midline structures. The associated dermatocranial elements (premaxillae, nasats, vomers, lacrimats, frontals) are partially or completely agenic. The orbitotemporal region and those BA derived elements that develop in close association with the nasal capsules (including the maxillae, palatines, and sphenoid) are deficient and malformed. The incus is fused to the ata temporatis of the alisphenoid. The mandibular BA1 elements are relatively spared though not perfectly normal. The elements of the calvarium are diminished and undermineralized. Hypoplastic otic vesicles seen at E 10.5 eventually yield small and incomplete otic capsules. Notably, the meninges chondri~,. BA2- and BA3-derived skeletal elements are either unidentifiable (e.g., the stapes) or severely malformed (e.g., the hyoid; Mendelsohn et at., I994a; Mark et al., 1995). R A R c ~ t - / - ; c e 2 + / - ; y -/- mutants, which are less severely affected than the complete double mutants, exhibit an ectopic pillar running parallel to the trabecular basal plate from the dysmorphic orbital cartilages

469 (i.e.. the pila postoptica) to the basisphenoid. Thus, many CNC-derived skeletal elements are altered in these mice. Less dramatic malformations are seen in the R A R f l -/- (all isoforms) mutants, which are exacerbated in the compound K 4 R a - / - ; f l -/- mutants This includes ocular defects and agenesis of the postoptic pillar and zygomatic process of the squamosat, hypoplasia of the ethmoturbinats and aberrant gonials (Ghysetinck et al., t 997). Moreover, the hyoid and Iaryngeal cartilages of the various compound mutants are malformed (Mendelsohn et al., 1994a: Ghyselinck et aI., I997). Various additional proteins are known to bind RA or retinol, including the cytoplasmic cellular retinol-binding proteins (CRBPt and II) and cytoplasmic retinoic acid binding proteins (CRABPI and It; Sporn et al., t994, Morriss-Kay and Ward, 19991). CRBPI and II likely act to store and regulate biologically active retinoids. The CRABPs do not appear to be essential for craniofacial development, as C R A B P I -/-" H -/- mutants are normal in finis respect (Gorw et at., t994; Lampron et al., t 995). Retinaldehyde dehydrogenase 2 ( R a t d h 2 ) h a s a high substrate affinity for retinaidehyde, an intermediate product of retinol oxidation to RA (Wang et at., 1996; D. Zhao et al., 1996). RaIdh2 expression has b u n correlated spatiotemporally with sites of RA synthesis (Niederreither et al., i997, i 999; Berggren et aL, 1999). R a t d h 2 -/- mice die at ---Et 0.5, exhibiting, among other traits, disruptions of otic vesicular and BA development (Niederreither e t a L , 1999). Although the isthmic organizer at the MB and R t/2 is apparently normal in the absence of RA synthesis (e.g., Ffg8, Pax2, Gbx2, Engrailed2, and M e i s 2 appear to be expressed normally), the caudal HB is altered throughout (as assayed by Ep,~A2, E p h A 4 , r~o.oJ3, o ~ regional .. H o x .~ genes. Krox-20, kreisler, 9 and W n t 8 a expression) and associated with increased neurepitheliat and ectomesenchymal cell death (Niederreither et al.,

200O). The spatiotemporal elaboration of RA signaling and cellular response continues to be clarified, in particular as it relates to the development of the olfactory axis (placode, nasal ectomesenchyme, and FB), optic and otic tissues, the rhombencephalon and CNC (e.g., Colbert et aI., I993; Mallo, 1997; Anchan et aI., I997" Choo et aI., 1998; Gavalas et at., t998" Whitesides et al., 1998; Dup6 et al., t999; Niederreither et aI., 1997; Zetterstr6m et at., 1999; Enwright and Grainger, 2000). For example, RA signaling is impaired in the olfactory and optic systems in the P a x 6 s probably represents a somatomammotrope intermediate celi type. Treatment of GH3 cells with GHRH causes differentiation into somatotropes and GH secretion. In contrast, treatment with nerve growth factor (NGF) promotes lactotrope differentiation and PRL production (Missate et al., 1994). Lactotrope proliferation is also stimulated by transforming growth factor c~ (TGF-c~), TGF-/3, and estrogen (Lloyd, 1983; McAndrew et aL, 1995; Phelps and Hymer, 1983; Hentges et al., 2000). The stimulatory effect of estrogen may be mediated through TGF-a. TGF-,B acts by stimulating production of FGFs from foiticuto-stellate cells. The FGFs probably act upstream of NGE Normal expansion of the lactotrope population requires thyroid hormone (StahI et at., 1999), although the mechanism of action is unknown. A variety of studies have indicated that Iactotrope differentiation is also promoted by epidermal growth factor, vasoactive intestinal polypeptide, and insulin (Felix et aL, t995; Goda et al., t 998; Kakeya et aL. 2000; Woods and Porter, t998). Many of these agents are likely to influence early cellular differentiation. While the majority of pituita~, hormones are secreted in response to positive regulation by the hypothatamus, tactotrope proliferation and protactin secretion are under negative control by dopamine. The inhibitory effect of dopamine on lactotrope proliferation was confirmed in mice with inactivating mutations in the dopamine transporter ( D a t l ) and dopamine receptor (Drd2) (Boss~ et at., 1997; Saiardi et at., I997). D a t l - / - animals have elevated dopamine levels that cause tactotrope hypoplasia, while Drd2 -/- animals have Iactotrope hyperptasia. Normal expansion of the somatotrope population requires thyroid hormone and GHRHR signaling (Mayo, 1996; Stahl et aL, 1999). Delivery of GHRH by the portal circulatopj system from the hypothalamus to the pituitary' normally results in GH secretion, enhanced Gh transcription, and increased proliferation of somatotrope cells. Somatotrope deficiency and ~owth insufficiency occur in mice and humans as a result of mutations in the gowth hormone-releasing hormone receptor gene (Ghrhr) (Baumann and Maheshwari, 1997; Wajnrajch et aL, I996). The receptor is G protein coupled such that GHRH binding causes a stimulation of adenylate cyclase and increase in intracellular cAMP levels. Conversely, constitutive activation of the GHRHR signaling pathway in humans and mice leads to somatotrope hyperplasia, GH-producing adenomas, gigantism, and acromegaly (Burton et al., 1991; Mayo et at., 1988; Stefaneanu and Kovacs, 1993). Close to 40% of GH-secreting tumors from

512 acromegatic patients are thought to have somatic mutations in a~, the GTP-binding subunit of the GHRHR associated G protein (Landis et at., 1990). The critical role of GHRHR signaling in the regulation of somatotrope proliferation serves as a general model for receptor-mediated regulation of cellular proliferation. Further studies are necessary to determine whether a common mechanism exists Ibr any of the pituitary celt types. The majority.' of thyrotropes arise from P i t l expressing progenitor cells. When high demand for thyrotropes occurs in neonates, thyrotrope proliferation appears to occur at the expense of the somatotropes and lactotropes (Kendall et aI., t995). However, in adult mice somatotropes may be recruited to produce thyrotropin, just as lactotropes apparently can arise from somatomammotropes. Thyrosomatotropes appear in response to hypothyroidism but disappear quickly when thyroid hormone is restored (Horvath et al.. 1990). Production of thyrotropin is stimulated by the hypothalamic peptide thyrotropin-releasing hormone (TRH). Recent studies have indicated that TRH is not necessary: for thyrotrope differentiation or for TSH production (Yamada et al., 1997). TRH-deficient mice have transient hypothyroidism, but an unknown compensatory mechanism results in elevated TSH. which supports catch-up growth. However. humans with TRH receptor mutations have profound hypothyroidism, su.,..estm., ~ " ~ that TRH receptor si~nalin_ .. .. is important for thyrotrope function. Targeted disruption of the TRH receptor gene may be necessary to assess the importance of the receptor on thyrotrope and differentiation (Collu et al., t997). Gonadotropes develop in hypothyroid mice. but thyroid hormone is required for expansion of the cell population (Stahl et al., 1999). Gonadotrope proliferation and function is also controlled by the hypothalamic peptide, gonadotropin-releasing hormone (GnRH). In mice lacking GnRH, gonadotrope cells are present but they produce vew little LH and FSH (Mason et al.. 1986a.b). Gonadotropes are also sensitive to the level of gonadal steroids and undergo hypertrophy and hyperplasia in gonadectomized animals (Ibrahim et al.. 1986). These results suggest that gonadotropes require both thyroid hormone and GnRH to respond to low levels of gonadal steroids. FGF2 has also been implicated as atrophic factor for gonadotropes (Schechter et al., 1995). N h l h 2 is expressed in embo, onic and adult pituitary, gland and in the developing nervous system (Good et at., 1997). Mutations in N h l h 2 affect gonadal development in males more severely than mutant females. The loss of function is attributed to gonadotrope dependence on N h l h 2 ; however, the relative importance of N h t h 2 for GnRH and gonadotropin production is not clear. Corticotrope expansion is sensitive to several mitogenic factors although more experiments are necessary" to clarify the role of each factor. Leukemia inhibitory, factor (LIF) is produced in the pituitary gland and has been shown to en-

111Organogenesis hance corticotrope differentiation (Stefana et at., t996). However. transgenic mice expressing LIF develop Rathke's cysts and exhibit hypopituitarism that affects all hormoneproducing cell types (Akita et aL, t997). Rathke's cysts are a common type of human pituitary adenoma, with cellular features suggesting alteration of cell fate. Cells within the cysts express some neural markers and are morphologically similar to ciliated epithelium of the airway. Analysis of LIF expression in development would be helpful for assessing the role of LIF in normal pituita~ development and in corticotrope differentation. Some cytokines stimulate ACTH secretion and POMC transcription, but their precise role in corticotrope differentiation is not clear. Interleukins 1, 2, 6, and 11 may regulate the development and function of differentiated pituitar?' cells in a paracrine mode (Auernhareaner and Melmed, 1999). CRH is mitogenic for pituitary corticotropes, but CRH-deficient mice exhibit normal corticotrope differentiation suggesting CRH is not a critical factor (Gertz et al., 1987; Muglia et al., 1995).

A. Mu!tihormonal Cells Multihormonal cell types exist in cells in normal tissue, in established cell lines, and in pituitar?" adenomas. The bipotential somatomammotrope and thyrosomatotrope have been discussed. In addition to these apparent normal precursor cell intermediates, some perplexing multihormonal combinations have been observed. For example, some corticotropes coexpress ACTH in conjunction with GH, PRL, or TSH (Childs et aI., 1989; Ishikawa et al., 1977). These multihormonal cells may represent minor alternative differentiation pathways or rare abnormal cells. Alternatively, the existence of these cells could indicate that the more specialized single hormone-producing cells are derived from a common multihormonal progenitor. However, developmental studies suggest that the specialized cells arise in distinct, spatially restricted regions. It is possible that renewal of specialized ceil types occurs differently thaa"i initial specialization and/or that the specialized cells are ve~, related, requiting only minor changes in gene expression to support expression of multiple hormones. Clearly, the nature of multihormonal cells requires further study.

IV. Conclusion The complete set of genes involved in inducing and specifying the hypophyseal placode, which forms Rathke's pouch, remains to be identified. These early steps in pituitar/patterning will be difficult to dissect with mouse genetics because many of the genes involved in early patterning have broad effects on structures arising from the anterior neural ridge. Celt-specific knock-outs and explant studies may be

20 Pituitary Gland Development the approaches that are best suited to explore the earliest steps directly, a l t h o u g h paradigms developed in other organs or other animals may be useful. An intriguing feature o f pituitary d e v e l o p m e n t is the initial organization of the celt types into discrete patches and the loss o f this spatial organization as the organ expands. These features suggest that c e i l - c e l l contacts or extracellular matrix may p r o v i d e an important role early in the differentiation process. Very little research has been done on the rote of extracellutar matrix in pituitary development. M a n y transcription factors critical for pituitary differentiation have been identified and placed within a genetic hierarchy. A challenge for the future is to m o v e from our simple understanding of the genetic hierarchy to demonstration o f molecular interactions in transcriptional control. A challenge in establishing molecular links arises due to the paucity of cell lines that can be induced to differentiate into more than one pituitary celI type. Moreover, none of the available cell lines expresses early markers like P r o p l or H e s x l . Thus, although genetics suggests a hierarchical relationship between H e s x l , P r o p l , and Pitt, molecular links have not been firmly established. The existence of additional genes regulating intermediate steps and additional coactivators is likely. Future d e v e l o p m e n t of pituitary-stage-specific c D N A libraries and use of gene array technology may be useful in identi~'ing additional target genes of the k n o w n transcription factors. Nevertheless, the pituitmD, stands out as an organ for which the differentiation markers are linked to known transcription factors, and the influence of specific signaling molecules on patterns of transcription factor expression has been established. It is encouraging that mouse models for hypopituitarism and hyperpituitarism correspond well to h o m o l o g o u s conditions in humans. In many cases discovery of genes in mice has led directly to molecular understanding of human birth defects. S o m e of the molecules demonstrated to have critical roles in development have been implicated in promotion o f pituitary tumors and adenomas. Hopefully the future will bring additional mouse models to help understand the basis for human disease, and the mechanisms controlling the specialization and proliferation of pituitary cell types.

Acknowledgments We have used official gene names wherever possible and refer readers to the mouse g e n o m e database for alternative names and additional references: http:/www.informatics.jax. org (Mouse G e n o m e Database). W~ have also used the standard nomenclature of italics for gene names with h u m a n genes being all uppercase and mouse genes with the first letter uppercase and the remainder lowercase. Proteins a p p e a r as all uppercase without italics.

513 References Acmmpora, D_ Mazan, S., Tuorto, F., Avantaggiato, ~, Trembta?~J. J., Lazzaro, D., Carlo, A. D., Mariano, A., Macchia. R E., Corte, G., Macchia. M, Drouin. J., Brfitet. R. mad Simeone, A. (1998). Transient dwarfism and hypogonadism in mice lacking Otxl reveal prepubescent stagespecific control of pituitm~" levels of GH, FSH and LH. Developmenz (Cambridge, UK) 125, I229-I239. Acampora. D., Postigiione. M. R~ Avantaggiato, V., Di Bonito, M., Vaccarino, E M.. Michaud, J., and Simeone. A. (t999). Pro~essive impairment of developing neuroendocrine celt lineages in the hypothalamus of mice lacking the Orthopedia gene. Genes Dev. I3, 2787-2800. Acher. R., and Chauvet, J. (1995). The neurohypophysiaI endocrine reguIatoD' cascade: Precursors. mediators, receptors, and effectors. Front. NeuroendocrinoL 16, 237-289. Akerblom, t. E.. Ridgway, E. C., and Mellon, R L. (i 990). An a-subunitsecreting cell line derived from a mouse thyrotrope tumor. MoI. EndocrinoL 4, 589-596. Akita, S.. Readhead, C., Stefaneanu, L., Fine. J.. Tampanaru-Sarmesiu, A., Kovacs, K.. and Melmed, S. (1997). Pituita,w-directed leukemia inhibiotD~factor transgene forms Rat~hke'scleft cysts and impairs adult pituitary function. J. Clin. Invest. 99, 2462-2469. Alarid, E. T.. Windle. J. J., Wh2,"te,D. B., and Mellon. R L. (1996). Ir~mortalization of pituitm2vcells at discrete stages of development by directed oncogenesis in transgenic mice. Development (Cambri(qe, UK) 122, 33i9-3329. Allen. R. G., Carey, C., Parker. J. D.. Mormad, M. T., Mellon. S. H., and Low. M. J. (1995). Targeted ablation of pituitary pre-proopiomelanocortin cells by herpes simplex virus-1 thymidine kinase differentially regulates mRNAs encoding the ach~enocorticotropinreceptor and aldosterone synthase in the mouse adrenal gland. MoL EndocrinoL 9, 1005-I0i 6. Atkin, S. L., Landolt. A. M.. Jeffreys, R. V., Diver, M.. Radcliffe. J.. and White, M. C. (1993). Basic fibroblastic growth factor stimulates protactin secretion from human anterior Ntuitary adenomas without affecting Menoma cell proliferation. J. Ctin. EndocrinoL Metab. 77, 831-837. Auernhammer, C., and Melmed. S. (1999). Interleukin-I 1 stimulates proopiomelanocortin gene expression and adrenocorticotropin secretion in corticotroph cells: Evidence for a redundant cytokine network in the hypothatarno-pituitmD'-adrenalaxis. Endocrinology (Baltimore) 140, 1559-t566. Banerjee, S. K., Sarkar, D. K., Weston, A. R. De, A.. and Campbell, D. R. (I997). Over expression of vascular endothelial grow~ahfactor and its receptor during the development of estrogen-induced rat pituitary tumors may mediate estrogen-initiated tumor angiogenesis. Carcinogenesis (London) 18, 1t55-1 t61. Baumann. G., and Mabeshwari, H. (1997). The Dwarfs of Sindh: Severe growth hormone (GH) deficiency caused by a mutation in the GHreleasing hormone receptor gene. Acta Paediat. 423, 33-38. Beamer. W. G.. and Cresswell, L. A. (1982). Defective thyroid ontogenesis in fetal hypothyroid (hyMhyt) mice. Anat. Rec. 202, 387-393. Behringer, R. R., Mathews. L. S., Palmiter, R. D., and Brinster, R. L. (1988). Dwarf mice produced by genetic ablation of growth hormoneexpressing cells. Genes Dev. 2, 453-46I. BentleF~ C. A., Zidehsarai, M. R, Grindley, J. C., Parlor< A. F.. Barth-HalI. S., and Roberts, V. J. (1999). Pax6 is implicated in murine pituitary endocrine function. Endocrine 10, 171-177. Bern, H. A., and Nicoll, C. S. (1%8). The comparative endocrinology of prolactin. Recent Prog. Horm. Res. 24, 681-720. Bodner, M., Castrilto, J.-L., Theill, L. E.. Deerink, T., Etlismmn, M.. and Karin, M. (1988). The pituita.~;-specific transcription factor GHF-1 is a homeobox-containing protein. Cell (Cambridge, Mass.) 55, 505-518. Borrelli, E. (! 994). Pitfalls during development: Controlling differentiation of the pituitary gland. Trends Genet. 10, 222-224. Borrelli, E., Heyman, R. A.. Arias, C.. Sawchenko. R E., and Evans, R. M.

514 (1989). Transgenic mice with inducible dwa~sm. Na.,ure (London) 339, 538-541. Boss& R., Fumagatli. F.. Jaber. M.. Giros. B.. Gainetdinov. R. R.. WetseI. W. C.. MissaIe. C.. and Caron. M. G. (1997). Anterior pituitmu: hypoplasia and dwarfism in mice lacking the dopamine transporter. Neuron 19, _/-138. Burbach. J. R. ,,an Schaick. H.. Lopes da Silva. S.. Asbreuk. C. H.. and Smidt. M. R (I 998). Hypothalamic transcription factors and the regulation of the hypothalamo-neurohyF~physial system. Adv. F_.xp.Meg BioL 449, 29-37. Burrows. H. L.. Birkmeier. T. S.. Seasholtz. A. F.. and Camper. S. A. (1996). Targeted ablation of cells in the pituitary primordia of transgenic mice. MoL EndocrinoL 10, 1467-1477. Burrows. H. L.. Douglas. K. R.. Seasholtz. A. F.. and Camper. S. A. (i999). Genealogy" of the anterior pituitary' gland: Tracing a family tree. Tre,vds En&)crinol. Metab. 10, 343-352. Burton. F. H.. Hasel. K. ~:. Bloom. F. E.. and Sutcliffe. J. G. (1991). Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transeene. :Vature (I_x)ndon) 350, 74-77. Calvo. B.. Bilbao. J. R.. Urrutia. I.. Eizaguirre. J.. Gaztambide. S.. and Castano. L. (I 998). Identification of a novel nonsense mutation and a missense substitution in the vasopressin-neurophysin II gene in two Spanish kindreds with familial neurohypophysea! diabetes insipidus. J. Clin. Endocrinol. Metab. 83. 995-997. Camper. S. A.. Saunders. T. L.. Katz. R. W. and Reeves. R. H. (1990~. The Pit-1 transcription factor gene is a candidate for the Snell dwarf mutation. Genomics 8, 586-590. Carbajo-Perez. E.. Motegi. M.. and Watanabe. Y. G. (1989). Cell proliferation in the anterior pituitary of mice during growth. Biomed. Res. 10, 275-281. Castrillo. J.-L.. Theill. L.. and Karin. M. ( 1991 ). Function of the homeodomain protein GHFI in pituitar5 ceil proliferation. Science 253, 197t 99. Chen. S.. Leger. J.. Garel. C.. Hassan. M.. and Czernichow. P. (1999). Growth hormone deficiency with ectopic neurohypophysis: Anatomical variations and relationship between the visibility of the pituitary stalk asserted by magnetic resonance imaging and anterior pituitar)' function. J. Clin. Endocrinol. Metab. 84, 2408-_,+1v~_'~. Childs. G. M. Vestlund. K. N.. and Unabia. G. (!989). Characterization of anterior pituitary target cells for arginine vasopressin: Including cells that store adrenocorticotrophin, thyrotrophin, and both hormones. Endocrinology (Baltimore) 125, 554-559. Cohen. L. E.. Wondisford, F. E.. Salvatoni. A.. Maghnie. M.. BruckerDavis. E. Weintraub. B. D.. and Radovick. S. (1995). A "'hot spot" in the Pit-1 gene responsible for combined pituitary hormone deficiency: Clinical and molecular correlates. J. Clin. Endocrinol. Metab. 80, 679684. Cotlu. R.. Tang, J., Castagne. J.. Lagace. G.. Masson. N.. Huot. C.. Deal. C.. Delvin, E.. Faccenda, E.. Eidne, K. A.. and Van Viler. G. (1997). A novel mechansim for isolated central hypothyroidism: Inactivating mutations in the thyrotropin-releasing hormone receptor gene. J. Ctin. Endocrinol. Metab. 82, 1361 - 1365. Couly. G. E. and Le Douarin. N. M. (I985). Mapping of the early' neural primordium in quail-chick chimeras. I. Developmental relationships between pIacodes, facial ectoderm, and proencephalon. Dev. Biol. 110, 422-439. Couly. G. F.. and Le Douarin. N. M. (1987). Mapping of the early neural primordium in quail-chick chimeras. II. The proencephalic neural plate and neural folds: Implications for the genesis of cephalic human congenital abnormalities. Def. Biol. 120, 198-214. Couly. G. F.. and Le Douarin. N. M. (1988). The fate map of the cephalic neural primordium at the presomititc to the 3-somite stage in the avian embryo. Development (Cambridge, UK) 103, Suppl.. 101-i 13. Crenshaw. E. B. I., Kalla. K.. Simmons. D. M.. Swanson. L. W. and Rosen-

I11 Organogenesis feld, M. G. (1989). Cell-specific expression of the prolactin gene in transgenic mice is controlled by synergistic interactions between promoter and enhancer elements. Genes Dev. 3, 959-972. Cushman. L. J.. Watkins-Chow. D. E., Brinkmeier. M. L.. Raetzman. k T., Radak. A. L.. Lloyd. R. V.. and Camper. S. A. (200I). Persistent Propl expression delays gonadotrope differentiation and enhances pituitary tumor susceptibility. Hum. Mole. Gene. 10. 114!-1153. Dasen. J. S.. and Rosenfeld. M. G. (t999). Combinatorial codes in signaling and synergy: Lessons from pituitary development. Curt. Opin. Genet. Dev. 9, 566-574. Dasen. J. S.. O'Connell. So M.. Flynn. S. E.. Treier. M.. Gleiberman. A. S., Szeto. D. R. Hooshmand. E. Aggmwal. A. K.. and Rosenfeld. M. G. (1999). Reciprocal interactions of Pit! and GATA2 mediate signaling gradient-induced determination of pituitary cell types. Cell (Cambridge. Mass.) 97, 587-598. Dattani. M. T.. and Robinson. I. C. (2000). The molecular basis for developmental disorders of the pituitary gland in man. C.~in. Genet. 57, 337-346. Dattani. M. T.. Martinez-Barbera. J.-R. Thomas, R Q., Brickman. J. M., Gupta. R.. MSrtensson. I.-L.. Toresson, H.. Fox. M.. Wales. J. K. H., Hindmarsh. R C.. Krauss. S.. Beddington. R. S. R. and Robinson. I. C. A. F. (1998). Mutations in the homeobox gene HESX]/Hesxl associated with septo-optic dyspIasia in human and mouse. Nat. Genet. 19, 125-I33. Dearden. N. M.. and Holmes. R. L. (1976). Cyto-differentiation and portal vascular development in the mouse adenohypophysis. Z Anat. 121, 551-569. Deiner. M. S., and Sretavan. D. W. (1999). Altered midline axon pathways and ectopic neurons in the developing hypothatamus of netrin-t- and DCC-deficient mice. J. Neurosci. 19, 9900-9912. de la Pompa. J. L.. Wmkeham. A.. Correia. K. M.. Samper. E.. Brown. S.. Aguiiera. R. J.. Nakano. T.. Honjo. T.. Mak. T. W.. Rossant. J.. and Conlon. R. A. (1997). Conservation of the Notch signalling pathway in mammalian neurogenesis. Derelopment (Cambridge. UK) 124, 11391148. De Moerlooze, L.. Spencer-Dene. B.. Revest, J., Hajihosseini~ M.. Rosewelt, I.. and Dickson. C. (2000). An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymaI-epitheiial signalling during mouse organogenesis. Development (Cambridge, UK) 127, 483-492. Denton. E. R.. Powrie. J. K.. Ayers, A. B.. and Sonksen, R H. (1996). Posterior pituitary ectopia and hypopituitarism~magnetic resonance appearances of four cases and a review of the literature. Br../. RadioL 69, 402-406. de Wied. D. (1999). Behavioral pharmacology of neuropeptides related to melanocortins and the neurohypophyseal hormones. Eur. J. Pharmacot. 375. 1-11. Drolet. D. W. Scully. K. M.. Simmons, D. M., Wegner. M.. Chu, K., Swanson. L. V~. and Rosenfeld. M. G. ( 199 i ). TEE a transcription factor expressed specifically in the anterior pituitary during emb~,ogenesis, defines a new class of leucine zipper proteins. Genes Dev. 5, I739-I753. Dubois. R. and Hemming. E (i 991). Fetal development and regulation of pituitary cell types. J. Electron Microsc. Techn. 19, 2-20. Dunger. D. B.. Broadbenc Vi. Yeoman. E., Seckl. J. R.. Lightman, S. L.. Grant. D. B.. and Pritchard. J. (11989). The frequency and natural histo~' of diabetes insipidus in children with Langerhans-cell histiocytosis. N. Engl. J. Med. 321, 1157-1162. E1Amraoui. A.. and Dubois. R M. (11993). Experimental evidence for the early commitment of the presumptive adenohypophysis. Neuroendocrinology 58, 609- 615. Elchebly, M.. Wagner. J.. Kennedy. T. E.. Lanctrt, C.. Michaliszyn, E., Itie. A.. Drouin. J.. and Tremblay, M. L. (i999). Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase o'. Nat. Genet. 21, 330-338.

20 Pituitary Gland Development Elkabes. S.. Loh. Y. P. Nieburgs. A.. and Wray~ S. (1989). Prenatal ontogenesis of pro-opiomelanocortin in the mouse central nervous system and pituitary.' gland: An in situ hybridization and immunocytochemical study. Brain Res. Dev. Brain Res. 46, 85-95. Ericson, J., Norlin, S., Jessett. T. M.. and Edlund, T. (I998). Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituita 9 Deveiopmenf (Cambridge, UK) 125, I005-I 0t 5. Feingold. M.. Shiere. E. Fogels. H. R., "-randDonaldson, D. (i969). Rieger's syndrome. Pediatrics 44, 564. Felix, R.. Meza. U.. and Cota. G. (I995). Induction of classical lactotropes by epidermal growth factor in rat pituita~D, celi cultures, Endocrinology (Baltimore) 136, 939-946. Frawley: L. S.. and Btoockfor. E R. (1989). Mammosomatotropes: Presence and functions in normal and neoplastic pituitaW tissue. Endocr. Rev. 12, 337-355. Gage. R J.. Brinkmeier. M. L., Scarlett, L. M.. Knapp. L. T., Camper, S. A.. and Mahon, K. A. (1996a). The Ames dwarf gene, df is required early in pituitaW ontogeny for the extinction of Rpx transcription and initiation of lineage specific cell proliferation. MoI. EndocrinoI. 10, t 570158I. Gage. R J.. Roller. M. L.. Saunders, T. L., Scarlett, L. M., and Camper, S. A. (1996b). Anterior pituitary, ceils defective in the cell autonomous factor. df undergo cell lineage specification but not expansion. Development (Cambridge. UK) 122, 151 - 160. Gage. E J., Sub, H., and Camper, S. A. (t999a). Dosage requirement of Pitx2 for development of multiple organs. Development (Cambridge, UK) 126, 4643-465 I. Gage. R J., Sub. H.. and Camper, S. A. (1999b). Review: The bicoid-related Pit_x gene family in development. Mature. Genome, 10, pp. 197-200. Gagtiardi, R C.. Bernasconi. S.. and Repaske, D. R. (I997). Autosomat dominant neurohypophyseal diabetes insipidus associated with a missense mutation encoding G l y 2 3 ~ > V a l in neurophysin Ill. J. Clin. Endocrinol. Metab. 82, 3643-3646. German, M. S.. Wang, J., Chadwick, R. B., and Rutter, W. J. (1992). Synergistic activation of the insulin gene by a LIM-homeodomain protein and a bsic helix-loop-helix protein: Building a functional insulin minienhancer complex. Genes Dev. 6, 2165-2176. Gertz. B. J., Contreras. L. N.. McComb. D. J.. Kovacs, K.. Tyrrell, J. B.. and Dallman. M. F. (1987). Chronic administration of corticotropinreleasing factor increases pituitary corticotroph number. Endocrinology (Baltimore) 120, 381-388. Gem'ins. R. Skoldenberg. E., and Claesson-Weish. L. (2000). Function of fibroblast growth factors and vascular endothelial growth factors -and their receptors in angiogenesis. Crit. Rev. OncoL Hematol. 34, 185I94. Gleiberman. A. S.. Fedtsova. N. G.. and Rosenfeld, M. G. (1999). Tissue interactions in the induction of anterior pituitar ,: Role of the ventral diencephalon, mesenchyme, and notochord. Dev. BioL 213, 340353. Goda. H.. Sakai. T.. Kurosumi. M.. and Inoue. K. (I998). Prolactinproducing cells differentiate from G0tG 1 arrested somatotrophs in vitro: An analysis of ceil cycle phases and mammotroph differentiation. Endocr. J. 45, 725-735. Godfrey. R, Rahal. J. O.. Beamer, W. G., Copeland, N. G.. Jenkins, N. A., and Mayo, K. E. (1993). GHRH receptor of little mice contains a missense mutation in the extracellu!ar domain that disrupts receptor func~ a . 7 ~ . ")'~ ~ ~. tion. Nat. Genet. 4, ."~v I Goluboff, L., and Ezrin. C. (1969). Effect of pregnancy on the somatotroph and the prolactin cell of the human adenohypophysis. J. Ctin. Ep,docrinoL Merab. 29, I533-I538. Good. D. J.. Porter, F. D.. Mahon. K. A.. Parlow, A. E, Westphal, H.. and Kirsch. I. R. (1997). Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene. Nat. Genet. 15, 397-40t.

515 Gordon, D. E, Haugen. B. R.. Sarapura. V. D.~ Nelson. A. R., Wood. W. M.. and RidgwaB E. C. (t 993). Analysis of Pit-1 in regulating mouse TSH/3 promoter activity in thyrotropes. MoL Ceil EndocrinoL 96, 75-84. Gordon, D. E. Lewis. S., Haugem B. R.. James, R., McDermott. M.. Wood. W. M., and Ridgeway. E. C. (I 997). Pit- I and GATA-2 inmract and functionaily cooperate to activate the thyrotropin/3-subunit promoter. J. BioL Chem. "~7"~ 24339-24347. Gothe. S.. Wang, Z.. Ng, L.. Kindblom, J. M., Barros, A. C., Ohlsson. C., Vennstrom. B.. arid For-rest, D. (19~ Mice devoid of all known th D roid hormone receptors are viable but exhibit disorders of the pituitary;thyroid axis, growth, and bone maturation. Genes Dev. 13, 1329-i 34 I. Heaney. A. R. Ho~,itz. G. A.. Wang, Z.. Singson. R.. and Metmed, S. (I999). Early involvement of estrogen-induced pitui~"y tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. ?v~t. Med. 5, I317-I321. Hentges. S., Boyadjieva. N.. and Sarkar, D. K. (2000). Transforming growth factor-beta 3 stimulates lactotrope cell growth by increasing basic vibroblast growth factor from foI!iculo-stellate cells. Endocrinology (Baltimore) 141, 859-867. Hermesz. E.. Mackem. S., and Ma.hon, K. A. (I 996). Rpx: A novel anteriorrestricted homeobox gene progressively activated in the prechordal plate. anterior neural plate, and Rathke's pouch of the mouse embwo. Development (Cambridge, UK) 122, 41-52. Hoeffler. J. R. Btcmckfor. E R., and Frawley, L. S. (t985L Ontogeny of prolactin cells in neonatal rats: Initial prolactin secretors also release growth hormone. Endocrinology (Baltimore) 117, 187-I95. Hooghe-Peters, E., BeIayew. A.. Herregodts, R, Velkeniers, B., Smets, G.. MaritaI. J., and Vanhaelst, L. (!988). Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary' cells: Possible role of dopamine. MoL EndocrinoL 2, 1I63-1168. Horvath, E., Lloyd. R. "v~.and Kovacs. K. (1990). Propylthiouracyl-induced hypothyroidism results in reversible transdifferentiation of somatotrophs into thyroidectomy cells. Lab. lnvest. 63, 511-520. ibrahim, S. N., Moussa. S. M., and Childs, G. V. (I986). MorphomeLric studies of rat anterior pituitary cells after gonadectomy: Correlation of changes of gonadotropes with the serum levels of gonadotropins. Endocrinology (Baltimore) 119, 629- 637. Ikeda. H., and Yoshimoto, T. (199 !). Developmental changes in proliferative activi~: of cells of the routine Rathke's pouch. Cell Tissue Res. 263, 4i-47. Ikeda. Y.. Luo. X.. Abbud, R., Nilson, J. H., and Parker, K. L. (1995). The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromediaI hypothalamic nucleus. MoL EndocrinoL 9, 478-486. Ingralham, H. A., Chen, R., Mangatam, H. J., Elsholtz, H. R, Hynn, S. E., Lin. C. R., Simmons, D. M., Swanson, L., and Rosenfeld, M. G. (1988). A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenoty'pe. Celt (Cambridge, Mass.) Sfi, 519-529. Ishikawa, H., Shiino, M., Arimura. A., and Rennets, E. G. (1977). Functional clones of pituit~: cells derived from Rathke's pouch epithelium of fetal rats. Endocrinology (Baltimore) 100, I227-1230. Japon, M. A., Rubinstein, M., and Low, M. J. (1994). tn situ hybridization analysis of anterior pituitaw hormone gene expression during fetal mouse development. J. Hisfochem. Cytochem. 42, 11 i 7-t 125. Jing, X., Ratty, A. K.. and Murphy, D. (1998). Ontogeny of the vasopressin and oxytocin RaNAs in the mouse hypothatamus. Neurosci. Res. 30, 343-349. Jorgenson, R., Levin, L., Cross, H., Yoder, E, and Kelly. T. (! 978). The Rieger Syndrome. Am. J. e~ed. Genet. 2, 307-318. Jost, A. (t954). Hormonal factors in the development of the ferns. Cold Spring Harbor &;mp. Quam. Biol. 19, 167-18t. Kakeya, T., Takeuchi, S., and Takahashi, S. (2000). Epidermal growth factor, insulin and estrogen stimulated development of prolactin-secreting cells in cultures of GH3 cells. Cell Tissue Res. 299, 237-243. Kapitoff, M.. Farkash, Y., Wegner. M.. and Rosenfetd, M. ( 1991 ). "v'ariable

516 effects of phosphoulation of Pit-1 dictated by the DNA res~nse elements. Science 253, 786-789. Karlstrom. R. O.. Talbot. W. S.. and Schier. A. F. (I999). Comparative synteny cloning of zebrafish you-~.oo: Mutations in the Hedgehog target Gli2 affect ventral forebrain patterning. Genes Dev. 13, 388-393. Kaufman. M. H. (19921). "'The Atlas of Mouse Development." Academic Press. San Diego. CA. Kawamura. K.. and Kikuyama. S. (I 995). Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonic,~s). Ceil Tissue Res. 279, 233-239. Kendall. S. K.. Saunders. T. L.. Jin. L.. Lloyd. R. V.. Glode. L. M.. Nett. T. M., Keri. R. A.. Nilson. J. H.. and Camper. S. A. (1991 ). Targeted ablation of pituitar/gonadotropes in transgenic mice. Mol. EndocrinoL 5, 2025 -_0_6. "~ "~ Kendall. S. K.. Samueison. L. C.. Saunders. T. L.. Wood. R. I.. and Camper. S. A. (1995). Targeted disruption of the pituitary glycoprotein hormone alpha-subunit produces hypogonadal and hypothyroid mice. Genes Dev. 9, 2007-2019. Kioussi. C. (1999). Pax6 is essential for establishing ventral-dorsal cell boundaries in pituitm~, gland development. Proc. ,~tl. Acad. Sci. U.SA. 96, 14378-14382. Kumar. T.. Wang. Y.. Lu. N.. and Matzuk. M. (1997). Follicle stimulating hormone is required for ovarian follicle maturation but not male fertilit). Nat. Genet. 15, 201-204. Labosky. R A.. Winnier. G. E.. Jetton, T. L.. Hargett. L.. Rvan. A. K.. Rosenfeld. M. G.. Parlow. A. E. and Hogan. B. L. (1997). The winged helix gene. Mf3. is required for normal development of the diencephalon and midbrain, postnatal growth and the milk-ejection reflex. Development (Cambridge. UK) 124, 1263-1274. Lala. D. S.. Rice, D. S.. and Parker. K. L. (1992). Steroidogenic factor 1. a key regulator of steroidogenic enzyme expression, is the mouse homolog of fi~shi tarazu-factor I. Mol. Er,docrinol. 6, 1249-1258. Lanctot. C.. Lamolet. B.. and Drouin. J. (t997). The bicoid-related homeoprotein Ptxl defines the most anterior domain of the emb~,o and differentiates posterior from anterior lateral mesoderm. Development (Cambridge, UK) 124, 2807-2817. Landis. C. A.. Harsh. G.. Lyons. J.. Davis. R. L.. McCormick. F., and Bourne. H. R. (1990). Clinical characteristics of acromegalic patients whose pituitary' tumors contain mutant Gs protein. J. Clin. EndocrinoL Metab. 71, 1416-1420. Lee. S. L.. Sadovsky. Y.. Swirnofi. A. H.. Polish. J. A.. Goda. R. Gavrilina. G.. and Milbrandt. J. (1996). Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (EgrI). Science 273, 1219 - 1221. Lew, D.. Brady. H.. Klausing. K.. Yaginuma. K., Theill. L. E., Stauber. C.. Karin. M.. and Mellon. R L. (1993). GHF-I promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice. Genes Dev. 7, 683-693. Li. H.. Zeitler. R S.. Valerius. M. T.. Small. K.. and Potter. S. S. (I996[). Gsh-I. an orphan Hox gene. is required for normal pituitary developmerit. EMBO J. 15, 714-724. Li. S., Crenshaw. E. B.. 3rd. Rawson. E. J.. Simmons. D. M.. Swanson. L. W:. and Rosenfeld, M. G. (1990). Dwarf locus mutants lacking three pituitary ceil types result from mutations in the POU-domain gene PitI. Nature (London) 347, 528-533. Lin. S.-C.. Lin. C. R., Gukovsky. I.. Lusis. A. J.. Sawchenko. R E.. and Rosenfeld. M. G. (1993). Molecular basis of the little mouse phenotype and implications for cell type-specific growth. Nature (London) 364, 208-213. Lipkin. S. M.. N~iiir. A. M., Kalla. K. A.. Sack. R. A.. and Rosenfeld. M. G. (I 993). Identification of a novel zinc finger protein binding a conserved element critical for Pit-l-dependent growth hormone gene expression. Genes Dev. 7, 1674-1687. Lloyd. R. "v: (1983). Estrogen-induced hyperplasia mid neoplasia in the rat

!11Organogenesis anterior pituitary gland: An immunohistochemical stud5'. Am. J. Pathol. 113, ! 98-206. Lloyd, R. ~v: (1993). Surgical pathology of the pituitary gland. In ~'Major Problems in Pathology" 0,/: A. Livolsi. ed.), p. 257. Saunders, Philadelphia. Lopez-Fernandez. L, Palacios. D.. Castillo, A. t.~ Toton, R. M.. Aranda. A., and Karin. M. (2000). Differentiation of iactotrope precursor GHFT cells in response to fibroblast growth factor-2. J. BioL Chem. (in press). Luo. X.. Ikeda. Y.. and Parker. K. L. (I994). A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell (Cambridge, Mass.)77, 481-490. Maki. K.. Miyoshi. I.. Kon. Y.. Yam~hita. T.~ SasakL N.. Aoyama. S.. Takahashi. E.. Namioka. S.. Hayashizaki. Y.. and K a r l . N. (I 994). Targeted pituitar : tumorigenesis using the human thyrotropin beta-subunit chain promoter in transgenic mice. MoL Ceil. Endocrinol. 105, 147-154. Markkula. M.. Kananen. K.. Paukku. T.. Mannisto, A., Loune. E., Frojdman. K., Pelliniemi. L. J.. and Huhtaniemi. I. (I9941). Induced ablation of gonadotropins in transgenic mice expressing Herpes simplex virus thymidine kinase under the FSH beta-subunit promoter. ;viol. Cell. EndocrinoL 11)8, 1-9. Mason. A. J.. Hayflick. J. S.. Zoelter. R. H., Young, W. S.. III. Phillips. H. S.. Nikolics. K.. and Seeburg. R H. (1986a). A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234, 1366-1371. Mason. A. J.. Pitts. S. L.. Nikolics. K.. Szonyi. E.. Wilcox, J. N., Seeburg, R H.. and Stewart. T. A. (1986b). The hypogonadal mou~: Reproductive functions restored by gene therapy. Science 234, 1372-1378. Mathis. J. M.. Simmons. D. M.. He. X.. Swanson. L. \~L. and Rosenfeld. M. G. (1992). Brain 4: A novel mammalian POU domain transcription factor exhibiting restricted brain-specific expression. EMBO J. I1, 2551-2561. Matzuk. M.. Kumar. T.. and Bradley. A. (1995). Different phenotypes for mice deficient in either activins or activin receptor type II. Nature (London) 374, 356-360. Mayo. K. E. ( t 996). A little lesson in growth regulation. Nat. Genet. 12, 8-9. Mayo. K. E.. Hammer, R. E., Swanson, L. Vv~.Brinster, R. L., Rosenfeld, M. G.. and Evans. R. M. (1988). Dramatic pituitar 5' hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene. Mol. EndocrinoL 2. 606 - 612. McAndrew, J.. Paterson, A. J.. Asa. S. L.. McCarthy. K. J., and Kudlow, J. E. (1995). Targeting of n'-ansforming growth factor-alpha expression to pituitary lactotrophs in transgenic mice results in selective lacton'oph proliferation and adenomas. Endocrinology (Baltimore) 136, 44794488. McCamhy, M. M.. and Altemus. M. (I997). Central nervous system actions of oxytocin and modulation of behavior in humans. MoL Meal Today 3, 269-275. Michaud. J. L.. Rosenquist. T., May. N. R.. and Fan, C. M. (i 998). Development of neuroendocrine lineages requires the bHLH-PAS transcription factor SIM I. Genes Dev. 12, 3264-3275. Minowa. O.. Ikeda. K., Sugitani. Y.. Oshima. T., Nakai. S., Katori. Y., Suzuki. M.. Furukawa, M.. Kawase, T.. Zheng. Y., Ogura. M.. Asad~ Y., V~,htanabe, K.. Yamanaka. H.. Gotoh, S.. Nishi-Takeshima, M., Sugimoto. T.. Kikuchi, T.. Takasaka. T.. and Noda. T. (I 999). Altered cochlear fibrocytes in a mouse model of D ~ 3 nonsyndromic deafness. Science 285, 1408 - 1411. Missale. C.. Boroni. F.. Sigala. S.. Zanellato. A.. Toso. R. D.. Balsari, A., and Spano. R (1994). Nerve growth factor directs differentiation of the bipotential cell line GH-3 into the mammotroph phenotype. Endocrinology (Baltimore) 135, 290-298. Mouse Genome Database (1996). "'Mouse Genome InformaticsY M. 3. !. The Jackson Laborato~,, Bar Harbor. ME. World Wide Web: URL: http ://www.informatic s.jax.org / Mugtia, L~ J.. Jacobson, L.. Dikkes. R. and Majzoub, J. A. (1995). Cortico-

20 Pituitary Gland Development tropin-releasing hormone deficiency, reveals fetal but not adult giucocorticoid need. Nature (London) 373, 427-432. Nakai. S., Kawemo, H., Yudate. T.. Nishi, M.. Kuno. J.. Nagata. A.. Jishage. K., Hamada. H.. Fujii, H., Kawamura, K.. et al. (1995). The POU domain transcription factor Brn-2 is required for the determination of specific neuronal lineages in the hypothalamus of Ne mouse. Genes De< 9, 3109-3121. Nemeskeo ~, A.. Nemeth. A.. Setalo. G.. Vigh. S.. and Hal~z. B. (19761< Cell differentiation of the fetm anterior pituit~y in vitro. Cel! Tissue Res. 170, 263-273. Netchine. I.. Sobrier. M. L.. Krude. H., Schnabel. D.. Maghnie. M.. Marcos. E.. Duriez. B.. Cacheux. ~, m "O

Lens

pit

r r .J

Soxl

v

o,4 ,ii

Primary lens fiber

I

Secondary

Soxl

c-Maf

Lens epithelium

!

c~-cry ++

pcry +++ y-cry +++

~

Pax6 FoxE2

(Sox2) (Six3) MafB Eyal

~-cry + !r

Figure 5 Morphological and biochemical changes during lens development. Expression periods of various transcription factor genes, indicated on the right, are correlated with morphological development of the lens shown on the left. Epithelium-specific and fiber-specific expression of these transcription factors are indicated in the scheme at the lower left comer. "Vnose in parentheses indicate a low expression level. Relative expression levels of c~,stallin genes (c~:) are also indicated by plus marks.

little turnover of crystallin proteins, and crystattins synthesized early in life are carried over throughout the life span. During lens development, expression of crystatlin genes occurs sequentially. In the mouse there are two a-cc~stalIin genes, six y-crystallin genes (Goring et at., I992), and a similar number of fl-crystallin genes, aA and aB crystallins are expressed from the stage of invaginating lens vesicle and continue to be expressed in both lens epithelium and lens fibers; by contrast, fi c~,stallins and y crystallins are expressed only during lens fiber development (Kondoh, 1999). In the chicken, y crystatlins are replaced by 6 crystallin (61 crystallin) coded by a single gene (Kondoh et aL, t991). In contrast to other crystallins in the mouse, 6-crystatlin expression in the chicken initiates at the lens ptacode stage and is highly augmented in the lens fibers. The c~,stallin genes

are potential targets of the transcription factors that play a role in lens development.

B. Lens Induction Ectodermal origin of lens and possible interaction with primitive retina had already been noted by the 19th century, but Spemann's experiment reported in 1901 using frog Rana fusca demonstrated for the first time the inductive influence of the retina on lens development. He demonstrated that when retina pfimordia are removed early enough before the contact to the head ectoderm occurs, no lenses develop. Lewis (I904) slightly later observed in flog and salamander embryos that tight contact between the optic cup and the head ectoderm is required for lens induction to occur. A

530 modem version of the Spemann experiment occurred genetically, in Ap2 lcnock-out mice, where optic vesicles were misoriented without contact with the head ectoderm, resulting in the absence of lens induction (Schorle et al.. I996: Zhang et al., 1996). Controversy surrounded whether the contact of the lens vesicle to the head ectoderm is required for initiation of lens development, with the variation in the conclusions stemming from the method of retina primordia ablation and interpretation of the data. It has been noted that retinal primordia of the forebrain extensively regenerates after tissue ablation. which may have caused ambiguous situations. In any event, contact of the optic vesicle with the head ectoderm is definitely required for initiation of lens development (Mizuno et aL. 1998: Zygar et al., 1998). Under a condition in which lens development somehow" initiates, it is noted that at least one of the GroupB Sox genes is activated in the region contacted by the optic vesicle. Sox2 in the case of the mouse, Sox2 and Sox3 in the case of the chicken (Kamachi et al., 1998), and Sox3 in the case of Xenopus (Zygar et al., 1998). When the optic vesicle makes contact with the head ectoderm. the surgical separation of these tissues is not possible without use of enzymes degrading extracellular matrices. Thus, if a secreto~: molecule is involved in lens induction. it must act within a short distance. For some decades, the optic vesicle was believed to induce a lens when apposed to any place of emb~'onic ectoderm. This belief, however, no longer exists because Grainger's group, employing a reliable cell-labeling technique, demonstrated that the old data were likely due to incomplete separation of the tissues before ~afting of the tissues (Hen~' and Grainger, 1987). The competence of the ectoderm to develop into lens tissue in response to the optic vesicle is confined to the head ectoderm. The entire mechanism that defines the competence has not yet been fully resolved, but evidence indicates that expression of Pax6 in the head ectoderm is highly involved. From before apposition by the optic vesicle, Pax6 is broadly expressed in the head ectoderm including the prospective tens area (Grindtey et aL, 1995" Li et at., 1994: Watther and Gruss. 1991), and this expression is not affected by the presence or absence of the optic vesicle (Kamachi et al., 1998" Li et al., 1994). In homozygous Sey mutant mouse embQ'os lacking Pax6 gene activity, optic vesicles are formed and. although slightly more expanded than normal vesicles, make contact with the head ectoderm but do not induce thickening of lens placode or any other lens traits (Grindley et al., 1995: Hill et al., 1991" Hogan et aL. 1986). Because both the optic vesicle and the responding head ectoderm express Pax6. it is not clear from the mutant phenotype which of the eye components is responsible for the absence of lens development. Using rat Sey mutant materials, an organ culture experiment was performed by recombining Sey homozygote head ectoderm

Iil Organogenesis and wild-tvpe optic vesicle, and vice versa (Fujiwara et aL, t 994). Lenses developed from the ectoderm if the ectoderm was derived from the wild type, regardless of the genotype of the optic vesicle; on the other hand, no lens was induced from the mutant ectoderm even by combination with wildtype optic vesicle. Therefore, Pax6 expression is an absolute requirement for the head ectoderm to be induced for lens development, but is not required for the inducing activity of the optic vesicle. Thus, Pax6 provides responsiveness of the head ectoderm to the inductive influence of the optic vesicle.

C. From Lens Placode to Lens 'vesicle Once Pax6-positive head ectoderm is contacted by the optic vesicle, the contacted area initiates expression of a few transcription factors: Sox2 (in the case of mouse), Six3, E y a l (Xu et al., 1997), P r o x l (Wigte et at., 1999), and Foxe3 (Blixt et al., 2000). Because Sox2 is commonly expressed among sensor" placodes (Uchikawa et al., 1999), it is speculated that it is involved in the placodal nature of the tissue. The problem of how" expression and function of these induced transcription factors is dependent on the activity of Pax6 in the head ectoderm is obscured because of involvement of Pax6 in multiple tissues and stages in eye development. Observations in Sev (Pax6) homozygotes could reflect either the cell autonomous effect of the absence of PAX6 in the ectoderm or an indirect effect caused by the defect of the CNS compartments. To circumvent the problem, Pax6 was inactivated only in the head ectoderm using a combination of a Cre recombinase transgene and LoxP-flanked Pax6 locus (Ashe~,-Padan et al., 2000). Cre recombinase was driven by the lens enhancer of the Pax6 gene itself, which is active in the head ectoderm before the stage of contact by the optic vesicle. This resulted in initiation of Pax6 expression in the ectoderm at E9, and in toss of Pax6 activi~' at E9.5 when the optic vesicle contacts with the ectoderm. Under the condition of ectoderm-specific Peer6 ablation, Sox2 expression was induced in the ectoderm, while expression of Six3 and P r o x t was lost. Therefore, at least at stages later than E9.5, expression of Sox2 is independent of Pax6, while Six3 and Prox] depend on Pax6 for their expression. Under the condition that Sox2 is induced in the head ectoderm in the absence of Pax6, no lens placode develops (Ashew-Padan et aL, 2000). An interesting comparison is the BMP4 mutant where Pax6 is expressed in the head ectoderm but Sox2 is not induced by contact with the optic vesicle, and lens placode fails to develop (Furuta and Hogan, 1998). These observations argue that both Pax6 and Sox2 are required for development of the lens placode. Kamachi et al., (200 t) demonstrated that Pa.x6 and Sox2 form a co-DNA-binding molecular complex, and this complex activates lens-specific genes and initiates lens placode development, thus providing the basis for the requirement of Pax6 and Sox2.

21 Development of the Eye The optic vesicle-contacted area of the head ectoderm area expressing Pax6 and Sox2 together begins to thicken and forms the tens placode. The placode invaginates and develops into the tens. When this invagination occurs, expression of c-Maf and c~ crystatlin begins (Kawauchi et aL, 1999; Kim et aI., t999: Ring et aI., 2000), and when the lens vesicle is formed. Sox2 expression largely ceases and is replaced by Soxt expression (Kamachi er al., 1998; Nishiguchi et aL, 1998). As the lens fibers differentiate, expression of fl and y crystallins initiates in the fiber compartment. The lens phenotypes of the Proxl knock-out mouse embryo and Foxe3 mutant mouse indicate that major functions of Proxl and FoxE3 correspond to later phases of lens development, in spite of the early onset of gene expression (Blixt et aL, 2000: Wigle et aL, 1999). It is notable that Six3 and Eyal are expressed from the early placodal stage of the tens in a Pa_r6-dependent fashion (Ashe~-Padan et aI., 2000: Xu et aL, 1997), but the significance of their expression in lens development has not yet been determined, in spite of the implied analogy with the case of toy-dependent expression of so, eya, and dac in Drosophila (Chen et al.. 1997: Pignoni et aL, 1997). Eyat knockout mouse does not show major eye defects except for an open eyelid (Xu et at., 1999); expression of Dach (mouse dac homolog) is neither PeLt6 dependent nor occurs at the placode stage of the lens (Hammond et at., 1998), while the effect of Six3 overexpression on lens development is not significant (Loosli et at., 1999).

D. Sox, Maf, and Crystaliin Gene Regulation SOX and MAF have been identified as the transcriptional regulators of the c~'stallin genes. Through the analysis of lens-specific regulation of the 6-crystallin enhancer of the chicken (Hayashi et aL, I987), an essential element was identified that is bound and activated by Sox proteins (Kamachi and Kondoh, t993" Kamachi et aL, 1995). This element is activated by GroupB SOX proteins, SOXt, SOX2. and SOX3, but not by other SOX proteins (Kamachi et aL, 1998, 1999). The same SOX proteins also regulate ycr?'stallin genes, the mammalian counterpart of 6 c~,stallin. A conserved and essential DNA motif in the promoters of the y-crystaltin genes is the binding site of the SOX proteins that activate the promoters (Kamachi et aL, t995). SOX1 strongly, and SOX2 and SOX3 modestly, activate these crystallin elements. Therefore, the transition from Sox2 to Soxl in the mouse lens development (Kamachi et aL, 1998) will augment y-crystallin expression. Sox2 -/- embryos die soon after implantation without providing information on eye development (Pevny et aL, 1998), but the phenotype of SoxI knock-out mice confirmed the essential function of SOX genes in cD'statlin gene regulation (Nishiguchi et aL, 1998). The major phenotype of Soxl null mutant mice is the arrest of lens development at the stage of

531 primary lens fiber elongation. Analysis of crystatlin gene transcripts indicates that all y-crystallin genes A to F are severely downregutated, while expression of cr and fl cD'stallins is not significantly affected. A low level y-crystaltin gene expression is observed at the initial stage, but becomes totally attenuated by birth. The arrest of morphological development may be accounted for by the absence of continued y crystallin synthesis, but loss of other strnctural components is not ruled out. In any case, expression profiles of y-crystatlin genes in Soxt null mutant mice must be understood considering the fact that Sox2, sharing almost the same characteristics, is expressed in the immediately prior phase of lens development (Kamachi et aL, 1998). It is likely that some y-crystallin gene expression is initiated by the effect of normal Sox2, but after attenuation of Sox2 expression during lens development, Soxl/2-dependent transcription of ycrystallin gene is lost in Sox I knock-out mice. In the mouse (and rat) lenses, (c-Maf) is expressed in the lens fibers and Mafb in the lens epithelium (Kawauchi et aL, I999; Yoshida et at., 1997). A third Mar family protein NrI is also expressed in late phases (possibly in the posmatal stage) (Kawauchi et aL, 1999; Liu et aL, 1996). Involvement of Maf family bZip transcription factors was indicated by the fact that Maf family proteins bind an element of chicken c~-crystatlin promoter, which shows lens specificity (Ogino and Yasuda, i998), c-Maf also binds the Maf-consensusrelated sequence in the y-crystallin promoters present betw'een TATA box and the Sox binding site, which was previously recognized as the yF- I binding site (Liu et aL, 1991). Retrospectively from the results of (Mar) knock-out mice, it was also shown that MAF binds the promoter sequences of some fi-crystallin promoters (Ring et aL, 2000). Three groups reported ~ e t e d disruption of the c-Maf gene (Kawauchi et aL, I999; Kim er aL, 1999; Ring et at., 2000). In these homozygous mice, lens vesicles are normally formed but elongation of primary lens fibers is largely inhibited. Lens defect is the major phenotype of the homozygous mice, although most of the animals are lost by weaning. There are some discrepancies concerning the effect of c-Maf null condition on expression of crystallin genes, but fine consensus woutd be that aA-crystatlin expression is reduced, ~B-cD'stallin expression less ~ffected, and expression of the majority of r - and y-crystallin genes significantly downregulated. It is also noted that c-Maf deficiency does not affect expression of other transcription factor genes, such as Soxt, Sox2, Pax6, Eyal, Eya2, or Proxl (Ring et aL, 2000). These results indicate that expression of/3 and y, crysta_llins is largely dependent on the activib" of c-Mar Considering the result of Soxt knock-out mouse, y-cuJstallin genes are dependent on both Sox and c-Maf regulations. Mar proteins heterodimerize with some other bZip-type transcription factors. ATF-4/CREB2 is one such example. The ATF-4 knock-out mouse develop microphthalmia due to apoptosis of the seconda_r5' lens fibers after Et 5 (Tanaka

532

I11Organogenesis

et aL. 1998). This phenomenon can be explained in the context of custaliin regulation by Mafrelated transcription factors. implying that M a f / A T F 4 heterodimers play a role late in lens development. In the chicken (Ogino and Yasuda, 1998), Maf family protein L-Maf ihas been expressed solely in early lens development, in contrast to multiple expression sites of other MaX protein genes (Ogino and Yasuda. !998). In addition, ectopic expression of L-Maf is reported to cause ectopic development of the tens tissue in the head ectoderm and cultured neural retina. The protein nature of L-Maf is similar to c-Maf. and its expression during lens development is almost identical to that of c-Maf. Considering the phenotype of c-Maf knock-out mouse. L-Maf may have a redundant function with c-Maf in the chicken lens. An analysis of ectopic lens development induced by LMar indicated that it is restricted to certain predisposed sites expressing Sox2 (Ogino and Yasuda. 2000). In the head ectoderm, the ectopic lenses arise in a wide stripe on the ventral side of the normal eyes where Sox2 is strongly expressed (Kondoh. 1999). The neural retina expressing Sox2 and transdifferentiating into lens by itself (Okada et al., 1979) is the one that responds to exogenous L-Mar. These observations and those described above areue for the model that the combination of a Mar Sox2. and Pax6 is a minimal requirement for lens development.

E. Maintenance of the Lens Epithelial Cell State Once a lens is made. Pax6 is expressed more stongly in the epithelial cell layer than in lens fibers (Kondoh. 1999). Although the certain crvstallin genes in tens epithelial cells can be activated by Pax6 (Sharon-Friling et al., 1998). fiberspecific/?-cwstallin genes are repressed by Pax6. This may account for the onset of/?-crystallin expression in lens fibers as following the derepression by Pax6 in the epithelial cells (Duncan et aL, 1998). Thus. Pax6 may function as a maintenance factor of the lens epithelium. A forkhead transcription factor gene Foxe3 is expressed in the lens epithelium and is mutated in dysgenetic lens (dyt) mouse mutant (Blixt et al., 2000). The mutations in the DNA-binding domain result in failure of the lens vesicle closure and in premature differentiation/apoptosis of the lens epithelial cells. FoxE3 thus represents another case of transcription factors maintaining the state of lens epithelium. In Xenopus, a forkhead gene Lens 1 related to FoxE1 seems to play an analogous role (Kenyon et al., 1999).

F. Genetic Requirements for Lens Fiber Development ProxI expression starts from the lens placode stage, and continues into later development with a preferential expression site at the transition zone from the lens epithelium to the

secondm,.,. .fibers, called the bow region (Oliver et aL, I9 9 o* ~ . . . Tomarev et aL, 1996). Lens development in the P r o x l nult mouse (Wigie et aL, 1999) is arrested at the vesicle stage more severely than the cases of Soxi or c-MafnulI lenses. In spite of this. expression of cwstallins is observed in the late vesicle stage, indicating that the defect of Prox] mutant lies not in the co~'staltin regulation but in the transition from the epithelium to the fiber state. In support of this, the posterior side of the mutant vesicle continues DNA synthesis without expression of Cdk inhibitors Cdknlb (p27 K.~p')and Cdkntc (p57K~p2), which normally occurs to arrest DNA synthesis in the fiber compartment (Zhang et aL, 1998); the expression of E-cadherin. normally restricted to the anterior epithelial compartment, persists to the posterior compartment (Wigte er al., 1999). Considering that P r o x l expression is prominent in the transition zone. it is conceivable that Proxl regulates the expression of a reception system to signals derived from the optic cup such as fibroblast growth factors (FGFs). Arrest of lens development at the lens vesicle stage has been found in aphakia mutant mice (Varnum and Stevens, 1968). Gli3 (Xt) mutants display analogous arrest of lens development at the vesicle stage (Franz and Besecke, 1991 ). In chimera mice derived from normal and aphakia embryos, aphakia cells are excluded from the lens, indicating cellautonomous defect of the aphakia homozygous cells (Liegeois et aL. 1996). Genetic mapping of the mutation revealed that it has a deletion in the 5' regulato W region of the Pitx3 gene causing a serious reduction of Pitx3 expression in the lens (Semina et aL, 2000). Thus, homedomain protein PITX3 is now registered as an essential transcription factor for lens development. In the case of Gti3, homozygotes display not only the lens defect but the abnormal retina development as shown in Fig. 3. Therefore, it is possible that the arrest of lens development in GIi3 homozygotes is secondary" to the defect of the retina.

G. Extracelluiar Signals in Lens Induction and Lens Development Little is known about the inductive signals for lens differentiation. BMP family proteins, though they themselves are not direct inducers of the lens, are shown to be involved in the lens induction process. BMP4 null embryos die when optic vesicle makes contact with the head ectoderm, but organ culture allows analysis of the following steps of eye development. It was found that Pax6 is expressed in the ectoderm, but Sox2 is not induced after the vesicle-ectoderm contact, resulting in the failure of lens induction. This defect is rescued by exogenous BMP4 (Furuta and Hogan, 1998). BMP7 null mutant embr?'os display an eye defect similar to Pax6 homozygotes: an initially expanded optic vesicle and absence of lens placode development, although not in full penetrance. A key to understanding this phenotype is the finding that Pax6 expression is impaired in the head ectoderm of the BMP7 mutant (Wawersik et at., 1999). These

533

2 t Development of the Eye BMP mutants suggest a genetic interaction between Pax6 and Sox2, which are essential elements of the initiation of lens development. FGFs are likely key players for induction of lens fiber differentiation. Strong support for this is the observation that a low level of FGF1/2 stimulated cell growth but a high level of the same growth factors induced fiber differentiation in lens epithelium explant placed in culture (McAvoy and Chamberlain, !989). Using transgenic mouse, various FGF proteins were overexpressed in the lens and found to be effective in inducing lens fiber differentiation in the epithelium compa~qment (Lovicu and Overbeek, t998). However, the expression pattern is compatible with regulation of tens development only for FGF1 and FGF2. These or uncharacterized new members of FGFs are believed to regulate fiber differentiation possibly through FGF receptor 2 (Lovicu and Overbeek, t 998). Other growth factors such as insulin-like growth factors (IGFs) (Beebe et al., 1987) and platelet-derived growth factors (PDGFs) (Brewitt and Clark, 1988) are also implicated in the lens development, but their precise roles remain to be defined. Experimental disturbance of these growth factors during eye development causes cataracts in a broad sense. Cataract is an opacity developed in the lens. The transparency of the normal lens cells is maintained by the monophasic state of the highly concentrated crystalIin and other lens proteins in the cytoplasm, which may be disturbed by aging process, imbalance of cu'staltins as exemplified by c~-cu'staIlin knockout mice (Brady et aI., 1997), or failure to regulate the ion composition. Association of the congenital cataracts with the latter two causes are often noted (Francis et at., 1999). Note that experimental cataract based on growth factor disturbance includes other cases, such as cell death in the lens and delamination of the epithelium.

H. Multiple Pathways of Lens Development Induction of the head ectoderm by the optic vesicle is not the only possible pathway of lens differentiation. There are three major instances where lenses arise from tissues distinct from the embryonic ectoderm. First, in certain amphibian and fish species, lens regenerates from dorsal iris or outer cornea after lens injury (Bosco, 1986; Eguchi, 1988; Yamada and McDevitt, 1984). In recent molecular analyses, it was demonstrated that the transcription factor genes employed in normal lens development are activated in these regeneration processes (Mizuno et aL, t999: Schaefer et aL, 1999). Second, neural retina of avian emb~'os readily "transdifferentiates" into lens cells under appropriate culture conditions (Okada et aI., 1979). It is speculated that sharing of a number of transcription factors between the lens and neural retina (e.g., PAX6, SOX2, SIX3) is the basis for this transition of the celt state (Kamachi et aL, t 998). The third and most intriguing instance is the recent discover5, that lens cells differ-

entiate from the primordium of the adenohypophysis, the epithelium of Rathke's pouch, in mutant animals where the Shah signal is impeded (Kondoh et aL, 2000). Examples are yot zebrafish mutants where dominant-negative GLI2 apparently interferes with all Gli activities (Kartstrom et al., t 999) and taIpi& cNcken mutant (Ede and Kelly, 1964) where the cells show no response to Shh (Lewis et aL, I999). It is known that adenohypophysis primordium expresses a number of transcription factors found in the tens, and it is speculated that Shh signaling through GLI proteins inhibits the potential of lens differentiation possessed by the adenohypophysis pfimordium (Kondoh et aL, 2000). Potentially, multiple developmental pathways give rise to lens differentiation. In all of these cases, expression of a set of transcription factors necessary' for lens development, either by de novo activation or by c ~ o v e r from a previous ceil state, seems to be the basis of lens differentiation through normal or "transdifferentiation" pathways (Kondoh et aL, 2000). The mechanism of normal lens induction dependent on the activity of retina primordium is thus the mechanism used to achieve precise positioning of the lens tissue in front of the retina.

IV. Conservation and Divergence of the Transcriptional Regulatory Systems in Eye Development The compound eyes of insects and the single eyes of vertebrates have long been viewed as a typical example of convergent evoIution where tissues of completely different origins exhibit the same function. However, the demonstration of Pax6 function in eye development across the animal kingdom indicates that compound eyes and simple eyes share some aspect of evolutionary origin (Gehring and Ikeo, t 999).

A. Pax6 and Eya/SixlDach Pathway Sey (small eye) mutants of the mouse have a defect in Pax6 function. In Drosophila, the ey (eyeless) gene also codes for a transcription factor with paired box and homeobox similar to Pax6, and its genetic defects result in apoptotic Ioss of the eye imaginal disks and in the absence of the eye tissues in adults: compound eyes as well as single eyes. Not only did the wild-type ~' transgene suppress the mutation, but its ectopic expression initiated ectopic development of the compound eyes in a few sites (Halder et al., I995), for example, antennae and the medial portion of the wings, which are correlated with the site of dpp expression (Chen et al., 1999). Furthermore, expression of mouse Pax6 in Drosophila using the same expression system as ey transgene similarly rescued the ey mutant phenotype or induced ectopic eye development, demonstrating the conservation of a Pax6-dependent mechanism of eye development (Halder

534

III O r g a n o g e n e s i s

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Figure 6 Regulatoo genes for eye development in Drosophila. and their homologs in vertebrates. Genetic interactions in eye development in Drosophila are indicated in the left panel, and expression of corresponding genes in mouseeyes are indicated in the fight panels. et al., 1995). Later analysis revealed that Drosophila has another Pax6-related transcription factor gene toy (twin o f eyeless) that is much closer to Pax6 and acts upstream of ey (Czerny et al., 1999). Evidence indicates that mouse Pax6 expressed in Drosophila mimics to)" rather than ey (Czerny et al., 1999). but the initial observation that Pax6 functions in the Drosophila eye system is significant. Production of ectopic eyes in Drosophila is not. however, the unique property of Pax6/ey gene activity. It has also been demonstrated that so (sin oculis) and eya (eyes absent) together (Pignoni et aI., 1997), or dac (dachshund) and eya together (Chen et aI., 1997) promote ectopic eye development after forced expression of these genes, in tissues more widely distributed than in the case of ey expression. So, Eya and Dac are thought to constitute a ternary complex in which the homeodomain protein So provides the DNAbinding component (Chen et al., 1997 Pignoni et al., 1997). This complex has a feedback loop with ey, where ey activates so and eya, and conversely eya and so activate ey. Toy is upstream of this feedback loop (Fig. 6). Therefore. any genetic manipulations that activate this regulato~ loop would result in ectopic eye development in the tissue areas where other conditions such as dpp expression (Chen et al., 1999) are met. In the mouse there are homologs of eya (Eyal to Eya3), so (SixI to SLr6), and dac (DachI and Dach2) identified. Lens placode expresses Eyal and Six3, while optic vesicle

expresses EyaI, Eya2, Eya3, Six3, and D a c h l (Hammond et al., 1998; Oliver et at., 1995; Xu et at., 1997). In the lens placode, Six3 expression is dependent on Pax6 (AsheryPadan et al., 2000). However, in the retina, which is considered to be analogous to Drosophila eye, expression of Eya, S/x, and Dach does not appear to be coordinated: ~ ' a t expression is dependent on Pax6 (Xu et at., I997), but that of Six3 or Dachl is not (Hammond et aI., 1998; Oliver et aL, 1995). Eyal, the major Eya in the eye, is dispensable for eye development in the mutant mouse (Xu et aI., 1999).

B. Ectopic Eye Development Inspired by the ectopic eye development induced by ey or Pax6 in Drosophila (Halder et el., 1995), several analogous attempts have been made in vertebrates. Overexpression of mouse Six3 in Medaka embryo resulted in infrequent lens development in otic vesicles (Oliver et el., 1996), which is partly ascribed to abundant expression of Sox2 in the otic vesicle (Kondoh, 1999). In Xenopus, it is reported that injection of RNA coding for FLAG-tagged PAX6 into cleavage stage embryos can induce expression of fib 1-crystallin in cell aggregates associated with a rostral ectoderm (Altmann et al., 1997). The same ~ o u p also reported that overexpression of Pax6 RNA could induce ectopic retina development with a sharp dependence on the amount of injected RNA (Chow et al., 1999), and the ectopic retinas are occasionally

21 Development of the Eye associated with ectopic lenses. As discussed before, overexpression of Six3 in Medaka (Loosli et aL, 1999) and Xenopus (Bemier et aI., 2000) resulted in ectopic retina development particularly in the midbrain tissue. Six6/Optx2(Six9)exhibits an analogous but more dramatic effect of ectopic retina induction (Bemier et at., 2000; Zuber et aL, I999). In this last case, ectopic retina is often associated with the lenses. All of these observations suggest that using Xenopus (and Medaka) emb~'os, overexpression of Pax6/Six3/Six6 can activate the endogenous program of retina development, presumably because embryo tissues of these animals are highly sensitive to this sort of genetic perturbation. It is remarkable that even in Xenopus embryos, ectopic retina/tens tissues develop only in the head region. It is the combined action of multiple transcription factors that regulates development of complex organs such as eye.

References Acampora, D.. Mazan, S., Laltemand, Y., AvantagNato, V., MauD', M., Simeone. A.. and Brulet. R (1995). Forebrain and midbrain regions are deleted in O t x 2 - / - mutants due to a defective anterior neuroectoderm specification during gastrulation. Development (Cambridge, UK) 121, 3279-3290. Ahmad. I.. Tang, L.. and Pham, H. (2000). Identification of neural progenitors in the adult mammalian eye. Biochem. Biophys. Res. Commun. 270, 517-521. Altmann, C. R.. Chow, R. L., Lang, R. A., and Hemmati-Brivanlou, A. (1997). Lens induction by Pax-6 in Xenopus laevis. Dev. Biol. 185, I19!23. Ang. S. L.. Jim O.. Rhinn, M.. Daigle, N., Stevenson, L., and Rossant, J. (1996). A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development (Cambridge, UK) 122, 243-252. Asheo'-Padan, R.. Marquardt, T., Zhou, X., and Gross, R (2000). Pax6 activib' in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes Dev. 14, 2701-2711. Barbieri, A. M., Lupo, G.. Bul%ne. A., Andreazzoli, M., Mariani, M.. Fougerousse, E, Consalez, G. G., Borsani, G., Beckmann, J. S., Barsacchi. G.. Ballabio, A., and Banff, S. (1999). A homeobox gene, vax2, controls the patterning of the eye dorsoventraI axis. Proc. NatL Acad. Sci. U.S.A. 96, 10729-10734. Beebe, D. C., Silver. M. H., Blecker. K. S., van Wyk, J. J., Svoboda, M. E.. and Zelenka, R S. (1987). Lentropin, a protein that controls lens fiber formation, is related functionally mad immunologicalIy to the insuIinlike growth factors. Proc. Natl. Acad. Sci. U.S.A. 84, 2327-2330. Bemier. G., Panitz. E. Zhou, X.. Holtemann, T, Gruss. R, and Pieler. T. (2000). Expanded retina temtory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus emb~'os. Mech. Dev. 93, 59-69. Blixt. A., Mahlapuu. M., Aitolm M., Pelto-Huikko, M., Eneft)ack, S., and Carlsson. R (2000). A fork_headgene, FoxE3, is essential for lens epithelial proliferation and closure of the lens vesicle. Genes Dev. 14, 245254. Bosco, L. (I 986). Transdifferentiation of ocular tissues in tarvaI Xenopus laevis. Differentiation (Berlin) 39, 4-I5. Brady, J. R. Garland, D.. Duglas-Tabor. Y., Robison, W. G., Jr., Groome. A.. and Wawrousek, E. E (I997). Targeted disruption of the mouse alpha A-cry'stNlin gene induces cataract and cytoplasmic inclusion bodies conmining the small heat shock protein alpha B-co~staIlin. Proc. Natl. Acad. Sci. U S.A. 94, 884- 889.

535 Brewitt, B., and Clark, J. L (I 988). Growth and transparency in the lens, an epithelial tissue, stimulated by' puIses of PDGF. Science 242, 777-779. Brown, N. L....Kanekar, S., Vetter, M. L., Tucker, R K., Gemzm D. L., and Glaser, T. (1998). Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis. Development (Cambridge, UK) 125, 4821-4833. Burmeister, M., Novak, J., Liang, M. Y, Basu, S., Ploder, L.; Hawes, N. L., Vidgen, D.. Hoover, R, Goldman, D., KaInins, V. I., Rodefick, T. H., Taylor, B. A., Hankdn, M. H., and Mc!nnes, R. R. (1996). Ocular retardation mouse caused by Chxl0 homeobox null allele: Impaired retinal progenitor proliferation and bipolar cell differentiation~ Nat. Genet. 12, 376-384. Chen, R., Amoui, M., Zhang, Z., and Mardon, G. (1997). Dachshund and eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila. Ceil (Cambridge, Mass.) 91, 893-903. Chen, R., Halder, G., Zhang, Z., and Mardon, G. (1999). Signaling by the TGF-beta homotog decapentaplegic functions reiteratively within the network of genes controlling retinal celt fate determination in Drosophila. Development (Cambridge. UK) 126, 935-943. Chiang, C., Litingtung, Y., Lee, E., "Young, K. E., Corden, J. L., Westphal, H., and Beachy, R A. (t996). Cyclopia and defective axial patterning in mice lackdng Sonic hedgehog gene function. Nature (London) 383, 407-413. Chow, R. L., Altmann, C. R., Lang, R. A., and Hemmati-Brivanlou, A. (1999). Paa~6 induces ectopic eyes in a vertebrate. Development (Cambridge. UK) 126, 42 t 3-4222. Coulombre, A. J., and Coutombre, L L (t964). Lens development, t. Rote of the lens in eye ~owth. J. Exp. ZooL 156, 39-47. Czemy, T., Halder. G., Kloter, U., Souabni, A., Geba-ing, W. L, and Busslinger, M. (I 999). ~'in of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. MoL Cell 3, 297-307. Drescher, U., Bonhoeffer, E, and Muller, B. K. (1997). The Eph family in retinal axon ~maidance. Curr. Opin. NeurobioL 7, 75-80. Duncan, M. K., Haynes, J. I., 2nd, Cvekl, A., and Natigorsky, J. (1998). DuN roles for Pax-6: A transcriptional repressor of lens fiber ceIIspecific beta-crystallin genes. Mol. Ceil. Biol. 18, 5579-5586. Ede, D. A., and Kelly', Vv\ A. (1964). I)evetopmentaI abnormalities in the head region of the talpid s mutant of the fowl. J. Embry,oL Exp. MorphoL 12, 161 - 182. Eguchi, G. (t 988). Cellular and molecular back~ound of Wo!ffian lens regeneration. Cell Differ Dev. 25(Suppl.), t47-158. Epstein, J. A., Gtaser.. T., Cai, J., Jepeal, L., Walton, D. S., and Maas, R. L. (1994). Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev. 8, 2022-2034. Favor, J., Sandulache, R., Neuhauser-Klaus, A., Pretsch, W., Chatterjee, B., Senti, E., Wurst, W:, Blanquet, V., Grimes, R, Sporle, R., and Schughart, K. (1996). The :mouse Pax2(tNeu) mutation is identical to a human PAX2 mutation in a family with renal-cotoboma syndrome and results in developmental defects of the brain, ear. eye, and Ndney. Proc. Nail Acad. Sci. [ZS.A. 93, 13870-13875. Fekete, D. M., Perez-MiTaetsanz, J., Ryder, E. E, and Cepko, C. L. (1994). CtonaI analysis :in the chicken retina reveals tangential dispersion of clonally related cells. Dev. Biol. 166, 666-682. Francis, R L, Berry, V., Moore, A. T., and Bhattacharym S. (1999). Lens biology: Development and human cataractogenesis. Trevxls Genet. 15, t91-I96. Franz. T.. and Besecke, A. (1991). The development of the eye in homozygotes of the mouse mutant Extra-toes. Anat. EmbD'oL 184, 355361. Fujii, H., Sam. T., Kaneko, S., Gotoh, O., Fujii-Kuriyama, Y., Osawa, K., Kato, S., and Hamada, H. (1997). Metabolic inactivation of retinNc acid by a novel P450 differentially expressed in developing mouse embryos. E;vIBO J. 16, 4163-4173.

536 F@wara, M.. Uchida. T., OsumS-Yamashita. N., and Eto, K. (t994). Uchida rat (rSey): A new mutant rat with craniofacial abnormalities resembling those of the mouse Sey mutant. Differentiation (.Berlin) 57, 31-38. Furukawa. T.. Kozak. C. A.. and Cepko, C. L. (I997a). Rax. a novel pairedtype homeobox gene. shows expression in the anterior neural fold and developing retina. Proc. Natl. Acad. Sci. L~S.A. 94. 3088-3093. Fumkawa. T.. Morrow. E. M.. and Cepko. C. L. (I 997b). Crx. a novel otxlike homeobox gene. shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell (Cambridge. Mass..) 91, 531541. Fumkawa. T., Morrow, E. M.. Li. T.. Davis. E C., mad Cepko. C. L. (1999). Retinopathy and attenuated circadian entrainment in Crx-deficient mice. Nat. Genet. 23, 466-470. Furuta. Y.. and Hogan. B. (1998). BMP4 is essential t'or lens induction in the mouse emb~,o. Genes Dev. 12, 3764-3775. Gallardo. M. E.. Lopez-Rios. J.. Femaud-Espinosa. I.. Granadino. B.. Sanz. R.. Ramos. C.. Ayuso, C.. Seller. M. J., Brunner. H. G.. Bovolenta. R. and Rodriguez de Cordoba. S. (1999). Genomic cloning and characterization of the human homeobox gene SIX6 reveals a cluster of SIX genes in chromosome 14 and associates SIX6 hemizygosity with bilateral anophthalmia and pituitary anomalies. Genomics 61, 82-91. Gan. L.. Xiang. M.. Zhou. L.. Wagner. D. S.. Klein. W. H., and Nathans. J. (1996). POU domain factor Bm-3b is required for the development of a large set of retinal ganglion cells. Proc. Natl. Acad. Sci. U.S.A. 93, 3920 -3925. Gan. L.. Wang, S. "~. Huang. Z.. and Klein, W. H. (1999). POU domain factor Brn-3b is essential for retinal ganglion cell differentiation and survival but not for initial celt fate specification. Dev. Biol. 210, 469-480. Gehring. ~,x,.,j.. and Ikeo. K. (1999). Pax 6: Mastering eye morphogenesis and eve evolution. Trends Genet. 15, 371-377. Goring. D. R.. Breitman. M. L., and Tsui. L. C. (1992). Temporal regulation of six c~.'stailin transcripts during mouse lens development. Exp. Eve Res. 54, 785-795. GradwohI. G., Fode. C.. and Guillemot. F. (11996). Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors. Dev. BioL 180, 227-241. Grindley. J. C.. Davidson. D. R.. and Hill. R. E. (1995). The role of Pax6 in eye and nasal development. Development (Cambridge, UK) 121, 1433-1442. Grun, E. Hirose. Y., Kawauchi. S.. Ogura, T.. and Umesono. K. (2000). ALDH6, a cytosolic retinaldehyde dehydrogenase prominently expressed in sensor5; neuroepithelia during development. J. Biol. Chem. 275, 41210-41218. Halder. G.. Callaerts. R. and Gehring. ~,~,':J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267, 1788- ! 792. Hallonet. M.. Hollemann. T.. Pieler. T.. and Gruss. R (1999). Vaxl. a novel homeobox-containing gene. directs development of the basal forebrain and visual system. Genes Dev. 13, 3106-3i 14. Hammond. K. L.. Hanson. I. M.. Brown. A. G.. Lettice. L. A.. and Hill. R. E. (11998). Mammalian and Drosophila dachshund genes are related to the Ski proto-oncogene and are expressed in eye and limb. Mech. Dev. 74.. 1"~ t .1-13 ~ Hatini. \(. Tao. W.. and Lai. E. (1994). Expression of winged helix genes, BF- 1 and BF-2. define adjacent domains within the developing forebrain and retina. J. Neurobiol. ~ , 1293 -1309. Hatta. K.. Takagi. S.. Fujisawa, H., and Takeichi. M. (1987). Spatial and temporal expression pattern of N-cadherin cell adhesion molecules correlated with morphogenetic processes of chicken emb~;os. Dev. Biol. 120,21" 9"~" Hayashi. S.. Goto. K.. Okada. T. S.. and Kondoh. H. (1987). Lens-specific enhancer in the third intron regulates expression of the chicken delta 1custallin gene. Genes Dev. 1.818-828. Hen~,'. J. J.. and Grainger. R. M. (1987). Inductive interactions in the spatial and temporal restriction of lens-forming potential in emb~'onic ectoderrn of Xenopus laevis. Dev. Biol. 12A, 200-214.

I11 Organogenesis Hill, R. E.. Favor. J.. Hogan. B. L., Ton, C. C., Saunders, G. E, Hanson, I. M.. Prosser. J., Jordan. T., Hastie. N. D., and ~ n Heyningen, V. (I 991 ). Mouse small eye results from mutations in a paired-like horneoboxcontaining gene. Nature (London) 354, 522-525: erratum: 355, 750 (I992). Hodgkinson. C. A.. Moore. K. J.. Nakayama, A.. Steingrimsson. E.. Copeland, N. G.. Jenkins. N. A.. and Amheiter. H. (1993). Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell (Cambridge, Mass..) 74, 395-404. Hogan. B. L., Horsburgh. G., Cohen, J., Hetherington, C. M., Fisher, G., and Lyon, M. E (1986). Smatl eyes (Sey): A homozygous lethal mutation on chromosome 2 which affects the differentiation of both tens and nasal placodes in the mouse. 3". Embo'ol. Exp. MorphoI. 97, 9 5 - ! I0. Huh. S.. Hatini. V., Marcus. R. C., Li. S. C., and Lai, E. (I999). Dorsalventral patterning defects in the eye of BF-l-deficient mice associated with a restricted toss of shh expression. Dev. Biol. 211, 53-63. Jean. D., Bernier, G.. and Gruss. R (1999). Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitar34 hypothalamic axis and the ventral optic stalk. Mech. Dev. 84, 31-40. Kamachi. Y.. and Kondoh. H. (1993). Overlapping positive and negative regulator3, elements determine lens-specific activi~" of the delta Ic~'stallin enhancer. Mot. Cell Biol. 13, 5206-5215. Kamachi. Y., Sockanathan, S.. Liu, Q., Breitman. M., Lovell-Badge. R., and Kondoh. H. (1995). Involvement of SOX proteins in lens-specific activation of crT,stalIin genes. EMBO J. 14, 3510-3519. Karnachi. Y., Uchikawa. M., Collignon, J.. LovelI-Badge, R.. and Kondoh, H. (1998). Involvement of Soxl, 2 and 3 in the early mad subsequent molecular events of lens induction. Development (Cambridge, UK) 125, 2521-2532. Kamachi. Y., Cheah, K. S.. and Kondoh, H. (I999). Mechanism of regulator: -target selection by the SOX high-mobili~'-group domain proteins as revealed by comparison of SOXI/2/3 and SOX9. Mol. Cell Biol. 19, 107-120. Kamachi. Y.. Uchikawa, M.. Tanouchi. A., Sekido, R.. and Kondoh, H. (2001). Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development. Genes Dev. 15, 1272-1286. Karlstrom. R. O.. Talbot, W. S., and Schier, A. E (1999). Comparative synteny cloning of zebrafish you-too: Mutations in the Hedgehog target gli2 affect ventral forebrain patterning. Genes Dev. 13, 388-393. Kastner. R. Mark, M., Ghyselinck. N.. Kxezel, W., Dupe, ~V:,Grondona, J. M.. and Chambon. P. (1997). Genetic evidence that the retinoid signaI is transduced by heterodimeric RXR/RAR functional units during mouse development. Development (Cambridge, UK) 124, 313-326. Kaufman. M. H. (1992). "'The Atlas of Mouse Development." Academic Press. London. Kawauchi. S.. Takahashi. S.. Nakajima. O.. Ogino. H.. Morita, M., Nishizawa. M.. Yasuda. K.. and Ya_mamoto. M. (1999). Regulation of lens fiber cell differentiation by transcription factor c-Maf..,1. BioL Chem. 274, 19254-19260. Kenyon, K. L.. Moody. S. A., and Jarm'ich, M. (1999). A novel forkhead gene mediates early steps during Xenopus lens formation. Development (Cambridge. UK) 126, 5107-5 t 16. Kim. J. I., Li, T., Ho, I. C., Grusby, M. J., and Glimcher, L. H. (I999). Requirement for the c-Maf transcription factor in c~,stallin gene regulation and lens development. Proc. NatL Acad. ScL ~:S.A. 96, 37813785. Kondoh, H. (1999). Transcription factors for lens development assessed in vivo. Curr. Opin. Genet. Dev. 9, 301-308. Kondoh. H.. Araki. I.. Yasuda, K., Matsubasa, T., and Mori, M. (t99t). Expression of the chicken "delta 2-crystatlin" gene in mouse cells: Evidence for encoding of argininosuccinate lyase. Gene 99, 267-27 I. Kondoh. H.. Uchkawa. M.. Yoda. H., Takedm H., Furutani-Seiki, M., and Karlstrom, R. O. (2000). Mutations in Gli mediated hedgehog signaling lead to lens transdifferentiation from the adenohypophysis anlage. Mech. Dev. 96, 165-I74.

21 Development of the Eye Koshiba-Takeuchi, K., Takeuchi, J. K.. Matsumoto. K., Momose, T., Uno. K.. Hoepker, V.. Ogura, K., Ta_kahashi. N.. Nakamura, H.. Yasuda, K.. and Ogura. T. (2000). Tbx5 and ~e retinotectum projection. Science 287, 134-137. Lewis. K. E.. Drossopoulou, G.. Paton, t. R.. MorTice, D. R., Robertson, K. E.. Burr, D. V~(, Ingham. R W., and Tickle, C. (1999). Expression of ptc and gli genes in talpid3 suggests bifurcation in Shh pathway. Development (Cambridge, UK) 126, 2397-2407. Lewis. VV: H. (I904). Experimental studies on the development of the eye in amphibia. I. On the origin of the lens. Rana patustris. Am. J. Anat. 3, 503-536. Li. H., Tierney. C.. Wen. L.. Wu, J. Y.. and Rao, Y. (1997). A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate. Developmenz (Cambridge, UK) I~M, 603-615. Li. H. S.. Yang, J. M.. Jacobson. R. D., Pasko. D., and Sundin, O. (1994). Pax-6 is first expressed in a region of ectoderm anterior to the early neuraI plate: Implications for stepwise determination of the lens. De~ Biol. 162, i81-194. Liegeois. N. J., Homer, J. W., and DePinho, R. A. (! 996). Lens comptementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo. Proc. Natl. Acad Sci. ~LS.A. 93, 1303-I307. Liu. Q. R.. Tini. M., Tsui. L. C.. and Breitman. M. L. (199i ). Interaction of a lens ceil transcription factor with the proximal domain of the mouse gamma F-c~'stallin promoter. Mol. Cell Biol. 11, 153i-1537. Liu, Q. R.. Ji. X.. Breitman, M. L.. Hitchcock, R F., and Swaroop, A. (1996). Expression of the bZIP transcription factor gene Nrl in the developing nervous system. Oncogene 12, 207-2I I. Loosli, F.. Winkler, S.. and Wittbrodt, J. (I 999). Six3 overexpression initiates the formation of ectopic retina. Genes Dev. 13, 649-654. Lopez-Rios. J., Gatlardo. M. E.. Rodriguez de Cordoba, S., and BovoIenta, R (I999). Six9 (OpLx2), a new member of the six gene family of transcription factors, is expres~d at early stages of vertebrate ocular and pituitary development. Mech. De,,'. 83, 155-I59. Lovicu. E J.. and Overbeek, R A. (t998). Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice. Development (Cambridge, UK) 125, 3365-3377. Macdonald, R., Barth, K. A.. Xu, Q., Holder. N.. Mikkola. I.. and Wilson. S. W. (1995). Midline signalling is required for Pax gene regulation and patterning of the eyes. Development (Cambridge, UK) 121, 3267-3278. Marquardt, T., Ashe~'-Padan, R.. Andrejewski, N., Scardigli. R., Guillemot. F., and Gruss. R (2001). Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105, 43-55. Marsh-Armstrong, N.. McCaffe~;. R, Gilbert, W., Dowting, J. E., and Drager. U. C. (I994). Retinoic acid is necessary for development of the ventral retina in zebrafish. Proc. Natl. Acad. Sci. L(S.A. 91, 72867290. Mathers, R H.. Grinberg, A.. Mahon. K. A., and Jamrich. M. (1997). The Rx homeobox gene is es~ntial for vertebrate eye development. Nature (London) 387, 603-607. Matsuo. I.. Kuratani. S., Kimura. C., Takeda, N., and Aizawa. S. (19951). Mouse Otx2 functions in the formation and patterning of rosLral head. Genes Dev. 9, 2646-2658. McAvoy, J. ~ , and ChamberIain. C. G. (1989). FibrobIast ~owth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development (Cambridge, UK) 1(!7, 22 i-228. McCaffery. R. Lee, M. O.. Wagner, M. A., Sladek. N. E., and Drager, U. C. (1992). Asymmetrical retinoic acid synthesis in the dorsoventraI axis of the retina. Development (Cambridge, UK) 115, 371-382. McCaffe~', R, Wagner, E., O'NeiI, J., Petkovich, M., and Drager, U. C. (1999). Dorsal and ventral rentinoic territories defined by retinoic acid synthesis, break-down and nuclear receptor expression. Mech. Dev. 85, 203-2t4. Mic. E A., Molotkov. A.. Fan. X.. Cuenca, A. E., and Duester. G. (2000). RALDH3. a retinaldehyde dehydrogenase that generates retinoic acid. is expressed in the ventral retina, otic vesicle and olfacto~' pit during mouse development. Mech. Dev. 97, 227-230.

537 Mizuno, M.. Takabatake, T., Tak~ashi, T. C.. and Takeshima. K. (1997). Pax-6 gene expression in newt eye development. De~: Genes EvoL 207, 167-176. Mizuno, N.. M ocNi, M., Takagi, C., Takahashi, T. C., Eguchi, G., and Okada, T. S. (1998). A critical role for the optic vesicle in lens develop, merit: a reinvestigation of free lens formation in Cynops pyrrhogaste~ Differentiation (Berlin) 63, 247-252. Mizuno, N., Mochii, M., Yamamoto, T. S., Takahashi, T. C., Eguchi, G., and Okada, T. S. (11999). Pax-6 and Prox I expression during tens regeneration from Cynops iris and Xenopus cornea: Evidence for a genetic prou a m common to emb~'onic development. DL~erentiation (Berlin) 65, 141-149. Mochii, M., Ono, T.. Matsubara, Y., and Eguchi, G. (1998). Spontaneous transdifferentiation of quail pigmented epithelial ceil is accompanied by a mutation in the Mitf gene. Dev. Biol. 196, t 45-159. Morgenbesser, S. D., Williams, B. O., Jacks. T., and DePinho, R. A. ( t 994). p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature (London) 371, 72-74. Morrow, E. M., Furukawa, T., Lee, J. E., and Cepla, C. L. (1999). NeuroD regulates multiple functions in the developing neural retina in rodent. Deveiop~nt 126, 23-36. Nishiguchi, S., Wood, H., Kondoh, H., Lovell-Badge, R., and Episkopou, V. (t998). SoxI directly regulates the garnma-cc~stallin genes and is essential t:or lens development in mice. Genes Dean 12, 776-78 I. Ogino, H.. and Yasuda, K. (1998). Induction of lens differentiation by activation of a bZIP transcription factor, L-Maf. Science 280, 115tt8. Ogino, H., and Yasuda. K. (2000). Sequential activation of transcription factors in tens induction. Dew Growt,h Differ. 42, 437-448. Ohsaki, K., Mofimitsu, T., Ishida, Y., Kominarni, R., and T~kahashi, N. (I999). Expression of the Vax family homeobox genes suggests multiple roles in eye development. Genes Cells 4, 267-276. Okada, T. S., Yasuda. K., Araki. M.. and Eguchi, G. (1979). Possible demonstration of multipotential nature of emb~onic neural retina by clonal cell culture. Dev. Biol. 68, 600-6 I7. O'Leary, D. D.. and Wilkinson, D. G. (1999). Eph receptors and ephrins in neural development. Curt. Opin. NeurobioI. 9, 65-73. Oliver, G., Sosa-Pineda. B., Geisendoff, S., Spana, E. R, Doe, C. Q., and Grass, R (1993). Prox 1, a prospero-related homeobox gene expressed during mouse development. Mech. Dev. 44, 3-16. Oliver, G., Mailhos, A.. Wehr. R., Copeland, N. G., Jenkins. N. A., and Gruss, R (1995). Six3, a routine homolog of sine ocuIis gene demarcates the most .anterior border of the developing neural plate and is expressed during eye development. Development (Cambridge, UK) 121, 40454055. Oliver, G., Loosli, E, Koster, R.. Wittbrodt, J., and Gruss, R (1996). Ectopic lens induction 9in fish in response to the murine homeobox gene Six3. Mech. Dev. 60, 233-239. Pei, Y. E, and Rhodin, J. A. (1970). The prenatal development of the mouse eye. Anat. Rec. 168, i 05 - 125. Perron, M., Kxnekar. S, Vetter, M. L., and Harris, Vv\ A. (1998). The genetic sequence of retinal development in the ciliary margin of the Xenopus eye. Dev. Biol. 199, 185-200. Pevny, L. H., Sockanathan, S., Placzek, M., and Lovelt-Badge, R. (1998). A rote for SOXi in neural determination. Development (Cambridge, UK) 125, ! 967-1978. Pignoni, E, Hu. B., Zavitz. K. H., Xiao, J.. Garri~-, R A., and Zipursky, S. L. (I 997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell (Cambridge, Mass.) 91, 881-89t" erratum: 92(4), 585 (t998). Pisano, M. M., and Chepetinsk?~ A. B. (1991). Genomic cloning, complete nucleotide sequence, and structure of the human gene encoding the major intrinsic protein (MIP) of the lens. Genomics I1, 981-990. Porter, F. D., Drago, J., Xu, Y., Cheema, S. S., WassiL C., Huang, S. R, Lee. E., Grinberg, A., Massalas, J. S., Bodine. D., Aft, F., and Westphat, H. ( t 997). Lhx2, a LtM homeobox gene, is required for eye, tbrebrain, and

538 definitive e~'t.ba-ocyte development. Development (Cambridge, ~:7(~124, 2935-2944. Ring, B. Z., Cordes. S. P., Overbeek. P. A.. and Barsh. G. S. (2000). Regulation of mouse lens fiber celt development and differentiation by the Mar gene. Development (Cambridge. UK) 127, 307-3!7. Sandilands, A., Prescott. A. R., Carter. J. M.. Hutcheson. A. M., Quinlan, R. A., Richards. J.. and FitzGerald. P. G. (1995). Vimentin and CP49/ filensin form distinct networks in the lens which are independentiy modulated during lens fibre cel! differentiation. J. Cell Sci. 108, i397i406. Sasaki. H.. Nishizaki. Y.. Hui. C.. Nakafuku. M.. and Kondoh. H. ~1999). Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: Implication of Gli2 and GIi3 as primary" mediators of Shh signaling. Development (Cambridge, UKj 126, 3915-3924. Schaefer. J. J.. Oliver. G.. and Henr~'. J. J. (1999). Conservation of gene expression during embryonic lens formation and cornea-lens transdifferentiation in Xenopus laevis. Dev. D3"n. 215, 308-318. Schedl. A.. Ross. A., Lee. M.. Engelkamp, D.. Rashbass. P., van Hevningen. V.. and Hastie. N. D. (1996). Influence of PAX6 gene dosage on development: Overexpression causes severe eye abnormalities. Cell (Cambridge, Mass.) 86, 71-82. Schorle. H., Meier, R. Buchert. M.. Jaenisch, R.. and Mitchell. R J. (1996). Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature (London.) 381,235-238. Schulte, D.. Furukawa. T.. Peters. M. A., Kozak. C. A., and Cepko. C. L. (1999). Misexpression of the Emx-reiated homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map. Neuron 24, 541-553. Semina. E. V. Murray. J. C.. Reiter. R.. Hrstka, R. E. and Graw. J. (2000). Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice. Hum. Mol. Genet. 9, 1575-1585. Sharon-Filling. R.. Richardson. J.. Sperbeck, S.. Lee. D.. Rauchman, M.. Maa.s.R.. Swaroop. A.. and Wistow, G. (1998). Lens-specific gene recruitment of zeta-crs'stallin through Pax6. Nrl-Maf. and brain suppressor sites. Moi. Ceil Biol. 18, 2067-2076. Sommer, L., Ma, Q., and Anderson, D. J. (1996). neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS. Mol. Celt. Neurosci. 8, 221-24I. Spemann, H. (1901). Ueber Correlationen in der Entwickelung des Auges. Verh. Anat. Ges. 15, 61-79. Steingrimsson, E., Nii, A., Fisher, D. E.. Ferre-D'Amare. A. R., McCormick, R. J.. Russell, L. B.. BurIey. S. K., Ward. J. M.. Jenkins. N. A.. and Copeland. N. G. (1996). The semidominant Mi(b) mutation identifies a role for the HLH domain in DNA binding in addition to its role in protein dimerization. EMBO J. 15, 6280-6289. Suzuki, R., Shintani, "F.. Sakuta. H.. Kato, A.. Ohkawara. T.. Osumi, N.. and Noda. M. (2000). Identification of RALDH-3. a novel retinaldehyde dehydrogenase, expressed in the ventral region of the retina. Mech. Dev.

98, 37-50. Takeichi. M. (199 I). Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, t451-1455. Tanaka. T.. Ysujimum, T.. Takeda. K., Sugihara. A.. Maekawa. A.. Terada. N., Yoshida. N.. and Akira, S. (I998). Targeted disruption of ATF4 discloses its essential rote in the formation of eye lens fibres. Genes Ceils 3, 801-810. Tassabehji, M.. New'ton. "V E.. and Read. A. R (1994). Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nat. Genet. 8, 251-255. Tomarev, S. I.. Sundin. O., Banerjee-Basu. S.. Duncan. M. K.. Yang. J. M.. and Piatigorsky. J. (1996). Chicken homeobox gene Prox I related to Drosophila prospero is expressed in the developing lens and retina. Dev. Dyn. 206, 354-367; erratum: Ibid. 207(1), 120 (1996). Tomita. K.. Ishibashi. M.. Nakahara, K., Ang. S. L., Nakanishi, S., Guillemot, E. and Kageyama, R. (1996). Mammalian hair2.,, and Enhancer of

1110rganogenesis spilt homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron 16, 723-734. Ton, C. C. T., Hirvonen. H., Miwa, H., Well, M. M., Monaghan, R, Jordan, T., van Heyningen, V., Hastie, N. D., Meijers-Heijboer, H., Drechsler, M., Royer-Pokora. B., Collins, E, Swaroop, A., Strong, L. C., and Saunders, G. E (199i). Positional cloning and characterization of a paired boxand bomeobox-containing gene from aniridia region. Celi (Cambridge, Mass.) 67, 1059-I072. Toy, J.. and Sundin. O. H. (I 999). Expression of the optx2 homeobox gene during mouse development. Mech. Din,. 83, 183-186. Tropepe. "v:. Coles. B. L., Chiasson. B. J., Horsford, D. J., Ella, A. J., Mclnnes. R. R.. and van der Kooy. D. (2000). Retina stem cells in the adult mammalian eye. Science 287, 2032-2036. Turner. D. L.. and Cepko, C. L. (I 987). A common progenitor for neurons and glia persists in rat retina late in development. Nature (London) 328, 131-136. Turner, D. L.. Snyder, E. Y.. and Cepko, C. L. (1990). Lineage-independent determination of cell ty'pe in the embwonic mouse retina. Neuron 4, 833-845. Uchikawa. M.. Kamachi, Y., and Kondoh, H. (1999). Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: Their expression during embryonic orga~nogenesis of the chicken. Mech. Dev. 84, 103 - 120. Vamum, D. S., and Stevens, L. C. (i 968). Aphakia. a new mutation in the mouse. J. Hered. 59, 147-150. Walther. C.. and Gruss, R (I 991 ). Pax-6, a murine paired box gene, is expressed in the developing CNS. Development (Cambridge, UK) 113, 1435-I'449. Wawersik. S., Purcell, R, Rauchman, M., Dudley, A. T., Robertson, E. J.. and Maas. R. (1999). BMPT' acts in murine lens placode development. Dev. Biol. 207, 176-188. Wigle. J. T.. Chowdhury, K.. Gruss, R, and Oliver. G. (1999). ProxI function is crucial for mouse lens-fibre elongation. Nat. GeneL 21, 3 1 8 322. Xu, E X., Woo, I.. Her, H., Beier, D. R.. and Maas, R. L. (I997). Mouse Eya homologs of the Drosophila eyes absent gene require Pax6 for expression in lens and nasal placode. Devetopmem (Cambridge. UK) 124, 219-23I. Xu. R X.. Adams. J.. Peters. H., Brown. M. C.. Heaney, S., and Maas, R. L. (1999). Eyal-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 9,3, 1 I3-117. Yamadm T.. and McDevitt, D. S. (1984). Conversion of iris epithelial ceils as model of differentiation control. Differentiation (Berlin) 27, 1-I2. Yoshida. A.. Rzhetsky. A.. Hsu. L. C.. and Chang. C. (1998). Human aldehyde dehydrogenase gene family. Eur. J. Biochern. 251, 549-557. Yoshida. K., Imaki, J., Koyama, Y.. Harada, T., Shinmei, Y., Oishi, C., Matsushima-Hibiya. Y., Matsuda, A., Nishi, S., Matsuda, H., and Sakai, M. (1997). Differential expression of mar-1 and maf-2 genes in the developing rat tens. Invest. Ophthalmol. Visual Sci. 38, 2679-2683. Yuasa, J., Hirano, S., Yamagata, M., and Noda, M. (1996). Visual projection map specified by topographic expression of transcription factors in the retina. Nature (London) 382, 632-635. Zhang. J., Hagopian-Donaldson, S., Serbedzija, G.. Elsemore, J.. PlehnDujowich, D., McMahon, A. R, Flavetl, R. A.. mad Williams, T. (1996). Neural tube, skeletal and body watl defects in mace lacking transcription factor AP-2. Nature (London) 381,238-241. Zhang. R. Wong, C.. DePinho, R. A., Harper, J. W.. and Elledge, S.J. (1998). Cooperation between the Cdk inhibitors p27(KIPl) and p57(tzd_P2) in the control of tissue growth and development. Genes Dev. 12, 3162-3 I67. Zuber. M. E., Perron. M.. Philpott, A., Bang, A.. and Harris, W. A. (19991). Giant eyes in Xenopus laevis by overexpression of XOptx2. CelI (Cambridge, Mass.) 98, 341-352. Zygar. C. A., Cook. T. L., and Grainger, R. M., Jr. (1998). Gene activation during early stages of lens induction in Xenopus. Development (Cambridge, UK) I25, 3509-3519.

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Development of the Mouse Inner Ear A m y E. K i e r n a n , *-I K a r e n P, S t e e l , * a n d D o n n a

M, Feketet

9MRC Institute of Hearing Research, Nottingham NG7 2RD, United ,Kingdom, .Department of Biological Sciences, Purdue UniversiO~ "WestLafm;ette, Indiana 47907

I. Introduction

tunately, we have learned a certain amount about the early development of the ear from studies in other vertebrates, where manipulation is much easier than in the mouse. Some of this work will be reviewed in the first part of this chapter. However, with the advent of molecular techniques in the mouse and other vertebrates, we are now beginning to scratch the surface of how a complex three-dimensional structure such as the inner ear is formed beyond the otic vesicle stage. By using principles learned from simpler structures such as the vertebrate limb and applying them to the ear, we are starting to develop models of how the broad regions of the ear might be patterned. In addition, by drawing parallels from well-studied genetic models such as Drosophila we are also gaining an understanding of how the fine-gained patterning of sensory cells may occu_r. One luclcy feature for unraveling mammalian inner ear development is that mice are particularly sensitive to vestibular defects and will exhibit strange behaviors, such as circling or head-shaking, if a vestibular defect exists. Thus, even genes that are not expected to be involved in ear development can be easily discovered, and sever~A examples can already be found in the literature. Unfortunately, genes involved in cochlear development cannot be uncovered this way unless they are also involved in vestibular development (which many a--e), although simple tests for severe hearing deficits can be used.

II. A n a t o m y of the Inner Ear III. Development of the Inner Ear IV. Early Development of the Otic Placode and Otocyst V. Pattern Formation in the Inner Ear VI. Sensory Differentiation VII. Neurogenesis VIII. The Stria Vascularis IX. Future Directions References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

I. I n t r o d u c t i o n The inner ear is one of the most complex and intricate structures that forms in mammals. Understandably, many developmental biologists in the past have avoided tackling this structure in exchange for simpler models, thereby leaving much of mammalian inner ear development a myste~': For~Present address: The Jackson Laborato~,, Bar Harbor. Maine 04609.

Mouse Development

539

Copyright 9 2002 Academic Pre~ All rights of reproduction in any form reserved.

!il Organogenesis

540

II. A n a t o m y of the Inner Ear The m a m m a l i a n inner ear is c o m p o s e d o f two diverse functional parts, the cochlea, which is the auditory portion of the ear, and the vestibule, which functions in detecting gravity and linear and rotational motion required for balance (Fig. 1 ). The s e n s o r ' receptor cell that performs these many diverse functions is the hair cell. Hair cells, along with their associated supporting cells, make up a sensor), patch, and six different sensory patches are located throughout the ear. Within the cochlea lies a single sensory patch, the organ of Corti. which is responsible for transducing sound waves into neuronal impulses. Two different types of hair cells are found in this organ, inner hair cells (IHCs) and outer hair cells (OHCs), which differ in both m o r p h o l o g y and function (Fig. tD). By sending information back to the brain. IHCs act as the traditional receptor cells in the organ of Corti. In contrast, the O H C s are motile cells that primarily receive in-

put from the brain and are thought to function as a cochlear amplifier (Datlos, 1 9 9 2 Davis, 1983). Within the vestibule there are five sensory organs that can be o f two types: cristae or maculae (Figs. 1B and i C). Cristae are humplike organs that tie at the base of each of the three semicircular canals. Maculae, o f which there are two saccular and u t r i c u t a r - - a r e flat organs located in the central region of the vestibule. As in the auditoc~" system, two types of hair cells are found in the vestibular organs, type I and tvpe II (shown in Fig. 1C), although the functional significance of having these two cell types is not yet clear. In addition to being located within the skull, the inner ear is itself encased in a bony shell known as the otic capsule. Within the enclosed epithelial c o m p a r t m e n t s of the ear is the endolymph, a specialized fluid with the unusual ionic composition of high [ K ' ] and low [Na+], an essential medium for normal hair cell transduction. B e t w e e n the otic capsule and the central e n d o l y m p h a t i c c o m p a r t m e n t s lies a fluid

Figure 1 Structure of the mouse inner ear. (A) Structure of the mouse inner ear showing the endolymphatic compartments from a lateral view. The cristae are shown in dark gray, the maculae in stripes, and the organ of Corti in light ~ay. (B-D) Schematic drawings of the three types of sensoo, epithelium found :in the inner ear (their locations in the ear are indicated in panel A). Support cells are shown in clark gray, nerve fibers in black, and the hair cells in white. The different types of hair cells and supporting cells are indicated, asc, anterior semicircular canal: c, crista: Cc, Claudius' cell: cc, common crus; cd. cochlear duct: Dc. Deiter's cells; ed, endolymphatic duct; es, endolymphatic sac: Hc. Hensen ceil: Ihc. inner hair cell: Isc. inner sulcus celt; lsc, lateral semicircular canal: m, maculae: oC. organ of Corti Ohc. outer hair cells: psc. posterior semicircular canal, pc. pillar cells; sac, sacculus; ut, utricle.

54t

22 Development of the Mouse inner Ear called perilymph, which has low [K§ and high [Na +], similar to most extracellutar fluids.

!11. Development of the Inner Ear The inner ear is first identifiable as a thickening in the ectoderm adjacent to the neural plate in the prospective hindbrain region. In mouse this thickening or placode can be observed between e m b ~ o n i c day E8 and E8.5, in an embryo with 4 - 1 t pairs of somites (Fig. 2A; Kaufman, 1992, pp. 5 0 - 5 t; Theiler~ 1989, stage 13). It is generally believed that the otic placode is entirely of ectodermal origin. How-

ever, a recent morphoIo~cal study has suggested that some of the otic placode may be derived from the neural folds in the chick (Mayordomo et aL, t 998), an assertion that will require detailed fate mapping to confirm. Once established, the pIacode begins to invaginate, forming a structure called the otic pit or cup (Fig. 2B" E9; 1 3 - 2 0 somites; Kaufman, I992, pp. 6 0 - 6 1 ; Theiler, 1989, stage 14). This pit continues to deepen until a complete vesicle or otocyst is f o r m e d (Fig. 2D; E9.5; 2 t - 2 9 somites; Kaufman, 1992, pp. 7 8 - 7 9 ; Theiler, 1989, stage 15). During the period of late otic pit and early otocyst formation, cells in the mnteroventral portion of the vesicle detaminate from the epithelium and migrate medially. These cells will coalesce to form the eighth cranial

Figure 2 Developmentof the mouse inner ear. (A-E) Schematic diagrams of the inner ear in cross section as it develops from otic placode (A), to otic pit (B, C), to otocyst (D, E) stages. The surrounding tissues that may play a role in the induction and subsequent development of the ear are indicated. Dorsal is up and lateral is to the fight. (F) Lateral views of paint-filled inner ears from otocyst stages and continuing at reTalar intervals until the adult form of the ear is attained at El7. ec, ectoderm (light gay); en, endoderm (light gray); m, mesenchyme (dark gray), n, notochord (white); he, neuroectoderm (dark gray stripes): oe, otic ectoderm (black): VIII, eighth cranial ganglia, shown mi~ating from the otic epithelium in (C). Bar in panel F = 200 #m. (Panel F was taken with permission from Morsli et aL, !998.)

!!10rganogenesis

542 eanelion and their peripheral dendrites will later innervate the sensory epithelium of the ear (Figs. 2C-E, Carney and Silver. 1983: Li et al., t978). After the otic vesicle stage of development, ear morphology becomes quite complex and is better viewed in wholemount situations (Fig. 2F; Morsli et al., 1998). Morphogenetic changes in the otic vesicle can first be observed by E 10.5 and these include the elongation of the ventral portion of the vesicle so that it resembles more of an oval shape, as welt as a small projection dorsally that will later develop into the endolymphatic duct and sac (Fig. 2E, Kaufman. 1992, pp. 116-117, Theiler, 1989. stage 17). In addition the epithelium does not show a uniform thickness at this time. because the ventral regions are much thicker than the dorsal regions. The semicircular canals evaginate dorsally and are formed by El3 (Fig. 2F), although they continue to grow and thicken until birth. The cochlea extends first ventrally (El 1.5) but then begins coiling at E l 2 and continues until E 17.5, by which time it has reached its full 1.5 turns (Fig. 2F: Morsli et al., 1998: Sher. 1971).

IV. Early Development of the Otic Placode

and Otocyst

A. Specification of the Otic Placodes The question of how and when the otic placodes are specified has been the subject of much study throughout the twentieth centu~;. Because the ear vesicle does not form when prospective ear ectoderm is grown in isolation or in foreign environments (see, for example. Gallagher et al., 1996; Jacobson, 1963; Waddington, 1937; Yntema, 1939), these studies have focused on which of the surrounding tis-

sue or tissues might be inducing its formation and at what point in development. The tissues that may potentially play a role in ear induction include the neural plate, mesoderm, notochord, and endoderm (Figs. 2A and 3). Prior to the advent of molecular techniques, these questions were primarily addressed through transplantation, ablation, and expiantation experiments in amphibia or chick. These studies revealed that otic determination is not the result of a single inductive event but rather takes place over a period of time and is likely to require a number of different inductive signals (Jacobson, 1966; reviewed in Torres and Giraldez, 1998). Questions of when instructive signals emanate from the surrounding tissues, and when the ectoderm becomes competent to respond to these instructions, were addressed by transplanting foreign or prospective ear ectoderm at different ages into the otic region of the amphibian e m b ~ o . These studies revealed that inductive signals probably occur between mid- and late gastrulation, because ear ectoderm during this time period showed a greater propensity to form otic vesicles than foreign ectoderm or ear ectoderm obtained from earlier stages (Galtagher et at., 1996; Yntema, t933). Furthermore. ectoderm derived from regions around the prospective ear was more likely to form ear vesicles than ectoderm from farther away, indicating that the first step in otic determination is the specification of an otic field (Fig. 3C). The experiments in amphibia by Jacobson (1963, 1966) have suggested that prior to the establishment of an otic field, there is first a placodal field, which includes a band of ectoderm that lies in a semicircle around the neural plate encompassing the prospective ear, nose, and lens placode (Fig. 3B). These experiments demonstrated that when head ectoderm is rotated 180 degrees, the sensou' organs will be respecified at their correct positions in relation to the e m b ~ o , indicating

Figure 3 Modelfor the stages of induction of the inner ear. (I-IV) Schematicdiagramsof dorsal views of the anterior portion of an early to late stage neurula amphibian embryo..Anterior is up. The first stage of the induction of the inner ear may be the specification of a placodal competent area (tI; red). Secondly, within the placodaI field, an otic competent area is specified (III; yellow), and finally an otic placode is formed within the otic field (IV; ~ay).

22 Development of the Mouse Inner Ear that the placodal ectoderm is multipotent during early development. Torres and Giraldez (1998) have taken this idea fiJrther and suggested that general piacodal competence is maintained by a set of genes (discussed below in the section titled Genes Expressed in the Otic Placode) that are then Iocaiized in unique combinations to either the nasal, lens, or otic areas. This issue of multiptacodaI competence has been addressed recently in the chicken through misexpression of the otic placode-inducing molecule Fgf3 (Vendrell et al., 2000). These experiments demonstrated that ectopic otic placodes could be induced in a large area of ectoderm that extends both anterior and posterior to the normal position of the otic piacode. However. otic ptacodes did not form in the most anterior ectoderm, including the region occupied by the lens and nasal placodes, suggesting chat some regions of the head ectoderm are not otic competent. However, Groves and Bronner-Fraser (2000) showed in the chick that anterior epiblast can form otic placodes when grafted in the correct place, suggesting that either the Fgf3 experiments were not performed early enough before otic competence is lost or other inducing factors are required. Recent evidence indicates that perhaps all ectoderm is competent to respond to specific placodai inducers in early emb~'os and that neither a general placodaI inducer nor a multiplacodat state may exist. Rather, it may be the loss of competence in all but a few patches that determines the final location of the neurogenic placodes. These patches are presumed to survive based on their proximity to inductive sources that are specific to the different ptacodes (Graham and Begbie, 2000). As such, the idea that all neurogenic placodes have a common developmental and evolutionary origin has been challenged precisely because the inductive mechanisms controlling their specification into different types of placodes are so variable. Classical transplantation studies have examined which of the surrounding tissues were responsible for the induction of the otic placode. These experiments were performed by explanting the prospective ear ectoderm in conjunction with one or a number of its potential inducers, or by ablating one or a number of its potential inducers. Results of these experiments established that, surprisingly, several of the surrounding tissues, including mesoderm, notochord, and neural tube, could induce an otocyst (see, for example, Jacobson, 1966; Kohan, 1944; Waddington, t937; Yntema, 1950). It was shown, however, chat the most normal differentiation of the otocyst occurred when several potential inducing tissues, such as mesoderm, endoderm, and neural tube, were included in the explant (Jacobson, t966). In addition, it was revealed that these tissues were not equipotent in their inducing ability; for example, neural tube by itself produced getter differentiation of the otic vesicle than mesoderm combined with endode~m. More recently, these questions have been addressed in zebrafish by transplants of germring tissue (early mesoderm and endoderm) or hindbrain tissues. These ex-

543 periments confirmed the emdier transplantation studies by showing that both types of tissues could induce ectopic otic vesicles (Woo and Fraser, 1997, 1998). Interestingly though, the hindbrain could not induce vesicles during its very early stages of development (80% epiboly), whereas germring tissue demonstrated this ability, suggesting that the nature and/ or amount of the inducing signals differs between these two tissues during development. "I'ne question of inductive influences has also been addressed through genetic studies in the zebrafish (Mendonsa and Riley, 1999). Using a number of zebrafish mutants in which one or a number of the potential inducing tissues was either missing or abnormal, Mendonsa and Riley (t999) have examined whether otic ptacode development was affected. Results of these experiments demonstrated that mutams, such as cyclops or one-evedpinhead, that have a partial or complete absence of prechordaI mesendoderm experienced a delay in otic placode development, and the morphology of the otic vesicle was abnormal. Moreover, the amount of the delay was dependent on the amount of prechordal mesendoderm that was missing. Mutations that affect the development of the notochord, such as no tail and floating heaa, did not affect the timing or morphology of the inner ear. The rote of the hindbrain was examined in the mutant vatentino, a mutation that affects rhombomeres 5 and 6 and is a homotog of the :mouse mutant kreisler. Interestingly, no delay was found in the appearance of the otic ptacode, although subsequent patterning of the vesicle was abnormal. These results have revealed an important role for the mesoderm in the correct timing of otic placode formation and support the manipulation studies that indicated an early rote for the mesoderm, although the identity of the mesoderminducing factor(s) remains unknown. However, the fact that the placode does eventually form in these mesoderm mutants suggests that other tissues (such as hindbrain) can induce otic ptacodes, albeit at a later time point. Indeed, a recent report performed in the chicken revealed that otic induction requires at least two signals: FGF19, expressed in the mesoderm, and Wnt8c, expressed by the neural tube (Ladher et al., 2000). This study demonstrated that only by combining these two molecules could previously uncommitted explanted ectoderm express a full complement of otic markers. However, despite the expression of several early otic markers, the induced ears did not produce hair cells. Presumably, the specification of hair cells from naive ectoderm requires additional factors, or the amount and timing of FGF19 and/ or Wnt8c expression in the cultures is inappropriate. Interestingly, FGF19 was found to be expressed in the neural tube at slightly later time points, providing a possible explanation for the observation that the hindbrain alone can eventually induce an otic placode. It wilt be importmnt to examine the expression of these two molecules in mice, to see whether they are likely to be playing a similar synergistic role in mouse otic induction.

544

II! Organogenesis

F i g u r e 4 Effects on the hindbrain and inner ear of mutations that disrupt the patterning of the hindbrain. Schematic diagrams of dorsal views of hindbrains and otocysts (ovoids) from wild-D'pe and various mutants. Rhombomeres (r) are numbered from rl to the spinal cord (sc). Dysmorphic otocysts are indicated by stripes. Colored rhombomeres indicate the presence of the expression of the designated gene. *Now: The yellow r6 rhombomere in the Krox20 mutant represents only the expression of Fgf3. since the expression of Krml was not investigated.

B. Role of the Hindbrain in Otic Patterning Mouse Mutants Perhaps the best evidence of the influence of the hindbrain on otic patterning comes from preexisting mouse mutants or, more recently, from mouse knock-outs that show defects in the development of both the hindbrain and otocyst (Fig. 4). This influence is proven most clearly in mutants such as kreisler and H o x a l -/- (Figs. 4B and 4C). in which the respective genes are never normally expressed in the ear. thereby demonstrating that the otic defects could only be derived from abnormal inductive signals. Other mutants. such as those deficient in certain retinoid signaling proteins (Figs. 4D and 4E), also show hindbrain and ear defects. Atthough it is possible that retinoic acid is required directly for early otocyst development, the location as well as type of defects reported in these mutants is consistent with the ear abnormalities resulting secondarily from hindbrain disturbances. As described in Chapter 6. during early development the hindbrain transiently displays repeating metameric units called rhombomeres that appear to function as compartments in the early hindbrain (Lumsden and Krumlauf, 1996). The otocyst develops adjacent to rhombomeres (R) 5 and 6 in normal animals, and so these are the rhombomeres most likely to play a role in ear development. Many genes expressed in the hindbrain appear to respect rhombomeric boundaries, thereby conferring each rhombomere with a unique genetic identity. It is therefore relatively simple to ascertain whether a rhombomere is missing or duplicated simply by examining gene expression patterns in the hindbrain. Interestingly, it is generally those mutants in which a rhombomere is missing or expanded that tend to affect the

development of the ear. Below are summaries of some of the hindbrain mutants that have been described m date and the effects of these mutations on the development of the inner ear (see also Fig. 4).

1. Kreisler The kreisler (kr) mutation was identified in an X-rav mutagenesis experiment because the mouse showed hyperactivity and circling behavior (Hertwig, 1942), indicative of a balance defect of the inner ear. Analysis of the kr,~r mutants showed that the hindbrain did not exhibit its usual rhombomeric bulges caudal to R3, and the otic vesicle was displaced laterally and subsequently developed into a large cystic structure (Deol, 1964; Hertwig, I942). Deol suggested, based on the transplantation experiments performed in birds and amphibia, that the malformed ear may be a secondary effect that is caused by defects present in the hindbrain. His theory, was proven correct when Cordes and Barsh (1994) cloned the gene at the kr locus, Krml, and showed that it was expressed in the hindbrain adjacent to the developing ear. but not in the otic vesicle itself. K r m l is a transcription factor of the bZIP family and its expression domain includes R5 and R6. The most recent analysis of the kr/kr hindbrain, using transgenic mouse lines carr?fing Hox/ lacZ reporters and by examining genes expressed in discrete rhombomeres, revealed that R5 failed to form and R6, atthough present, was abnormal (Fig. 4B; Manzanares et aL, 1999). According to Deol (1964) sensoEv areas do form in kr/kr animals, usually maculae and some cristae, although their size and position is abnormal. Additionally the otic capsule was rarely complete and often displayed gaps on the medial side of the cochlear duct.

22 Development of the Mouse Inner Ear 2. H o x a l

Two different knockouts of the Hoxat gene were produced (Chisaka et al., t 992; Lufkin et aI., 1991) and both showed defects in the hindbrain and inner ear, as welt as in some of the derivatives of the neural crest. Analysis of the hindbrain in these mutants revealed that R5 failed to form, R3 was expanded, and R4 was reduced (Fig. 4C; Carpenter et aI., 1993; Mark et al., I993; Rossel and Capecchi, t999). The otic vesicles of the mutants were displaced laterally and rostrally and developed adjacent to r4, rather than in their normal position next to R5 and R6. During subsequent development of the mutant ears, the endolymphatic ducts failed to form and. similar to kr mutants, the ears developed into large cystic structures, although much variation was observed. Some sensor' development was reported to occur, although it included primarily vestibular epithelium such as cristae and maculae similar to the kr/kr mutant (Lufkin et al., 1991 ). Double knock-outs of both Hoxat and Hoxbl, which led to a more sizable expansion of R3 and an absence or severe reduction of R4 in addition to R5, were not reported to have more severe otic defects than H o x a l mutants alone, although the ear abnormalities in the double mutants were more penetrant (Gavalas et at., I998; RosseI and Capecchi, 1999).

3. Mutants Deficient in Retinoic Acid Signaling Components Retinoic acid (RA) is a well-known signaling molecute that appears to be involved in the patterning of numerous regions of the developing embryo including the hindbrain (Morriss-Kay and Ward, t999). Several recent knockouts that affect RA signaling have produced patterning defects in the hindbrain and inner ear. The analysis of these rhombomeric defects (location and type) as well as the similarities of the inner ear defects to other hindbrain mutants (Kr and H o x a I ) suggests that the otic abnormalities are likely to be due to the hindbrain defects. In double kmock-outs of the retinoic acid receptors, Rara and Rarb, R5 appeared enla,~ed and R5/6/7 boundaries were abnormal (Fig. 4D: Dupe et al., t 999). Interestingly, ectopic otic vesicles were often observed. These otic vesicles formed from an enlarged otic pit, which may be induced due to an enlargement of R5. However, further development of these ectopic vesicles was not supported and the ear subsequently developed normally in these mutants. In addition to these receptor knock-outs, RA production has also been affected through disruption of the retinatdehyde dehydrogenase 2 (Raldh2) gene, an enzyme required for much of the RA synthesis in the embwo. In addition to other defects (Niederreither et at., t 999), this knock-out resulted in a severe disruption of the caudal hindbrain (Niederreither et al., 2000). Specifically, the rhombomeres posterior to R2 did not appear to form normally and gene expression studies suggested there was an "anteriorization"

545 of the caudal hindbrain. As observed in other hindbrain mutants, the otic vesicle was small and displaced laterally (Fig. 4E). Unfortunatel}~ an ~atysis of the mature labyrinth was precluded because the embryos died at E10.5. However. gene expression studies of the otic vesicle using the regionatized homeobox-containing genes, Pax2 and H~rLr3, showed there were likely to be severe problems in otic patterning, because Pear2 expression was absent and Hmx3 transcripts failed to localize normally in mutant vesicles. 4. Fgf3: An Ear Inducing Molecule? One of the best candidates for at least one of the signals emanating from the hindbrain is a member of the fibroblast growth factor family, FGF3. FGF3 was originatl) postulated to be involved in ear induction because it showed strong expression in the otic region of the hindbrain during the period of early ear development (Wilkinson et at., t988). In vitro experiments in chick demonstrated that ofic vesicle formation could be prevented using Fgf3-targeted antisense otigonucleotides and antibodies (Represa et al., 1991). More recently it was shown that misexpression of Fgf3 in the chick caused ectopic otic placodes to form (Vendretl et al., 2000). These experiments provide strong evidence for a role for FGF3 as an otic inducer. However, a targeted disruption of the Fgf3 gene in the mouse displayed no defects in otic vesicle formation, atthough its subsequent patterning was severely disrupted (Mansour, 1994). These conflicting results can be explained in two ways: (1) The mouse and chicken differ in the identity of their otic inducer(s) or (2) another molecule can compensate for FGF3's normal inducer role in the mouse knock-out. Although the latter explanation cannot be ruled out, expression studies suggest that the former may be true because in the mouse Fgf3 expression in R5 and R6 is not heightened until the otic ptacodes are already morphologically identifiable (McKay et at., 1996). In contrast, Fgf3 expression in chickens is observed in the hindbrain at a time more compatible with otic induction (Mahmood et at., 1995). The otic phenotype in Fgf3 -/- mouse mutamts was similar in some respects to the other hindbrain mutants in that the endotymphatic duct (ED) failed to form. However, the phenotype did not appear to be as severe as those described for kr, Hoxal, and RA mutants. Early, the morphology of the otic vesicle appeared normal in that it was not displaced or small. Later, most of the ear structures were present but were described as distended and swollen, suggesting endolymphatic hydrops. These observations led Mansour (t994) and colleagues to speculate that the role of FGF3 produced by the hindbrain is to induce the formation of the endolymphatic appendage, and on failing this in Fgf3 -/- mutants, endolymph production is not properly regulated and the ear becomes swollen. A complication of this interpretation is that Fgf3 is also expressed in the otic vesicle, indicating that a patterning mechanism autonomous to the otic vesicle may be responsible for the otic defects present in Fgf3 -/- mu-

546 tants. However, McKay et aL (t996) argue that in tact it is the expression in the hindbrain that is critical for ED formation. because kr/kr mutants, which also fail to form an ED, showed decreased expression of Fgf3 in the hindbrain but not in the otic vesicle. Further support for a role for FGF3 in the development of the ED has been demonstrated by overexpression of the gene in chicken, which resulted in an elongated ED (Vendrell et al., 2000). A recent knock-out of the IIIb isoform of the Fgy?2 gene revealed that the effects of Fgf3 in the ear are likely to be mediated via this receptor, since some of the otic defects (including absence of an ED) observed in these mutant mice are very similar to those described for Fgf3 -/- mutants (De Moerlooze et al., 2000). When taken together, although these hindbrain mutants reveal a role for the hindbrain in otic development, its role in the induction of the otic placode or vesicle remains controversial, since vesicles, albeit abnormal, form in all the hindbrain mutants. It is possible, however, that a mutation that interferes with the otic-inducing signal has not yet been discovered. Furthermore, in Rara and Rarb double mutants, an expanded otic pit forms next to an enlarged R5. suggesting the two phenomenon are causally related. One possibility is that the hindbrain, although it does not initiate otic placode formation, can influence the size of the placode or otic pit. This interpretation would be consistent with the fact that when R5 (or R5 and R6) is missing, the otic vesicle is small. These data suggest a particularly key role for R5 involvement in the signals emanating from the hindbrain, although this interpretation may be influenced by the fact that R5 is the rhombomere that is most often missing. Furthermore a disruption of the Krox20 gene results in a loss of R3 and R5, but the otic vesicle develops normally (Fig. 4F). This apparent discrepancy can be explained in two ways: (1) R6. which appears unaffected in Krox20 mutants, is sending the required signals: or (2) the required signals could be sent from R5 prior to its loss. since R5 is initially established but then later lost in Krox20 -/- animals (Schneider-Maunou~ ~et al., 1993, 1997; Swiatek and Gridley, 1993). Further analysis of the role of the hindbrain in ear induction would be facilitated by better analysis of the mutant ear phenotypes on a similar genetic background, because it is not clear exactly which mutations produce the most severe otic phenotypes. It is clear, for example, that FGF3 is one of the signals coming from the hindbrain since its absence disrupts otic patterning. It is likely, however, that FGF3 is not the only signal emanating from the hindbrain since all the other mutants, such as kr/kr, Hoxal -/-. Ralh2 -/-, Rara-/-/ Rarb -/-, exhibit more severe otic defects despite the fact that F J 3 expression is reduced or missing in their respective hindbrains. Gene expression studies on the mutant otic vesicles, such as those performed in the Ralh2 -/- mutants, would further elucidate the effects of hindbrain signaling on otic patterning.

111Organogenesis C. Genes Expressed in the Otic Placode As yet, despite the number of mouse (Steel, 1995" Steel et al., 2001) and zebrafish (Malicki et aL, 1996; Whitfield et at., 1996) mutants that have been examined for ear defects, no gene has been found to be essential for otic placode formation. This suggests that there is either some functional redundancy regarding ptacodal specification or that the gene(s) involved is/are also essential for early emb~'ogenesis, such that the mutations cause very early lethality. A number of genes are expressed at placodal stages (see Bussoli et aL, 2000: Torres and Giraldez, 1998), although only a handful are expressed early enough and in a sufficiently nomregionalized manner (i.e., throughout the otic placode) to represent good candidates for placode specification. The majority of genes found to mark the early otic placode in its entirety are transcription factors, mostly homeobox-containing, including Dlx3 (chicken; Pera mad Kessel, 1999), Pax2 (chicken; Groves and Bronner-Fraser, 2000; zebrafish; Krauss et at., 1991), Pax8 (Xenopus; Heller and Brandli. 1999), LmxI (chicken; Giraldez, t998; Torres and Giraldez, 1998), Sox9 (mouse" Heller and Brandli, 1999), Sox2/3/21 (chicken" Groves and Bronner-Fraser, 200,0; Uchikawa et al., 1999), Six4 (chicken; Esteve and Bovolenta, 1999), Gbx2 (chicken; Sharnim and Mason, 1998), and Gata3 (chicken: Sheng and Stem. 1999). In addition to these transcription factors, two secreted factors, Fgf3 (mouse; Mahmood et aL, 1996; McKay et aL, 1996) and Bmp7 (chicken: Groves and Bronner-Fraser, 2000; Oh et aL, I996), and a receptor, Epha4 (mouse; Nieto et at., t992), are also expressed throughout the placode at early time points. Interestingly, several of these genes, including Dlx3 (Akimenko et aI., 1994), cSix4 (Esteve and Bovolenta, 1999), and cGata3 (Sheng and Stem. 1999) have been reported to mark the entire placodal area (Fig. 3B) before being restricted to the otic field. These expression data lend support to Torres and Giraldez's model (1998) in which each placode (ear, lens, or nose) is specified by a unique combination of genes that are first expressed throughout the entire placodal region, but then are subsequently restricted to one or two of the placodes.

V. Pattern Formation in the Inner Ear One remaining challenge is to determine the rules by which gene expression patterns get interpreted and convetted into complex pattern formation in the ear. It seems obvious that the process of patterning will be manifested by differential spatial and temporal control of cell behavior (such as cell shape changes or migation), as well as cell number (involving boLh proliferation and programmed cell death). Descriptive details about the control of cell number continue to be refined and are discussed first. However, the

22 Development of the Mouse Inner Ear m e c h a n i s m s by which these processes are controlled, the in-

volvement of celt-cell signaling, and the site(s) of action and range of diffusible signals remain murky. The degee to which patterning information arises directly within the ear epithelium versus being specified and/or maintained from the surrounding tissues, especially the mesenchyme, remains an open question.

A. Ear Morphogenesis and the Control of Cell Proliferation and Death Morphogenesis and growth of the inner ear is undoubtedly influenced by cell number, which will be manifest through regulation of cell proliferation and cell death. As the otic placode deepens to form a cup and ultimately pinches off to form a vesicle, celt numbers continue to rise. The topology of dissociating the otic vesicle from the overlying ectoderm requires epithelial cells at the junction to change their cell-cell associations, and in fact this process is accompanied by a focus of celt death at the junction (Alvarez and Navascues, 1990; Lang et at., 2000; Marovitz et aI., 1977; Represa et at., 1990). As the ear pro~esses through the critical stages of ear morphogenesis, cell proliferation and ceil death are spatially regulated. These two processes have been compared systematically in the chick ear (Lang et at., 2000). One surpfsing finding was that differential outgrowth of the endolymphatic duct, canal plates, and cochlear duct was not necessarily correlated with increased ceil proliferation in the growing regions at early stages. Rather, the ventral half of the vesicle contains a disproportionate number of the dividing cells, whereas outgowth of the dorsal half appears to involve thinning of the epithelial surface with only a modest increase in cell proliferation. The ventral proliferation associated with cochlear duct outgowth in mouse is discussed below. As morphogenesis proceeds, the spatial patterning of proliferation gets progressively more complex, as might be expected. Some of the regions of reduced ceil proliferation can be explained by the early withdrawal of sensor" organs from the cell cycle. Others can be correlated with areas that will undergo programmed cell death (such as the ventromedial wall of otocyst and, in the chick, the fusion plates of the semicircular canals). The function of programmed cell death, particularly within the ventromedial wall where it is especially robust in birds and mammals, remains a mystery. This focus of cell death is located near the proximal end of the cochlear duct mad, intrigaaingly, is not apparent in lower vertebrates that lack a cochlea (Bever and Fekete, t 999).

B. Semicircular Canal Morphogenesis Morphogenesis of the semicircular canals is a stunning example of complex tissue remodeling to generate rat.her pre-

547 cise three-dimensional structures, In general, a canal arises initially as an outpocketing from otocyst, called a canal pouch. T h e two verticaI canals develop from a single dorsally directed pouch, whereas the horizontal (lateral)canal develops from a separate laterally directed pouch (Fig. 2E El2). To form a canal, two apposing surfaces approach each other over a relatively broad area near their centers and then fuse into a single epithelial layer. Experiments in the frog suggest that the driving force for the approach phase is dependent on hyaturonan secretion from the canal plates into the underlying mesenchyme (Haddon and Lewis, 199 i). In the mouse this fusion process begins first in the anterior canal, followed closety by the posterior and then lateral canal (Martin and Swanson, t993). Once fusion has occurred, the fusion plate cells disappear to leave only the rim around the outside of each canal (Fig. 2E El3). In the chick, this rim (the duct proper) has an increased level of celt proliferation as the canal g o w s significantly in size. In the canal fusion plates of the chick ear, pr%~ammed ceil death appears to play- an important role in removal of fusion plate cells (Fekere et aI., 1997). However, in the mouse this remodeling may be accomplished by a different mechanism, because extensive programmed cell death appears to be lacking in this locate (Martin and Swanson, I993; Nishikori et al., 1999). Instead, cells of the fusion plate may be resorbed into the expanding rim of the canals. It has been shown recently that the laminin-like molecule netrin 1 (Ntnl) plays a critical role in the canal fusion process (Salminen et at., 2000). This result came as something of a surprise because prior to this report, netrins were known primarily for their rote in axon guidance. Mice generated from a gene-trapping experiment were identified as having a disruption in the N t n t gene and did not develop lateral or posterior canals, and the anterior canal was small. During development the fusion process did not proceed normally in any of the canals in the mutant. Expression studies showed that N t n l is expressed in the central portions of the canal, consistent with a proposed role in the canal fusion process. However, the fact that the anterior canal does form in the N t n I mutants, albeit abnormally, suggests that Ntnl cannot be the sole mediator of the canal fusion event.

C. Gene Expression Domains and Morphogenesis There is an expanding list of genes that are expressed in or around the otic epithelium in spatially restricted patterns (Fig. 5) prior to overt morphogenesis (e.g., canal formation or cochlear duct elongation) and long before overt cellular differentiation (e.g., recognizable hair cells). A majority" of these genes contain homeoboxes, and thus are presumed to be transcription factors. Genes that exhibit regionalized expression at early stages should be considered candidates for

548

111Organogenesis

Figure 5 Potential compartments in the early otocyst. The mouse otic vesicle is represented as a hollow sphere with proposed compartment boundaries segregating it into dorsN-ventrat, anterior-posterior, and medial-lateral halves. (Top) The expression domains of several genes known to be involved in ear morphogenesis are shown from a lateral perspective. The precision of the compartment boundaries with respect to the gene expression domains is hypothetical, as in most cases the degree of overlap between the different genes has either not been definitely established or is not as precise as that shown (see text). (Bottom) A schematic of the inner ear pheno~pes that result when the gene is disrupted, shown from a lateral perspective. Structural defects that are highly penetrant are indicated either as altered or missing structures. When the penetrance of a structural defect or deficiency is variable, the affected region is indicated with gray shading, but appears normal in morphology. See text for references and fur'daer details. ac. anterior crista: asc. anterior semicircular canal: cc. common crus: csd. cochlear saccular duct: ed, endolymphatic duct: es. endolymphatic sac: lc. lateral crista: lsc. lateral semicircular canal: oC. organ of Corti: pc. posterior crista: psc. posterior semicircular canal: S. saccular macula: U. utriclar macula: usd. utricular saccutar duct.

22 Development of the Mouse Inner Ear the regulation and specification of patterning information. Experimental support for such a rote is currently limited to a handful of cases in which gene knock-out gives an ear phenotype; these are discussed below. Genes that are normally expressed in the otocyst itself may be easier to interpret with respect to these deletion phenotypes, since the requirement for the gene product is more likely to be ceil autonomous, particularly in the case of transcription factors. Genes that are expressed exclusively in the surrounding mesenchyme, but still can alter patterning in the otic epithelium, lend support to the idea that rnesenchymatepithelial interactions also play an important role in the growth and patterning of the membranous labyrinth.

1. Genes Expressed in Ectoderm That Affect Ear Morphogenesis Thus far, most of the genes found to affect early ear morphogenesis are expressed within the otic epithelium. These genes are expressed in asymmetrical domains in the very early otocyst (and several are expressed prior to this). The fact that their expression domains at otocyst stages roughly correspond to the location of the defect when the gene is removed (Fig. 5) suggests that these early expression domains may play an important role in the gross patterning of the inner ear. For example, Pear2 is expressed in the medial and ventral portions of the otocyst, and two mouse mutants that affect the Pax2 gene show a complete agenesis of the cochlea, a ventral ear structure (Favor et al., 1996; Tortes et al., t996). Similarly, mice deleted for Dlx5 (expressed dorsally) do not develop the anterior and posterior semicircular canals, structures that are located in the dorsal part of the adult inner ear (Acampora et aI., 1999" Depew et aI., 1999). Loss of Hmx3, which is normally expressed dorsolaterally, can affect all three semicircular canals, although the lateral and posterior canals are most often abnormal ( H a d e s et aI., 1998). A milder allele showed absence of only the lateral crista and ampullae as well as a continuity between the saccular and utricular maculae, perhaps reflecting a failure of the utriculosaccular duct to undergo constriction (Wang et al., 1998). OtxI is expressed in a discrete wedge in the posterior-ventral-lateral part of the otocyst. Deletion of Otxl leads to loss of the lateral canal and crista, as well as the utriculosaccular and cochleosaccular ducts. V~3aen the Otxl knock-out is combined with the loss of one Otx2 allele (Otx2"/-), most of the defects are more penetrant and more severe, particularly the cochlear dysmorphogenesis. An effort was made to see whether the Otx2 protein could substitute for Otxl by cloning the human OTX2 gene into the disrupted Otxl locus of the knock-out mouse (Morsli et al., 1999). OTX2 was able to partially rescue all but the lateral canal pheno~;pe, suggesting a unique role for the OTX I protein in lateral canal morphogenesis.

549 2. Genes Expressed in Mesoderm That Affect Ear Morphogenesis It is generally accepted that the mesoderm play's an important role in sculpting the inner ear epithelium, although exactly how this is done and which genes p l ~ a rote remain largely unknown. A study in the chicken demonstrated the importance of the mesenchyme by showing that when otic vesicles, devoid of mesenchyme, were transplanted to foreign mesenchyme, they failed to undergo any of the major morphogenetic events, such as cochlear outgrowth or semicircular canal formation (Swanson et at., 1990). Interestingb, although most of the overt morphogenesis of the inner ear failed to occur in the transplants, most cellular differentiation, including sensor,; proceeded normally. Unfortunately, despite the probable importance of the mesenchyme, there is only one mouse inner ear mutant, the Pou3f4 mouse l-mock-out, in which the affected gene's exclusively mesenchymat expression pattern may suggest a role in mesenchyrnal-epithelial interactions. Pou3f4 is expressed in the ventral mesenchyme at early otocyst stages, although later its expression spreads throu~_hout the entire periotic mesenchyme. Pou3f4 mutants show hypomorphic bone formation, manifested as a thinning of the otic capsule and a widening of certain bony foramena (Phippard et al., t 999). A possible disruption of mesenchymal-epitheliat interactions is suggested by the hypomorphic development of the cochlear duct, which does not express Pou3f4 yet fails to complete its normal 1.75 turns in most Pou3f4 knock-out rrdce. It is possible, however, that Pou3f4 is not involved directly in mesenchymal-epithetial interactions, but that its effects are derived secondarily due to failure of proper mesenchymat differentiation. For example, the report of abnormal spiral ligament cells, which are derived from the mesenchyrne, associated with reduced endocochlear potential in the cochlear duct, suggests an alternative mechanism for the coclatear defects (Minowa et al., 1999).

3. Genes Expresso:t in Ectoderm and Mesoderm That Affect Ear Morphogenesis At least two examples are known thus far of mutants whose genes are expressed in both the ectoderm and mesoderm, complicating the interpretation of whether the inner ear defects are due to failed mesenchymal inductive interactions or whether they are due to failed otocyst-autonomous patterning events. However, in the case of Prxl and Prx2 double knock-outs, which lead to toss of the lateral canal, thickening of the anterior and posterior canals, and a reduction in the size of the otic capsule, it is likely that the defects arose due to failed mesenchymal-epit.helial interactions, because both genes only colocalize in the lateral periotic mesenchyme (ten Berge et aI., 1998). In the case of the Eyat gene, which is expressed in both the ear ectoderm at early otocyst stages and later throughout

550 the mesenchyme, it is more difficult to resolve whether its main effect on inner ear morphogenesis is mediated through its ectodermal expression, inductively through its mesenchymal expression, or both. Total loss of expression of Eyat results in one of the most severe inner ear phenotypes described to date. as the otocyst fails to undergo all structural specializations, including formation of the otic ganglion (Xu et at., 1999). The defect is greater than what might be predicted by the gene's early expression domain, which is restricted to the ventral half of the vesicle and the cells of the developing eighth cranial ganglion at E 10.5 (Kalatzis et al.. 1998). Nonetheless. the temporal expression of the gene in ectoderm versus mesenchyme suggests that the profound morphogenetic block in the knockout is due to a direct effect in the ectoderm rather than an indirect effect from the mesenchyme. A hypomorphic allele (Eyal b~ has also been identified that results in a severe truncation of the cochlear duct, while leaving the vestibular portion of the ear relatively intact (Johnson er al.. 1999). Eyal is of particular interest because it underlies an autosomal dominant human syndrome, branchio-oto-renal (BOR) syndrome, which causes early developmental defects in all three divisions of the ear (outer. middle, and inner) as well as kidney defects (Abdelhak et aI., 1997). In the chick it has been shown that Bmp4 may play an important role in signaling from the mesenchyme, as mesenchymally implanted beads soaked in noggin, a Bmp4 antagonist, impaired inner ear morphogenesis (Chang et al., t999: Gerlach et al.. 2000). The noggin beads seemed to exert their strongest effect on the semicircular canal development, consistent with Bmp4 expression in the mesenchyme surrounding the future canal portion of the otocyst. However. Bmp4 is also expressed within the epithelium of the ear. making it unclear exactly which Bmp4-expressing areas were impaired by the noggin beads.

D. Regionalized Expression of Genes and Possible Compartments It is worthwhile to consider some aspects of the regionalized gene expression domains in the context of pattern formation. Once the neural progenitors have emigrated from the inner ear. the remaining otocyst segregates into approximately seven gross structural elements: the endolymphatic duct/sac, the three semicircular canals with their ampullar2s enlargements, the utricle, the saccule, and the cochlear duct. One senso~ ~"organ will arise within each of the compartments, with the exception of the endolymphatic duct/sac. One simplistic mechanism to specify the major parts of the ear would be to put each under independent genetic control. In such a model, the early otic vesicle would consist of a mosaic of seven distinct compartments, each giving rise to one of the seven structures recognized in the adult. There would be cochlea-speci~ing genes, saccule-specifying

I11Organogenesis genes, and so on. In the parlance of the field, such genes would be called selector genes, because they select a specific identity for the cells that express them that is ultimately interpreted as patterning (Lawrence and Strah!, i996). In the extreme, each piece of the mosaic would be expected to represent a lineage-restricted compartment, defined by differential gene expression and reinforced by precise lineagerestriction boundaries across which cells would not mix. The question to be asked is whether our current knowledge of lineage compartments, gene expression domains, and the effects of gene knock-outs supports such a model for the ear. With respect to the existence of lineage compartments, there are no data of sufficient precision to draw any conclusions. In the mouse, Li et ai. (t978) cultured sectors of the otic vesicle, and followed their subsequent differentiation to generate a specification map for the mouse otocyst. Their data are consistent with a gross regionalization of the otocyst: dorsal structures (such as the endolymphatic duct and semicircular canals) arise from the dorsal otocyst, while ventral structures (the cochlea and saccule) arise from the ventral otocyst. Similar generalities can be made for anterior versus posterior and medial versus lateral tissues, but there is significant overlap in the fates of the eight dissected sectors. Interpretation of these results may be confounded by the phenomenon of emb~onic regulation, whereby when one part of an embryonic field is removed, and another can alter its prospective fate to partially or completely fill in the missing part. Another concern is that it might be necessaps for different parts of the otocyst to signal to one other in order for the normal pattern to manifest itself. Growing portions of the otocyst in isolation would necessarily disrupt such inductive signaling. Direct fate mapping data, whereby small groups of cells are labeled and allowed to develop in situ, are needed but are only just beginning to be compiled for other species, including the chicken (Brigande et aI., 2000a). With respect to gene expression, a precise description of potential compartments is also lacking. Side-by-side comparisons of gene expression domains in single organisms are needed to address this issue with certainty. Relatively few" genes have been explicitly compared in the mouse with the goal of determining whether there is overlap or whether the genes meet at precise boundaries. For example, three different members of the Iroquois family of transcription factors appear to have different, but partially overlapping, expression domains in the E9.5 ventral otocyst (Bosse et at., 1997). One day later, the three genes are expressed throughout the otocyst. Likewise, Otxt and Otx2 display nested expression domains in the ventrotateral otocyst at E9.5 (Morsti et at., 1999). Along the medial-lateral axis, Hmx3 (a lateral gene), Epha4 (Sek), and Pax2 (medial genes) have nearly mirrorimage expression patterns, although they apparently overlap slightly at the limits of their expression in the dorsal and ventral otocyst at E 10.5 (Rinkwitz-Brandt et aI., t 996).

22 Development of the Mouse Inner Ear

55t

Figure 6 Dynamic patterns of expression of markers of the sensory, organs during development. Expression data were accumulated from several different sources (see text for details), so the exact stage cannot be specified. A and B are lateral views of the otocyst. *Note: Jagt is probably expressed at El0.5 based on other species, but the pattern in mouse has not yet been demonstrated. A, anterior: ac. anterior crista: ass, anterior semicircular canal: co. cochlea: D. dorsal; ed. endolymphatic duct: KOCs. K611iker'so~an ceils; H/CCs, Hensen's and/or Claudius" cells: IHCs. inner hair cells: L. lateral; tc. lateral cnsta: lss. lateral semicircular canal: OHCs, outer hair cells; .pc. posterior crista; pss, posterior semicircular canal: sac. saccular macula: SCs, supporting cells: ut, utricular macula. (The outline of the organ of Corti in panel C was reprinted from Lim and Rueda. 1992. Copyright 9 I992. with permission from Elsevier Science.) Although the cochlea arises from the ventral otocyst, it is difficult to identify a cochlea-specific c o m p a r t m e n t based on the current set of k n o w n gene expression domains. M o r e likely, cochlear d e v e l o p m e n t requires a more complex arrangement and is defined by several genes. It is notable that the epithelial-derived components of the cochlear duct are divided longitudinally into a number o f distinct tissue types (e.g., modiolus, inner suicus, inner hair cells, tunnel of Corti, outer hair cells, outer sulcus, stria vascularis, and Reissner's membrane). Some of the nested and/or partially overlapping gene expression domains in the ventral otocyst appear to presage what wilt become the long axis of the cochlea. This raises the possibility that specific combinations of genes may generate unique domains that are required to pattern the Iocation and/or specification of the distinct anatomical components observed later (as seen in Fig. 6C). In contrast to the overlapping gene expression domains that seem to predominate in the ventrM otocyst, the dorsal otocyst may be more orderly. Kiernan et al. (I998) showed that the endolymphatic duct of the chicken arises just medial to a lateral d o m a i n defined by S O H o t , as shown schematically in Fig. 7. Brigande et at. (2000b) further showed that the S O H o l domain meets the PAX2 domain at the dorsal pole of the ear, with no overlap in the expression of the two genes. That is, they appear to define a mediolateraI (ML) boundary at the dorsal pole. Recent fate mapping of the chicken otic cup suggests that there is a M L lineage boundary at this same location, and it intersects an AP b o u n d a r y that bisects the endolymphatic duct (Brigande et at., 2000b). These lineage boundaries are positioned such that c e i l - c e l t signaling across them might be involved in specifying the

location of e n d o l y m p h a t i c duct outgrowth. At the present time, there is no direct evidence to support or refute this idea. In conclusion, the possibility that the otocyst is segmented into compartments, and that these c o m p a r t m e n t s acquire their identity through selector genes, is still only a

F i g u r e 7 Model for sensow organ specification. One possible scenario for how the different sensoD' organs of the mouse inner ear mav be induced by their proximity to the compartment boundaries shown in Fig. 5. This figure is primarly derived by analogy to studies in the chick, where both the endoiymphatic duct and ~veraI sensow o~ans were shown to arise at the mediolateraI (ML) boundary' (Brigm'~deet al., 20CA)b;Kiernan et a!., 1997). In the mouse, the lateral crista has been shown to form at the bounda,D of the OtxI expression domain in anteroventrolateral otocyst (Morsli et ai., 1999). An anterior-posterior (AP) boundmD' that bisects the endolymphatic duct was demonstrated by fate mapping the chick otic cup (Brigande et al., 2000b). AC. anterior crista: AR anterior-posterior: DK dorsal-ventral: ED, endolymphatic duct: LC, lateral crista: ML, medial-lateral: OC. organ of Corti: PC. posterior crista: S, saccule: U, utricle.

I11Organogenesis

552 working model. The fact that the loss-of-function phenotypes often manifest missing structural elements is intriguing. Ultimately, though, the best evidence in support of the model would be if genetic manipulations could be designed that gave rise to homeotic transformations, that is, phenotypes in which one part of the ear is substituted for another. Barring that. it will be important to establish with certainty that there are precise lineage boundaries that correspond in some meaningful way to the gene expression boundaries. and that the two are mutually interdependent for appropriate pattern formation.

E. Compartment Boundaries and the Specification of Sensory Organs There is a stronger case to be made for a role for gene expression boundaries and the specification of sensoo' organ location. During early ear patterning in the mouse, several genes appear to label some or all of the sensoo' organ anlage prior to overt differentiation (Fig. 6B), including the secreted factor, Bmp4 (marking cristae" Morsli et aI., 1998), the Notch-pathway modulator Lunatic fringe (marking maculae and the organ of Corti: Morsli et al., 1998). and the Notch ligand, Jaggedl (marking all the sensor?; regions: Morrison et al., 1999). Together these markers have pushed back the time at which sensory anlage can now be recognized, and they suggest that the specification of sensow fate is one of the earliest events in ear morphogenesis. Shortly after otic vesicle closure, at about the time that ganglion cells begin to emigrate ventrally and the endolymphatic duct begins to evaginate dorsally, two distinct loci of Bmp4 gene expression can be found at the anterior and posterior poles of the otocyst (Fig. 6A). Interestingly. Bmp4-positive foci originate near the edge (or boundar?') of several broader gene expression domains. including Lunatic fringe in the anteroventral otocyst (Fig. 6A) and S O H o l (an Hmx gene family member) in the lateral domain in the chick (Kiernan et al., 1997). The anlagen of the lateral crista is located at the boundary of the Otxl domain (Morsli et al.. 1999). These data provide circumstantial evidence in support of the idea that the specification of sensory organ location may be mediated by the intersection of boundaries of genes with broader domains of expression. This was formally proposed as a theoretical possibility (Fekete, 1996). Here we update our model of the placement of the organs with respect to known or presumed gene expression boundaries (compare Figs. 5 and 7); we emphasize that these relationships have only been definitely demonstrated with respect to PAX2. SOHo-!. or Otxl (Fekete and Gao. 2000; Kiernan et aI., t997" Morsli et al., 1999). Note that the boundaries may be established and maintained by multiple genes, such that elimination of any one gene may not necessarily result in the absence or mislocation of a particular sense organ that arises at that boundary'.

The model is based on the idea that short-range signals may diffuse across the boundaries that dictate a sensory organ fate to the ceils receiving the signal. Diffusion across a single boundary would be expected to specify an elongated sensory patch (such as shown for the cochlea and utricle). In contrast, the intersection of two orthogonalty arrayed boundaries might generate a focal region where signals arising from the two compartments are colocalized, and the responding tissue might then be specified to generate a spatially punctate organ (such as the cristae). This model could explain both the positioning and the overall shape of the initial sensory field that is generated. Finally, the responding tissue might derive further information about the. type of sensory organ to generate by the combination of genes expressed in the larger compartment of which it is a member.

VI. Sensory Differentiation Prior to differentiation, hair cells and supporting cells undergo their final mitosis in the cochlear and vestibular sensory regions between E11.5 and birth, although auditow and vestibular sensory' regions exhibit different spatiotemporal patterns of terminal division and differentiation. In the cochlea there is a gadient of hair and supporting cell terminal mitosis starting at the apex at approximately E 11.5, and continuing around the coil until, finally, the hair ceils at the base undergo their final division between E14.5 and E15.5 (Ruben, 1967). These data led Ruben (1967) to hypothesize the existence of a zone of mitosis at the base of the cochlea, near the saccule, in which the first cells to emerge from this zone would be the cells at the apex and the production of increasing numbers of cells would account for the outgrowth of the cochlea. Marovitz and Shugar (1976) showed that this zone exists by demonstrating that cells in mitosis were only observed in this region, independent of the time point in development that was examined. Interestingly, differentiation proceeds in the opposite direction; hair cells in the base exhibited the first signs of differentiation immediately after their final mitosis, followed by the hair cells in the middle and apical regions (Lim and Anniko, 1985; Sher, 1971 ). In addition to this longitudinal gradient of differentiation, there is also a horizontal ~adient of differentiation from the modiolar to the lateral side of the cochlea, in which the IHCs begin differentiation before the OHCs. In the vestibular sensory regions there is a center to periphery gradient of terminal mitosis (Sans and Chat, 1982), with the peak of terminal mitosis occurring around E 13.5-i 5.5 (Ruben, 1967). Unlike the organ of Corti, however, differentiation in the vestibular epithelia appears to follow the same spatiotemporat pattern as that observed for the final mitosis (Mbiene and Sans, 1986). At present we do not know the signals that initiate and regulate hair cell differentiation in these prescribed patterns,

2 2 Development of the Mouse Inner Ear and as yet gradients of signaling molecutes in these different sensory patches have not been found. One obvious candidate for this differentiation signal was the invading afferent nerve fibers, as their m-rival was coincident with the first signs of hair celt differentiation (Sher, t 971). However, depriving the sensory epithium of innervation does not affect hair cell differentiation, demonstrating that the afferent nerve fibers are not the cue for hair cell differentiation (Corwin and Cotanche. 1989: Swanson et al., i 990; ,van De Water, 1976).

A. The Sensory Mosaic A Model of Lateral Inhibition? The sensory epithelium of the inner ear is composed of two basic celt types, the hair cell and the supporting cell. Exactly how these two cell t:ates are specified is unknown; although it has been shown that that the decision appears to be a binau one made at or after the final division, as lineage analysis has demonstrated that two-cell clones contain both a hair cell and a supporting cell (Fekete et al., 1998). These two ceil types are arranged in a highly organized and reproducible pattern, such that one hair cell is always surrounded by supporting cells. This alternating pattern of cell types led several researchers to hypothesize that the hair/supporting celt decision may be made through lateral inhibition, a process in which a cell that adopts a certain cell fate inhibits its neighbors from adopting the same cell fate (Corwin et al., 1991 Lewis, t 99 t). Early indications that this mechanism may be operating in the sensory epithelium came from studies in the embryonic cochlea in which nearby supporting cells were found to differentiate into hair cells after laser ablation of individual hair cells (Kelley et aL, 1995). More recently an immortalized supporting celt line has been shown to give rise to hair cells (Lawlor et aL, 1999). These data suggest that supporting cells have the ability to differentiate into hair cells (at least early in development), but normally do not do so due to lateral inhibition supplied by neighboring hair cells. In Drosophila and Caenorhabditis elegans, lateral inhibition has been shown to be mediated by cell-ceil interactions using the Notch-signaling pathway (Artavanis-Tsa_konas et al., 1995). The vertebrate homologs of the many components of the Notch-signaling pathway have been cloned and show expression in many vertebrate systems including the ear (Chan and Jan, 1999). By applying the principles learned from Drosophila about lateral inhibition and Notch signaling, a simple model of lateral inhibition can be devised to explain the hair and supporting cell patterns within the sens o ~ epithelium of the ear (Figs. 8Ai-iii, Adam et al., 1998" Lewis, 199 I). In this model, cells in the sensory epithelium represent an equivalence group and express both Notch and Delta (or any Notch ligand). For unknown or stochastic reasons. certain cells in the epithelium begin to express higher levels of the ligand and activate Notch in the surrounding

553 cells. This initial imbalance is amplified due to a inhibitory; feedback loop in which activation of Notch results in a downregulation of Delta. High levels of Notch activation eventually cause a downregulation of prosensow genes within these cells, causing them to differentiate as supporting cells, while cells expressing Delta differentiate as hair cells. Expression studies demonstrating that Notch-signaling components were expressed within the expected cells in the sensory epithelium provided the first strong evidence for lateral inhibition within the inner ear. For example, the Notch ligands De#a-like homotog ] (Dll]) and Jagged2 (Jag2) are expressed in the nascent hair cells within the organ of Corti (Fig. 6C; Lanford et aL, 1999; Morrison et al., t999), and Notcht is expressed within the supporting cells (Lanford et aL, 1999). In addition, the spatiotemporal expression patterns of Notcht, DtIt, and Jag2 during development are consistent with a role in cell fate decisions. Notchl is expressed initially throughout the organ of Corti at E t 4.5, but is downregulated in hair cells as they differentiate. In addition, both DIlt and Jag2 are expressed first within the inner hair cells between E 14.5 and E 15.5, and then later in the outer hair cells by EI6.5 (Fig. 6C), reflecting the normal horizontal gradient of differentiation that is known to occur. DtI1 has also been shown to be expressed in the nascent hair cells of the vestibular epithelium as early as E 12.5 in the cristae. One aspect of the expression patterns that is not consistent with the original model is that, of the Notch ligands examined thus far that localize to hair cells (Dit] and Jag2), neither is initially expressed throughout all cells of the epithelium. Therefore, it remains unclear exactly how these Notch tigands are expressed specifically in hair ceils. One possibility is that there is an initial bias~possibly through lineage-based determinants such as N u m b ~ a s to which cells will express Dttl or Jag2 and become hair cells (Campos-Ortega, 1997: Jan and Jan, 1998). tf the simple model for lateral inhibition within the inner ear is correct, disruption of Notch signaling should lead to an overproduction of hair cells at the expense of supporting cells. Unfortunately the proposed role for DIll within the ear has not yet been tested, because mice deleted for Dill die during early embryonic life (Hrabd de Angelis et aL, 1997; Swiatek et al., 1994). However. Jag2 knock-out mice survive long enough to study sensory differentiation within the ear; and these mice showed extra rows of both inner and outer hair cells (Fig. 8C; Lanford et aL, 1999). Interestingly, the normal spacing between hair cells was preserved, indicating that most of the supporting cells were present. These data suggest that either Jag2 is not the primary Notch ligand regulating the decision between hair cells and supporting cells in the organ of Corti, or perhaps more likely, there is some redundancy in the system (such as Dlll). Similar to Dill knock-outs, the Notch I knock-outs also die ve~' early in development (Swiatek et aL, 1994). Recently, however, the cochleas of Notch l heterozygotes were examined, and this

554

I11 Organogenesis

Figure 8 Model for creating the sensor" mosaic and pheno~'pes of mutants that disrupt the mosaic and/or sensory differentiation. ( A - G ) Schematic diagrams of surface views of the organ of Corti. Inner hair cells are identifiable by the crescent shape of their stereocilia and outer hair cells are depicted by their V-shaped bundles. Pillar cells are depicted as elongated cells that lie between the first row of outer hair cells and the row of inner hair cells. ( A - I ) All the cells of the epithelium are initially equipotent (white cells) and express Notch and Delta (or any Notch ligand). (ii) For unknown or stochastic reasons, certain cells begin to express the membrane-bound ligand Delta (black) at higher levels than in surrounding cells and activate Notch (gray) in the these cells. Notch and Delta negatively feed back on each other, so that cells that express higher levels of Delta, downregulate their levels of Notch and vice versa, thereby allowing initially small differences between cells to become amplified. (iii) Notch activation results in a signaling cascade that eventually leads to a downregulation of proneural or prosenso~' genes within these cells, causing them to differentiate as supporting cells (tan), while cells expressing more Delta differentiate as hair cells (red). ( B - I ) Mutants that show disruptions in the cellular pattern of the organ of Corti due either to defects in cell fate decisions (B-E), defects in differentiation of cell ~'pes (F-H), or defects in the control of proliferation (I). Pink cells in F are cells that express some hair cells markers but not others. The straight black lines on some of the hair cells represent the abnormal hair bundle morphology. + RA or - RA refers to experiments in which retinoic acid has been added ( + ) or depleted ( - ) in the o ~ a n of Corti. See text for references. PC, pillar cells: HC. hair cells.

22 Development of the Mouse Inner Ear analysis revealed that extra rows of outer hair celt were formed (Zhang et al., 2000), demonstrating that the gene dosage of the Notch t product is important to produce the correct pattern in the organ of Corti. A more dramatic result has been demonstrated using a Notch-directed antisense oligonucleotide in cultures of the developing rat organ of Corti, in which several extra rows were obtained for both inner and outer hair cells (Zinc et at., 2000). A similar, although slightly tess pronounced effect was obtained using a Jag i-directed oligonucteotide (Zine et at., 2000). This was an interesting result considering that, unlike the other Notch ligands, Jag! is expressed in the supporting cells of the organ of Corti (i.e.. the same cells as Notch 1), making its role somewhat unclear. These data indicate that Jag] may play a role in promoting Notch activation rather than suppressing it (lateral induction). A knock-out of Hesi, a homolog of the Drosophila haio' and enhancer-of-split genes and a probable downstream target of Notch, also results in a slight excess of hair cells in the utricle and organ of Corti (Zheng et al., 2000). Interestingly, although the Lfng knock-outs show no cochlear defects, the double knock-out of both Jag2 and Lfng suppresses the effect of the Jag2 knock-out on the inner hair cells (Zhang et at.. 2000). These results indicate that LFng probably acts to suppress Jag2-independent signaling, such as signaling via Dill or Jag1. Extra hair cell rows appear to be a common effect of disruption of the Notch-signaling components in the cochlea. Interestingly, extra rows are known to occur sometimes even in control cochleas, particularly at the apex. This suggests that the patterning of the hair cells into only four rows is a tenuous feat, and only achieved when all the signaling components are working correctly. As previously mentioned, lateral inhibition is thought to result in the downregulation of proneural genes, which in Drosophila involves members of the basic helix-loop-helix (bHLH) transcription factor family mad includes the genes of the achaete-scute complex and atonal (Campuzano and Modolell, 1992). Within the inner ear, sensor' regions express a homolog of the Drosophila gene atonal, Math1, and Mathl -/- mutants do not produce hair cells (Fig. 8B; Bermingham et al., 1999). The sensory" regions of these mutants showed that supporting cells were present, although it was not clear whether they were present in geater numbers. However, the absence of excess cell death indicated that there may have been a cell fate switch from hair cells to supporting cells in Math1 -/- inner ear epithelia. In addition, overexpression of ?r in the cochlea in which perinatal cells from the greater epithelial ridge were converted to hair cells indicates that Math1 alone can decide the hair cell fate (Zheng and Gao, 2000). Interestingly, it has been shown more recently that extra hair cells cannot be produced in cochleas that are overexpressing Hesl, suggesting there is a balance bem'een Math I and factors that inhibit hair cell differentiation such as Hesi (Zheng et aI., 2000).

555 Simpte lateral inhibition, however, cannot explain all aspects of sensor" patterning, particuIarly in the mammalian organ of Corti where the epithelium is much more organized and specialized than in the vestibular organs or the sensory epithelia of other vertebrates. Many questions remain unanswered including these: What timits the sensory' domains? How are inner hair cells generated before outer hair cells? How are inner and outer hair cells generated in such perfect rows? Examination of the hair cells and supporting ceils in the chicken cochlea revealed that the sensor~ mosaic is perfected by ceil rearrangement (Goodyear and Richardson, t997). However, the mammalian organ of Corti shows near-perfect alignment of hair cell rows from ve~/early in their development (E16.5; A. Kiernan, unpublished observations), indicating that much of the patterning in this organ may' occur prior to hair cell differentiation. Extra rows of either or both inner and outer hair cells occur in Jag2 -lcochleas (Lanford et at., 1999), Notcht +/- (Zhang et at:, 2000) cochleas, and in cochleas that had been exposed to retinoic acid (Fig. 8C; Kelley et aI., 1993), indicating that many of the cells outside the normal sensor' domain have the ability to adopt the hair cell fate, but are normally prevented from doing so by as yet unknown mechanisms. Interestingly, the expression domains of a number of other genes in the Notch pathway" as well as other signaling molecules during hair cell genesis in the cochlea suggest a more complex control over when and where hair ceil differentiation takes place than classical lateral inhibition. For example Jaggedl (Jag1), another Notch Iigand, and Lunatic fringe (Lfng), a molecule that modulates the Notch pathway (Lwine, 1999), are initially expressed throughout the prospective sensory," epithelium of the organ of Corti, and later become restricted to the supporting cells (Fig. 6C; Morrison et al., 1999; Morsli et aI., 1998). These genes may be involved in defining the borders of the sensory regions, because it is becoming increasingly clear that Notch signaling is involved in establishing boundaries between fields of cells (lateral induction) as well as playing its more weii-Known role of selecting between alternative cell fates (ArtavanisTsakonas et aL, I999; Bray, 1998). Interestingly, Bmp4 is expressed along the lateral watt of the cochlea early in development adjacent to Lfng and Jagl expression, marking the boundary between the outer hair cell region and the future Henseffs mad Claudius' cell region (Fig. 6C; Morsli et aI., 1998; Takemura et at., 1996), raising the possibility that this molecule too may" be involved in defining sensory/ nonsensory borders.

B. Hair Cell Differentiation Once the decision to become a hair celt is made, possibly through lateral inhibitor' mechanisms, a number of steps still remain to be taken before a mature hair cell is formed. An ever-increasing number of early markers of hair cell

111Organogenesis

556 differentiation have been identified. These include the transcription factor Pou4f3 (Erkman et aL, 1996; Xiang et at'., 1997), structural markers such as myosin VIIa and myosin VI (Chen and Segil, I999; Xiang et aL. t 998), and calciumbinding proteins such as cairetinin, catmodulin, and parvatbumin (Dechesne et aL, 1994; Xiang et aL, t 998; Zheng and Gao, 1997). Probably one of the earlier genes required for hair cell differentiation is the gene encoding the POU domain transcription tact or Pou4f3 (Btvz3.l/Brn-3c). A targeted disruption of this gene resulted in what appeared to be a total failure of hair cell differentiation, because none of the cells displayed stereocilia or nuclei in the correct hair cell position in the lumenal layer of the epithelium (Erkman et aL, 1996: Xiang et aL. 1997). These results suggested that perhaps Pou4f3 played a role in the initial fate determination of hair cells. However. further analysis earlier in development revealed that hair cells were initially formed in the Pou4f3 -/-~ sensory organs, since there were some cells that expressed early markers of hair cell differentiation, including myosins VI and VIIa as well as calretinin and parvalburain (Fig. 8F: Xiang et al., 19981). Furthermore. approximately 25% of the cells in the epithelium at E16.5 did show some morphological characteristics of hair cells, including the correct position of the nuclei. Numbers of these hair celllike cells were reduced over time. suggesting that these cells were gradually dying, and these results were consistent with the increased levels of cell death that were observed. Expression studies have shown that Pou4f3 is expressed immedi-

atety after the hair cells become postmitotic, and some expression was observed in cells present in the supporting cell layer. These data suggest that Pou4f3 may be expressed even before prospective hair cells migrate from the lower level o f the epithelium to the lumen. As previously mentioned, myosin VIta and myosin VI, two unconventional myosins, also mark the nascent hair cells very early in their development. The onset of their expression has been shown to occur i day after Pou4f3, and expression of these myosins was never observed in the supporting celt layer, indicating these myosins are expressed only after lumenal migration (Xiang et aL, 1998). Mutations in Mvo7a and Mvo6 have been identified in the mouse mutants shakerl and Snell's waltzer, respectively, and both mutants show stereocilia defects in the cochlea (Self et aL, 1998. I999). Interestingly, these mouse mutants displayed different phenotypes, indicating they are likely to be playing different roles in stereocilia development. For example, shaker l mutants showed early disorganization of the stereocilia (Fig. 9D), whereas Snell's waltzer mutant hair cells demonstrate early stereocilia fusion (Fig. 9C). Thus, myosin VIIa appears to be involved in hair bundle organization, whereas myosin VI seems to be involved in maintaining stereocilia integrity, possibly through anchoring of the apical membrane. A third unconventional myosin, myosin X'v, may also be involved in stereocilia development (Probst et aL, 1998). Shaker2 mutants, which are deaf and circling and display

Figure 9 Scanningelectron micrographs of the inner hair cells of several myosin mutants. (A) Control at posmataI day (P) 20 demonstrating the normal appearance of the inner hair celt :bundle morphology. (B) Shaker2 homozygous mutant at P30. Note the short, stubby sterec~ilia, multiple rows. and V shape of the bundle, which is not normally observed in mature inner hair cells. (C) SnetFs waltzer homozygote inner hair cells at P7. Note the fused stereocilia. (D) Shaker1 (Myo7a*'se`) homozygote inner hair cells at P I2. Note the disorganization of the stereocilia. Bar = 5/xm.

557

22 Development of the Mouse Inner Ear short, stubby stereocilia (Fig. 9B), were shown to have a defective M y o ] 5 gene. tn addition to the short stereocilia, shaker2 homozygotes aIso displayed tong abnormal actincontaining structures that protruded from the base of the inner hair cells. These results indicate that myosin XV may play a more general rote in actin organization in the hair ceil, although it is not clear as yet whether myosin XV is required during development or for tater maintenance of the actin cytoskeleton or both. However. M y o 1 5 transcripts have been detected as early as E t 5.5 in the mouse cochlea (Anderson et aI.. 2000), and mice and humans who have this defective gene are deaf at an early stage, indicating it is likely that myosin XV is required during the development of the hair cell. Stereocitia production is clearly a complex process and undoubtedly involves numerous other genes in addition to those myosins already mentioned. A potentially important involvement of the extracellular matrix in hair bundle devel, opment has been implicated by the finding that stereocilia are not produced normally in the utricles of mice deficient in c~s/3~ inte~in (Littlewood Evans and Muller, 2000). In addition, several mouse mutations, including Jackson shaker (js), bustling ( v ' " ), and tailchaser (Tlc), have been described as specifically affecting hair bundle formation, and all have been localized to distinct regions of the genome, indicating many different genes are involved (Kiernan et al., 1999; Kitamura et aI., I992" Moriyama et aL, 1997; Yonezawa et aI., 1996, 1999). In addition to these mouse mutants, several zebrafish mutants have also been found to display abnormal hair bundle morphology (Nicotson et at., t998). Unfortunately, despite the number of mutations that affect stereociliagenesis, hair bundle development has not been welt studied in the mouse. StereociIia development has been more thoroughly investigated in the chicken cochlea where it was revealed that bundle growth could be divided into three stages consisting of two phases of elongation separated by a phase of increasing stereocitia width (Tilney et al., 1992). In contrast, hair bundle studies in the hamster cochlea have shown that growth in both dimensions happens concurrently (Kaltenbach et al., 1994), suggesting that stereocilia growth in the chicken and mammals happens somewhat differently. Nonetheless, certain features of stereocilia development appear similar among all types of hair cells and between mammals and birds. These features include an initial ove~roduction of stereocilia, increased growth of the stereocitia nearest the kinocilium so that graded heights are achieved, and finally, reabsorption of the excess stereocilia. In addition to Notch signaling, retinoic acid signaling also appears to play an important rote in the development of the organ or Corti, although it is not vet clear whether it plays a role in cell fate decisions or in early differentiation events (Raz and Kelley, 1999). Several components of the RA pathway are present in the mouse cochlea including retinoic acid (Dolle et al., i994; Kelley et aL, 1993; Raz and Kelley, 1999; Romand et al., 1998), its receptors (Dolle et at., 1990), cel-

lular retinot binding protein (CRBP; Dolle et al., 1990), and cellular retinoic acid binding protein (CRABP; Dolle et al., 1990). Exogenous RA causes supernumera~' hair cells to develop in the organ of Corti in a time- and dose-dependent manner (Fig. 8C; Kelley et aL, t993). These new hair cells did not arise from cell division, indicating that tLA. may.. play a role in the celt fate decisions within the cochlear epithelium. Recently, retinoic acid receptor expression has been examined in more detail within the ventral cochlear epithelium and all show a dynamic pattern of expression during the time period of hair and supporting ceil differentiation (E 13 - 17; Raz and Kelley, 1999). RARc~, RXRc~, and RXRy are initially expressed widely throughout the ventral cochlear epithelium at E t4, but by E 17 were upregulated in the region of the organ of Corti. By P3, all receptors were expressed at Ngher levels in hair cells than in supporting cells. Specific blocking of the RARc~ receptor or in the production of RA results in a significant decrease in the number of cells that deveIop as hair cells (Fig. 8D; Raz and Kelley, t999). Furthermore, those small number of cells that did differentiate as hair cells did not exhibit normal cellular or bundle morphologies. Examination of early markers of hair celt differentiation showed that both myosin VIIa and Pou4f3 expression were disrupted. Interestingly, myosin VI expression was not abnormal. In contrast to the experiments with exogenous RA. these data suggested that RA may not be involved in the initial fate determination of hair cells, since hair cells begin to form normally in cochleas that have been blocked for RA signaling, as assessed by myosin VI expression. However, further work needs to be done to confirm this result, because RA signaling may not be blocked completely in these cultures, and myosin VI m ~ 7 be activated normally even with very low levels of RA.

C. Supporting Cell Differentiation Unfortunatel}~ supporting cell differentiation has not been studied in as much detail as hair cell differentiation. Because of this, many of the roles for the supporting cells throughout the inner ear are not known, although one important role for them seems to be in secreting the extracelIular structures, such as the rectorial membrane, otolithic membranes, or cupulae, that lie above the sensory epithelium in the cochlea, macutae, and cristae, respectively (Lim and Rueda, 1992; Ritey and Grunwald, 1996). Unlike the vestibular regions, the supporting celts within the cochlea are quite specialized and bear different names depending on their morphology and position (Slepecky, 1996). For example, the pillar cells are the supporting cells that develop between the inner and outer hair cells and form the tunnel of Corti. Their differentiation, which occurs primarily posmatall3~ leads to a wide separation between the inner and outer hair cells. Deiter's cells, another supporting celt type, surround the base of the outer hair cells, and extend processes m the lumenal surface. Immedi-

I11Organogenesis

558 atety next to these cells toward the lateral walt are Hensen's cells, followed by Claudius" cells. Immediately surrounding the inner hair cells are the inner border and phalangeat cells and medial to these cells are the cells of the inner sulcus. The differentiation of these supporting cells appears to depend on the correct differentiation of the hair cells, since in mutants where hair cell differentiation is disrupted, such as in the Math l -/- and the Pou4J3 -/- cochleas, many of the supporting cells do not exhibit their normal morphology (Bermingham et al.. 1999: Erkman et at.. 1996: Figs. 8B and E: Xiang et al.. 1997). As previously discussed, several early markers of the supporting cells have recently been found (Fig. 6C) including Lfng (Deiter's and pillars" Morsli et al., 1998), J a g l (Deiter's, pillars, and K611iker's organ cells: Morrison et al.. 1999). and B m p 4 (Hensen's and/or Claudius' cells" Morsli et at.. I998). In addition, the p75 low-affinity neurotrophin receptor has been shown to specifically mark the pillar cells in developing rats (Wqaeeler et aI.. 1994). The roles of these genes in the development of supporting cells is not yet known, but their identification as early markers of these different cells types will be helpful in the analysis of present and future mouse mutations that show disruptions in the differentiation of the organ of Corti. In addition to these developmental genes, a number of cytoskeletal proteins have been found to be specific to certain types of mammalian supporting cells (Slepecky. 1996), including cytokeratin and modified tubulin, although the specificity and onset of expression of most of these proteins have not been thoroughly investigated in the mouse. Pillar cells have been shown to require FGF signaling for their differentiation, since mice with a disruption in the fibroblast growth factor receptor 3 (Fgfr3) gene were deal" and did not have normal pillar cells (Fig. 8E: Colvin et al.. 1996). The pillar cells in F g f r 3 - ' - cochleas appeared immature and displayed a Deiter-like appearance. Several other cochlear regions, including the spiral vessel and the mesothelial cells beneath the organ of Corti. appeared immature in F g f r 3 - / cochleas, and outer hair cell innervation was reduced, indicating that FGF signaling is required for several aspects of cochlear maturation. Fgfr3 has been shown to be expressed in the pillar and Deiter's cells (Pirvola et al., 1995), demonstrating that the pillar cell abnormality is likely to be a primary' defect of the Fgfr3 disruption. The ligand that normally activates the FGFR3 receptor is unknown, as are the cells that produce it.

D. Control of Cell Proliferation in the Organ of Corti Unlike the sensory epithelia of other vertebrates and to some degree the mammalian vestibular organs, the mature mammalian organ of Corti shows no capacity for regeneration after damage due to environmental or genetic factors (Stone et at., 1998). The permanent toss of hair cells is tikely

to be a major factor in the pathogenesis of genetic and environmental deafness, and much interest has been focused on the possible reasons for this lack of regenerative ability. Recently, two studies of the organ of Corti in a mouse devoid of the cyclin-dependent kinase inhibitor, p27 Kipl, have revealed at least one of the factors contributing to the organ's lack of proliferative ability (Chen and Segit, t999; Lowenheim et al., 1999). The onset of p27 Kip'~ expression was shown to occur sometime between E12.5 and EI 4.5 in the nascent organ of Corti (Chen and Segil, I999), consistent with the time of withdrawal from the cell cycle (Ruben, 1967). Initially, p27 Kip1expression was observed throughout the sensory region, but by E16.5 expression was concen, trated in the supporting cell region, indicating that once hair cells start to differentiate they downregutate expression of p27 Kipl (Chen and Segil, 1999). p27 K~p~continued to be expressed in supporting cells throughout adult stages (Lowenheim et al., 1999). Both studies reported an increase in the mitotic activity of the organ of Corti prenatally and tO a lesser degree postnatally. This mitotic activity resulted in an increased number of supporting cells in the p 2 7 ~>.'-/- cochleas, although only one study reported excess hair cells (Fig. 8G" Chen and Segil, 1999). This difference was probably due to the fact that many of the hair cells were dying and Lowenheim et aI. (1999) only examined the organs at adult stages. These results suggest that p_, ~v K~p~is an important factor in the regulation of cell proliferation in the mammalian cochlea. Tight control of ceil proliferation appears to be necessary for the correct cytodifferentiation and functioning of the organ of Corti, since the structure of the p27X~,':-/cochleas was abnormal and the mice were severely hearing impaired (Lowenheim et aI., 1999). However. inactivation of the p 2 7 K~p~gene did not result in completely uncontrolled growth in the organ of Corti, particularly at postnatal stages, indicating other factors are involved. Indeed, recent evidence suggests that p I9 ~"ka4 (another cell cycle regulator) may partially compensate for the loss of p27 K~p~because double-null mice showed increased mitotic activity in the hair cell region after birth when compared to p 2 7 xes mutants alone (Chen et al., 2000).

VII. Neurogenesis Sensor" neurons can arise from two different sources: neural crest or placode. Although fate mapping of the otic ganglia has not yet been performed in the mouse, studies using quail-chick chimeras have shown that in birds the afferent neurons that innervate the ear (the eighth cranial ganglia) are largely otic placode derived, although the neural crest contributes to the glial population (D'Amico-Martet and Noden, 1983). Unfortunately little is known about how the prospective neurons are specified within the otic vesicle, although recent molecular evidence implicates an involvement

22 Development of the Mouse Inner Ear of the Notch-signaling pathway. Expression data have shown that DttI is expressed in the anteroventraI portion of the otocyst (Ma et aL, 1998; Morrison et at., I999), in agreement with the site of neurobtast emigration (Carney and Silver, 1983). This expression has been studied in more detail in the chicken, where DeltaI expression has been shown to coincide with early neuronal markers, although the two are never coexpressed within the same cell, suggesting that Deltal is downregulated prior to neuronal differentiation (Adam et at.., 1998). Based on expression, Dllt appears to be used in both hair celt and neuronal production. However, it is unlikely that the same proneural gene is used in both cases, because studies of Math] mutants have not reported a reduction in the neuronal population (Bermingham et al., 1999). However, studies have shown that another atonal-related gene, neurogenic differentiation 3 (Neurod3 or Ngnl), is required for the generation of neuronal precursors within the inner ear (Ma et aL, 1998). Neurod3 was shown to be expressed in the ventral portion of the otocyst, in the same regions as Delta l, and mice devoid of Neurod3 did not develop any sensor 5, neurons (Ma et al., 1998). Interestingly, Delta1 expression was not present in the Neurod3 -/- otocysts, indicating that Neurod3 positively regulates Dettat, as would be expected of a proneuraI gene. A more recent analysis of the Neurod3 -/" inner ears demonstrated that the sensor?" patches were smaller, particularly the saccular maculae (Ma et al., 2000). This result raises the intriguing possibility that some hair cells and sensory neurons share a common progenitor, although lineage analysis has not yet addressed this question. Another bHLH gene, Neurodl, appears to be important for neuronal development because Neurodt mutant inner ears show a severe reduction in their innervation (Liu et at., 2000). Developmental studies revealed that this reduction was due to failed delamination of the neuroblasts rather than specification, indicating that Neurodl is downstream of Neurod3. One area of neuronal development in the ear that remains largely unknown is the method by which outgrowing neurites find their way back to the ear to innervate their correct targets. As yet no mutants have been identified in which the neurons are generated correctly, but subsequently cannot find their hair cell targets. In vitro studies have shown that the otocyst secretes a factor that promotes neurite outgrowth, although the identity of this factor is not yet known (Bianchi and Cohan, 1993). One interesting observation made by Carney and Silver (1983) is that axon outgrowth seemed to occur along the same path that the neuroblasts use to migrate. Thus, they hypothesized that the neurobtasts express or secrete a factor or matrix as they are migating, thereby guiding the neurites back to their correct target. This hypothesis would require that hair cells and neurons be generated from the same region of the otocyst, which may be the case at least for some sensory, areas, since DeItal (marking the neurogemc area) and Lfng (marking at least some of the senso~

559 areas) are both expressed in the anteroventraI portion of the early otocyst (Morrison et aL, 1999; Morsli et aL, 1998). Once the axons arrive at the otocyst, their survival depends on one of two neu_rotrophic substances, neurotroplain 3 (NTF3/NT3) and brain-derived neurotrophic factor (BDNF), which are secreted by the sensory epithelium. Mice that lack both NTF3 and BDNE or their receptors, NTRK3 (TrkC) and NTRK2 (Trk~), respectively, demonstrate no afferent innervation at birth (Emfors et aI., 1995; reviewed in Fritzsch et al., 1995, t997), although one study reported only a 40% toss of afferent neurons (Minichielto et al., 1995). However, single k~nock-outs of either Bdnf/Ntrk2 or N(3/Ntr'k3 had different effects on the ear. For example, Bdnf or Ntrk2 null mutants showed a large reduction in the number of vestibular ganglion cells, which resulted in a complete absence of afferent innervation to the semicircular canals, and reduced ivmervation to the macutae (Bianchi et at., 1996; Conover et at., 1995; Ernfors et aI., 1994, 1995; Fritzsch et al., 1995; Minichielto et al., 1995; Schimmang et aL, 1995). The effects on the cochlea are somewhat controversial, because one group reported an absence of innervation to the outer hair cells (Schimmang et aL, I995), whereas another group only found a reduction in the apical portion of the cochlea (Fritzsch et aL, 1997). In contrast, NzT~3 or Ntrk3 null mutants showed a severe toss of spiral ganglion cells but only a mild reduction in the number of vestibular neurons (Ernfors et aL, 1995; Farinas et at., 1994; Minichiello et aL, t 995; Schirrmaang et aI., 1995). These differences can be explained in part by the differentiaI expression patterns displayed by B d n f and N0r in the senso~q regions of the ear, since B d n f alone is expressed in the cristae of the semicircular canals (Pirvota et al., t992, t 994). Most of the other sensory areas express both Bdnfand Nrf3 (Schecterson and Bothwell, 1994; V~eeler et aL, 1994; Ytikoski et al., 1993), suggesting that some neurons may require a specific factor (Fritzsch et aL, 1998). However, recent analysis of the expression patterns of B d n f and Ntf3 during development has suggested this is not the case, since both neurotrophic factors initially show dynamic, nonoverlapping expression patterns in the developing cochlea (Fritzsch et aL, 2000). These data indicate that the specific losses gnat are observed in the mutants are due to lack of expression of either B d n f or N rf3 factor at a specific time, rather than a requirement for a specific factor. Preliminary results from a transgenic mouse in which B d n f replaces Nrf3 showed full rescue of the N~f3 phenotype, supporting the hypothesis that inner ear neurons do not specifically require one neurotrophin over the other (Coppola et aL, 2000).

VIIi. The Stria Vascularis The stria vascularis lies on the lateral wall of the cochlear duct, and has a vital role in generating the high resting

560 potential, the endocochtear potential, in the endolymph filling the scala media. It also contributes to the unusual highpotassium, low-sodium content of the endolymph. As its name suggests, the stria vascularis contains an extensive capillavy bed, presumably to supptv the high-energy demands of generating the endocochlear potential. The remainder of the stria is composed of three ceil layers of divergent origins. The marginal cells line the lumenal surface and are derived from otic epithelium. The basal cells lie several cells thick on the lateral (outer) side of the stria, next to the fibrocytes of the spiral ligament, and these are derived from the mesenchymal cells that surround the otic vesicle at early stages of development. Scattered between the marginal and basal cell layers lie the intermediate cells. These are derived from migratory neural crest and are a specialized form of melanocyte (Hilding and Ginzberg, 1977: Steel et at.. 1992). As with other cochlear structures, the stria in the basal turn develops ahead of the apical turn. The stria first appears as a patch of electron-dense cuboidal epithelial cells sitting on a basal lamina on the cochlear wall opposite the organ of Corti..&round the time of birth, these future marginal cells start to extend fine processes from their basolateral membranes, and these ultimately begin to push through to interdigitate with processes from the basal cells, while the basal lamina between these two layers breaks down. The basal cells at the same time are condensing from the previously loosely packed mesenchymal cells, distinguishing them from the fibrocytes of the spiral ligament, which remain loosely packed. The fine membranous processes of the marginal cells next fill with mitochondria, and extend further down into the basal cell layer, and gap junctions can be seen to develop between adjacent basal cells. Blood vessels are incorporated into the stria during this period (Hilding, 1969, Kikuchi and Hilding, t 966; Steel and Barkway, 1989: Thorn and Schinko, 1985). The melanocytes migrate as melanoblasts from the neural crest, and can be detected in the mesenchyme in the vicinity of the otic vesicle from around E l 0 using a specific melanocyte marker (Cable et aI., 1995" Steel et al.. 1992). During the next 2 or 3 days, they accumulate in clusters in the mesenchyme immediately next to the developing stria, and by E16.5, the labeled melanoblasts become very closely associated with the stria. In young mice, the melanocytes took ve~, similar to normal melanocytes elsewhere in the body, growing extensive dendrites that become filled with melanosomes. However, as the mice get older, their strial melanocytes appear to become more specialized, apparently producing fewer melanosomes, and in some strains of mouse. some of these cells accumulate large amounts of electrondense material (Cable and Steel, 1991). The function of the melanocytes is not known, but in mutants which have no melanocytes in their strias, such as several alleles of dominant spotting (Kit) and steel (Mgf), no endocochlear potential is generated (Cable et al., 1994; Steel

111Organogenesis et ai., t 987). These data suggest that melanocytes are essential for the generation of this resting endocochlear potential, and explain the link between coat spotting and deafness seen in many different mammals including humans. In the absence of melanocytes, the marginal cells extend their basolateral processes as usual, but these fail to interdigitate extensively with the underlying cells, and after some week.s these processes retract (Steel and Barkway. 1989). The abnormal development and function of the stria in these spotting mutants is not caused by lack of pigment, because the stria develops and functions normally in albino animals. Melanocyte deficiencies are likely to be the cause of deafness in humans with Waardenburg syndrome, because all of the genes that have been shown to have a role in this disease, including PAX3, MITK ED,?~, and EDNRB, are known to be involved in neural crest development (Read and Newton, 1997). Apart from the genes involved in melanocyte development, little is known about the molecular basis of strial development.

IX. Future Directions Clearly our understanding of the molecular processes that underlie inner ear development in the mouse remains in its infancy. However. due in large part to the contribution of studies of mouse knock-outs and mutants (Fekete, 1999), we are starting to gain a rudimentary, understanding of how a complex structure such as the inner ear might be patterned. Large gaps in our knowledge remain including the answers to the following questions" WEhat is/are the molecule(s) that induce ear formation? How are the parts of the ear specified and are boundaries important? How large a role does lateral inhibition play in creating the cellular pattern in the sensor,,, regions of the inner ear? Because the inner ear is a fairly latedeveloping structure in the mouse, some of these questions may be unanswerable by straightforward loss-of-function studies due to early lethality, highlighting the importance of creating conditional mutants. Additionally, the issue of genetic redundancy can be addressed through creating combinations of mouse mutants, such as those created for the neurotrophins and their receptors. Misexpression experiments are also needed, especially regarding the question of boundaries. For example, moving these boundaries or creating new ones may be more informative than removing them altogether. Finally, because of the ease with which vestibular mutations and severe hearing deficits can be identified in the mouse, phenotypic approaches should be useful for identifying genes involved in ear development, such as large-scale mutagenesis schemes (see, for example, Brown and Notan, 1998; Hrab6 de Angelis and Bailing, 1998). A mutagenesis approach witl not only increase our catalog of genes involved in ear development, but also enrich for the types of mutations that can be pro-

22 Development of the Mouse Inner Ear d u c e d f o r e a c h g e n e , t h e r e b y c r e a t i n g b o t h h y p o m o r p h i c and h y p e r m o r p h i c alleles that m a y give new i n s i g h t s into g e n e function.

Acknowledgments We would like to thank Doris Wu t'or donating the picture of the paintfilled mouse inner ears (Fig. 2F) and Yehoash Raphael for donating the shaker2 scanning electron micrograph (Ng. 9B). \ ~ would also like to thank Bernd Fritzsch and Rob Grainger for helpful discussions regarding various parts of the chapter and Michele Miller Bever for valuable comments on the entire manuscript. This work was supported by the European Commission (contract CT97-2715). the MRC, and Defeating Deafness. D.M.R is supported in part by research grant 1-FY99-0483 from the March of Dimes Birth Defects Foundation and by the NtH (I3(202756).

References Abdelhak, S., Kalatzis, "v:,Heilig, R., Compain, S., Samson, D., Vincent, C.. Well. D., Cruaud, C., Sahly. I.. Leibovici, M., Bitner-Glindzicz, M...Francis. M.. Lacombe. D., Vigneron, J., Charachon, R., Boven. K., Bedbeder, P., Van Regemorter. N.. Weissenbach, J., and Petit, C. (1997). A human homologue of the Drosophila eyes absent gene underlies branchio-otorenal (BOR) syndrome and identifies a novel gene family. NaL GeneL 15, 157-I64. Acampora, D.. Merto. G. R., Paleari. L.. Zerega, B., Postiglione, M. P.. Mantero. S., Bober, E., Barbieri, O.. Simeone, A., and Levi, G. (1999). Craniofacial, vestibular and bone defects in mice lacking the distal-lessrelated gene dlx5. Development (Cambridge, UK) 126, 3795-3809. Adam. J., Myat. A.. Roux. I. L.. Eddison. M., Henrique, D., Ish-Horowicz, D.. and Lewis, J. (I 998). Cell fate choices and the expression of Notch, Delta and Serrate homoiogues in the chick inner ear: Parallels with Drosophila sense-organ development. Development (Cambridge, UK) 125, 4645-4654. Akimenko, M. A.. Ekker. M., Wegner, J., Lin, W.. and Westerfield, M. (I994). Combinatorial expression of three zebrafish genes related to distal-less: Part of a homeobox gene code for the head. J. Neurosci. 14, 3475-3486. Alvarez. I. S.. and Navascues, J. (1990). Shaping, invagination, and closure of the chick embryo otic vesicIe: Scanning electron microscopic and quantitative study. Anat. Rec. 228, 315-326. Anderson. D. W:, Probst, F. J., Belyantseva. t. A., Fridell, R. A., Beyer, L.. Martin. D. M.. Wu, D., Kachar. B., Friedman, T. B., Raphael, Y., and Camper. S. A. (2000). The motor and tail regions of myosin XV are critical for normal structure and function of auditor" and vestibular hair cells. Hum. MoL Genet. 9, 1729-11738. Arr~vanis-Tsakonas, S., Matsuno, K., and Fortini, M. E. (t 995). Notch signaling. Science 9,68, 225-232. b~-tavanis-Tsakonas, S., Rand. M. D., and Lake, R. J. (1999). Notch signaling: Cell fate control and signal integration in development. Science 284, 770-776. Bermingham. N. A.. Hassan. B. A.. Price. S. D., Vollrath, M. A.. Ben-Arie, N.. Eatock, R. A., Belten, H. J.. Lysakowski, A., and Zoghbi, H. Y. (1999). Math 1" An essential gene for the generation of inner ear hair cells. Science 284, 1837-t841. Bever, M. M.. and Fekete. D. M. (I 999). Ventromedial focus of ceil death is absent during development of Xenopus and zebrafish inner ears. J. Neuro~3'tol. 28, 78 t-793. Bianchi, L. M., and Cohan, C. S. (1993). Effects of the neurotrophins and CNTF on developing statoacoustic neurons: Comparison with an otocyst-derived factor. Dev. Biol. 159, 353-365. Bianchi, L. M.. Conover. J. C., Fritzsch, B., DeChiara, T., Lindsay, R. M., and Yancopoutos. G. D. (1996). Degeneration of vestibular neurons in

56t late emb~'ogenesis of both heterozygous and homozygous BDNF null mutant mice. Development (Cambridge. UK) 122, 1965-1973. Bosse, A., Zutch, A., Becker, M. B., Torres, M.. Gomez-Skarmeta~ J. L.. Modolell, J., and Grass, R (t 997). identification of the vertebrate Iroquois hc~meob,ox gene family with overlapping expression during early development of the nervous system. Mech. Dev. 69, 169-181. Bray, S. (t998). Notch signalling in Drosophila: Three ways to use a pathway. Semin. Cell Dev. Biol. 9, 59 t-597. Brigande. J. 'v~.Item L. E., and Fekete, D. M. (20Cg)a). A fate map of chick otic cup closure reveals lineage boundaries in the dorsal otocyst. Dev. Biol. 227, 256-270. Brigande, L V., Kieman, A. E., Gao, X., Item k E., and Fekete, D. M. (2000b). Molecular genetics of pattern formation in the ear: Do compartment boundaries pl~; a role'? Proc. NatL Acad. Sci. U.S.A. 97, 11700-11706. Brown. S. D. M., and Nolan, R M. (1998). Mouse mutagenesis-systematic studies of mammalian gene function. Hum. MoL Genet. 7, I627-I633. Cable, J., and Steel, K. R (1991). Identification of two ~'pes of melanocyte within the stria vascuIaris of the mouse inner ear. Pigm. Cell Res. 4, 87-iOI. CaNe, J., Huszar, D., Jaenisch, R., and Steel K. R (i 994). Effects of mutations at the W to,cus (c-kit) on inner ear pigmentation and function in the mouse. Pigm. Cell Res. 7, I7-32. Cable, J., Jackson, I. J., and Steel, K. R (1995). Mutations at the W Iocus M'-fectsu~'ival of neural crest-derived melanocytes in the mouse. Mech. D~. 50, I39-150. Campos-Ortegm J. A. (t 997). Asymmetic division: Dynastic intricacies of neurobtast division. Curt. Biol. 7, R726-R728. Campuzano, S.. and Modolell, J. (1992). Patterning of the Drosophila nervous system: The achaete-scute gene complex. Trends Genet. 8, 2022O8. Carney, R R., and Sitver. J. (I983). Studies on cell migration and axon guidance in the developing distal auditory" system of the mouse. Z Comp. NeuroL 215, 359-369. Cmq3enter, E. M., Goddard, J. M., Chisaka, O., Mantey, N. R.. and Capecchi, M. R. (1993). Loss of Hox-At (Hox-l.6) function results in the reorganization of the murine Nndbrain. Development (Cambridge, UK) 118, 1063-I075. Chan, Y. M., and Jan, Y. N. (1999). Conservation of neurogenic genes and mechanisms. Curt. Opin. NeurobioL 9, 582-588. Chang, ~ 2 Nunes, E D., De Jesus-Escobar, J. M., HarIand, R., and Wu, D. K. (I 999). Ectopic noggin blocks sensor" and nonsenso~" organ morphogenesis in the chicken inner ear. Dev. Biol. 216, 369-38t. Chen, R, and Segit, N. (1999). p 2 7 ( ~ p 1) links cell proliferation to mo~hogenesis in the developing oNan of Corti. Development (Cambridge, UK) 126, 158t-1590. Chen, R, Zindy, E, Roussel, M., and Segit, N. (2000). Synergistic action of Ink and Cip/Kip CDK inhibitors in the developing organ of Corti. Assoc. Res. OtotaryngoL 23 (Abstr. No. 510): also available at: http:// www, aro.org. Chisaka, O., Musci, T. S., and Capecchi, M. R. (I992). Developmental defects of the ear, cranial nerves and hindbrain resulting from targeted disruption of the mouse homeobox gene Hox-l.6. Nature (London)355, 516-520. Cotvin. J. S., Bohne, B. A., Harding, G. "v~C,McEwen, D. G., and Ornitz, D. M. (I996). Skeletal overgro~th and deafness in mice lacking fibroblast ~ow~h factor receptor 3. Nat. Genet. 12, 390-397. Conover. J. C., Erickson, J. T., Katz, D. M., Bianchi, L. M., Poueymirou, W. T.. McClain, J., Pan, L., Helgren, M., Ip, N. Y., Botand, R et aL, (t 995). Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT4. Nature (Lopag.on) 375, 235-238. Coppola, V., Kucera, J., Patko, M. E., Martinez-De Velasco, J., Lyons, V~LE., Fritzsch, B., and Tessarotlo, L. (2000). Dissection of NT-3 functions in vivo by gene replacement strategy. Soc. Neurosci. Abstr Available at: http:/Isfn.schotarone.com.

562 Cordes. S. R, and Barsh. G. S. (1994). The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor. Ceil (Cambridge. Mass.) 79, 1025-1034. Co~vin. J. T.. and Cotanche. D. A. (I989). Development of locationspecific hair cell stereocilia in denervated embD'onic ears. J. Comp. :\'eurol. 288. 529-537. Corwin. J. T.. Jones. J. E.. Katayama. A.. Kelley. M. W.. and Warchoi. M. E. (19911). Hair cell regeneration: The identities of progenitor ce!ls, potential triggers and instructive cues. In "'Regeneration of Vertebrate Sensor' Cells" (G. R. Bock and J. Wheian. eds.), pp. I03-130. Wilev. Chichester. Dailos. R (I992). The active ct~h!ea. J. Neurosci. 12. -,..~,7S_--;585. D'Amico-Martel. A.. and Noden, D. M. (i983). Contributions of placoda! and neural crest cells to a\ian craniM peripheral ganglia. A,n. J. Anat. 166, ~ 5 - 4 6 8 . Davis. H. (1983). An active process in cochlear mechanics. Hear. Res. 9. 79-90. Dechesne. C. J.. Rabejac. D.. and Desmad~l. G. (1994). Development of calretinin immunoreactivity in the mouse inner ear. J. Comp. Neuroi. ~M6, 517-529. De Moerlooze. L.. Spencer-Dene. B.. Revest. J.. Hajihosseini. M.. Rosewell. I.. and Dickson. C. (2C~3). An important role for the ItIb isofonn of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Developmen,: r UK) 127, 483-492. Deol. M. S. (1964). The abnormalities of the inner ear in kr mice. J. Ernbrwol. Exp. Morphol. 12, 475-490. Depew. M. J.. Liu. J. K.. Long. J. E.. Presley. R.. Meneses. J. J.. Pedersen. R. A.. and Rubenstein. J. L. (! 999). Dlx5 regulates regional development of the branchial arches and sensor;, capsules. Development (Cambric~oe. UK) 126, 3831-3846. Dotle, R. Rube~e. E.. Lero.v.R. Morriss-Ka.v.G.. and Chambon. R (1990). Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential pattern of transcription during mouse organogenesis. Development ;Cambrid.ge. UK) 110, 1 I ' " 1151. Dolle. R, Frautob. 'vl. Kastner, R. and Chambon. R (I994). Developmental expression of routine retinoid X receptor (RXR) genes. Mech. Dev. 45, 91-104. Dupe. V.. Ghyselinck. N. B.. Wendling. O.. Chambon. R. and Mark. M. (1999). Key roles of retinoic acid receptors alpha and beta in the patterning of the caudal hindbrain, pharyngeal arches and otocyst in the mouse. Development ( Cambridye. UK) 126, 5051-5059. Erkman. L.. McEvillv. R. J.. Luo. L.. Rvan. A. K.. Hooshmand. E. O'Connell. S. M.. Keithle?. E. M.. Rapaport. D. H.. Ryan. A. F.. and Rosenfeld. M. G. (1996). Role of transcription factors Brn-3.1 and Brn-3.2 in auditor and visual system development. Nature (London) 381, 603606. Ernfo~. R. Lee, K. E. and Jaenisch. R. (I994). Mice lacking brain-derived neurotrophic factor develop with sensory deficits. Nar.ure (London) 368, 147-150. Ernfors. P.. Van De Water~ T.. Loring. J.. and Jaenisch. R. (1995). Comp!ementar)" roles of BDNF and N~-3 in vestibular and auditor? development. Neuron 14, 1153-1164. Esteve. R. and Bovolenta. R (i 999). cSix4, a member of the six gene family of transcription factors, is expressed during placode and somite development. Mech. Dev. 85, I61-165. Farina.s.I., Jones. K. R.. Backus. C.. Wang. X. Y.. and Reichardt. L. F. (1994). Severe sensor and sympathetic deficits in mice lacking neurotrophin-3. A'amre tLondon) 369, 658-661. Favor. J.. Sandulache. R., Neuhauser-Klaus. A.. Pretsch. W.. Chatterjee. B.. Sent't. E.. Wurst. W.. Blanquet. \~. Grimes. R. Sporte, R.. and Schughart. K. (1996). The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a fatuity with renal-coioboma syndrome and results in developmental defects of the braim ear. eye. and kidney. Proc. Natl. Acad. Sci. U.S.A. 93, I3870-I3875.

II! Organogenesis Fekete, D. M. (1996). Cell fate specification in the inner ear. Curt. Opin. NeurobioL 6, 533-54 I. Fekete~ D. M. (1999). Development of the vertebrate ear: Insights from knockouts and mutants. Trends Neurosci. (in press). Fekete. D. M.. and Gao, X. (2000). A model t-or compartments and boundaries in the developing inner ear. In "'Molecular and Celt Biology of the Ear" (D. Lira. ed.). Plenum. New York (in press). Fekete. D. M., Homburger, S. A., Waring, M. T.~ Riedt, A. E., and Garcia, L. F. (1997). Involvement of programmed cell death in morphogenesis of the vertebrate inner ear. Development (Cambridge, UK) 124, 2451246I. Fekete. D. M.. Muthukumar. S.. and Karagogeos. D. (11998). Hair cells and supporting cells share a common progenitor in the avian inner ear. J. Neurosci. 18, 7811-782 I. Ffitzsch. B.. Silos-Santiago. t.. Smeyne. R.. Fagan, A. M.. and Barbacid, M. (I995). Reduction and loss of inner ear innervation in trkB and trkC receptor knockout mice: A whole-mount DiI and scanning electron microscopic analysis. Audiol. Neurosci. 1, 40 i - 4 ! 7. Fritzsch. B.. Silos-Santiago. I.. Bianchi. L. M.. and Farinas. t. (I997). The role of neurotrophic factors in regulating the development of inner ear inne~,ation. Trends Neurosci. 20, 159-164. Fritzsch. B.. Barbacid, M.. and Silos-Santiago, I. (t998). The combined effects of trkB and trkC mutations on the innervation of the inner ear. Int. J. Dev. Neurosci. 16, 493-505. Fritzsch. B.. Silos-Santiago. I.. Farinas. I., and Jones. K. (200"3). Neurotrophins and neurotrophin receptors involved in supporting afferent inner ear innervation. In "'The Neurotrophins" (I. Machete, ed.), pp. 19 r Satzburger and Graham. Gallagher, B. C., HenDy. J. J., and Grainger. R. M. (1996). Inductive processes leading to inner ear formation during Xenopus development. Dev. Biol. 175, 95 - 107. Gavalas. A.. Studer. M.. Lumsden. A.. Rijli, E M., Krum!auf R., and Chambon, R (1998). Hoxal and Hoxb 1 synergize in patterning the hindbrain, cranial nerves and second phar?/'ngeal arch. Development (Cambridge. UKI 125, 1123-I 136. Gertach, L. M., Hutson, M. R.. Germilter. J. A., Nguyen-Luu. D., Victor. J. C., and Baratd, K. E (2000). Addition of the BMP4 antagonist, noggin. disrupts avian inner ear development. Development (Cambridge, ~LK) 127, 45-54. Giraldez. F. (1998). RegionaIized organizing activity of the neural tube revealed by the regulation of imxI in the otic vesicle. Dev. Biol. 203, 189-200. Goodyear. R., and Richardson. G. (1997). Pattern formation in the basilar papilla: Evidence for cell rearrangement. J. Neurosci. 17, 6289-630t. Graham. A.. and Begbie. J. (2000). Neurogenic placodes: A common front. Trends Neurosci. 23, 313-3 ! 6. Groves, A. K., and Bronner-Fraser, M. (2000). Competence, specification and commitment in otic placode induction. Development (CambrMge, UK) 127, 3489-3499. Haddon, C. M., and Lewis, J. H. (1991). Hyaluronan as a propellant for epithelial movement: The development of semicircular canals in the inner ear of Xenopus. Development (Cambridge. UK) 112, 541-550. HadD's. T., Braun. T.. Rinkwitz-Brandt, S.. Arnold. H. H., end Bober, E. (1998). Nkx5-1 controls semicircular canal formation in the mouse inner ear. Development (Cambridge, UK)125, 33-39. Heller. N.. and Brandli. A. W (1999). Xenopus Pax-2/5/8 or~,hotogues: Novel insights into Pax gene evolution and identification of Pax-8 as the earliest marker for otic and pronephric cell lineages. Dev. Genet: 24, 208 -=.'~19. Hertwig. R (1942). Neue Mutationen und Koppellungsgruppen bet der Hausmaus. Z. indukt. Abstamm. Vererbungs!. 8I), 327-354. Hilding, D. A. (! 969). Electron microscopy of the developing hearing organ. Laryngoscope 79, 1691-1704. Hilding, D. A.. and Ginzbe~, R. D. (1977). Pigmentation of the stria vascularis. The contribution of neural crest melanocytes. Acre Oto-Lao, ngol. 84, 24-37.

22 Development of the Mouse Inner Ear Holme. R. H.. Bussoli, T. J.. and Steel. K. R (200!). Table of gene expression in the developing ear. Available at: http://www.ihr.mrc.ac.uk/ hereditary/genetable/search.html. Hrab6 de AngeIis. M., and Bailing, R. (1998). Large scale ENU screens in t~he mouse: Genetics meets genomics. Mutat. Res. 400, 25-32. Hrab6 de Aneetis. M.. McIntyre. J.. 2rid. rand Gossler, A. k 99,,). Maintenance of somite borders in mice requires the Delta homoiogue Dttl. Nature (London) 386, 717-721. Irvine. K. D. (t 999). Fringe. Notch. and m~ing developmental boundaries. Curt'. Opin. Genet. De,,. 9, 4 3 4 - ~ 1 . Jacobson. A. G. (i963). The determination and positioning of the nose. lens, and ear. I. Interactions with the ectoderm, and between the ectc,derm and underlying tissues. Ji Exp. 7~ol. 154, 273-283. Jacobson. A. G. (1966). Inductive processes in embuonic development. Science 152, 25-34. Jan. Y. N., and Jan, L. Y. (1998). Asymmetric cell division. Nature (London.) 392, 775-778. Johnson. K. R.. Cook, S. A.. Erway, L. C.. Matthews, A. N.~ Sanford, L. R. Paradies, N. E.. and Friedman. R. A. (1999). Inner ear and kidney anomalies caused by IAP insertion in an intron of the Eyal gene in a mouse model of BOR syndrome. Hum. ?Vlol Genet. $, 645-653. Kalatzis. \~. Sahly, t.. EI-Amraoui, A.. and Petit, C. (1998). Eyal expression in the developing ear and kidney: Towards the understanding of the pathogenesis of Branchio-Oto-Renal (BOR) syndrome. Dev. l~.,n. 213, 486 -499. Kaltenbach. J. A.. Falzarano, R R.. and Simpson, T. H. (I994). Postnatal development of the hamster cochlea. II. Growth and differentiation of stereocitia bundles. J. Cornp. NeuroL 350, 187-198. Kaufman. M. H. (1992). "'The Atlas of Mouse Development." Academic Press. San Diego. CA. Kelley, M. W., Xu. X. M.. Wagner. M. A.. Warchol. M. E., and Corwin. J. T. (1993). The developing organ of Corti contains retinoic acid and %rms supernumerary hair cells in response to exogenous retinoic acid in culture. Development (Cambridge. UK) 119, 1041-1053. Kelley, M. W.. Talreja. D. R.. and Corwin, J. T. (1995). Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from emb~'onic and neonatal mice. J. Neurosci. lS, 3013-3026. Kiernan, A. E.. Nunes, E. Wu. D. K.. and Fekete. D. M. (11997). The expression domain of two related homeobox genes defines a compartment in the chicken inner ear that may be involved in semicircular canal formation. Dev. Biol. 191, "~15-229. Kiernan, A. E.. Zalzman. M., Fuchs. H.. HraM de Angelis. M.. Baliing, R.. Steel. K. R, and Avraham. K. B. (1999). Tailchaser (Tlc): A new mouse mutation affecting hair bundle differentiation and hair ceil supr J. NeurocytoL ~A~,969-985. Kikuchi. K.. and Hilding. D. A. (1966). The development of the stria vascu!aris in the mouse. Acta Oto-Lac:ngoL 62, 277-291. Kitamura. K.. Kakoi. H.. 5:bshikawa, Y.. and Ochikubo, E (1992). Ultrastructural findings in the inner ear of Jackson shaker mice. Acta OtoLao'ngoL 112, 622-627. Kohan, R. (I944). The chordomesoderm as an inducer of the ear vesicle. C. R. Acad. Sci. URSS 45, 3 9 - 4 I. Krauss. S., Johansen, T.. Korzh, "v:, and Fjo~, A. ( 1991). Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development (Cambridge. UK) 113, 1 t93-1206. Ladher. R. K.. Anakwe, K. U., Gm'ney, A. L., Schoenwo!L G. C., and Francis-West. R H. (2000). Identification of synergistic signals initiaing inner ear development. Science 2 ~ , 1965-I967. Lanford. P. J.. Lan, Y., Jiang, R., Lindselt. C., Weinmaster, G.. Gridley, T., and Kelley, M. W. (1999). Notch signalling pathway mediates hair ceil development in mammalian cochlea. Nat. Genet. 21, 289-292. Lang. H., Bever, M. M., and Fekete. D. M. (2000). Cell proliferation and ceil death in the developing chick inner ear: SpatiaI and temporal patterns. J. Comp. NeuroL 417, 205-220. Lawlor. P.. Marcotti, W. Rivolta, M. N.. Kros, C. J., and Holley, M. C. (1999). Differentiation of mammalian vestibular hair cells from con-

563 ditionally immortal, postnatal supporting cells. Z Neurosci. 19, 94459458. Lawrence. R A.. and Stmhl. G. (1996). Morphogens, compartments, and pattern: Lessons from drosophila'? Celi (Cambridge, Mass.)85, 951961. Lewis. J. (199 t ). Rules for the production of sensor,, cells. In "'Regeneration of Vertebrate Sensou Ceils" (G. R. Bock and J. Whelam eds.), pp. 25-53. Wiley, Chichester. Li. C. W., Van De Water, T. R.. and Ruben, R. J. (1978). The fate mapping of the eleventh and twelfth day mouse otocyst: An in vitro study of the sites of origin of the embr?~onic inner ear sensor?, stpactures. J. MorphoL 157, 249-267. Lira, D. J.. and Anniko, M. (11985). Developmental morphology of the mouse inner ear. A scanning electron microscopic observation. Acta Oro-I~c,~ngol., SuppL 4 "~_.., t-69. Lim. D. J., and Rueda, J. (1992). Structural development of the cochlea. In ....Development of Auditory and Vestibular Systems 2" (R. Romand, ed.), pp. 33-58. Elsevier. Amsterdam. Litt lewcx~ Evans, A., and Muller, U. (206,"3). Stereocilia detects in the sensor5' hair cells of the inner ear in mice deficient in integrin alpha8beta I. Nac GeneL 2,4, 424 - 428. Liu. M.. Pereira. E A., Price, S. D.. Chu, M.. Shope. C.. Himes, D., Eatock. R. A., BrownelI, W. E., LysakowskL A., and Tsm, M. J. (200,9). Essential role of BETA2/NeuroDI in development of the vestibular and auditory systems. Genes Dev. 14, 2839-2854. Lowenheim, H.. Furness. D. N.. Kil, J., Zinm C.. Gultig. K., Fero. M. L.. Frost. D., Gummer, A. W.. Roberts. J. M., Rubel. E. "~, Hackney, C. M., and Zenner. H. R (1999). Gene disruption of p27(Kipl) allows cell proIiferation in the postnatal and adult organ of corti. Proc. Nail Acad. Sci. US.A. 96, 4084-4088. Lufkin, T.. Dierich. A.. LeMeur, M.. Mark. M.. and Chambon, R (1991). Disruption of the Hox-l.6 homeobox gene results in defects in a region corresponding to its rostraI domain of expression. Celt (Cambridge. Mass.) 66, I 105 - 1t 19. Lumsden. A., and KrumlauK R. (1996). Patterning the vertebrate neuraxis. Science 274, i t09-1 ! 15. Ma, Q., Chen, Z., del Barco Barrantes, t., de la Pompm J. L.. and Anderson, D. J. (I998). Neurogeninl is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20, 4 6 9 482. Ma, Q.. Anderson, D. J., and Fri~sch, B. (2000). Neurogenin I null mutant ears develop fewer, morphologically normal hair cells in smaller sensor' epithelia devoid of innervation. J. Assoc. Res. Otolao, ngoL 2, 129I43. Mahmood, R.~ Kiefer, R. Guthrie, S., Dickson. C., and Mason, I. (I995). Multiple roles for FGF-3 during cranial neural development in the chicken. Development (Cambridge, UK) 121, 1399-14 t0. Mmbmood, R., Mason, t. J., and Morriss-Kay, G. M. (1996). Expression of Fgf-3 in relation to hindbrain segmentation, otic pit position and phawngeai arch morphology in normal and retinoic acid,exposed mouse embryos. Anat. Embc~oL 194, !3-22. Malicki. J.; Schier. A. F.. Solnica-KrezeI. L., Stempte, D. L., Neuhauss, S. C., Stainier, D. Y., Abdelil',~h, S., Rangini, Z., Zwm"tkruis. E, and Driever, W. (1996). Mutations "affecting development of ~ e zebrafish ear. Development (Cambridge, UK) 123, 275-283. Mansour. S. L. (t994). Targeted disruption of int-2 (fgf-3) causes developmental defects in the tail and inner ear. Mol. Reprod. Dev. 39, 62-67: discussion: pp. 67-68. Manzanares, M., Trainor, P. A., Nonchev, S., Ariza-McNaughton, L., Brodie. J., Gould, A.. Marshall, H., Morrison, A.. Kwan. C. T., Sham, M. H., Wilkinson, D. G., and Krumlauf. R. (I999). The role of kreisler in .segmentation during hindbrain development. Dev. Biol. 211,220-237. Mark, M., Lufldn, T.. Vonesch, J. L.. Ruberte, E., Olivo, J. C., Dolle, P.. Gorry, P.. Lumsden, A., and Chambon, P. (1993). Two rhombomeres are altered in Hoxa-I mutant mice. Development (Cambridge, UK) 119, 319-338.

564 Marovitz, W. F., and Shugar, J. M. (t976). Single mitotic center for rodent cochlear duct. Ann. Otol., RhinoL, Lao,ngol. 85, 225-233. Marovitz, ~,~LF.. Khan, K. M., and Schutte, T. (I977). Ultrastmctural development of the early .rat otocyst. Ann. OtoL, Rhinoi., LacmgoI. 86, (Suppl.), 9-28. Martin, R, and Swanson, G. J. (1993). Descriptive and experimental analysis of the epithelial remodetlings that control semicircular canal formation in the developing mouse inner ear. Dev. Biol. 159, 549-558. Mayordomo, R.. Rodriguez-Gallardo. L.. and AIvarez. I. S. (I998). Morphological and quantitative studies in the otic region of the neural tube in chick embu'os suggest a neuroectodermal origin for the otic piacode. J. Anat. 193, 35-48. Mbiene, J. R. and Sans. A. (1986). Differentiation and maturation of the sensor?.' hair bundles in the fetal and postnatal vestibular receptors of the mouse A scanning electron microscopy study. J. Comp. Neurol. 2"M, 271-278. McKay, I. J.. Lewis, J.. and Lumsden. A. (1996). The role of FGF-3 in early inner ear development: An analysis in normal and kreisler mutant mice. Dev. Biol. 174, 370-378. Mendonsa, E. S.. and Riley. B. B. (1999). Genetic analysis of tissue interactions required for otic placode induction in the zebrafish. Dev. Biol. 206, I00-1 12. Minichiello. L.. PiehI. F.. Vazquez. E.. Schimmang. T.. Hokfelt. T.. Represa. J.. and Klein. R. (1995). Differential effects of combined trk receptor mutations on dorsal root ganglion and inner ear sensor, neurons. Development (Cambridge. UK) 121, 4067-4075. Minowa. O.. Ikeda. K.. Sugitani. Y.. Oshima. T.. Nakai. S.. Katori. Y.. Suzuki. M.. Furukawa. M.. Kawase. T.. Zheng. Y.. Ogura. M.. Asada. Y.. Watanabe. K.. Yamanaka. H.. Gotoh. S.. Nishi-Takeshima. M.. Sugimoto. T.. Kikuchi. T., Takasaka. T.. and Noda. T. (1999). Altered cochlear fibrocytes in a mouse model of D ~ ' 3 nonsyndromic deafness. Science 285, 1408 - 1411. Moriyama. K., Hashimoto, R., Hanai. A.. Yoshizaki. N., Yonezawa. S.. and Otani, H. (1997). Degenerative hairlets on the vestibular sensou" cells in mutant bustling (BUS/Idr) mice. Acta Oto-Lao'ngol. 117, 20-24. Morrison. A.. Hodgetts. C.. Gossler. A.. Hrab6 de Angelis. M.. and Lewis. J. (1999). Expression of Deltal and Serratel (Jaggedl) in the mouse inner ear. Mech. Dev. 84, 169 - 172. Morriss-Kay. G. M.. and Ward. S. J. (1999). Retinoids and mammalian development. Int. Rev. Cyto!. 188, 73 - 131. Morsli. H.. Choo. D.. Ryan. A.. Johnson. R.. and Wu. D. K. (1998). Development of the mouse inner ear and origin of its sensor, organs. J. Neurosci. 18, 3327-3335. Morsli. H., Tuorto. E, Choo. D.. Postiglione. M. R. Simeone. A.. and Wu. D. K. (1999). Otxl and Otx2 activities are required for the normal development of the mouse inner ear. Development (Cambridge. UK) 126, 2335-2343. Nicolson. T., Rusch, A.. Friedrich. R. W. Granato. M.. Ruppersberg, J. R, and Nusslein-Volhard. C. (1998). Genetic analysis of vertebrate sensor' hair cell mechanosensation: The zebrafish circler mutants. Neuron 20, 271-283. Niederreither, K., Subbarayan. "~(, Dolle. R, and Chambon. R (1999). Emb~'onic retinoic acid synthesis is essential for early mouse postimplantation development. ~ t . Genet. 21, --444-448. Niederreither. K., Vermot, J.. Schuhbaur. B.. Chambon. R. and Dolle. R (20001). Retinoic acid synthesis and hindbrain patterning in the mouse emb~:o. Development (Cambridge, UK) 127, 75-85. Nieto. M. A.. Gilardi-Hebenstreit. R, Charnay. R. and Wilkinson, D. G. (I 992). A receptor protein tyrosine kinase implicated in the segmental patterning of the hindbrain and mesoderm. Development (Cambridge, UK) 116, 1137-1150. Nishikori, T., Hatta T.. Kawauchi, H., and Otani. H. (1999). Apoptosis during inner ear development in human and mouse emb~,os: An analysis by computer-assisted three-dimensional reconstruction. Anat. Ernbo:ol.

200, 19-26.

I!i Organogenesis Oh. S. H., Johnson, R., and Wu, D. K. (1996). Differential expression o f bone morphogenetic proteins in the developing vestibular and auditor" sensor" organs. J. NeuroscL 16, 6463-6475. Pera, E., and Kessel. M. (1999). Expression of DLX3 in chick emb~,os. Mech. Dev. 89, t 89-193. Phippard, D., Lu, L., Lee, D., Saunders, J. C., and Crenshav~; E. B., 3rd (I999). Targeted mutagenesis of the POU-domain gene Bm4/Pou3f4 causes developmentaI defects in the inner ear. J. ~r 19, 5 9 8 0 5989. Pir~ola. U.. Ylikoski. J.. Palgi, J.. Lehtonen. E.. Arumae, U.. and Saarma, M. (1992). Brain-derived neuro~'ophic factor and neurotrophin 3 mRNAs in the peripheral target fields of developing inner ear ganglia. Proc. Natl. Acad. Sci. U.S.A. 89, 99 ! 5-9919. Pi~ola. U.. Arumae. U.. Moshnyakov. M., Palgi, J.. Saarma, M., and Ylikoski. J. (1994). Coordinated expression and function of neurotrophins and their receptors in the rat inner ear during target innervation. Hear. Res. 75, 131 - 144. Pir~'ola. U.. Cao. Y.. Oeltig, C., Suoqiang, Z.. Pettersson, R. E, and Ylikoski, J. (I995). The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea. Proc. Natl. Acad. Sci. U.S.A. 92, 9269-9273. Probst. E J., Fridell. R. A.. Raphael. Y.. Saunders, T. L.. Wang, A., Liang, Y.. Morell. R. J.. Touchman. J. W., Lyons. R. H.. Noben-Trauth. K.. Friedman. T. B.. and Camper. S. A. (1998). Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science 280, 1444 - 1447. Raz. Y.. and Kelley. M. W (1999). Retinoic acid signaling is necessary for the development of the organ of Corti. Dev. Biol. 213, 180-t93. Read. A. R. and Newton, V. E. (1997). Waardenbu~ syndrome. J. Med. Genet. 34, 656-665. Represa, J.. Leon. Y., Miner. C.. and Giraldez. E (1991). The int-2 protooncogene is responsible for induction of the inner ear. Nature (Lorurlon) 353, 561-563. Represa. J. J.. Moro. J. A., Gato. A., Pastor. E, and Barbosa, E. (t990). Patterns of epithelial celt death during early development of the human inner ear. Ann. Otol., Rhinol., La~'ngol. 99, 482-488. Riley. B. B., and Grunwald. D. J. (!9%). A mutation in zebrafish affecting a localized cellular function required for normal ear development. Dev. Biol. 179, 427-435. Rinkwitz-Brandt, S., Arnold, H. H.. and Bober, E. (1996). Regionalized expression of Nkx5-1. Nkx5-2. Pax2 and sek genes during mouse inner ear development. Hear. Res. 99, 129-138. Romand, R.. Sapin. V., and Dolle. R (1998). Spatial distributions of retinoic acid receptor gene transcripts in the prenatal mouse inner ear. J. Comp. Neurol. 393, 298-308. Rossel. M.. and Capecchi, M. R. (1999). Mice mutant for both Hoxa I and Hoxb 1 show extensive remodeling of the hindbrain and defects in craniofacial development. Development (Cambridge, UK) 126, 5027-5040. Ruben, R. J. (I 967). Development of the inner ear of the mouse: A radioautographic study of terminal mitoses. Acta Oto-Lao, ngol.. Suppl. 220, 1-44. Salminen. M.. Meyer. B. I., Bober. E., and Gruss. R (200(3). Netrin I is required for semicircular canal formation in the mouse inner ear. Development (Cambridge, UK) 127, 13-22. Sans, A.. and Chat. M. (1982). Analysis of temporal and spatial patterns of rat vestibular hair ceil differentiation by tritiated thymidine radioautography. J. Comp. Neurol. 206, 1-8. Schecterson, L. C.. and Bothwell, M. (1994). Neurotrophin and neurotrophin receptor mRNA expression in developing inner ear. Hear. Res. 73, 92-100. Schimmang, T.. Minichielto, L., Vazquez, E., San Jose, I., Giraldez, E, Klein, R., and Represa, J. (I 995). Developing inner ear sensor" neurons require TrkB and TrkC receptors for innervation of their peripheral targets. Development (Cambridge, UK) I21, 3381-3391. Schneider-Maunou~', S., Topitko, R, Seitandou, T.. Levi, G., Cohen-

22 Development of the Mouse Inner Ear Tannoudji. M.. Pournim S.. Babinet. C.. and Charnay, R (1993). Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. Cell ( Camb ridge. Mass.) 75, t t 99 - t 214. Schneider-Maunouu. S.. Seitanidou, T.. Charna> E. and Lumsdem A. (!997). Segmental and neuronal ~chitecture of the hindbrain of Krox20 mouse mutants. Development (Cambridge, UK) 1~4, 1215-I226. Self. T.. Mahony. M.. Fleming. J., Walsh, J.. Brown, S. D., and Steel. K. R (I 998). Shaker-1 mutations reveal roles for myosin VIIA in both development mad function of cochlear hair cells. Development (Cambridge. UK) 125, 557-566. Self. T.. Sobe, T.. Copeland. N. G., Jenldns, N. A., Avraham, K. B.. and Steel. K. R (1999). Role of myosin VI in the differentiation of cochlear hair cells. Dev. Biol. 214, 331-341. Shamim. H., and Mason. I. (t998). Expression of Gbx-2 during early development of the chick emb~'o. Mech. Dev. 76, t57-159. Sheng, G.. and Stem. C. D. (1999). Gata2 and gata3: Novel markers for early embryonic polarity and %r non-neural ectoderm in the chick embwo. Mech. Dev. 87, 213-216. Sher. A. E. (I 97I). The embryonic and postnatal development of the inner ear of the mouse. Acta Oto-Lar3;ngoL, Suppl. 285, 1-77. Slepecky, N. B. (1996). Structure of the mammalian cochlea. In "The Cochlea" (E Dallos. A. N. Popper. and R. R. Fay, eds.). Vol. 8, pp. 4 4 - I 2 9 . Springer-Verlag. New York. Steel. K. R (1995). Inherited hearing defects in mice. Annu. Rex,. Genet. 29, 675-701. Steel. K. R. and Barkw~,. C. (I 989). Another role for melanocF~es: Their importance for normal stria vascutaris development in the mammalian inner ear. Development (Cambridge, UK) 107, 453-463. Steel. K. R. Barkway. C.. and Bock. G. R. (I 987). Strial dysfunction in mice with cochleo-saccular abnormalities. Hear. Res. 27, 11-26. Steel. K. R. Davidson. D. R.. and Jackson, I. J. (t992). TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development (Cambridge, UK) 115, 111 I - 1119. Steel. K. R. Erven, A.. and Kieman. A. E. (2001). Mice as models for human hereditary deafness. In "Springer Handboook of Auditory Research" (B. J. Keats, A. N. Popper, and R. R. Fay,, eds.). Springer, New York (in press). Stone, J. S.. Oesterle. E. C.. and RubeI, E. \~,((1998). Recent insights into regeneration of auditor, and vestibular hair cells. Curt. Opin. NeuroL 11, 17-24. Swanson. G. J., Howard, M.. and Lewis. J. (t990). Epithelial autonomy in the development of the inner ear of a bird embry'o. Dev. Biol. 137, 2 4 3 257. Swiatek, R J., and Gridley, T. (1993). PerinataI lethally, and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev. 7, 2071-2084. Swiatek. R J.. Lindsell. C. E., del Amo. E F.. Weinmaster. G,, and GridIey. T. (1994). Notchl is essential for postimplantation development in mice. Genes Dev. 8 , "10/-J " " 19. Takemura. T., Sakagami, M.. Take bavashi. K., Umemoto, M., Nakase, T.. Takaoka. K.. Kubo, T.. Kitamura. Y., and Nomura, S. (1996). Localization of bone morphogenetic protein-4 messenger RNA in developing mouse cochlea. Hear. Res. 95, 26-32. ten Berge. D., Brouwer. A.. Korving, J., Martin, J. F., and Meijlink, E (1998). Prx I and Prx2 in skeletogenesis: Roles in the craniofacial region, inner ear and limbs. Development (Cambridge, UK) 125, 3831-3842. Theiler, K. (1989). "The House Mouse." Springer-Verlag, New York. Thorn, L., and Schinko, I. (i985). Light- and eleckronmicroscopic investigations of the development of the stria vascularis in the ductus cochleavis of the guinea pig fetus. Acta Anat. 12,4, 159-166. Tilney, L. G.. TiIney, M. S.. and DeRosier. D. J. (1992). Actin filaments, stereocilia, and hair cells: How cells count and measure. Annu. Rev. Cell Biol. 8, 257-274. Torres, M., and Giraldez, F. (1998). The development of the vertebrate inner ear. Mech. Dev. 71, 5-21. 9

565 Tortes. M.. Gomez-Pardo, E., and Gruss. R (!996). Pax2 contributes to inner ear patterning and optic nerve trajectow. Development (Cambridge, UK) 122, 3381-3391. Uchikawa. M., Kamachi, Y., and Kondoh, H. (1999). Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: Their expression during embry'onic organogenesis of the chicken. Mech. Dev. 84, 103- I20. Van De Water, T. R. (1976). Effects of removal of the statoacoustic ganglion complex upon the growing otocyst. Ann. OtoL, Rhino& LaungoL 85, 2 - 3 ! . Vendrelt, M. Carnicero, E., Giraldez, E. Alonso, M. T., and Schimmang, T. (200-0). Induction of inner ear fate by FGF3. Development (Cambridge, UK) 127, 2011-2019. Waddington, C. H. (1937). The determination of the auditou p!acode in the chick. J. Exp. Biol. 14, 232-239. Wang, ~(. Van De Water, T.. and LuNin, T. (1998). Inner ear and maternal reproductive defects in mice lacking the Hmx3 homeobox gene. Development (Cambridge, UK) 125, 621-634. Wheeler, E. E, Bothwell, M., Schecterson, L. C.. and yon Bartlhetd. C. S. (1994). Expression of BDNT and NT-3 mRNA in hair cells of the organ of Corti: Quantitative analysis in developing rats. Hear. Res. 73, 46-56. Whitfield. T. T., Granato, M., van Eeden, F. J., Schach, U.. Brand. M., Furutani-Seiki. M., Haiffter. R, Hammerschmidt, M., Heisenbe~, C. R, Jiang, Y. J., Kane, D. A., Kelsh. R. N.. Mullins, M. C., Odenthal, J., and Nusslein-Volhard. C. (1996). Mutations affecting developmen~ of the zebraafish inner ear and lateral line. Development (Cambridge, UK)123, 241-254. Wilkinson, D. G., Peters, G.. Dickson, C.. and McMahon, A. R (1988). Expression of the FGF-related proto-oncogene int-2 during gastmlation and neurutation in the mouse. EMBO or. 7, 691-695. Woo, K.. and Fraser. S. E. (t997). Specification of the zebrafish nervous system by nonaxiaI signals. Science 277, 254-257. Woo. K., and Fraser. S. E. (1998). Specification of the hindbrain fate in the zebrafish. Dev. Biol. 197, 283-296. Xiang, M., Gan. L., Li, D., Chert, Z. Y.. Zhou, L., O'Malley, B. "~:. Jr.., Klein, W:, and Nathans, J. (t997). Essential role of POU-domain factor Brn-3c in auditory' and vestibular hair cell development. Proc. Natl. Acad. Sci. U.S.A. 94, 9445-9450. Xiang. M., Gao, W: Q., Hasson. T., and Shin, J. J. (1998). Requirement for Brn-3c in maturation and survival, but not in fate determination of inner ear hair cells. Development (Cambridge, UK) 1 ~ , 3935-3946. Xu, R X., Adams, J., Peters, H., Brown, M. C., Heaney, S., and Maas, R. (1999). Eyal-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat: Genet. 23, 1 t 3-117. YlikosM. J., Pirvota. U., Moshnyakov, M., Palgi, J., Ammae. U.. and Saarma, M. (1993). Expression patterns of neurotrophin and their receptor maRNAs in the rat inner ear. Hear. Res. 65, 69-78. Yntema, C. L. (1933). Experiments on the determination of the ear ectoderm in the embwo of the Amblvstoma punctamm. J. Exp. ZooL 65, 317-357. Yntema. C. L. (t939). Self-differentiation of the heterotopic ear ectoderm in the embwo of the Arnbtystoma punctatum. J. Exp. ZtooL 80, I - i 5. Yntema. C. L. (I950). An analysis of induction of the ear from foreign ectoderm in the embry'o of Ambiysmma punctatum. J. Exp. ZooL I13, 211-244. Yonezawa. S., Nodasaka, Y.. Kamada, T.. Fujita, S. C., Kato, K., Yamada, Y., Ogasawara, N., and Shoji, R. (1996). Cochlear histopathology of the mutant bustling mouse. BUS/Idr. Acta Oto-La~,ngoL 116, 409-416. Yonezawa, S., Yoshiki, A., Hanai, A., Matsuzaki, T., Matsushirna, J., Kamada, T., and Kusakabe, M. (1999). Chromosomal localization of a gene responsible for vestibulocochlear defects of BUS/Idr mice: Identification as an atlele of waltzer. Hear. Res. 134, 116-122. Zhang, N., Martin, G. V., Kelley, M. W., and Gridley, T. (2000). A mutation in the lunatic fringe gene suppresses the effects of a jagged2 mutation on inner hair cell development in the cochlea. Curt. Biol. 10, 659-662.

566 Zheng. J. L., and Gao. ~ Q (1997). Analysis of rat vestibular hair cell development and regeneration using calretinin as an early marker. J. Neurosci. 17, 8270- 8282. Zheng. J. L., and Gao. ',~ Q. (2000). Overexpression of math I induces robust production of extra hair ceils in postnata! rat inner ears. Nat. Neurosci. 3, 580-586.

III Organogenesis Zheng, J. L., Shou, J.. Guillemot, E, Kageyama, R., and Gao, Vv: (2000). Hes 1 is a negative regulator of inner ear hair cell differentiation. Development (Cambridge, UK) 127, 4551-4560. Zinc, A., "van De Water, T. R., and de Ribaupierre, E (2000). Notch signaling regulates the pattern of auditor' hair ceil differentiation in mm-nmais. Development (Cambridge, UK) 127, 3373-3383.

Integumentary Structures Carolyn Byrne a n d M a t t h e w H a r d m a n School of Biological Sciences, Universi~" of Manchester, Manchester M13 9PZ United Kingdom

I. Introduction

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v

II. Mature Skin I. I n t r o d u c t i o n

!!1. Non-Neural Embryonic Ectoderm IV. Stratification

The integument, derived from the Latin integumentum meaning a "covering," includes the skin and its appendages--hair, nails, and glands. The integument provides the principal barrier between internal body structures and the environment. Part of the intense drive to understand integument biology arises from the need for treatments for human dermatological conditions and the feasibility of gene therapy in this most accessible of organs, expanding interest in transdermal delivery of DNA and drugs, and the need to appreciate and treat problems of immature skin in premature infants. A range of cosmetic applications will also arise from understanding the development and biology of integument and it appendages. Skin appendage morphogenesis and epidermal terminal differentiation, resulting in formation of the protective stratum corneum and barrier, are currently the subject of intensive research. This chapter places these events in the context of skin development from early organogenesis to birth. Studies of ma~,rn-r~aiian integument development benefit from both the tractability of mouse to genetic manipulation and a long histo~, of research exploiting experimental accessibility of chick skin. In addition, murine developmental studies and transgenic/gene knock-out models are providing

V. Dermal Development VI. Epidermal Appendage Morphogenesis VII. Model for Follicle Formation: The First Dermal Signal VIII. Follicle Spacing IX. Follicle Morphogenesis and Differentiation X, Follicle Morphogenesis and Follicle Cycling XI. Molecular Parallels between Skin Tumorigenesis and Skin Development XII. Early Terminal Differentiation Xlll. Regulation of Transit to Late Stages of Terminal Differentiation XIV, Late Terminal Differentiation: Formation of Stratum Corneum and Skin Barrier XV. Periderm Disaggregation XVi, Conclusions and Future Directions References Mouse Development

567

Copyright 9 2002 Academic Press All rights of reproduction in any form reserved.

Table I Comparison of Key Stages during Skin Development in Mouse, Human, and Chicken _- __

Moird

--

_- - - _-. -.-

.

Il111l1:111~' -

___-

~

~~IllL~cIl~

__

'l'hc iIcr's

I 7 If) 12 14 I 3 I5 14 20 20 27

23 24- 25 2s 20 26

x5 x

10 0 X5 0 5 9 12 12 I 5 I5 I 6 17 17-IX IX

I .s 2.6 1.0- 2.3 I .7 - 2.5 2.6 7 7. 0 .1 9.I

12.2 -14.2 14.2- 17.X 17.8

10 22 x I0 I0 12 22 23

N(I stiigiiig syhlelll till. lct;il period

21 55 14 21 21 2x 5 s 70 7 0 00 100

147 16X 175 1x2 210

2 2x

1 2 2 5

21 61 hl I10 I20 200 230 240 250 2x0

05 I 15 2 2 25

I S f) x I4 IF

17 175 175 1x5

25 4 5 7 0 7 2.6 I I 7 15-21 flO (15

70 75 7s xo

569

23 Integumentary Structures significant insight into human disease. Hence, developmental stages and descriptions are cross-referenced among species (Table I). Last, an extensive literature documents regulation of epidermal proliferation and terminal differentiation in adult skin, including provision of structural proteins or "markers" for specific epidermal differentiative stages. Cultured keratinocyte models closely replicate adult keratinocyte terminal differentiation and are yielding insights into regulation of differentiation. Sequential change during epidermal development broadly mirrors spatial change from basement membrane to the stratum corneum in mature skin (Fig. 1). Hence, the developing and differentiating systems are compared.

Ii. M a t u r e Skin The primary purpose of mature s ~ n is to provide a protective barrier to the environment. In addition, it forms appendages (hairs, nails, glands) that provide sensory, thermoregulator,, and sociosexual functions. During development, ectoderm- and mesenchyme-derived (or cranial neural crestderived; Le Douarin et aL, 1993) dermis are juxtaposed to produce skin. Skin comprises an outer epidermal layer in contact with basement membrane, and an inner dermis (Fig. I). lit is the epidermal component that provides b a m e r function via the outer, impermeable stratum corneum. The dermis provides support and nourishment.

Expression Domains

Figure I Diagammatic representation of adult skin. Swacturally the skin can be envisaged as three components: dermis, basement membrane, and epidermis. The epidermis can be further subdivided into tS~urI~'ers, each representing a specific terminal differentiation stage. The domains of expression for structural proteins specific to particular layers or strata are shown. An adult histological section is included, clearly demonstrating the specific skin layers. GAG, glycosaminoglycan: K5, KI4, K1. K10, keratins 5, 14. 1, and t0; PR pars papillaris; PR. pars reticularis: Tgase. trmnsglutaminase.

570 Skin serves additional functions, including an essential immunoregulatory role (Robert and Kupper, 1999; Kupper, 1996), and respirator,, secreto~ ~,excretoD, and biosynthetic roles (e.g., biosynthesis of vitamin D). Keratinocytes, the major epidermal cell, derive from stem cells located in a basal proliferative epidermal layer (Watt and Hogan, 2000; Fuchs and Segr6, 2000). Keratinocytes derived from stern cell division, termed transit a mpli~'ing cells, undergo limited proliferation in the basal layer then withdraw from the cell cycle. In response to unknown cues. the G0/G~ basal layer keratinocytes downregulate basement membrane integrin receptors, lose contact with basement membrane, and are displaced outward. Outward-bound keratinocytes alter their program of gene expression to produce protein and lipid components of the outer, protective stratum corneum. Keratin, the major protein product of epidermal keratinocytes, protects epidermis from mechanical stress (Fuchs, 1996) and participates in formation of stratum corneum (Candi et at., 1998). Stratum comeum cells (squames) are anucleate and flattened, filled internally with ag~egated keratin matrix, and surrounded by a tough, impermeable comified envelope that is cross-linked to external lipid. Outer squames are shed into the environment to be replaced by keratinocytes migrating from the basal layer. This dynamic cycle of keratinocyte production, differentiation, and shedding, is called terminal differentiation or keratinization. One of the key challenges in the epidermal field is to understand how the balance between proliferation and differentiation is achieved. Epidermal cells adhere to each other mainly via adherens junctions and desmosomes (intercellular adhesion junctions) and to basement membrane via integrin receptors. Basement membrane is anchored to underlying dermis via anchoring fibrils (Fig. 1). The principal dermal cells, fibroblasts, sit in a matrix of collagen, elastic fibers, and fibronectin. Epidermis and dermis interact during development and the adult hair cycle to produce hair, gland, and nail skin appendages. The epidermal-dermal interface can comprise a series of ridges and hollows, particuarly in nonhairy skin. Dermal protrusions into the epidermis are known as dermal papillae and epidermal protrusions into dermis are called rete ridges. Gross architecture of skin is important because it has been proposed that dermal papillae/fete ridges provide microenvironments for epidermal stem cells and transit amplifying cells (Cotsarelis et al., 1990. and references within; Jensen et al., 1999: Lavker and Sun, 2000). Evidence for stem cell rnicroenvironments in skin derives from selective labeling of slow-cycling stem cells in epidermis, and localization of cells with surface properties (e.g., integvin levels) that have been defined as characteristic of stem cells via in vitro studies (Jones et al., 1995" Tani et aI., 2000). Recently a follicular origin for epidermal stem cells has been proposed (Taylor et at., 2000; Lavker and Sun, 2000) in hair-bearing skin. In vivo stem cells are slow cycling and

iti Organogenesis hence retain DNA label, permitting detection. Follicular stem cells reside in the "bulge" area of the hair follicle (Cotsarelis et aL, 1990; Morris and Potten, 1999) and are bipotent, or able to differentiate along both epidermal and follicular pathways. Follicular bulge-derived stem cells can migrate horizontally into interfollicuiar epidermal locations where they provide a source for epidermal renewal.

II!. N o n - N e u r a l E m b r y o n i c E c t o d e r m Epidermis derives from the outer, surface cell layer (ectoderm) of the postgastrulation emb~'o. Ectoderm consists of a single layer of histologically undifferentiated epithelial cells. Single-layered nonneural ectoderm stratifies regionally to produce an outer surface layer of flattened cells, the periderm (Fig. 2; Theiler, 1989, stages 16-20; Kaufman, 1992, Plate 46, a-d). Periderm is a transitory embryonic ectodermal celt layer that provides an interface between embryo and anmiotic fluid during most of epidermal development in utero. Periderm will develop and differentiate in tandem with underlying ectoderm and will be shed into the amniotic fluid later in gestation (Boneko and Merker, 1988; Hoyes, 1968). A marker protein for periderm is keratin 6 (Mazzalupo and Coulombe, 2000. and references within). The function of periderm is still unknown. It has been proposed to play a protective role. sheltering the fragile, underlying ectoderm. In humans exfoliated periderm mixes with secreted sebum to form vernix caseosa, a slipper5; white epidermal coveting thought to act as a lubricant during birth (Agorastos et al., 1997). Periderm may play an analogous role in mouse. Occasionally in humans periderm persists until birth, forming a fetal "cocoon." A child born with this is termed a collodion baby (Akiyama et aI., 1997; O M I M ~ , MIM numbers 245130 arid 604780). Invariably, in these cases, the underlying epidermis is abnormal. Periderm appears to have an interactive role with amniotic fluid during development. The outer surface of the cells are blebbed, and covered with microvilli to increase cell surface area, and transport vesicles are evident intracelluarly. Periderm could absorb nutrients from the amniotic fluid to supply epidermal demands prior to formation of the skin capillary network (Hoyes, t 968). Early ectoderm resembles simple epithelial cells both morphologically and biochemically, because it produces simple epithelial-specific keratins K8 and K18 (Jackson et at., i981" Moll et al., 1982: Thorey et aI., t993), though the functional significance, if any, is obscure because keratin 8/18 knockout mice do not display obvious skin developmental defects (Baribault et al., 1994; Magin et at., 1998). The regional nature of postgastrulation skin becomes apparent at this stage. Ectoderm morphology varies with body site, an extreme example being the thin, stretched ectoderm overlying the neural tube. This results in developmental de-

23 Integumentary Structures

57 !

Figure 2 Diagrammaticrepresentation of epidermal development from single-layered ectoderm (E8.5-I0) to stratified postnatal skin (P0+). (a) Epidermis derives from single-layered non-neural emb~'onic ectoderm. Initial ~itoses are parallel to the skin resulting in ectodermal expansion. (b) At around E9 regional stratification gives rise to an outer layer, the pefiderm. (c)Around EI2 the orientation of basal layer mitoses changes and many are now at fight angles to the epidermis, which results in stratification and formation of the intermediate layer. (d) By EI5 cells from the intermediate layer enter terminal differentiation and markers such as K1/KI0 are induced. (e) By El6 terminal differentiation is essentially complete, marked by the appearance of a specialized outer epidermal layer, the stratum corneum (arrowhead). Toludine blue-stained histological sections accompanying each stage demonstrate dramatic increase in epidermal thickness from single-layered ectodeml (an'ow) to adult (bracket) and characteristic structural changes.

lay manifested as a middorsat stripe of lagging skin persisting to very late (E 16) in gestation. Other changes (periderm formation and induction of K5 and K14 keratins characteristic of mature epidermis; Fig. 1) occur regionally (Byrne et al., 1994). Regional ectodermal development could be due to (1) ectodermal heterogeneity, possibly mediated by H o x genes that are expressed regionally in ectoderm (Chuong et at., t 990; Couly and Le Douarin, 1990; Hunt et aL, 1991; Kanzler et at., 1994). Murine branchial ectoderm expresses the H o x code corresponding to underlying neural crest-derived tissue (Hunt et al., 1991) and it has been proposed that surface ectoderm coveting craniofacial primordia forms part of the ectomeres or developmental units comprising the central nervous system, neural crest and surface ectoderm (Couly and Le Douarin, 1990). (2) It could also be a result of programming by heterogenous underlying mesenchyme. As described below for follicle morphogenesis, development of skin and its appendages is regulated by underlying dermis. Dermis derives from diverse mesodermal sources (sornites, lateral plate, neural crest).

Ectodermal cells also display regional morphogenetic activity. Ectodermal signaling to underlying tissue, such as limb mesoderm (Johnson and Tabin, 1997), nept-~c duct (Obara-Ishihara et aL, 1999), or lateral plate mesoderm (Fun~,ama et aL, 1999), is necessary for organ specification, determination, or morphogenesis. At this stage of development ectoderm mad mesenchyme act as a regionally heterogeneous signaling organ rather than an inert surface coveting.

IV. Stratification Further stratification (Theiler, 1989, stages 2 0 - 2 3 ; Kaufman, 1992, Plate 46, c - f ) produces an intermediate proliferative ectodermal l~'er, sometimes called the stratum intermedium (Hansen, 1947; Sengel, 1976; Fig. 2). However, the resultant muttilayered structure still bears little resemblance to adult epidermis. Stratification also occurs regionally. Transcriptional control of stratification could be mediated by p63, a homolog of the tumor suppressor p53, because epidermis of homozygous p 6 3 gene-targeted mice

572 remains single layered (Mills et aI., 1999: Yang et at., 1999), probably arresting at E9.5 (Theiler, 1989, stages 16-I7). After stratification, mitoses are present throughout the basal layer, intermediate layer and periderm. It is probable that early embryonic stratification is achieved by mitotic activity, rather than via cell migration as in mature epidermis (Smart, 1970; SengeI, 1976). In postnatal epidermis and single-layered embryonic ectoderm, mitoses are mainly oriented with their spindle at right angles to the basal lamina or basement membrane, so that mitotic activity contributes to expansion of the basal layer (Fig. 2;t. Hence, during earliestectodermal development, mitotic activity contributes to ectodermal expansion that can accommodate the rapidly expanding embD;o. However, concomitant with emb~'onic stratification the orientation of a majority of mitotic spindles becomes parallel to the basal lamina, so that mitotic activity leads to stratification (Smart, 1970; Fig. 2). Embryonic proliferative stratification differs radically from stratification associated with terminal differentiation, where withdrawal from the cell cycle precedes stratification. Mitotic spindle orientation will revert to early embwonic pattern later in development, as ectoderm transits to terminally differentiating epidermis. During this phase of development, the proliferative activity of basal and intermediate layers is paralleled by basal and intermediate layer expression of keratins K I4 and K5 (Byrne et al., 1994). These keratins are usually regarded as "markers" of the proliferative basal layer in mature epidermis (Fig. l" Fuchs, 1996), so K5/K14 expression in the suprabasal layer (upper epidermal layers, not in contact with basement membrane) emphasizes the unique nature of this transitory emb~,onic skin stage. Adultsuprabasal-associated keratins (K1 and K10, Fig. 1) are not yet present. During embryogenesis expression patterns of keratins reflect differentiative changes but probably have no functional significance until birth when epidermal keratin null mice show stratum-specific blistering (Fuchs, 1996: Magin, 1998). In addition, the transit to stratification is marked by a change in integrin gene expression and protein localization. Integrins are heterodimeric (a and/3 subunit) transmembrane glycoproteins proposed to function in adhesion, cell survival, mi~ation, and epidermal terminal differentiation (Watt and Hertle, 1994; Fuchs et al., 1997). The c~6/3~subunits form part of the specialized hemidesmosome receptor for extracellular laminin from the basement membrane (Fig. t) and bind the intermediate filament network intracelluarly, while c~3/3~ subunits also provide receptors for extracellular laminin but bind the actin cytoskeleton intracelluarly. The c~6/3~ integrins adopt a pericellular organization in basal cells prior to stratification, then relocate to the basement membrane zone during stratification, concomitant with hemidesmosome formation (Hertle et al., 1991). Relocation probably correlates with adoption by ectodermal cells of Iaminin-based adhesion (Di Persio et al., 2000)

Iil Organogenesis

V. Dermal Development Dermal influences control appendage (hair, nail, gland, teeth) development and epidermal differentiation (Sengel, t976; see below), so development of dermis is key to understanding development of the integument. Dermis has diverse origins. Murine dorsal dermis derives from the dermamyotome compartment of somites (somitic mesenchyme); ventra! and limb dermis derive from lateral plate mesenchyme (somatic mesenchyme)" and much cranial mesenchyme defives from neural crest (Couly et al., 1992: Osumi-Yamashita et al., 1994). Skin appendages, such as hair, glands, and scales, are characteristic of each body region. Dermis controls regionalspecific appendage development and transplantation experiments show that this ability is acquired by mesenchymal tissue extremely early in chick development (Hamburger and Hamilton, 1951, stages 14-23 or 2 - 4 days of chick development; Mauger, 1972 Dhouaily, 1998). Since early stages of appendage formation are conserved in mouse and chick (see below), it is possible that analogous early regional mesenchymal specification occurs in mouse. In addition, chick neural tube progTams this regional appendage-forming activity in somitic mesoderm (Mauger, 1972), although the mechanism is unknown. The cellular component of dermis consists mostly of fibroblasts, which are responsible for secretion of dermal constituents. The extracellutar space between dermal fibroblast cells, with its network of macromolecules, constitutes the extracellular matrix (ECM). Mature dermis is composed primarily of fibroblast cells, collagen, elastin fibers, and an interfibritlar glycosaminoglycans (GAG)/proteoglycan gel that imparts massive water holding capacity (Fig. I). Collagen (predominantly type 1 in adult) imparts tensile strength. Interconnecting the collagen bundles are networks of e!astin fibers, composed of elastin and fibrillin, which restore the normal collagen structure following deformation. Epidermal nutrient requirements are supplied entirely by the dermis, which also acts to cushion against mechanical inju~'. The aqueous portion of the ECM permits rapid diffusion of nutrients, hormones, and metabolites and creates tu~or pressure to withstand considerable compressive force. GAGs, linked to protein as structurally diverse proteoglycans, assemble into chains and form gets of varying pore size. These act like sieves to regulate molecular traffic. They also play a major role in chemical signaling, binding various secreted signaling molecules, enzymes, and inhibitors (Keene et at., 1997). Specific proteoglycans play important roles in regulating dermal architecture. For example, d e c o r i n knock-out mice exhibit fragile skin due to abnormal fibril cross-linking (Danielson et at., 1997). Collagen fibrils of multiple subtypes aggregate to form fibers or bundles (Vuorio and de Crombragghe, 1990).

23 Integumentary Structures Dermal fibers mainly comprise collagens ~'pe I, tH, with minor components, such as v p e V (Mauger et aL, 1987). Type 1 collagen is predominant in adult skin. Type III collagen is repo~,.d to predominate in human fetal skin (e.g., Epstein, 1974), however, both collagen I and III transcripts are expressed coordinately in dermal precursors from ve~ " early in mouse development (E8.5; Theiler, t989, stage 13; Niederreither et aL, 1994). The multitude of dermatological disorders resulting from collagen defects (Uitto, 1999; e.g., OMIM, MIM numbers 120t 80 and I20I 50) combined with specific transgenic mouse models highlight the complex nature of fibrillogenesis. Type III collagen knock-out mice develop serious skin lesions (Liu et aL, 1997) and distribution and size of all types of collagen fibrils is abnormal, confirming a rote for type III collagen in mediating fibrillogenesis during development. Knock-out mice for the rvJnor type V collagen exhibit a similar phenotype of weakened skin and severe scarring (And,mkopoulos et aL, 1995); however, these mice specifically display reduced adult collagen I fibril formation, implicating t,fpe V collagen in the r%malation of type I collagen fibril formation. Murine dermal development has been systematically described (Van Exan and Hardy.. 1984) and categorized into four developmental stages. Like epidermal stratification, dermal maturation is regional in mouse and human, where cranial dermis is first to differentiate (.a&tan et aL, 1999). However, in contrast to epidermis, developing dermis displays an outward-to-inward developmental gTadient, that is, fibroblasts and mat--ix adjacent to the basement membrane are more mature than those in the deeper derrojs. The boxed material provides a systematic description of the stages of murine dermal development (Van Exan and Hardy, 1984) derived from the whisker pad, a region that develops precociously. Dermal development over the remainder of the body is regional and m ~ be retarded in comparison. The dermis greatly increases in thickness as it matures (around a sevenfold increase from stage 1 to stage 4, depending on body region) and stratifies, Wing rise to the presumptive h ~ e r m i s (Smith and Holbrook, I982; Van Exan and Hardy, 1984). Dermal expansion leads to decreased fibroblast density. This produces gaps that are filled by newly .synthesized fibers and fibrils resulting in a remafmably constant dermal density throughout development. At the epidermal-dermal junction, extracellular matrix organizes into a thin sheet, the basement membrane (Burgeson and Christiano, 1997; Bruckner-Tuderman, t 999). The basement membrane prevents contact between dermal fibroblasts and the overlying keratinocytes, selectively irur'Jbiting molecular sig-naling. It contains Iaminin, nidogen, heparan sulfate proteoglycan, the specialized network-forrning type IX.: and anchoring fibril formAng type VII collagens, and ori~nates from components secreted by both keratinocytes and fibroblasts.

573

IntegTin a~, a3 and a5 subunits dimeri_ze with a fl~ subunit in keratinocytes to form receptors for collagen, laminin-5, and fibronectin extracellularly mad attach the actin network inl~aceliularly. The arfl4 subunits form part of the hemidesmosomes, birding larainin extracellularly and intermediate filamewts inside the keratinocyle. In mature skin integrins localize to the basal sin-face of basal layer kemfinocytes (Or or are expressed peficeltularly in basal cells (aft1 heterodimers). Keratinocytes have been reported to display heterogeneous/3i subunjt levels, with stem cells located within ill-rich regions of epidermAs and transit amplifying ceils (proliferative stem cell progeny) displaying lower levels of fi~ integTin, which is inactive in keratinocytes committed to terminal differentiation (Watt and Hogan, 2000). I n t e r n receptors play a role in development of basement membrane. Mice lacking ce3 integrin, a component of the major kemtinocyte laminin r~_eptor a3/31, present defect~s in

574 basement membrane formation (DiPersio et al., 1997) starting at the point when skin has stratified and is about to enter terminal differentiation. Mice lacking c~5 integrin, part of the c~5/3~ fibronectin receptor, are also embryonic lethal with mesodermat defects (Yang er al., t993) and are probably defective in fibronectin matrix assembly during development (Vv~nnerberg et al., 1996). Mice lacking/3,, integrin present detects in processing of laminin-5 (Brakebush et al., 2000: Raghavan et ai.. 2000).

VI. Epidermal Appendage Morphogenesis During the stratification period in mice. epithelial appendages (hairs, nails, glands, teeth: Theiler. 1989. stages 21-25: Kaufman. 1992. Plate 46. e-l) form from ectoderm and mesenchyme by a well-characterized program of cell movements and proliferative change (Fig. 3). The initial stages of all appendage development (e.g., stages 0-3: Davidson and Hardy. 1952: Fig. 3) are similar, whereas later stages diverge to give rise to different appendage types. Appendage development involves a binary' decision by ectodermal cells regarding epidermal or appendage fate, followed by morphogenesis and differentiation. In situ hybridization and immunohistochemicaI studies show that major signaling molecules associated with follicle morphogenesis are conserved between mouse and chick (see below), and mouse-chick tissue recombinations can successfully support early stages of appendage development (Dhouailly et al.. 1998). Hence, the following description of follicle formation will draw from both systems. Traditional approaches (Sengel, 1990) involved use of epithelial-mesenchymal separation followed by recombination of tissue from different developmental stages, body sites, or species to decipher contribution of each tissue to appendage development. Morphological analysis of the appendage formed by the epithelial-mesenchymal recombinants can demonstrate the controlling tissue at each developmental stage because follicle morphology and density vary over the body surface in a species and developmentalstage-specific manner (Fig. 3). These studies showed that follicle morphogenesis involves a scheme of reciprocal interaction or signaling between mesenchyme-dermis and epithelium (Sengel, 1976; Hardy, 1992: Dhouailly et al., 1998; Chuong and Widelitz, t998; Paus and Cotsarelis, 1999; Paus et at., 1999a). Mesenchyme signals first to ectoderm with the message "make an appendage," and it seems that any ectodermal tissue (and some nonectodermal epithelial tissues) is competent to respond. This primary" message is probably transient and can operate across species. Ectoderm responds to the primary message by forming an ectodermal placode or cluster of elongated cells (Fig. 3, stage 0). The ectodermal placode becomes partly autonomous and can signal back to

tl! Organogenesis unspecified dermis to render it morphogeneticatly active. Mesenchymal cells aggregate under the ptacode and form a signaling center that persists in the mature follicle as the dermal papilla (Wessells, I965; Senget, I976; Fig. 3). Subsequent dermal messages to ectoderm can include information (and apparently detailed instructions) about making regionspecific and species-specific appendage types. Subsequent morphological change accompanying follicle formation is described in Fig. 3 using the staging of Hardy (t969), as modified by Paus et at. (t 999a). Recently many of the moIecular players in this story have been identified (Oro and Scott, 1998).

Vii. Model for Follicle Formation: The First Dermal Signal Initiation of follicle formation is controlled by dermis. The succession of events in follicle formation has been dissected in chick epidermis because here appendages form sequentially within a two-dimensional array of rows and files called feather "tracts" (Fig. 4), whereas the pattern and sequence of follicle formation is less ordered and defined in mice (except for specialized areas such as the vibrissae pad; Fig. 5). Placodes form first in an initial row. A wave of appendage-specifying ability or "morphogenetic activity" has been proposed to propagate from row to row across the tract, with new rows induced by the preceding row (Sengel, 1976; Patel et al., 1999), and experimental evidence for waves is provided using spatially separated incisions to disrupt waves in embryonic skin organ culture (Patel et al., 1999). Alternatively, a temporal differentiation gradient is proposed across the array (Davidson, 1983) or a moving "'morphogenetic stripe" (Jiang et aI., 1999). The morphogenetic stripe confers competence for follicle formation as underlying mesenchymal cell density increases. This latter theo~ receives experimental support from tissue reconstitution experiments where disaggregated and reconstituted mesenchymal cells from a tract support simultaneous follicle formation as soon as a permissive concentration of mesenchymal cells is provided (Jiang et aI., 1999). Propagation of the "morphogenetic stripe" or establishment of the temporal differentiation gradient still requires explanation. The mode of follicle formation via waves or differentiation gradients, as illustrated by feather morphogenesis, is probably conserved in mammals. Murine vibrissae follicles form as an array (Fig. 5; Kaufman, i992, plate 49). Embryonic hair follicle formation occurs first at day 13 (Theiter, 1989, stage 21) on the shoulder in mouse and successive waves of follicle formation move caudally, anteriorly, ventrally, and dorsally (Mann, 1962; Kaufman, 1992). Waves of guard hairs (t)qotrichs or monotrichs; Fig. 5) appear first, followed by awls at day t 6 (Theiler, 1989, stage 24) and auchennes/zigzags at day 18 (Theiler, 1989, stage 26). A cra-

23 Integumentary Structures

575

Figure 3 The eight stages of hair follicle development described by Davidson and Hardy (1952). Stage 0: Ectodermal placode formation. Stage t: Derm-~ fibroblasts aggegate below the epidermal placode. Stage 2: Prominent hair :germ. Stage 3: Hair peg cavity formed by invasion of aggregated dermal fibrobIasts. Stage 4: Inner root sheath cone apNars. Stage 5: Bulbous peg stage, inner root sheath elongates and the bulge appears. Stage 6: Early sebaceous gland, hair shaft appears and dermal papilla becomes enclosed. Stage 7: Further elongation and hair shaft development. Stage 8: Hair shaft emerges. Note the significant increase in length following stage 5. Inset, post-stage 3 upward movement and differentiation of files of cells from the germinative matrix population (pink) gives rise to all the structures of the mature hair follicle (see key). nial-to-caudal wave of follicle formation also occurs in human infants (Holbrook et al., 1988). T h e presence of waves of developmental change is also important w h e n considering follicle cycling and late terminal differentiation o f epidermis (see below).

The identity of the first dermal signal is unknown, however, the signal could be a W n t molecule or another activator o f / 3 - c a t e n i n signaling ( N o r a m l y et al., 1999). W n t s are a family of evolutionarily c o n s e r v e d secreted factors h o m o l o gous m the Drosophila wingless protein (Peifer and Polark5s,

Figure 4 Model of feather tract patterning proposed by Jung e~ al. (1998) and Noramly et al. (t999). This model incorporates both lateral inhibition and reaction-diffusion mechanisms. (a) Within each tract a long-range dermal inductive signal crosses the skin as a moving morphogenetic front (arrows). (a. b) The dermal inductive signal induces ectodermal feather placodes, initially within a single row in a rostral-to-caudal progression. Induced ectodermal feather placodes express a local profollicular signal that determines placodal size and a diffusable inhibitor" signal that determines placodal spacing by lateral inhibition (see e). (c. d) New placodes only form when the long-range inductive signal passes the individual zones of placodal inhibition.

Figure 5 Pelage and vibrissae are the best characterized and most frequently studies of the eight hair ~]~es present in the mousse. The mouse coat. composed of pelage hairs and coveting the majori~; of the body, presents severn hair types. Fine hairs (auchene. zigzag) are thinner, with less medulla space and one or more bends. The coarser guard and awl hairs are thicker and significantly longer with more medulla cells. Note the smaller size compared to mystacial vibrissae hair. Mouse vibrissae follicles can be subdivided into major mystacial (colored), secondary (blue shades), and supernumerary (black) groups. In an El2 mouse emb~,o the individual vibrissae display a caudorostral development gradient (Van Exan and Hardy, 1984). The most mature follicles (Davidson and Hardy. 1952, stage 1) are at the caudal edge of the whisker pad in horizontal rows 3 to 5 and vertical row number 6. Conversely, the least mature follicles (stage 1) are t2~ose at the rostralmost edge of the whisker pad.

577

23 Integumentary Structures 2000; Nusse, 1999). Signaling by Wnt stabilizes/3-catenin, which then translocates to the nucleus where it can interact with Lefl/TCF (lymphoid enhancer-binding factor 1/T-celtspecific transcription factor) to regulate expression of downstream genes. Experimental activation of epithelial/3-catenin signaling (by expression of N-terminal truncated, constitutively stabilized forms) induces ectopic follicles in both mouse and chick skin (Gat et aI., I998; Noramly et aL, t999). Conversely, downregulation of /3-catenin signaling (through Lefl knock-out; van Genderen et aL, 1994) results in loss of vibrissae arid some pelage follicles in mice (Fig. 5), whereas expression of Lefl in epidermis of transgenic mice can cause ectopic follicle formation (Zhou et al., 1995). Nuclear-localized/3-catenin, indicative of/3-catenin signaling, appears diffusely across the skin of the feather tract as one of the earliest indicators of feather formation (Noramly et aI., t 999). As the placodes form,/3-catenin signaling concentrates at the nascent placodes. Experimental activation of ectodermal fl-catenin signaling in chick skin not only results in new" placode formation in skin areas that could not normally support follicle formation but induces a battery of downstream genes normally associated with follicle formation (Noramly et aI., 1999). It is proposed that the initial dermal signal that induces ectodermal/3-catenin signaling has been experimentally short-circuited, resulting in ectoderm that has become an autonomous signaling center able to support follicle formation, a stage predicted by early skin biologists. The identity of the initial dermal signal awaits experimental verification. Nuclear-localized fl-catenin expression is not the only molecular change that occurs ahead of the wave of ptacode formation. Bmp2, BmpZ Lnfg, Wnt7a, and follistatin (Jung et al., 1998; Crowe et al., t998; Noramly and Morgan, 1998; Noramly et aI., 1999; Widelitz et aI., 1999; Patet et aI., 1999) also appear diffusely in the ectoderm prior to placode formation. In fact, ectopic fi-catenin signaling is reported to induce Bmp2, Bmp7, and Lnfg (Noranaly et aL, 1999) and ectopic follistatin expression to induce Bmp2, Bmp4, Bmp7. and Shh (Patel et aL, 1999). These molecules will later localize to the ectodermal placode. In addition, the Notch family members and their ligands (Notch], Notch2, Delta1, Serratet, and Serrate2) are expressed diffusely first, then later localize to the placode (Chen et al., t 997; Crowe et al., 1998). The appearance of these molecules in a diffuse wave in the ectoderm has led to the proposal that the initial dermal inducer acts homogeneously and uniformly on tract ectoderm (Fig. 4) rather than inducing single follicles in a localized manner. If so, then spacing of the follicles would be mediated by a separate localized response of ectoderm (Jung et al., 1998; Norm-r@ and Morgan, 1998; Noramly et at., 1999" see below).

VIII. Follicle Spacing Murine pelage follicles are regularly spaced during development and are initially patterned hexagonally, as in chick skin (see Zhou et al., 1995). Spacing of follicles and placodes within arrays is thought to be self-organizing and operates by lateral inhibition from previously formed placodes (Claxton, 11966; Ede, i972; Davidson, t983). Molecules that localize to the nascent follicles include Fgf4, Bmp2, 4 and 7; follistatin, noggin; Wnt7a, Writ 1t" WntlOb; Notchl and 2 / Delta and Shh and patched (Ptch) (Bitgood and McMahon, t995: Chuong et aL, I996; Ting-Berreth and Chuong, 1996; Crowe et aI., 1998; Jung et aI., t998" Morgan et at., t998; Noramly al-td Morgan, 1998" St. Jacques eta!., 1998; Patet et al .... 1999). Ectopic expression studies show that Fgf4, foItistatin, noggin, and Shh are activators of follicle development, while BMPS are follicle inhibitors. The profollicular FGFS are thought to antagonize B MPS. Follistatin and noggin bind to BMP molecules axld inhibit BMP "proepiderreal" activits; in a manner rerpSniscent of early neural induction (Normaly and Morgan, 1998" Jung et aL, 1998; Patel et aI., t 999; Jiang et at., 1999; Botcb2=:arev et ai., t 999). Cm-rent models for fotlicte spacing place both activators and inhibitors within a single source and propose differences in diffusion capabilities between activators arid inhibitors (Fig.. 4). Activators act locally, while inhibitors diffuse over a greater distance. This concept involves application of the reaction-diffusion model for generation of a repetitive pattern to skin (Nagorcka and Mooney, 1985). Activators determine the size of the follicle domain, while inhibitors define follicle spacing. Locally acting Bmp inhibitors (follistatin, noggin) neutralize Bmp activity within the placode, leaving follicle activators (e.g., FGF4) to predominate (Fig. 4). Brnps, the follicle inhibitors, are proposed to have geater diffusion capabilities and are dominant and active in the interfollicular space where they inhibit additional follicle formation within their zone of influence. The ability of Bmps to diff-ase over distances as high as 20 ceil diameters (Nellen et at., 1996) lends some support to this theory. In contrast, follistatin bound to the GAG heparin sulphate in the extracellular matrix would be loca2[Iy sequestered (Hashimoto et aL, 1997). Activity of factors within the follicle could be reinforcing. For example, follistatin upregutates profollicutar F j 4 expression (Patel et ai., I999). The model, largely derived in chick, has receiwM confirmation in mouse by demonstration of retarded follicle formation in noggin knock-out mice and opposing and interacting effects of exogenous Noggin and BMP4 on folIMe size in mouse embryonic skin organ culture (Botchkarev et aL, 1999). A number of simplified models for follicle induction have been proposed (e.g., Fig. 4; Jung et aL, 1998" Noramly and Morgan, 1998; Oro and Scott, t998; Jung and Chuong,

578

111Organogenesis

1998" Noramly et aI., 1999) that axe compatible with early models derived from epitheliat-mesenchymal recombination experiments. The first dermal message is homogeneous (consistent with diffuse streaks of signaling molecules preceding the placode induction wave). However. both positive and negative signals within the ectoderm restrict response to the primary dermal signal. Locally acting positive signals and diffusing negative signals derive from the placode. The negative signal is proposed to be BMP2. which patterns the ectoderm via lateral inhibition from placodal sources (Jung et al., 1998: Noramly and Morgan. 1998). Positive signals counteract the negative signals locally and provide new sources of positive signaling molecules that reinforce the proplacodal message and induce early morphogenesis.

IX. Follicle Morphogenesis and Differentiation Follicle morphogenesis and differentiation are being studied in mouse (Philpott and Paus. 1998) using skin culture models and the theories verified in transgenic and knock-out mice. The same molecules involved in follicle induction and spacing are reused in later stages of follicle morphogenesis. A full molecular description is beyond the scope of this report. However, analysis of the role of Shh. one of the best characterized molecules, in follicular differentiation illustrates how information from multiple sources gives an integrated view of the functional role of SHH. After establishment of the placode, Shh signaling within the epithelial component of the follicle is proposed to control proliferation and epithelial growth in hair/feathers (TingBerreth and Chuong, 1996: Morgan et al., 1998: St. Jacques et aI., 1998: Chuang and McMahon, 1999: Jung et al., 1998) and additional epithelial appendages (e.g., Dassule and McMahon, 1998; Jung et at., 1999). S h h -/- murine embryos show histologically normal follicular development during early stages of hair follicle morphogenesis (Chuang and McMahon. 1999; St. Jacques et at., t 998). Epithelial placodes and adjacent mesenchymal aggregates form, but subsequent morphogenesis is arrested, despite expression of some advanced-stage follicle differentiation markers. Reduction in proliferation is seen within the invaginating ectoderm. SHH promotes keratinocyte proliferation as well as opposing p21-mediated withdrawal of differentiating keratinocytes from the cell cycle in culture models (Fan and Khavafi, 1999). This is consistent with the reduction in proliferation in knock-out mice. Expression studies bear out this proliferation-inducing role for SHH in late follicle development. Shh is expressed in epithelial cells, first within the central region of the plaque, and later in the invaginating epithelial cells that maintain contact with the mesenchymal aggregate as it matures and forms the dermal papilla; that is, it is expressed

within cells that will eventually form the epithelial matrix (Fig. 3). Prolonged expression within the developing matrix cells could indicate repeated or sustained signaling. As the matrix matures, SHH changes expression patterns and. in hair. localizes to specific matrix lineages that give rise to an inner root sheath compartment (Bitgood and McMahon, 1995" Fig. 3). Hence, during late stages of hair development, Shh probably adopts a new role in elaboration of the inner root sheath lineages. A specific role for Shh in late stages of feather or hair maturation is also indicated by expression analysis (Ting-Berreth and Chuong, 1996; Morgan et aI., 1998). However, functional confirmation is frustrated by growth arrest or distortion at early stages of appendage development in S h h misexpression studies in chick and embryonic lethality of S h h -/- mice.

X. Follicle Morphogenesis and Follicle Cycling In adult mice follicles undergo cycles of regression and re-formation called the hair cycle. The adult hair cycle consists of anagen (growth phase), catagen (regression phase), and telogen (resting phase; Paus and Cotsaxetis, t 999). During catagen the lower portion of the follicle regresses via apoptosis of keratinocytes and movement of the dermal component (dermal papilla) up beneath the bulge (Fig. 3" Paus and Cotsarelis, 1999). Molecular interaction between the dermal cells and the bulge will result in formation of the new follicle. It is the length of anagen that determines hair length and this varies over the surface of the mouse and between strains. For example, F g f 5 -/- mice have an extended anagen reproducing the long-haired pheno~pe of fine angora mouse mutant (Hebert et al., t994), while other transgenic/knockout mice can have shorten hairs resulting from truncated anagen (e.g., Charpentier et al., 2000, and references within). Human conditions (alopecia, hirsutism; e.g., OMIM, MIM numbers 300042 and 142625) can arise from irregularities in the cycle. Regulation of follicle cycling is the subject of intense speculation and a "hair cycle clock" is proposed (Paus et al., 1999b). During postnatal development in the mouse, the first few hair cycles are synchronous then later become async~onous, as in human. The progression of follicles through the stages of the hair cycle occurs regionally in adults, resulting in waves of follicle cycling. Postnatal moving fronts or waves associated with follicle cycling (cranial to caudal, and ventral to dorsal) are well documented in adult mouse (Powetl and Rogers, 1990, and references within). Many of the molecular players associated with follicle development are also involved in regulation of follicle cycling. Onset of anagen may reproduce the changes associated with follicle development (signaling between dermal and epithe-

23 Integumentary Structures tia cells). For example, ectopic Shh expression in adult mice forces resting stage (telogen) follicles back into the gowth phase (anagen" Sato et al., 1999).

XI. Molecular Parallels between Skin Tumorigenesis and Skin Development Constitutive activation of two of the major skin development signaling pathways (Shh and Wnt pathways) leads to skin tumors in adult. Adult epidermis and its appendages continually cycle, providing ample opportunity for pathway activation to reprogam cell fate. Mutations in the SHH receptor Ptch underlie basal cell Nevus (Gorlin) syndrome (OMIM, MIM number 109400), a disease causing developmental defects and increased frequency of tumors, such as basal cell carcinoma (Hahn et aL, 1996, 1999; Johnson et aI., 1996). In addition, ptc mutation or allele loss occurs at high frequency in sporadic basal cell carcinomas (BCC; Johnson et at., 1996; Gailani et aL, 1996). ptc is a negative regulator of Shh signaling and can be considered a tumor suppressor. Conversely, Shh pathway activators should predispose to BCC. In fulfillment of this prediction it was found that overexpression of Shh in transgenic mouse skin results in "tumorlike" epidermal growths emanating from follicles with histological and behavioral similarities to BCC (Oro er at., 1997). Similar results are obtained in human (Fan et al., 1997) and chick skin (Ting-Berreth and Chuong, 1996; Morgan et aL, 1998). Hence, pathway activation seems to predispose to tumorigenesis via increased proliferation and expansion of developing or cycling hair follicles. This is consistent with the role of Shh in follicle development. GLI 1 transcription factor activation is a downstream outcome of Shh signaling and, interestingly, overexpression of GLI1 in amphibian skin also induces epithelial tumors, with similarity to mammalian BCC (Dahmane et at., t997). This strongly suggests that pathway activation, by any means, underlies BCC. Therefore, mutations that activate fine Shh pathway should cause tumors. A conserved Shh mutation has been detected in BCC and breast carcinoma (Oro et al., 1997: OMIM, MIM number 602968), and activating mutations in smoothened (smo, a membrane receptor normally repressed by ptc binding, but activated after Shh binding to ptc) occur in BCC (Xie et aL, 1998). Hence, Gilt transcriptional activation of (as yet unknown) downstream genes may underlie BCC. To date, kaaown targets activated by Glil include Ptch, Gill itself, and Hnf3fi (Foxa2) in certain tissues. The Wnt signaling pathway also appears upregulated in epithelial tumors. Stabilization of fl-catenin, through truncation of the fl-catenin phosphorylation site, causes ectopic follicle formation as described previously, but also causes tumors in transgenic mice (Gat et aL, t998). A variety of tumors result from constitutive signaling through stabilized

579 fl-catenin, most notably colon cancers where mutation in APC (adenomatous polyposis c o l i ~ a pathway component invotlved in degradation of/3-catenin; OMIM, MIM number 175100) can promote stabilization and constitutive signaling (Kinzler and Vogelstein, 1996). Constitutive activation of the pathway through fi-catenin stabilization can also result in tumors in a variety of celt types (Gat et at., t998, and references within) and skin pilomatricoma tumors show a high frequency of mutation in the N-termAnal phosphorylation site of fl-catenin that targets fi-catenin for degradation (Chart et aI., 1999; OMIM, MIM number 132600). The downstream targets of/3-catenin signaling involved in tumorigenesis are uncertain. Identification of fl-catenin/ LEF/TCF downstream targets Xnr-3 (McKendry et aL, 1997), Siamois (Brannon et at., i997; Fan et al., 1998), Twin (Laurent et at., 1997), and possibly Noggin (Tao et al., 1999) reflects fl-catenin-mediated specification of dorsal body tissue, because these molecules locate to and induce Speman organizer activity, though Noggin has been shown to have a role in skin development. Examples of additional direct downstream genes include the proto-oncogene cMyc (He et at., t998) and cvcIin Dt, both regulators of cell cycle progression (Tetsu and McCormick, 1999; Shtutrnan et aL, 1999). This is consistent with the potential for nonmembrane B-cawnin to act as an oncogene (Eastman and Grosschedt, 1999: Nusse, t 999).

XII. Early Terminal Differentiation Following stratification in mouse (E t 4 - I 6, Theiler, i 989, stages 22-24; Kaufman, 1992, Plate 46, e - j ) basal ectodermal cells enter a program of terminal differentiation, marldng the point where ectoderm begins to resemble postnatal epidermis. Terminal differentiation is the process whereby mitotically active cells in the basal layer cease proliferation, downregutate integaqn receptors, lose contact with basement membrane, and move to the surface (Fig. 1). Su~'ace-bound ceils alter their program of gene expression to produce protein and lipid required for assembly of surface stratum corneum. Stratum corneum is the tough, wateriml:uermeabte structure that provides the major environmental barrier in postnatal epidermis and its production is the endpointwand apparent purpose--of epidermal terminal differentiation. Adoption of a terminal differentiation program is associated with imposition of the mature pattern of marker distribution. Hence, K_5/K14 keratin mm-kers become basally associated and K t/KI0 keratins are induced in suprabasal layers (Fig. 1). Regulation of embryonic transit to terminally differentiating epidermis is not understood. However, by analogy with control of t~rninal differentiation in adult skin it seems probable that the process is mediated by a change in the intracellutar calcium level resulting in cell cycle withdrawal

580 (Dotto, 1999). Addition of calcium to cultured murine keratinocytes induces withdrawal from the cell cycle followed by induction of differentiation markers (Hennings et al., 1980). There is an extracellular calcium gradient in differentiated layers of adult skin, peaking at the outer granular layer (Menon et al., 1992; Forslind et al., 1997; Mauro et aI., 1998) that probably mediates terminal differentiation. However, establishing when the calcium gradient forms during development is hampered by insensitivity of detection methods. An epidermal calcium gradient has been demonstrated in fetal rats (Elias et al., 1998a). although the gradient is first detected late in terminal differentiation. A calcium-sensing membrane receptor has been detected in murine epidermis (Oda et al., 2000, and references within). Knock-out of a form of this receptor, which is most abundantly expressed in basal keratinocytes, results in defects in differentiation and downregulation of epidermal loricrin, a late differentiation marker (Oda et al., 2000: Fig. 1). However, the role of this receptor in transduction of the calcium signal cannot be fully appreciated yet as the gene has an additional splice variant unaffected in the knock-out. Withdrawal of keratinocytes from the cell cycle is also regulated by c-Myc (Gandarillas and Watt, 1997), which could thereby stimulate onset of terminal differentiation. Myc-Max heterodimers predominate in the epidermal basal layer and Mad-Max heterodimers predominate suprabasally (Gandarillas and Watt, 1995 Hurlin et al.. 1995" Vastrik et at., 1995).

XIII, Regulation of Transit to Late Stages of Terminal Differentiation Terminal differentiation in culture models appears to have two phases: an early phase, marked by cell cycle arrest and induction of the keratin stratification markers K1 and K10. and a late phase characterized by upregulation of late terminal differentiation markers (e.g.. loricrin, filaggrin: Fig. 1). Evidence for distinct stages includes, first, ability to pharmacologically dissect skin differentiation in cultured keratinocytes. Induction of early stage markers (K1 and Kt0) is phorbol ester independent, whereas late stage markers can be induced by the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate via activation of protein kinase C: Dlugosz and Yuspa, 1993). Secondly, a rise in p21 accompanies keratinoctye cell cycle withdrawal soon after calcium induction of differentiation. However, p21 protein levels later fall. This fall in p21 protein level is necessary because p21 inhibits late stage terminal differentiation (Di Cunto et at., 1998). If induction of differentiation in keratinoc~e culture represents a useful model for fetal skin development, then adoption of the late phase of terminal differentiation probably corresponds to transit to late terminal differentiation at E 1516 (Theiler stages 24-25) during mouse embryogenesis.

tli Organogenesis This stage is marked by morphological change and appearmnce of a granular layer in fetal skin. Late marker induction (e.g., toricin) occurs in fetal skin at this time (Byrne et aI., t994: Bickenbach et at., t995). The calcium gradient peaks at the granular layer in adult skin and calcium signaling, probably through protein kdnase C induction, causes downregulation of early terminal differentiation markers, transit to late stages of terminal differentiation, and induction of late markers (Dlugosz and Yuspa, 1993). Cholesterol sulfate, a metabolite of epidermal cholesterol, is also most abundant in the granular cells of adult epidermis and. in cultured keratinoctyes, cholesterol sulfate induces expression of PKCr/ and 6 isoforms and terminal differentiation (Ohba et at., t 998). PKCrl is predominantly expressed in differentiating epithelial tissues, including the upper granular layer of epidermis (Kuroki et at., 2000). Cholesterol sulfate itself activates terminal differentiation in cultured cells and. via PKCr/or 6 isoforms, induces T G a s e l gene expression. As described below, activation of TGase 1 enzyme is key to the formation of the epidermal bmq'ier, the endpoint of epidermal terminal differentiation. IKKe~ (Chuk) null mice appear developmentally arrested between terminal differentiation stages, that is, after epidermal stratification and expression of early stage keratin differentiation markers, but before onset of late terminal differentiation and barrier formation (Hu et al., I999; Takeda er al., 1999: Li et al., 1999). IKKc~ is a component of the IKB kinase that phosphorylates cytoplasmic It, 7" 180. 342. ~4-0 . 352.36-4 Buch. C.. 162, 170, 177 Buchanan, J. A.. 438,454, 490 Buchberg, A. M., 341. 352, 359 Buchert, M., 79, ]04, 453,486 Buck. C. A.. 162, 170, 177. 200, 201,209, 9219, 230 Buckert. M., 529. 537 Buckingham, M., i31. 132, t40, 141,144, t45, 147, I48, 258, 260, 263, 264, 268, 272, 273, 276, 277, 34t, 343, 9.~4:~. o:)6. 361, 362

Buckingham, M. E.. 140, 141,148, 258, 267,269, 274., 275, 344, 362 Buckler. A. J..~:~.. 364. 374. 375,392, 399. 4 t 5 . 4 t Z 419 Budnik, V., 334, 361 Buehr, M.. t89. 189, 382, 389, 39] Buffinger, N., 140. 144, 263, 2 73 Buj-Bello, A., 402. 420

Bulfone, A.. 76. 77.79. 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 95, 97, 98, 99. ! 03,. 104, 382, ~8~"/.. 388. . . 390. . 4,,_.'~'~ 437,450, 451,470, 47 I, 472, 481, 493, 525,535 Bulfone-Paus. S., 588 Bullejos, M., 389 Bullen, R J., 357, 364, 387, 390, 568,584 Bullock, S. L., 407,417 Bulotsky, M. S., 136, 137, t47 Bumcrot, D. A., 105, 140, t4I, 145. 147. 262, 264, 275, 470, 490 Bundman, D. S., 580, 581,582. 583,584, 586, 587 Bundy, J., 350, 370 Bunton, T., 353, 356, 367 Burbach, J. R, 506. 513 Burch, J. B. E.. 316, 326, 336, 339, 342, 344, 360, 362?, 367 Burd, G. D., 77, 84, 98 Burdine, R. D., 338, 353,354, 355, 356. 357, 359, 370 Burgeson, R. E., 573,584 Burgess. R.. 135.t38.137, 145, 250, 267, 273 Bi.irglin, T. R.. 479, 482 Burgos, M., 388,391 Burgoyne, R S., 372. 377, 378, 379, 383, 386, 387, 389, 392, 393

Burhan, A. M., 86, 104 Burke, A. C., 129, 142, t45. 305. 328 Burke. D., 452, 466, 482 Burke, R., 577, 587 Burki. K.. ~_1 326 Burley, S. K., 11.16, 525.538 Burting, A.. 26. 35

60 t

Burmeister, M., 527, 535 Burn, J., 357, 361 Burnham, Vv: M., 247, 252 Burris. T. R, 379, 389 Burrow's, H. L., 502, 511,513 Burr, D. x,~, 388, 392, 534. 536 Burton, E J., 511,514 Burton, R B. J., 34t, 359 Burtscher, H., 282, 295 Buscher, D., 83, 9& 470, 482, 580, 581,583, 587 Bush, S. M., 334, 335, 336, 337, 339, 365 BushwelIer, J. H., 204, 210, 220, 233 Buskin, J. N.. 257, 276 Busstinger, M., t 15, 125, 204, 209, 428, 475,497, 533, 535 BussoIi, T. J., 546, 561 Butcher, R. L., 22, 33 Butler, C. M., 372, 386, 389 Butler, K., 303,330 Butler, R M., 436, 456, 482 Butler-Brown, G., 344, 362 Buttitta, L. A., t41,147 Butzler, C., t 73, 175 Buxton, J. A., 227, 23I Buxton, R, 422, 426, 449, 487 Buyukmumcu, M., 573,584 Buyuksat, I., 47, 51 Buzney, E. A., 193, 206, 207, 246, 250, 270, 27 i, 274 Byerty, K. A., 398, 419 Byrd, C. A., 77, 84, 98 Byrne, C., 440, 498, 568, 571,572, 577. 580, 582, 583, 584, 586, 58Z 589

Byrne, M. C., 318, 328, 583,587 Byskov, A. G., 373, 390

C Cable, J., 560, 561 Cabral, L. J., 3 ! 3,324 Cabrera, C. "v:, 257,276 Cacheiro, N. L. A., 22, 34 Cacheux, V, 508, 516 Cachianes, G., 223, 230 Cadalbert, L., 583, 586 Cagan, R., 263, 275 Cai, C., 336, 368 Cai, J., 528, 535 Cai, L., 245,249 Cai, N., 91, 97 Cai, Y., 240, 250, 4t4, 415,418, 419, 420 Cai, Z. L., 20I, 204, 207 Caille. t., 236, 237, 2.44, 245,248, 249 Caillol, D., 303,324 Cain, K. T., 22, 34 Cairns, L., I85, 189

6

0

2

A

u

Calafat. J.. 583,589 Calarco, R G., 23, 26. 33 CaIautti, E.. 580. 585 Calfton, M.. 219. 231 Call. K. M.. 415, 4]7 Callaerts. R, 533,536 Callahan. R., 226, 230 Calley, J.. 185.1(~9. ]90 Callihan. B.. 263.274 Calo. C.. 442. 443.494 Calvari. C.. 382. 387.388.390 Calvert. J. T.. 227.232 Calvet. J. R, 414, 4 i 7 Calvio, C.. 400. 417 Calvo. B., 506. 514 Calvo. K. R.. 479. 488 Calvo. W.. 195.208 Camakaris. J.. 443.482 Cambon. B.. 271. 273 Camerino. G.. 372. 378. 379. 380. 382. 384. 387. 388, 390, 391. 393 Cameron. H. A.. 247. 249 Campbell. D.. 287. 293. 504. 513 Campbell. I. K.. 3 I, 34. 115. 124 Campbell, K.. 82. 83.84, 88. 89, 91.9& 103, 105, 106. 110. 114. 120. 125, 161. I79, 241.2"44.249, 428. 453. 49]. 497 Campbell, M.. 354. 359 Camper, S.. 6. 19. 85. I06, 353.354. 355.361. 378. 379. 393. 433,478,485. 502. 507. 508. 509.511.512.513. 5t4, 515, 5]8. 556. 557, 561. 564 Campion. E., 14, I5 Campione, M.. 341. 345. 347.348. 350. 351. 354. 355,356. 359. 361 Campos-Barros. A.. 511.512.518 Campos-Ortega. J. A., 134, 136. 138. 145, 147. 148, 149, 55.~. 561 Campuzano. S.. 555.561 Camus, A.. "~ 3.4. 40. 44.50, 132. 148, 4 ,v'~ 427. 482, 496 Canals, J. M., 91.98 Candi, E., 570. 58 t, 584, 588 Candia, A. E. 135. 145, 260. 267,275 Caniggia, I.. 173. 174. ] 75 Canipari. R.. 86. 103, 188. I90 Cann. G.. 266. 276 Canning, D. R.. 303, 329 Cano, A., 355.359. 423. 482 Cantley, L. C., 227,232 Cantor, A. B.. 343, 369 Cao. L.. 350. 370 Cao, Q., 10. 11, 18 Cao. w: H., 432, 450, 465,489 Cao, X.. 346. 364 Cao. Y., 558, 564 9

~-,,

9

,-,-'-.

t

h

o

r

Index

Capco, D. G., 8, 13, 16, ]Z 18. 27, 33, 34 Capdevila, J., 338, 354, 356, 357,359 Capdevitta, J., 83, 98 Capecchi, M. R., 1t7, 119, 125, I66, 179, 305.3I 1,32Z 427, 428, 443.466, 471,474, 483, 490, 496, 544, 545, 56t. 564 Capehart, A. A., 350~ 365 Capel, B., 373, 377, 378, 381. 382, 383, 387,389, 390, 391, 392. 393 Cap~as. S.. 9. 16 Caplen. N. J.. 14. 16 Caprioli. A., 199.20 I, 207. 1987 Capron, E, 94, 99 Caras. I.. 84, 102 Carbajo-Perez, E.. 511,514 Cardell, E. L.. 453,467, 494 Cardiff. R. D.. 171, 175 Cardin-Girard. J. E, 171.173, ] 76 Cardoso. W. V.. 311.312, 313, 315. 323, 3261, 327, 329 Carey. C., 510. 513 Carey. J. C.. 478.495, 507.5I 7 Carey, M. L.. 475.481 Carey, R. M.. 412. 420 Caric, D., 87.94, 98, 105 Cart. M.. 116, I25 Carl. T. F.. 423, 482 Carlisle. C.. 378. 390, 392 Carlo. A. D.. 188, 189, 190, 508.513 Carlson, B. M.. 395.417 Carlson. G. J.. 45 t, 453. 474, 489 Carlson, L., 442, 484 Carlsson, B.. 167, 178 Carlsson, L.. 320. 325, 409, 419 Carlsson, R, 479, 487, 527.530, 532, 535 Carlton. M. B., 7. ]6, 160. 167, 178, 203,210, 453,482 Carmeli, C., 141. 144 Carmeliet. R. 166, 176. 203.207, 223, 225,226, 228, 229, 321.325 Carney, R R., 542, 559, 561 Carnicero, E., 543, 545, 546, 565 Carninci, R, I 1, t 2, ]8 Caron. M. G.. 341,352, 362, 51 t, 513 Carpenter. E. M.. 545. 561 Carraway, K. E., 227.232 Carre-Eusebe. D., 372, 387,392 Carri~re, C.. 506, 508. 509, 518 Carrion. D., 353,357, 365 Carroll, R M.. 15, 18 Carroll, S. B., 471,493 Carroll. T. J.. 395,420 Carrozzo. R.. 94. 103 Carson. D. D., 156, 175 Carson, J. L., 38, 52 Carter, J. M., 527, 530, 531,537

A u t h o r Index

Carter. Vr J., 268, 269, 274 Carthew, R. Vv\, 14, 15, 17 Carver, B., t 6 t, 178 Carver-Moore, K., 203, 207, 223, 229, 287, 293, 402, 4]8 Casagranda, E, 266, 275 Casares. E, 91,103 Casarosa, S., 83, 88. 89. 90, 95.98. 99, ]05 Casci, T., 466. 482 Cascio. S., 165. ]74, 303. 323 Cases, S., 161,175 Casey, B., 47, 51. 357,359, 361,363 Casey, E. S.. 303.323, 329 Casiano, D. E., 376, 389 Caskey, C. T., 94. 103 Caspary, T., 168, 176 Casper, R. E, 13, 17 Cassidy, R. M., 91, t01 Cassie, C.. 243. 249 Cassini, A., 26, 33 Castagne, J.. 512, 5t4 Castano, L.. 506. 514 Casteilla. L., 271,273 Castitla. L. H., 226, 227.233 Castillo, A. I.. 510, 516 Castillo. M. M.. 345. 348. 360 Castrillo, J.-L., 509, 513, 514 Catala, M., 39, 40, 50, 200. 209 Care, R. L.. 374, 385. 389 Cattanach, B. M., 378, 387, 390 Caudy, M., 257, 276 Caveda, L.. 226. 228 Cavenee, W., 415, 4t7 Caviness, V. S.. Jr., 87, 92, 94, 9& 103, 105 Cayrol, C.. 303,324 Cecchi. C., 92, 102 Cecchini, M. G., 443, 485 Celeste, A. J., 338. 363, 467, 498 Censulto, R, 121, t24 Centanni, J. M., 7, 19 Cepko, C. L.. 87, 88, 98, t00, 243,252, 343, 345,348, 349, 350, 351,352. 358, 359, 521,525,527, 535, 53Z 538 Cereghini, S., 165, 174, 303,323 Cerretti, D. R, 91. 106 Chada, K., t 88, t90 Chadwick, R. B.. 509. 515 Chae. S.-S., 227; 230, 231 Chae. T., 94, 98 Chai, N.. 23.33, 166, 167, t 75 Chai. Y., 228, 229, 246, 249, 426. 482 Chakravarti, A., 479, 482 Chalepakis, G., t 13. 125 Challice, C. E., 345, 369 Chamberlain, C. G.. 532, 537 Chamberland, M., 478, 489

603

Chambers, A. E., 262, 275 Chambers, I., 23, 34, 160, 166, ] 78 Chambon, R, 40, 47, 5I, 9t, 101, ]04, 109, 1t7, tt8, 119, t22, 123, 125, 172, i73, 179, 305,310, 312, 327. 329, 34t, 346, 347, 352, 355. 357, 359, 362, 366, 368, 369. 427.4.~.~, ~ 443.449. . . . . . 451.,4 ~ . ~ 8 , 469, 470, 474, 479~ 484, 485, 489, 490. 491, 492, 494, 525,536, 5~4-f4,545~ 557, 562, 563, 564 Champigny, C., 167, 179 Chan, E. R, 579, 584 Chan, S., 117, 118, 124 Chain S. K., 478, 490 Chan, W., 164, 175, 302, 324 Chan, Y., 452, 494 Chan, Y. M., 553,561 Chandra, S., 443, 449, 479, 495 Chang, B., I36, t49 Chang, C., 11, 17, I0], 525,538, 585 Chang, C. R, 476, 482 Chang, C. Y., 3 t 8, 326 Chang, D., 101, t20, 125 Chang, D. K., 378, 392 Chang, D. T., t41, t45, 287, 292 Chang, H., 226, 227, 229, 336, 352, 359 Chang, H. H., 451,475, 488 Chang, L., 87, ]04 Chang, W., 550, 56I, 580, 583,588 Channon, K. M., 227, 231 Chapman, D. L.. 1.~_. t ~ , 135. t37. 145 Chapman, V. M., 157, ] 75, 178 Chapouton, R, 83, 98 Charachon, R., 398, 416, 550, 56t Chareonvit, S., 422, 424, 435, 437, 475, 476, 492 Chafite, J., 342, 344, 349, 359, 365 Charles, R., 348, 360 Charlieu, J. R, 391, 416, 419 Charnas, L. R., 453,487 Charnay, R, 91,100, 1i8, t 19, 124, 125; 135, 14Z 468, 492, 51 O, 518, 546, 564, 565 Charpentier, E., 578, 585 Charron, R, 342, 343, 359, 360, 366 Charron, J., 171, 173, t 76, 318, 324 Charron, R., 342, 366 Chastant, S., 9, 15 Chastre, E., 27 I, 277 Chat, M., 552, 564 Chatterjee, B., 522, 527, 535, 549, 562 Chatterjee, V. K., 31 i, 324 Chau, T. C., 222, 231 Chaussain, J. L., 376, 391 Chauvet, J., 500, 513 Chawengsaksophak, K., 160, t 67, 174, 175, 305,307, 323 Chazaud, C., 40, 47, 51, 346. 347, 355, 359

604 Cheah, K. S., 281. 283,293, 380, 38t, 389, 39t, 392, 531, 536 Checa. N., 91, 98 Cheema, S. S., 87, 91. 103, 508, 517. 521,527, 537 Chegini, N., 47, 51 Chelly, J.. 94, 99 Chem L.. 164. 177 Chen, A., 3 t, 33, 40, 45, 50, 338.360 Chen, C. M., 169, t 75, 177 Chen. C. T., 91, t03 Chen, C. W., 577, 585, 589 Chen. C.-Y.. 342, 343,359, 367 Chen, D.. 268. 275, 378. 390 Chen, D. Y.. 227. 229 Chen. E. Y.. 441. 488 Chen, E. 227.229 Chen, G.-Q., 346, 364 Chen, H., 203,207, 223. 229. 313,323. 479. 482, 488 Chen, J., 90, 95. 106. 203,209, 313.323, 341. 342. 343. 352, 359, 362. 363, 510. 518 Chen, J.-N., 339, 351,355,359. 368 Chen, L.. 286, 292, 293 Chen. L.-H.. 577. 583.584 Chen. R. 556. 558.561 Chen, Q. J.. 291,292 Chen, R.. 509. 515. 530. 533.535 Chen, S.-H., 7, 19, 79.83, 89.90, 92. 95, 98, 102. 103 Chen, S. L.. 334, ~4_ 34.~, 359, 361 473 474. 489. 507, 514 Chen. Vvl S.. 164. 165.175. 177, 179, 185.189, 303.316. 323, 329. 453. 487 Chen, X.. 290. 292, 479. 482 Chen, Y.. 167, 174, 176, 437, 440, 482, 498 Chen, Z., 322, 326. 341. 348, 349, 352, 359, 368, 435.490, 559.563 Chert, Z. F., 473,482 Chen. Z.-F., 79. 98, 218, 225,229. 232, 243.249 Chen, Z. Y.. 556. 558. 565 Chenevix-Trench, G., 287.292, 470. 486 Cheng, A. M., 47, 50, 78. 104, 171,173. 175, 178 Cheng. C., 338.36I, 580, 586 Cheng, H. T.. 433.476. 477. 478. 489 Cheng. L.. 188. 189, 190 Cheng, R E. 257, 258. 273. 277, 349. 362 Cheng, S., 45, 50, 78, 100 Cheng, S. H., 121, t24, 451. 453, 470, 471.49] Cheng, S. Y., 343,360 Cheng, Y.. 186, 190 Chenleng, C., 336, 339, 342, 368 Chenn, A., 92, 94, 98 Chepelinsky, A. B., 528, 537 Cheraud, Y., 267. 274 Cheresh, D. A.. 223.228, 229 Chernoff, G. E, 79, ]00

A u t h o r Index Cheshier, S. H., 245,249 Chetty, R., 415,420 Cheung, A., 468, 495 Chhabra, A., 452, 492 Chi, C. L., 466, 491 Chi, D.-C., 136, 137, t47 Chi, M. M., 269, 274 Chia, ~3L,240. 250 Chiang, C.. 43.50, 78, 80, 85.86, 9& 10], 120, I21,123, I25, 14I, 144, 145, 260, 265,273, 287, 292, 312, 319, 323, 326, 439, 451,470. 482, 521,535 Chiang, L. C.. 453.486 Chiannikulchai. N.. 341,369 Chida. K., 580, 58K 588 Chida. S.. 22.33 Chidambaram, A.. 287, 292, 470, 485, 486 . .173. . 174, ~ 9 , .~" ~ 41, 342, 343, 344, 345, Chien, K. R.,. t72, 352. 356. 357. 358, 359, 36t, 362, 363, 366, 36Z 368 Chien, Y. H., 192, 194, 207. 245, 250 Chik. K. ~,u 121. 124, 451. 453.470, 471,491 Chikamori, M., 79, 99 Childs, G. V.. 512, 514. 515 Chin. A.. 351. 355,359 Chin. E. R.. 269, 273, 277. 342. 369 Chin. J. R.. 223.233, 465,491 Chin. M. T.. 347.359 Chin. N., 384. 385, 393 Chin, Wi '&:, 469, 498 Ching, A.. 6, t 7 Chiovato, L., 311,326 Chiquoine, A. D., 183, 189 Chirgwin, J. M.. 321,328 Chisaka, O., 408, 4 t Z 427, 443,474, 483, 544, 545,561 Chisholm, J. C.. 23, 26, 34, 156, 175 Chiumello, G.. 378.387, 389 Cho, E. A.. 408,409, 410, 417 Cho, E. S.. 402. 417 Cho, J., 91.97, 286, 294 Cho, K. W., 40, 50, 323, 477,482, 483 Cho, K.-Y., 579, 587 Choe. S.. 354, 367 Choi. C. Y., 334. 343,361 Choi, J., 263. 274 Choi. K., 200, 207, 217, 232 Choi, T., 443,495 Cholly, B., 117, 122 Chong, J. A., 243,249 Choo, D., 469, 478, 483, 49t, 54 t, 542, 550, 55 t, 552, 555, 558, 559, 564, 579 Choudhar?', K., 88, 98 Choudh~', A., 341. 369 Chourrout, D., 116. 125 Chow, L. T., 412.417 Chow, M., t40, 144

A u t h o r Index

Chow'. R. L., 533.535 Chowdhury, K., 311, 32t, 32Z 329, 398, 41Z 451,453, 475, 490, 495, 496, 527, 530, 532, 538 Chowdu~1, K., 183. t90 Choy, B., 243, 249 Christ, B., I28, t30, t3t, I32, t40, 144, ]45, I46, ]47, 254, 255,263,264, 266, 272, 273, 276, 277 Christensen, E.. 504, 518 Christensen. L.. 422, 4.~ ""~. 440. 450. 9451 , 471 , 472, 484, 493

Christiano, A. M., 573, 584 Christians, E., 9, 19 Christiansen. J., 28 t, 294, 470, 485 Christiansen, J. H., t 72, 173, 17K 336, 337, 365 Christie, S., 416, 419 Christoffels, V. M., 341,343, 345,347, 348, 350, 351,355, 356, 359, 366 Christofori, G., 322, 324 Christy, B. A., 349, 358 Chu, K., 502, 514 Chu, M.. 559, 563 Chua, S. C., Jr., 443,497 Chua-Couzens, J., 91,101 Chuang, R T., 578, 583. 585 Chui, D. H., 202, 209 Chun, Y. K., 354, 365 Chung, R T., 315,324 Chung, U. I.. 285, 287,292, 293 Chuong, C. M., 571,574, 577, 578, 579, 585, 586, 58;7 589 Church, D. M., 286, 294 Chwoe, K. Y., 341,360 Chyung, J. H., 282, 293 Ciau-Uitz, A., 198, 200, 207 Ciemerych, M. A., 27, 33 Ciesiolka, T., 183, t 90 Ciliberto, G., 318, 324 Cinnamon, Y., t31,132, 140, 145, 146, 255,256, 273:. 275 Cirillo, L. A.. 316, 324 Ciruna. B. G.. 133, 145, 167, 175, 303, 317, 324 Claesson-Welsh, L., 504, 515 Clapp, D. W, 243, 244, 249 Clark, I., 185, 189 Clark. J. i., 532, 535 Clark. R. A., 223,228, 287, 293 Clark, W. R., 319, 328 Clarke, A. R., 416, 4]9, 441. 488 Clarke, D. L., 236, 237, 244. 245,246, 248, 249, 250. 318, 324

Clarke, J. D.. 422, 426, 449. 487 Clarke, L. A., 452, 453,481 Clarke, T. R., 381,382, 393 Clar5r, D. O., 9 I, 101 Clausen, J. A., 87, 94, 105 Ciauss, I., 318. 328

605

Claxton, D. E, 202, 208 Ctaxton, J. H., 577, 585 Cleary, M. L., 443, -449, 476, 479, 482, 487, 495 Cleaver, O. B., 348, 359 Ctemente, H. S., 200, 201,209, 220, 232 Clement-Jones, M., 387,390 Ctements, D., 166, 176, 303,325 Clements, M., 31, 34, 46, 47, 48, 51, 3 t 1, 316, 327 Ctendenin, C., 341,343, 352, 368 Clevers, H., 92, 100, t72, ]75 Clifford, J., 469, 49i Clifford, V., 379, 387, 393 Ctifton-Bligh, R. J., 31 t, 324 Ciiment, S., 337, 359 Clore, G. M., 377, 393 Closson, V., 226, 230 Clotman, E, 226, 229, 321,326 Clout, D. E. W., 341,345, 347, 348, 350, 359 Clouthier, D. E., 432, 465,478, 483, 498 Coates, M. I., 436, 495 Cobos, I., 84, 85, 102, 1 I6, 124, 428, 490 Cobourne, M., 440, 494 Cobrinik, D., 483 Cochran, J., 347, 364 Cockell, M., 321,324 Cock.roft, D. L., 29, 32, 33, 34, 156, 157, ] 75, t 76 Coerver, K. A., 44, 5t Coffey, R. J., 169, t 79 Coffin, J. D., 286, 293, 453, 483 Coffin-Collins, R A., 465,483 Coffinier, C., t 65, 175 Cogan, J. D., 509, 517 Cogliati, T., 376, 389 Cogswelt, C. A., 239, 250 Cohan, C. S., 549, 561 Cohen, A., 237,252 Cohen, D., 405,417 Cohen, J., 23, 27, 34, 35, 84, 10t, 45 t, 475,486, 527, 528, 530, 536 Cohen, J. H., 319, 329 Cohen, L. E., 509, 514 Cohen, M. M., 442, 485 Cohen, R. I., 167, I7t, 173, 179 Cohen, S., 465,483 Cohen, S. M., 471,483 Cohen-Gould, L., 34 I, 362 Cohen-Tannoudji, M., 84, 98. 10~ 386, 387, 390, 546, 564, 565

Cohn, D. H., 286, 294 Cohn, S. A., 454, 483 Colbert, M. C., 469, 483 Cote, T. J., 314, 324 Cole, W., 443,495 Cote, W: G., 288, 293

6

0

6

A

u

Coleman. J. R.. 136, 146, 3 t 6. 317,325 Coletta. M.. 14I. 144, 206, 207, 246. 249, 250, 271,273, 274 Colledge. W\ H., 7. I6, 160, 167. 178. 203.210, 453.482 Collen. D., 166. 176. 203.207, 223,225. 226. 228, 229, 321.325 Cotler. B. S.. 223,230 Coller. J. M.. t 1, I6 Colligan, J.. 377. 390 Collignon, J., 43, 44. 45.46.50, 354. 355. 357. 359, 386. 387,390, 468 497, 529.._,0. 531 ~. 4, 536 Collins. A. E.. 92. t02 Collins, C. C., 347. 364 Collins, E S.. 202. 208. 347. 366, 528.538 Collins, N. S.. 260. 267. 275 Collins. S.. 316. 325 Collins. T.. 343. 352. 360 Collu. R 519 514 Colman. D. R.. 408.419 Cotmenares, C., 260, 268. 272. 273, 468. 482 Coltey, M.. 140. 141. 142. I47. 263.264. 266. 276 Coltev. 9"~ 4,..~. "~' 426. 427. 433.483. 572. . R M. . I28. 145, 4_,;. 585 Colvin. J.S..286.292. 293. 313. 314. 315.324, 558,56I Comley, M., 4 t 5.420 Comoglio. R M.. 266. 275 Compain, S.. 398.416. 550. 561 Compernolle. V.. 226. 229 Complton, J. G.. 570. 584 Compton, D.. 224, 230, 231 Concordet. J. R. 78. 101, 262.268, 276 Conkie, D., 317.32 7 Conley, R B.. 164, 177 Conlon, F. L., 45, 47.50. t33, 137, 145, 338, 355.359, 468. 483 Conton, R. A., 31.33, 42, 46, 49, 116, 122. 135, 136. 137, 139. 145, I48. I49. 323. 468.478. 481, 483. 509. 514 Conne, B.. 10. 18 Conner. A. S.. 193.207 Conner. D. A.. 343.350. 35 I. 352. 353.357. 359 Conner, J. M.. 91, 97 Conners. J. R.. 32t. 328 Connolly. D.. 264, 272 Connolly, D. J.. 45, 52 Connors, H.. 246, 250, 318. 326 Conover. J. C.. 549, 559. 561 Consalez. G. G., 525,535 Consortium, T. E. E K. D., 414. 417 Constam. D. B.. 50 Constanti, A.. 91. 102 Contreras, L. N.. 51 "~ 515 Conway, S. J.. 350. 370, 475.483 Conway-Campbell. J., 281,293, 381. 392 Cook. D. L., 356, 359 .,

.-,

9

"---,

t

h

o

r

Index

Cook, D. M., 400, 418 Cook, G., 225,229 Cook. M.. 4vv 427. 433 487 Cook, S. A.. 94. 105, 550, 563 Cook. T. L.. 529. 538 Cooke. J., 45, 52, 139, 142, t45, 146, I85, 188, 189, 262, 263. 264, 272, 274, 354, 355,356, 362, 364, 366, 423, 492 Cookingham, J., 442. 443, 494 Cooper. B., 336, : : 9 . :42. 368 Cooper, E. C., 94, 100 Cooper. G. M.. 7. 14. i6, 18 Cooper. J. A.. 94. ]00, I01, 104 Cooper. L.. 281. 294, 377, 389 Cooper. M. K.. 78, 98, 470. 483 Copeland. N. G., 78.79, 81.85, 99, 100, 103, 168, 169, 173. 175, 176. 179, 193, 204, 208, 321. 328, 341,342, 345,348, 349, 352, 359, 360, 365, 366, 411. 418, .443, 467, 476. 482, 485, 488, 508, 5t5, 521. 525,533, 536, 537, 538. 556. 565 Copeland, T., 7, I& 183.184, 185. 189 Copp, A. J.. 156. 170. 175, t83, 189, 350, 362, 475,483, 486 Copperman, A. B., 174. 178 Coppola, V., 559. 562 Corballis, M. C., 351,356. 360 Corbel, C.. 201. 209 Corbi. N.. 160. 180 Corbin, J.. 170. 174 Corbin, x/:, 136, 145, 148 Corden, J. L.. 43.50, 78. 80, 85, 86, 98, 12t, 123, 14I, 145, 260, 265,273, 319, 323, 439, 451,470. 482, 521,535 Cordes, S. R, i 18, 124, 527, 530, 531,537, 544, 562 Cor6. N., 376. 389, 390, 453.481 Corfas. G.. 94, 104, 343.369 Corley, L. S., 47 t, 493 Cormier. E. 198, 200, 201. 207 Cornelison. D. D., 271,273 Cornett. C. V.. 22, 34 Corotto, E S.. 237,249 Corrales, J. D., 338, 353, 354, 356, 357, 370 Correia. K. M., 509, 514 Corsi. A. K.. 341. 360 Corsin, J.. 432. 435,483 Corte, G.. 48, 49, 78, 81, t01, t 10, I14, 12t, 122, 124, 478, 481, 508.5t3 Cortes, F.. 201,208 Corwin. J. T.. 553,555,557, 562, 563 Cossu. G.. 131. 140, 141,144, 145, 148, 149, 206, 20Z 246. 249, 250. 258,260, 263, 2642 268, 271,272, 273, 274, 277 Costa. M.. 263. 274 Costa, R. H., 82, 102, 3 I3, 3t6. 32 t, 324, 328,. 330 Costa, T., 347, 364

A u t h o r Index

Costantini. F., 204, 209, 402.419 Costantini, L. C., 9 I, 98 Cota, G., 51 t, 514 Cotanche, D. A., 553. 562 Cotsarelis, G., 570, 574, 578, 585, 588 CottrilI, C., 357, 358 Coucouvanis. E., 45, 50 Coughlin, S. R., t69, 175 Coulling, M.. 157, 177 Coutombe. A., 271,277 Coulombe, R A.. 570. 587 Coulombel. L., 194, 200, 20 I, 207. 209, 220, 232 Coulombre. A. J.. 519. 535 Coulombre, J. L., 519. 535 Couly, G. F., 77.84, 85, 98, 128, 145, 422, 423, 426, 427, 433,435,483, 502, 507, 514, 569. 571,572, 585, 587 Cousins, E M., 203, 20Z 226, 229, 467, 484 Couso, J. R, 136, 145 Coutinho, A., 196, 207 Couvineau. A., 285, 293 Cova, L.. 235, 242. 244, 250 Coward, R, 378,390 Cowin, R. 578, 585 Cox, D. R., 287, 293, 579, 586 Cox, R.. 269. 275 Crabtree, G. R., ! 64. 177 Crackower. M. A., 12t, t24, 451,453.470, 47 t, 491 Craggs, G.. 10, 17 Craig, A. W., 11, 16 Craig, C. G., 235, 236, 244, 245.251 Craig, S., 206. 209 Crair. M. C., 92, 106 Crandall, E. D., 3 t 3,324 Cranley, R. E., 424, 442, 443. 482 Cremer, H., 322, 324 Crenshaw, E. B. I., 509, 514 Crenshaw, E. B. Ill, 506, 509. 5161, 51Z 549, 564 Cressman, D. E., 318, 324 Cresswell, L. A., 500, 513 Crews, S.. 85, 99, 225,229 Crisp, M. S., 311,324 Crispino, J. D.. 343, 352. 360 Critcher, R.. 380. 393 Critser, E. S., 26, 34 Croisille, L., t 94, 207 Croissant. J. D.. M_. 359, 360, 369 Crompton, T., 288. 294, 442, 443, 453, 493 Crosby, J. L., 40, 43, 49 Cross, A. J., 247, 249 Cross, H., 507, 515 Cross, J. C., 163, 168, 169, 170, 171,173, 174, 174, t75, 176, 177, 178, 179, 349, 350, 352, 355, 360, 367' Crossin, K. L., 134, 148 Crossley, J. M., 40, 52, 115, 116, 123, 125

607

Crossley, R H., 84, 85, 99, I02, 113, t 16, 123, 124, 132, 145, 317, 324, 337, 340, 360, 428, -44.-9,466, 483, 490 Croucher, S. J., 435,483 Crowe, D. L., 314, 315, 329 Crowe, R., 577, 585 Crowlev, D., 223, 22& 230 Crowley, W. E, 500, 518 Crozet, E, 134, t 35, 138, 139, 146 Cruaud, C., 398, 416, 550. 561 Crumine, D., 580, 583,588 Crumsine, D., 580, 58 t, 585, 586 Crystal, R G., 579. 588 Cserjesi, R, 102, 135, t45, 262, 267,273, 342, 349, 350, 360. 362, 36& 451,473, 476, 483, 495 Csete, M., 239, 241, 25t Cubadda, Y., 343, 360, 361 Cudennec, C. A., 201,207 Cuenca, A. E., 525, 537 Cuesta, R., 1 I, 17 Cui, S., 313, 32& 4t3,419 Cui, Y., t 1, 12, 17 Cullander, C., 580, 582, 585, 587 Cultinan, E. B., 9, I0, t 7 Cumano, A., t 93, t 94, t 95, t 96, 199, 207.. 220, 229 Cunha, G. R., 30I, 326 Cuntiffe, H. E., 398, 419 Cunniff, K., 204, 207 Cunningham, J. M., 411,419 Cunningham, M. L., 468, 485 Cupples, R., 402, 417 Curran, T., 94, 99, 103, 104, 313, 327 Cuttle, P. D., 264, 265, 266, 269, 272, 273, 276 Curtis, A. R. J., 357, 364 Cusella-De Angelis, G., 206, 20Z 246, 249, 263, 27 t, 274, 277

Cusella De Angelis, M. G., 246, 250, 271,273 Cusmai, R., 94, 99 Custer, R. R, 245,249 Cutforth, T., 90, 92, 95, 98 Cuzin, E, 391 Cvekl, A., 532, 535 Cybulsky, M. I., 162, 170, t 76 Czernichow, R, 321,327, 507, 514 Czerny, T., 533,535

D Dabeva, M. S., 318, 324 Dabovic, B., 376, 393 Dacic, S., 452, 484 da Costa, L. T., 86, t 00, 579, 586 Daegelen, D., 262, 268, 2 76 D'Agati, V., 402, 419 Dmhl, E., 348, 360, 398,417, 475,483, 498

608 DahI, N., 506, 517 Dahm. L. M., 226, 230 Dahmane, N., 86, 99, 579. 585 Dahmann, C., 1i2, 123 Dai, D.. 426, 495 Dai, R, 121, ]23 Dai, V~. 3 ~,'~ 354. 366 Daigle, N.. 31,33. 46.49. 451. 478.48], 521. 527.535 Daikoku. S., 79. 99 Daitey, L., 160, I80, 283,292 Dakodis, A., 341,367 Dalcq, A. M., 21.23, 27.33 Dale, B., 23.34 Dale, B. A., 575.586 Dale, E., 15.17 Dale, J. K., 78, 81, 99, 134, 138, 139, 147 Dale, L., 203.207. 239, 249 Dale, T.. 440. 494 Dallman, M. E. 512, 515 Dallos. R. 540, 562 Daly, T. J.. 224. 23I Damay, M. D., 353,357.365 Damert. A., 228. 230 D'Amico. M.. 86, ]04, 579. 588 D'Amico-Martel, A., 434, 483, 558. 562 D' Amore, R A., 226. 230 Damour, O.. 585 Danielian, R S., 84. I01, 116, 117. 123. 124. 489. 577, 578. 583. 589 Daniels. K. J.. 161, 179 Danielson. K. G.. 572. 583.585 Danilchik. M., 44. 50 Danilenko. D. M.. 312. 327 Danks, D. M., 443,482 Danos. M. C.. 356, 364 Danpure, C. J.. 6. 16 Danse. J. M.. 453,482 Danto, S. I.. 313, 324 D'Apice, M. R.. 40, 46. 52, 79, t04, 478. 495 D'Arcangelo, G.. 94. 99, !04 Dardik. E B., 341,343, 352. 368 D' Argati, C.. 415.420 Darimont. B. D., 379, 390 Darling, S., 189, 189 Darnell, J. E., Jr.. 164, 165, 175, 17Z 179, 303, 316, 323, 324, 329 Das, G. D.. 237, 248. 248 Das, S. K., 156. 177 Dasen, J. S., 85, 99, 506, 508, 509, 510. 514, 518 DasGupta, R., 577, 579, 586 Dashner, R., 282, 294, 465,492 Dassute, H. R., 439, 440, 441. 483, 577. 578, 583,585, 589 Daston, G. R, 161, 178 Daszuta, A., 247,249

Author Index Date, I., 247, 249 Dathe, V., 266, 2 76 Datson, N. A., 478, 495, 507, 517 Dattani, M. T., 3 t, 33, 47, 50, 45 I, 476, 483, 500, 506, 508, 509, 514 Dattatreyamurty, B., 4 t 6, 420 Daubas. R. -6~, 264. 273 Dausman. J.. 427. 484 Davenne. M.. 117.119. 123 Davey, A., 22, 34 David, D. J., 453.466, 474, 498 Davidson, B. R, 3, 4, 50, 422, 427, 482 Davidsom D., 84, 86, 94, 99, t00, 104, 399, 419, 474, 483, 574. 575,576, 577,585 Davidson, D. R., 435, 45 i, 468, 475,476, 485, 491, 52 t, 522, 527, 530, 536, 560, 565 Davidson, E. H., 6, 16, 21, 33 Davies, A. M., 183. 190, 402, 420, 434, 435, 48R 497 Davies, J. A.. 396, 403,405,417, 419 Davies, R J. A., 469. 484 Davies, R. C., 400. 417 Davies, T. C., 170, t 76 Davies, T. J.. 156, 176 Davis. A. M.. 235,236, 237, 245, 249, 336, 363 Davis. C. A., 1t 2, 113.115, 123 Davis. C. L., 344, 360 Davis D. L., 342, 360 Davis E. C., 226, 230 Davis E C., 527, 535 Davis H., 540. 562 Davis M. B., 86, 102 Davis N. M.. 85.102, 339, 343. 347. 359 Davis R. J., 87, 101, 257, 258, 268, 274, 277, 399, 417 Davis R. L., 257, 258, 273, 275, 277, 512, 516 Davis S., 224, 229, 231, 232 Davis. xL. 162. 170, 176 Davis W.. Jr.. 9, 11, 12. 16 Davisson, M. "IF..94, 105 Dawid, I. B., 78, 103, 374, 390 Dawson, K., 169, 170, t 73, 174, t 78, 349, 355,367 De, A., 504, 513 Deacon. T. ~:, 89, 99 Deal. C., 512, 514 de Amo, F. E, 553,565 Dean. J.. 14. 15, 16 Dean, M., 287, 292, 470, 485, 486, 579, 586 Dean, W. L., 9, 18, 26, 33 Deanehan, J. K.. 194, 206, 209 De Angelis, L., 271,273 DeAngelis, R. A., 318, 324 Dearden, N. M., 504, 514 Debase, H. T., 321. 324 de Beer, G., 421. 442, 449, 457, 471,484 Debey, R, 9, 14, 15 9 I.)

~

Author Index

609

Deblandre, G. A., 226, 232 de Boer, R A. J.. M.. 345 _~48,366 de Bold, A. J., 343, 350. 351,352, 359 de Bold, M. L. K., 343, 350, 351,352. 359 de Bmijn, M., 194, t 99, 20 t, 204, 207 de Bmijm M. R. T. R., t96, t 99, 200, 201,202, 207 De Caestecker, M. R, ~~9. 368 de Carlos, J. A., 84, 90, 95, 99, 102, i05 de Celis. J. F.. 136. 145 Dechesne, C. J., 556, 562 DeChiara. T. M.. 166, 175, 292, 292, 549, 561 Decimo, D., 3 I2. 327 D6cimo, D.. 469, 485 Declercq, C., 203,207, 223. 228 Deconinck, A. E., 219, 232, 341,343, 352, 369 de Crombrugghe, B., 79, 106, 28t, 282, 283, 284, 285,287, 289. 292, 293, 294, 295, 380, 389, 476, 498, 572, 589 deCrombrugghe, B.. 451,453,473, 476, 493, 498 de Crombrugghe, B., 573,588 de Cuevas, M.. 7. 18 Deed. R. ~:, 1O, 17, 185, 188, I89 Deerinck, T., 453, 486, 580, 58 t. 583, 586 Deerink, T., 509. 513 de Fatima Bonaldo, M., 390 De Felice, M.. 311,324, 326 de Felici, M., 86, t03, 188. 189. 189, t90 Defize. L. H.. 285,293, 442, 443, 489 DeFrances, M. C.. 318, 327 Degen, J. L., 318, 328 Degenhardt, K., 334, 348, 367 Degenstein, L., 577, 579, 586 Degnin, C., 267, 277 De Grandi, A., 382, 387, 388, 390 Deguchi, K., 204, 209, 220, 232, 288, 293, 442, 443, 453, 488 Deguchi, R., 8, 16 deHaan, G., 245, 249 DeHaan, R. L., 340. 343,344, 351,360, 367 Dehart, D. B., 38, 52 Dehbi, M.. 399, 417 Deimling, J., 313,328, 413, 4t9 Deiner. M. S., 506, 514 Deissler, K., 354, 355,359 Dejana, E.. 226, 228, 229 De Jesus-Escobar, J. M., 238, 252, 468, 498, 550, 561 de Jong, E, 334, 341,345, 347, 348, 350, 359, 360, 366 de la Chapelle, A., "441. 488 de la Cruz, J., 357, 359 de la Cruz, M. V., 344. 345, 348, 355, 360 De La Guardia. R. D., 388, 391 Delaisse, J. M., 291,292 Delaney, S. J., t72, 173, 177. 336, 337,365 Delannet, M., 424, 496 de ta Pena, J., 354, 367 9

.

3

~

,

de ta Pompa, J. L., 135, 136, 137, 139, t45, 147, 34t, 370, 435,468, 490, 495, 509, 514, 559, 563 de La Porte, S., 271,277 De Larco, J. E., 465,484 Delassus, S., t 94, 195, 207 det Barco, J. L.~ 135, t45 del Barco Barrantes, I., !36, t37, t39, 145, 435,490, 559, 563 ~'~'~ det Barrio, M. G.. 355 ~ 359, 4_~, 482 DeIbridge, M. L., 387, 390 DeIezoide, A. L., 286, 294 Detfini, M., 262, 263, 273 Delhase, M., 453, 486, 580, 58 I, 583, 586 DeLise, A. M., 280, 292 Delius, H., 44, 50, 325, 327 DeLoia, J. A., 11, 12, 18 Delorme, B., 348, 360 Deltas, C. C., 4t4, 418 Deltour, L., 320, 324 De Luca, L. M., 346, 36Z 589 Delvin, E., 512, 514 Demarchez, M., 585 de Maximy, A. A., 315,324 DeMayo, E J., 310, 3t5, 321,322, 326, 327 DeMelto, D. E., 314, 324 Demers, J., 163, t 75 de Meulemeester, M., 174, 174 Demignon, J., 262, 268,276 De Moerlooze, L., 312, 324, 505,514, 546, 562 De Montellano, R O., 582, 584 de Moor. C. H., 10, 11, 18 Den, J. M., 283, 284, 289, 294 Denda, S., 410, 418 Denef, C., 5 t I, 517 Denetclaw, W., Jr., 219, 229, 255,273, 334, 360 Deng, C.~ 167 171.173, 179. 286, 292. 294, :~.~,484 Deng, C.-X., 226, 227, 23I, 233, 286, 293, 313, 329 Deng, H., 580, 583,588 Deng, J. M., 40, 44, 52, 281,282, 289, 292, 328, 380, 389 Deng, K. Y., 402, 404, 4 t8, 453, 479, 488 Deng, Y., 347, 364 Dengler, T. J., 225, 231 Denhez, F., 467, 49t den Hollander, N. S., 285, 293 Dennefeid, C., 305, 329 Denning, M. E, 585 Dennis, J. E., 34 t, 362 Denson, L. A., 316, 324 Denton, E. R., 507, 514 Denyn, M. M. E J., 334, 366 DenzeI, A., 288, 294, 442. 443, 453, 493 Deo, R. C., 11, 16 Deol, M. S., 544, 562 DePatma, G. E., t 1, 12, ] 7 9~

~

.

.

.

.

.

4

-'~

61 0 DePamphilis, M. L., 5, 9, I0, t Z t9 Depew, M. J., 79, 86, 92.99, 100, 105, 172, 175, 290. 292, 422.432, 435, 443.449, 450, 45 t, 453,466. 471. 472, 473.475,477,478.479. 484, 495, 497, 549, 562 DePinho, R. A.. 528, 532,536, 537. 538 de Ribaupierre, F., 555,566 De Robertis. E. M.. 3 I, 33, 39, 40, ~ , 48, 49, 50, 5t, 52, ~ ' . "~ 7 ~ 78. 98, t41 144,, J23, 338. 358, 359, 366..~8,39_~. 432. 473, 477, 482, 483, 485, 498 Derom. C.. "~ 33 Derom. R.. 22, 33 DeRose. M.. 312. 327 DeRosier. D. J.. 557, 565 DeRuiter, M. C.. 333.360 de Ruiter. M. C.. 226. 229 Derynck. R.. 226. 229. 280. 292. 465. 491 Desai. N.. 31.33. 40. 45.50. 338, 360 Desai. S. Y.. 260, 268.272. 468. 482 de Santa Barbara, R. 381. 390 De Santis, M.. 338, 367 De Santo. R.. 40. 51 de Sauvage. F.. 402. 420 Deschamps. J.. 376. 393 Deschet. K.. II 6. 125 Desclozeaux. M.. 381. 390 Desguerre, I.. 94. 99 DeSimone. D. W.. 424. 481 Desmadryl. G., 556. 562 Desmarquet. C.. 118. 125 De S medt, V., 1,4, 8, I6 De Sousa, R A., 11.12. I6 De Souza. E S.. 2.4 de Souza, R, 452, 498 des Portes. V.. 94. 99 D'Esposito, M.. 290, 294, 450. 471. 472, 473.495 Detrich, H. W. III. 213,232 Dettman. R. V~(.219. 229, 334. 360 Deuchar. D. C., 354. 359 Deutsch. G.. 316. 320. 324 Deutsch. U., 224, 228. 232. 398. 417, 475.481, 484 Devaney, E., 83, 86, 100 de Vant6ry, C.. 7, 16 de Vargas, C.. 475,485 Deville. ES.-S.. 236. 249 Devine, W: R, 94, 106 de Viragh, R A., 582, 583, 587 Devon, K., 380, 392 Devor, D. E., 173, t76 Devoto, S. H., 265, 269, 273 Devriend, T. K., 398,419 de Vries. W. N., 13, 15, 16, 18 Dewar, K., 380, 392 Dewerchin. M.. 166, 176, 225, 226, 229. 321. 325 Dewhirst, M. "~,V,227, 23t

A u t h o r Index de Wied, D., 500, 514 de Winter, J. P.. 385. 389 de Wit, D., 465,498 Dexter, M.. t 85, 188, 189, t 94, 207 Dey, C. R., 3 t 3, 330 Dey, S. K., 156. I75, 177, i78 Dhanasekaran, N., I2, 13, 18 Dhouaiily, D., 571,572, 574. 585, 587 Diamonti, A. J., 227, 232 Dias, M. S., 40, 50, 317,328 Diaz, E. M., 42, 51, 158, 177 Di Bonito. M.. 95, 9Z 506. 513 Di Carlo, A., 478.48I Dichoso, D.. 341. 360 Dickinson, M. E., 117, 121. 123, 423,484 Dickman, E. D.. 342, 368 Dickson, C.. 84. 102, 312. 324, 505.514, 545, 546. 562, 563, 565 Dickson, K. S., 10, t5 Dickson, M. C.. 203, 207. 226~ 229, 467, 484 DiColandrea, T.. 581,585 Di Cunto, F., 580. 585 Di Donna, S., 140, 145. 258. 263.273 Diekwisch. T. G.. 433.476, 477, 478, 489 Dierich, A., 40. 46, 47, 52, t 14, 117, 123, 125, 310, 32 t, 325, 427, 433,435.443. 449, 451,453,469, 474, 478, 479. 484. 485. 489, 490, 491, 494, 544, 545,563, 583, 586 Dieteche, C M. '~ 4 Dieterlen-Lievre, F., 195, 196, 198, 199, 200, 201,206, 207, 208, 209, 219, 220, 229, 232 Dietrich, R, 122. 124 Dietrich, S.. 140. 145, 260. 263. 264, 265, 266, 273, 277 Dietz, H. C.. 347,360, 453,495 Diez-Roux. G., 219. 229 Dighe, A. S., 287, 293 Digilio. M. C., 356, 365 di Giovanna, J. J.. 570, 584 Dikkes. R, 94. 98, 510. 512. 516 Dikoff. E. K.. 82, 105 Di Lauro, R., 85, 103, 311,326, 330 Dillehay, L. E., 225, 231 Dillon, N., 202. 207 Dilullo. C., 263,274 DiMario, J. X., 269, 273 DiMario, S., 271,273 DiMattia. G. E., 509. 517 Dimitrov, S.. 14, 15 DiNardo. S.. 188, 189 Dinchuk. J. E., 156. 177 Ding, J., 31.33, 40, 45, 50, 267. 272, 338, 353,354, 356, 357,360, 370. 434, 467, 493 Ding, Q., 1121, 123, 124, 312, 327 Ding, X. Y., 579, 589

Author Index

DiPersio, C. M., 572, 574, 585 Di Rocco, G., 334, 335,336. 337, 339, 365 Diver, M., 505,513 Dixon, J. E.. 402, 420 Dixon, K. E.. 185, 190 Djabali. M., 376, 389, 390, 453.48] Djian, R, 582, 583, 585 Djikeng, A.. 15, ]8 Dlugosz, A. A., 169. 179. 580, 585 Do, M. S.. 467.492 doAmaral. C.. 292. 294 Dobbs, L., 314. 327 Dobi. A.. 9 I, 103 Dobson, C.. 203.210 Dobyns, W2 B., 94. t00, t03 Dockter, J. L., 140, 145 Dodd. J., 45.52, 78, 8 I, 99, 403,418 Dodds, S. G.. 349, 358 Dodig, M.. 452, 484 Doe, C. Q., 340, 361, 532, 537 Doetsch, F.. 95.99, 236, 237, 2"44, 245, 248, 249 Doetschman. T., 434, 453,467, 493, 494 Doherty, D., 136, 145 Doherty, R. 116, 125, 425,484 Dohlman. H. G., 45.50 Dohse, M., 246, 250, 318, 326 Doi, H.. 11, 12, 17, 422, 423, 424, 425,435, 437, 45 I, 475, 476, 478, 490, 492, 493, 572, 588 Doig, J., 4t6, 419 Dolci, S., 188. 189 Dol16, R, 40, 47, 51, 117, 123, 305, 310, 326, 329, 34t[, 346, 347, 352, 355,359, 366, 427. 433, -443,449, 450, 451,453, 469, 470, 474, 479, 484. 485, 489, 490, 491, 492, 494. 545,557. 562, 563, 564 Dolmetsch, R. E., 8, 16 Domashenko, A. D., 10, 16 Domeier, M. E., t 65, 176, 316, 325 Domen, J., .~ "7,6, 393 Dominguez, M., 470, 484 Dominguez-Steglich, M. A., 281,292, 380, 390 Dommer..ues, M.. 194, 207 Donahoe. R K.. 47, 50, 381,382, 385, 390, 393 Donaldson, D., 507, 5t4 Dong, J., 174, t 77 Doniach, T., 44, 50, 108, I23 Donis-Keller, H., 321. 325 Donlan, D. E, 443.482 Dono, R., 86, 99, 338, 360 Donoghue, D. J., 452, 453, 466, 485, 497 Donoghue, M. J.. 84, 99 Donovan, M. J., 3 t 4, 326, 4 lO. 4i8 Donovan, R J.. 7, 19, 185. 188, I89, t89, 190, 236, 24.6, 249

Donoviel. D. B., 347, 352. 360

6] I

Dony, C., 282, 295 Dooher, G. B., 442, 443,494 Dor, Y., 225,229 Dorai. H., 4t6, 420 Dorin, J., 451,453,474, 489 Dorn, G. Wi H, 453, 483 Dorovkov, M., i 5, I8 Dortland, B., 201,204, 207 Doss. J. B., 292, 294 dos Santos Silva, E., 3 t 6, 323 Dotto, G. R, 580, 585 Dou, C. L., 78.81, 82, 86, 99 Douglas, K. R., 502, 513 Douglas, R., 206, 209 Douglass, E. C., 398,417 Dove, L. E, 405, 407, 41Z 4t9 Dowd, M., t21,123, 203, 20Z 223, 229 Dowhan, D. H., 342, 343,359 Dowling, J., 572, 583, 585, 586 Dowling, J. E., 525, 537 Downing, D. T., 58t, 582, 585, 587. 589 Downing, J. R., 201,204, 207, 209, 220, 23t Doyen, A., 166, 178 Drager. U. C., 345, 346, 347, 366, 36Z 469, 482, 492, 498, 525, 537 Drago, J., 87, 91,103, 402, 4JR 508, 51Z 521,527, 537 Drake, C. J., 223, 229 Drake, D. R, 84, 97, 435,469, 476, 481 Draper, B., 240. 243, 251 Drescher, U., 525,535 Dreschler, M., 528, 538 Dresser, D. W., 38t, 390 Dressier, G. R., 160, 178, 373. 374, 385, 393, 398, 399, 409, 402, 403, 408, 409, 410, 41Z 418, 419, 420, 475, 484, 493, 496

Dreyer, J. L., 9 t, 106 Driancourt, M.-A., 510, 518 Driever, W., 395, 417. 546, 563 Drinkwater, C., 40, -44, 50, 323, 338, 358 Driscoll, M., t 5, 18 Driver, S. E., 14, 16 Drolet, D. \u 502, 514 Drossopoulou, G., 534, 536 Drouin, J., 478, 481, 489, 507, 508, 509, 510, 513, 514, 516, 517. 518

Druck, T., 290, 294, 450, 47 t, 472, 473, 495 DruffeI-Augustin, S., 92, 104 Druker, B. J., 361 Drum, H., 312, 327 Drummond, I. A., 395,415, 417, 418 DrybuNh, L. C., 438, 484 D~sdale, T. A., 339, 342, 347, 360, 362, 369 D' Souza, R., 573, 588 Du, D., 469, 470, 486

61 2 Du, Y., 509, 517 Duan. D. R., 227, 229 Duband. J.. 424. 496 Dubois, R, 320. 324, 504, 514 DuBois. R. N.. 287,293 Duboule. D.. 85 103. _',0~ 309. 3 I0. 326. 330. 450, 484 Dubourg, C.. t 98. 199.201. 207 Ducy, R. 288.289. 292, 294 Dudley. A. T.. 3.4. 82. 99. 105. 171.175. 408, 417, 451. 467. 484, 495, 532. 538 Dudley, C. E. _-.."'~7,__~79 Duester. G.. 525.537 Duff. R. S.. 236. 249 Dufort. D.. 43.50. 158. 165.175. 303. 304. 316. 324 Dufton. C., 423.482 Duglas-Tabor. Y. 528. _.~.~.535 Dub. E M.. 227. 229 Duke. M.. 347, 360 Dulac. C.. 201,208 Dulac, O.. 94. 99 Dull. M., 440, 471. 488 Dulor, J. R, 271,273 Dutuc, I., 308, 310, 324 Duman, R. S.. 247.249 Duman. S.. 573.584 Dumenil. D., 205.207 Dumont. D. J., 218. 223.224. 228. 229, 230 Duncan. A. M. V., 224. 229 Duncan. M. K.. 188. 190, 532.535, 538 Duncan. R. R. 8.16 Duncan. S. A., 164. 165.175, 17Z 302, 303, 316. 317.323, 324, 326, 32Z 340. 343. 347. 352. 365 Dunger, D. B.. 506, 514 Dunlop, L. L., 484 Dunn, A. R., 194, 208 Dunn. I.. 388. 392 Dunn. L. C.. -442. 443.494 Dunn. M. A.. 185.190. 243.249 Dunn, N. R., 183, 190, 3 t l , 3 1 2 , 3 1 3 , 3 1 4 . 3 1 5 , 3 2 5 , 3 2 9 , 336. 363 Dunn. R.. 305.329, 453.467. 496 Dunne, C.. 236, 252 Dunner. S.. 264. 274 Dunwoodie, S. L., 135. 145. 324. 339. 348, 360, 368 Dup6, V., 91.10t, I 17, 123, 451,468, 469, 484, 485, 490, 525,536, 545,562 Dupin, E.. 236, 237.241. 244. 249. 250 Duplan Perrat. E. 585 Duprat, A. M., 303.324 Duprez, D.. 140. 141. 142. 146, 147. 148. 262. 263. 264. 266, 2 73. 2 74, 2 77, 336. 358 Dupuis, S., 90. 95, 106 Durand. B.. 469. 485 Durbin. J. E., 286, 292

A u t h o r Index Durham-Pierre, D., 6, 17 Duriez, B., 508, 516 Durocher, D., 342, 343, 360 Duronio, R. J., t 70, 175 Durviaux. S. M., t66, ] 76. 32 t. 325 Dussel, R., 86, 99 Dutt, R. 196. 202. 210 Dutta, A.. 251 Duxson. M. J.. 131. 132, 149 Dwarki, V. J.. 258, 277 Dwivedi, R. S., 318. 328 Dyer. R.. 354. 355.364, 451. 489 Dymecki. S. M.. 109, 110, 123, 228, 229 Dyson, E., 341. 352. 368 Dyson, J.. 378. 387.390 Dyson, N.. 483 Dziadek, M.. 314. 327 Dzierzak. E.. 192, 194. 195, 196, 199, 200, 201,202, 204, 207. 208. 209. 220, 231, 317,327

E Eagleson, G. W., 77, 99 Eagleson, K. L., 84, 101 Eales. N. B.. 473.484 Easley, K., 582. 583,585 Easter, S. S., 85, 102 Easter. S. S., Jr., 85, 102 Eastman, Q., 579, 585 Eatock. R. A., 555. 558, 559, 561, 563 Eaves. C. J., 242, 245,251, 252 Ebendal. T., 9 I, 105 Ebensperger, C., 140, 144, 145, 147, 255, 263. 273 Eberhardt, C.. 20.~, 207. 2,._~, 228 Eberspaecher, H.. 281,295, 473,476, 498, 573, 588 Ebron, M. T., 161, 178 Eccles, M. R., 398, 419, 453,487 Echelard. Y.. 78, 99, 121,123, 14I, 145, 470, 484 Echenne, B., 94, 99 Eckert, R. L., 58t, 588 Ecochard, V., 303,324 Economides, A. N., 44, 50, 338, 362 Economou, A., 377, 390 Eddison, M., 553, 559, 561 Eddy, E. M., 27, 33 Ede, D. A., i28. 137, I39. 146, 470, 484, 534, 535, 577, 585 Edelberg, J. M., 219, 228, 229 Edelman. G. M., 134. 148 Edelmann. W., 43.50, 415,420 Edgar, B. A.. 169. 170, t 75 Edlund, H., 319, 320, 321,322, 323, 325, 326 Edlund, T.. 78.79, 80. 8i, 85, 87, 99, 104, I08, t20, 123, 124, 141,148, 319, 320, 321,323, 325, 504, 505,506, 514

Author Index

Edmondson, D. G., 260, 262, 273. 274, 333, 342, 360 Edom-Vovard, E, 266, 273 Edwards, J. G., 343, 360 Edwards, M. C., 468, 485 Edwards, R. G., 7, 16 Ee, H. C., 321,328, 451,475,494 Egarter, C.. 174, 177 Eggermont, E.. 281,293. 381,391 Eglitis, M. A.. 26, 33, 35, 206, 207 Eguchi, G., 525,529, 53 t. 534, 535, 537 Ehmke, H., 86, 99 Ehrlich, J. S., 451. 476, 493 Ehrtich, M. E., 91, lOI, 505,517 Ehrman, L. A., 339, 360 Eichele, G., 287, 294, 336. 342, 346, 360, 36& 370, 469, 470. 486 Eicher, E. M.. 311,323, 378, 382. 389, 390, 392 Eichmann, A., 200, 20Z 208 Eichmuller, S., 574, 588 Eickholt, B. J., 425,484 Eidne, K. A., 512, 514 Eiholzer, U., 509, 517 Eimon, R M., 44, 50, 338. 362 Eisen. J. S.. 265,269, 273 Eisenbach, L., 467, 492 Eisenberg, L. M.. 339. 360 Eisenman. R. N., 580. 586 Eisenstat, D. D., 79, 87, 88, 89, 90, 91, 95, 9Z 99, 450. 470.471,481, 484 Eizag~airre, J., 506, 514 Eizuru. Y.. 473,498 Ekblom, R, 407, 410, 41 I, 41Z 418, 420 Ekker, M., 89, 106, 450, 470, 471. 473, 48], 496, 498, 546, 561

Ekker, S. C., 470, 489 E1Amxaoui, A., 504, 514 E1-Amraoui, A., 550, 563 el Bahi, S., 476, 477. 496 Elbaum, M., 94, I04 Elchebly, M., 510, 514 Eldadah, Z. A., 347,360, 453, 495 E1-Deiw, W. S., 34 I, 370 Elder, F. E B., 352, 354, 366 Elefanty, A. G., 203,209, 343, 3619 Elenius, K., 156. 178 Elger. M.. 395,417 E1 Ghouzzi, V., 452. 453,484 Elia, A. J., 136, 137, 139, 145, 341,370, 468, 495 Elias. R M., 570, 580, 581,582, 583, 584, 585, 586, 58Z 588 Elinson, R. R, 335,362 Elkabes, S., 502, 514 Elkahloun, A. G., 347, 366 Eikins, J. A., 357, 358 Elkins, R., 347,360

6 13

Elledge, S. J., 287, 294, 468, 485, 528, 532, 538 Ellies, D. L., 450, 47t, 496 Elliot, D. A., 34 I, 348, 349, 356, 358, 362, 367 Eltiott, C., t 73, t 79 Elliott, D. A., 348, 349, 352, 353, 357, 358 Ellis, K., 15, 15 Ellisman, M., 453,486, 509, 513, 580, 581,583, 586 Elmer, E., 247, 249 Elsemore, J., 79, 106, 529, 538 Elsholtz, H. R, 509, 515 Elwood, N. J., 203, 209 Emerson, C. R, Jr., 140, 141,144, 258, 260, 263,264, 265, 267, 272, 274, 276 Emmen, J. M., 386, 393 Emmert-Buck, M. R., t 73, t 76 Emmons, S., 185, t89 Emonard, H., 573, 587 Emoto, H., 3 t I, 312, 313, 314, 315, 323, 325 Emoto, N., 465,498 Emura, M., 3 I5, 324 Enderich, J., t 70, 173, 179, 238, 252 Enders, A. C., 156, 175 Enders, G., 188, 190 Endo, N., 288, 293 Enerback, S., 479, 48Z 527, 530, 532, 535 Engel, J., 408, 4t8 Engel, W., 386, 388, 392, 393 Engelkamp, D., 391, 528, 537 Engels, L. J., 193, 207 Engemann, S., 9, I8 Engsig, M. T., 29 t, 292 Enomoto, H.. 289, 294 Enomoto-Iwamoto, M., 289, 294 Ensini, M., 109. I23 Enver, T., 194, 199, 200, 20t, 207, 210 Enwright, J. E III, 435, 469, 476, 484 Enyeart-VanHouten, D. L., 381,392 Ephmssi, A., 185, 189 Epifano, O., ! 4, 16 Episkopou, V., 2, 4, 9I, 102, 527, 530, 531,537 Eppig, J. J,, 8, t0, 11, i2, tZ 18 Epstein, D. J., 78, 79, 99, t02, t15, 120, 12t, 123, I24, I41,145, 428, 470, 475,484, 491 Epstein, E. H., 573,579, 583,585, 586, 588 Epstein, E. H., Jr., 287, 293 Epstein, J. A., 268, 273, 353, 357, 365, 528, 535 Erdelyi, M., 185, 189 Erdjument-Bromage, H., 405,407, 416, 417 Eren, R., 196, 207 Erickson, C. A., 237, 249, 423, 424, 484 Erickson, R. L., 27 t, 276 Erickson, R. R, 14, t5 Ericson, J., 43, 50, 78, 79, 80, 81, 82, 85, 88, 98, 99, I20, 123, 474, 494, 504, 505, 506. 514

61 4

A u t h o r Index

Eriksson. E S., 237, 247, 248, 249 Erkman, L., 556, 558, 562 Erlander. M. G., 12, ] 7 Ertebacher. A., 280, 292 Erler. T., 167, 174 Ernfors. R, 559. 562 Ernsberger, U.. 238, 251 Ernst. H., 350, 3 70 Ernst. M. K.. 7.19. 188, 189 Ernst. R. 376. 390 Ernst. R. E.. 194. 206. 209 Erven. A.. 546, 565 Erway, L. C.. 550. 563 Escalante-Alcalde, D., 43.50 Escary, J. L., 205. 207 Esni, E. 322. 324. 409. 419 Essner. J. J.. 47.50 Esteban. L., 171, 173.178 Esteve. R. 546. 562 Esumi. E.. 260. 276 Esumi. H.. 310. 327 Etheridge. A.. 351. 364 Etkin, A.. 318. 328 Etkin. L. D.. 8. 17 Eto, K., 422, 424. 425.435.437, 438, 451,453. 475,476. 48Z 492, 493, 527. 530. 535. 572. 588 Eto, Y., 321. 324 Evans. H. M., 211. 229 Evans. J. R. 7.16 Evans. M. J.. 7.16, 26, 32.35, 158. 160. I66. 167. I78. 179, 203, 210, 303.3 I3.324, 327. 453,482 Evans. R. M., 172. 173.174, 341. 346. 352. 354. 357,358, 363. 367, 368, 468.469. 489, 490. 511. 513, 516 Evans. S., 334, 336. 339. 342. 360. 361. 368 Evans, T.. 281. 294. 316. 326. 339. 342. 343. 346. 347.361, 362, 363 Evrard. Y. A.. 136. 137. 138, 145 Evsikov. S. V.. 26. 27.33 Exalto. N.. 174. 177 Externbrink, A.. 347. 363 Ezer. S.. 441,488 Ezine. S.. 205,207 Ezfin. C.. 511, 5t5

F Fabian, B. C.. 337, 361 Fabri, L., 40, -44. 50, 323, 338, 358 Faccenda. E., 512, 514 Factor. S., 353, 357, 365 Faerman, A.. 258, 263,267. 274 Faessler. R.. 183, 187. 188, t89 Fagan, A. M., 91, 99, 559, 562 Fagerstr6m, C., 91, t05, 120, 125

Fahrig, M., ,.0~, 20Z ')93 228 Faielta, A., 422, 427, 433, 487 Fainzilber, M., 40:2, 420 Fairclough, L., 303, 323, 329 Fair6n. A.. 84. 105 Falchetto. R., 226. 230 Falck. R, 173, 178 Fallon. J. E, 471. 493 Fallon, J. R., 11.19, 121. t25 Faloon. R. 217.232 Falzarano. E R.. 557. 563 Familari, M., 14. 16 Fan, C. M., 40, 44. 51, 85, 95, 99, 102, t40, t4i, I42, 145, 147, 225,229, 260, 263, 264, 265,266, 274, 275, 276, 287, 292, 506, 516 Fan. H., 578. 579, 585 Fan, M. J.. 415,418 Fan. W.. 226. 232 Fan. X.. 415,418, 525,537 Fancher. K. S.. 15, 16 Fang. G.-H.. 218. 230 Fang. J.. 279. 292. 427, 484 Fang. M.. 402. 417 Fangman. J.. 2, 4 Fannon, A. M., 408, 419 Fantes, J.. 361 Fantuzzi. G.. 314. 324 Faq, C.-M.. 141,147 Faravelli, L.. 235.242, 244, 250 Farese, R. V.. Jr.. 161.175 Farih-Sips, H., 285,293 Farinas, I., 91, 92, 100, 10t, t72, 175, 239, 249, 402, 418, 440. 471. 488, 497, 549, 559, 562, 583,589 Farkash. Y.. 510,515 Farlie, R G.. 453,486 Farrance, I. K.. 262. 274, 277 Farrell, A. R. 350, 360 Farrell, M. J.. 14, 17 Farrington, S. M., 200, 206 Fassler, R.. 204, 20Z 284, 293, 317, 324, 427. 484, 574, 583,584, 585, 589 Fatkin, D.. 343, 350, 351,352. 357, 358, 359 Faust, C.. 31.34 Faust. N.. 194. 207 Faustinella. E. 94, 103 Fausto. N., 318. 324, 329 Favier, B.. 585 Favor, J., 94, 104, 451,475,486, 522, 527, 528, 530, 535, 536, 549. 562 Fawcett. E., 442. 456, 484 Fawcett. J. W,. 134, 148 Fazeli, A., 318. 325 Fazleabas, A. T., 156, 175 Featherstone, M., 118, I24

'

Author

i

n

d

e

Federici, R., 314, 330 Fedtsova, N. G., 504. 505, 515 Feger. G., 136, 145 Fegert, R, 316. 323 Fehon, R. G.. 136, 145 Feijen, A., 47, 50, 92, t06, 467, 484 Feingold, K. R., 580, 58 I, 584. 585, 586, 587 Feingold, M.. 507.514 Feinstein. S. C., 9 t, 98 Fekany, K., 78, 104 Fekete, C. N.. 375,389 Fekete, D. M.. 527,535, 547, 548, 550, 551,552, 553, 560, 561, 562, 563 Feldman, B., 166. 175, 313, 314, 315,324 Feldmeyer, D., 92, 104 Felice, M. D., 311. 326 Felix, R.. "443, 485, 511,514 Fellous, M., 375,376, 382. 387. 389, 391. 392 Feneley, M., 348, 349, 352, 353. 357, 358 Feng, X. H., 226, 229 Fenner, M. H., 339, 368 Ferguson, B., 441,488, 491, 497 Ferguson, C.. 434, 437, 438, 440, 451. 484, 496 Ferguson, E. L., 336. 370 Ferguson, M. W. J., 434, 437, 441,467, 490, 493, 496, 568, 582, 583,586 Fernandez-Salguero, R, 3 t 1, 312, 326, 474, 488 Fernandez-Teran. M.. 349, 360 Fernaud-Espinosa, I., 525,535 Fernyak, S. E., 269. 273 Fero, M. L.. 558, 563 Ferrante. E., 378. 087. " 389 Ferrara, N., 203, 20Z 223,229, 230, 291,292 Ferrari, C.. 509, 5I 7 Ferrari, G., 206, 20Z 246. 249, 271,274 Ferre-D' Amare. A. R.. 525, 538 Ferreira. V., 203, 20Z 223,228 Ferreiro, B.. 77.99 Ferrero. G. B., 357. 361 Ferretti, E., 443.449, 478. 479, 482, 484 Ferretti. R, 422. 426, 449, 487 Ferri, G. L.. 86. 103, 188, I90 Ferrier. M. L., 403,418 Fessler, L. I., 205, 209 F6siis. L.. 582. 588 Fetka, I., i73, 179 Fetka, L., 428, 475, 497 Fidler, I. J., 172. 173, 180 Fielder, T. J., 286, 294 Fiering, S., 157, 180 Figdor, M. C., 75, 99 Filosa, S., 40, 43, 50, 477. 484 Filvaroff, E. H., 280, 292 Fine. J., 512, 513

x

6

1

5

Fine, L. G., 404, 420 Finegold, M. J., 44, 51, 246, 250, 3 t5, 318, 326, 32Z 374, 385,389 Finger, J. N., 6, 17 Fink, I. L., 343, 360 Fiocco, R., 246, 250 Fiolet, J. W. T., 343, 345, 348, 366 Fiorenza, M. I"., 10, 16 Fire, A., t4, t6... 1Z 185, 190, 243, 252, 34t, 360 Firmin, D. N., 35 I, 363 Firulli. A. B., I68, 175, 342, 343, 344, 347. 349, 352, 361, 365 Firulli, B. A., 347, 349, 361 Fischer, A., a5~, a~6, 361 Fischer, K.-D,, 203, 209, 2 t 7. 224, 231, 232, 318, 327 Fischer, L., 280, 292 Fischer, W. H., 286, 292 Fischman, D. A., 219, 228, 231 Fishell, G., 78, 8!, 84, 88, 89, 99, 100, 101, 102, 103, 1I0, 121,123, 124, 241,244, 249, 251 Fisher, A., 376, 390 Fisher, C., 575, 586 Fisher, D. E., 525,538 Fisher, G., 84, 101, 45t, 475,486, 527, 528, 530, 536 Fisher, L. J., 247,252 Fisher, M., 424, 495 Fisher, R. I06 Fisher, S. A., 34 i, 361, 369 Fisher, S. J., t63, 170, 173, 174, 174, 175, 176, 177 Fishmm-a, D. A., 34 t, 362 Fishman, G. I., 334, 348, 367 Fishman, M. C., 213, 232, 339, 344, 345, 35i, 355, 359, 361, 368, 395, 417 Fissore, R. A., 8, 16 FitzGerald, R G., 527, 530, 531,537 Fjose, A., 546, 563 Flaherty, L. A., 451,476, 493 Flamme, I., 21 I, 215,220, 228, 229, 230, 232 Flanagan, J. N., 381,392 Flanders, K. C., 226, 230, 3 t4, 325 Flandin, R, t 0, 18 Flannew, M. L., 168, 169, 175, 179, 349, 360 Flavell, R. A., 79, 87, 100, 101, 106, 529, 538 Flax., J. D., 86, 104 Neenor. J., 14, 16 Fleiget, L., 343,347, 361 Fteisch, H. A., 443, 485 Fteischman, R. A., 204, 20Z 245, 249 Fleming, J., 556, 565 Fleming, R. J., 136, t45 Fleming, S., 399, 416, 419 Fleming, T. R...a, 34, 156, t58. t 75 Fleming, W. H., 244, 252 Flenniken, A., 119, 125, 423, 424, 494

61 6 Fletcher. C. F., 85, 99, 321,328 Fletcher, J. M., 407,417 Fliedner, T., 195,208 Fliering, S., 356, 360 Ftiniaux. I.. 585 Flint, A. F., I93, 206, 207. 246, 250. 270. 271,274 Flint. O. R, 128, 137, 139. I46, 470, 484 Flores-Delgado, G., 31 t, 329 Floridia, G., 378. 387. 389 Florkiewicz. R. Z.. 453.483 Flower. V~ H., 442, 484 Flugge, G.. 247, 250 Flynn, J.. 263,274 Flynn, L. M.. I61. 178 Flynn. S. E., 506. 508. 509. 514, 515. 518 Fode, C.. 83.88.89.90, 95.98, 99, 435.484, 490, 526. 536 Foged, N. T.. 29 t. 292 Fogels. H. R., 507. 514 Fogo. A.. 305.329, 403.418 Fogo, A. B.. 453.467. 496 Foidart. J. M.. 573.587 Foitzik, K., 574, 588 Foley. A. C.. 48.50, 427.484 Foley. K. R. 580. 586 Folkman. J.. "~v0 ;~)9 Follette. R J.. 170. 175 Follettie, M.. 40. 49, 323 Fong, G.-H.. 217. 223,227. 229 Fong, T. A.. 217. 220, 229 Fontaine-Perus, J., 267,274. 335,337. 340. 358 Forbes. A.. 185.186. I89, 190 Ford. L. R, I1.16 Forehand. C. J.. 469. 482 Foreman, R. K.. 468, 488 Forlani, S., 9, 16 Forrest, D., 510. 515 Forsberg, H., 134, 135. 138, 139. 146 Forslind, B., 580, 586 Forster, A., 203.210 Forstrom, J. W.. 467, 495 Fortini, M. E., 136. 146, 553.561 Fossett. N., 334. 343.361 Foster. D. N.. 349. 362 Foster, E S., 109, 125 Foster, J. A., 7, 16 Foster, J. W., 281. 292, 377, 380, 381,386. 388. 390, 391, 393 Foti, G.. 263,274 Fougerousse, E, 525,535 Foulquier, E, 303,324 Fournier-Thibautt. C., 264, 266, 273 Fox, C., 8, 17 Fox, C. H., 311.312, 326, 474, 488 Fox, G. M., 402, 417

Author Index Fox, H., 322, 326 Fox, J. W., 94, t00 Fox, M., 47, 50, 451,476, 483, 508, 5t4 Fraboulet, S., i34, 1138, 139, 147 Fraccaro. M.. 378, 387, 389 Frain, M., i ! 8, 1 t 9, t24, 125, 468, 492 Francesca, T., 114, 122 Francis, E, 94, 99 Francis, M.. 398,416, 550, 561 Francis, N., 239, 249 Francis, R J., 533. 535 Francis-West, R H., 140, 141. I42, 147, 238, 251, 263, 264, 266. 276, 422, 467, 481, 484, 577, 578, 579, 587 Franco. D.. 341,343.344. 345.347. 348, 350, 35 I, 355, 356, 359, 361, 362 Franco Del Amo, E, t35, 136, 137, 148 Francomano, C. A., 286, 292, 452, 453, 481 Frank, E.. 237, 249 Franke, W. W:. 570. 586 Frankel, Wi N.. 136. 137, 147 Franklin. G., 173. 178 Frankovsky, M. J., 342, 359 Franz, T.. 83, 84, 99, 258, 263,264. 266, 272, 274, 475, 482, 522. 527. 532, 535 Franzoso, G., 442. 484 Frasch, M., 334. 336, 340. 358, 361, 363, 364, 370 Fraser, E C., 443,495 Fraser, R, 202, 20Z 210 Fraser, S. E., 40, 52, 118, t I9, 122, 123, 237, 243, 244, 249, 252, 424, 425, 488, 495, 543, 565 Fraulob. V., 310, 329, 557,562 Frawley, L. S., 511,514, 5t5 Frazer-Abel, A. A.. 505, 517 Frazier-CierpiaI, L., 237, 249 Frederich. M. E., 307, 324 Frederick. L., 287,294 Fredga, K.. 388. 390, 391 Freedman, M., 202, 208 Freeman. A., 575, 576, 577. 578, 588 Freeman, G., 313,324 Freeman, M., 466, 482 Freeman, T. B., 247, 251 Freer. A. M.. 121,124, 451,453.470, 471. 491 Freidman, T. B., 557, 561 Freidrich, G. A., 341,352, 359 Freie. B., 243, 244, 249 French, M. C., 398, 419 Freshney, R. I., 317.32 7 Freund, J. N., 308.3 t 0, 324 Freund, N., 403,418 Frick. H., 442, 485 Friday, R. V., 166. 176, 303, 325 Fridell, R. A., 556, 557, 561, 564 Friedman, R., 453,467,494, 550, 563

A u t h o r Index

Friedman, T. B., 475.481, 556. 564 Friedrich, R. W.. 557, 564 Friedrich, U., 281,293, 381,391 Friend, D. S., 318, 328 Fries. R., 264, 274 Friesel. R. E., 453, 492 Frischauf. A. M., 377, 388, 393, 415.4I 7 Frisen, J., 236, 237.244, 245, 246, 248, 249, 250. 3 t 8, 324, 402. 419 Frist. N. L., 26.34 Fritsch, R.. 83.85.9 I, 105 Fritschy, J. M., 84. I03 Fritz, D. T., 11, 16 Fritzsch, B., 84. I01, 1t6, 1t7, 124, 489, 549, 559, 56I, 562, 563

Froelick, G. J., 374, 385,389 Froesch. E. R., 506. 517 Frohman, M. A.. 79, 98, 243, 249 Frojdman, K., 5 t 1,516 Frolichsthal, R, 235, 242, 244, 250 Fromentat-Remain, C.. 310, 329 Fromm. S. H., 437, 498 Frost, D., 558, 563 Frost, H. M., 442, 485 Frotscher. M., 92, 104 Fruth, E. E., 318, 324 Frutiger, S., 321,326 Fu, X. Y., 286, 293, 294 Fu. Y., 334, 348, 361 Fuchs, E., 247,250, 271,274, 440. 498, 570, 571,572, 574, 577, 579, 580, 581,583, 584, 585, 586, 588, 589' Fuchs. H.. 551,552, 557, 563 Fiichtbauer, A., t0, 12, 16, 135, t46 Fiichtbauer. E. M.. 10, 12, 16 Fugo, N. W.. 22, 33 Fuhrmann, G., 160, 175, 183, 190 Fujii, H., 47, 51, 89, 95, 99, 102, 506, 516, 525, 535 Fujii, S., 343,344, 351,367 Fujii, T., 374, 390, 508, 517 Fujii-Kuriyama, Y., 525,535 Fujimori, K. E., 90, 105 Fujimori, T., 133, I35, 137, t49 Fujimoto, S., 305, 307,328 Fujisawa, H., 223,230, 528, 536 Fujisawa-Schara, A., 258, 271,272, 274, 275 Fujita, J.. 189. 189 Fujita, K., 189, 189 Fujita, S. C., 557, 565 Fujita, T., 336, 365, 443,453,487 Fujiwara, M., 312, 328, 422. 424, 435,437, 451,475, 476, 492, 505, 51Z 527, 530, 535 Fujiwara, Y., 203,204. 207, 209, 210, 219, 224, 228, 232, 341,343. 352, 369 Fukai, N.. 292. 294

61 7

Fukamachi, H., 308, 324 Fukiishi, Y., 437, 485 Fukuda-Taira, S., 316, 317,324 Fukumoto, S., 347, 359 Fukumura, D., 218 _2.~, 2125.229, 230 Fukushige, T., t 65, 175, t 7Z 316, 325 Fukuyama, M., 165, 180, 316, 330 Fuller-Pace, E, 84, 102 Fulton, D., 225, 23I Fults, D. V~, 287, 294 FumagaIli, E, 511, 5t3 Funahashi, J., 112, 114, 115, 1i6, 123, ]24 Funayama, N., 57 I, 586 Fundele, R., 9, 17, I8 Fung, S., 403,418 Funke, B., 353, 357, 365 Furley, A., 78, 8 i, 99 Fudonger, K., 318, 327 Furness, D. N., 558, 563 Furness, J. B., 167, t 74, 305, 323 Fumkawa, M., 321,324, 50,'5,516, 549, 564 Furakawa, T., 521,525, 527, 535, 537 Fummoto, T., 135, t 46 Furuta, Y., 7, ]6, 82, 86, 99, 287, 292, 305, 3 t 2, 314, 3 i5, 323, 329. 453,467, 496, 504, 505,507, 508, 5t0, 51& 530, 532, 535 Furutani-Seiki, M., 78, 104, 532, 534, 536, 546, 565 Fuse, N.. 169, 175 Fushiki, S., 58 I, 582, 583,587

G Gabet, C. A., 27, 33 Gabow; R A., 414, 4t8 Gabriel, H. D., 173, t 75 Gad, J. M., 129, i42, 146, t62, 174 Gadi, I., 156, ] 79 Gaffield, W, 78, 99, 101 Gage, E H., 235, 236, 237. 241,244, 246, 247, 248, 249, 250, 251, 252

Gage, R J., 353, 354, 355, 361, 433,478, 485, 507, 508, 509, 511,514, 515 Gagliardi, R C., 506, 515 Gaiano, N., 78, 89, 99, 100, t 10, 123 Gailani, M. R., 287, 292, 470, 486, 579, 586 Gaillard, D., 271,277 Gainer, H., 435, 498 Gainetdinov, R. R,, 51 t, 513 Gaio, U., 353, 356, 361 Gajewski, K., 334, 343, 361, 363 Galacteros, E, 376, 391 Gatante, R E., 3 t0, 321,324, 325, 327 Galceran, J., 83, 86, 92, t00, t 72. 175, 440, 471,488 Galindo, J. E., 12, t 7

61 8 Gall, J. A., t 94, 208 Gallagher, B. C., 542, 562 Gallahan, D., 226. 230 Gallardo, M. E.. 522, 525, 535, 537 Galli. R.. 235,242. 244, .~46, 250 Gallicano. G. I.. 8. 13.16. 17. 27.33, 34 Gallione. C. J.. 227, 231 Galliot. B.. 475.485 Gallo. M.. 40. 49. 323 Galloway, A. C., ,_794,232. 504, 517 Galvin. B. D., 453,485 Galvin. N., 314. 324 Gambarotta. A.. 89, 106. 450. 471. 498 Gan. L.. 136, 137. 138. I45, 527. 535. 536. 556. 558.565 Gan, O. I., 199. 208 Gandarillas, A.. 580. 586 Gandolph, M. A.. 415.420 Gangadharan. U.. 281. 295 Gannon. E H.. 271. 276 Gannon. M.. 324 Gans, C., 421,427, 457,485 Gansmuller, A.. 40. 43.50. 94. 100, 451,453.469. 477. 484, 489 Gao. J.. 219. 233 Gao. L. Y.. 284. 294. 442. 443.497 Gao, M.. 11.16 Gao, Q. S.. t 66, 179 Gao, W: Q., ) ~ . z~6. 565. 566 Gao, X.. 136. 149, 548.551. 552. 561,562 Gao. Y. H., 288. 293. 442. 443.453.488 Garber. M.. 91.99 Garbutt. C. L.. 26, 34 Garcfa. C.. 84, 95. I02, 105 Garcia. L. F., 547, 562 Garcia Bellido. A.. 136. 145 Garcia-Cardena. G.. 225.231 Garcia-Castro, M.. 121. 125. 187. 189, 355.356.361 Garcia Delgado, C., 453,486 Garcia-Fern~indez, J., 305,323 Garcia-Garrido. L.. 219. 228.23I Garcia-Martinez, V., 317.328, 336, 344. 361, 362 Garcia-Porrero, J. A., 195, 196, 200, 207 Garcfa-Verdugo. J. M., 90. 95, 102, t06 Garcia-Wijnen, C. C., I94, 208 Gardahaut. M.-E, 335, 337, 340. 358 Gardiner. A.. 334. 335,336. 337. 339. 365 Gardiner. C. S., 509. 518 Gardner, A.. 352. 354, 366 Gardner, D. G.. 343,364 Gardner. J., 292. 294 Gardner, R. L., 25, 26, 27, 28, 29, 30, 31.32, 33, 34, t56, 157. 158. 161. 166, I76, I78, 182. 183. 189, 2 0 2 . 2 t 0 Garel, C.. 507.514 Ga_rel, S., 91, I00

A u t h o r Index G ~ n k t e , J., 292, 294 Garland, D., 528,533,535 Garnier, J. M.. 469, 485, 489 Garofalo, S., 286, 294 Garratt. A. N., 353, 356, 36] Garrels, J. I.. 11, I7 Garretson. D. C.. 307.324 Garrett. K. L.. 260. 269, 27 !. 274, 275, 277 Garrick, D., 376, 390 Garrity, R A.. 399. 419. 530. 533~ 537 Ga~wod, D. R.. 405, 417 G~tner. A.. 83.98 ~" 237, 244. 245. 248, 249 Garzia-Verdugo, J. M. . ~.~6. Gasca. S.. 50, 121,123 Gashier, A., 400, 418 Gaspar, M. L.. 386. 390 Gassmann, M.. 173, 174. 175, 225,230, 424, 425.485 Gat. U.. 577. 579, 584, 586 Gato, A.. 547. 564 Gattner. K.. 415,420 Gattone, V. H. II, 415,417 Gaub. M. R. 469, 490, 49t Gaudenz. K., 452. 453.481 Gaunt. S. J.. 40. 50, 376. 390, 477,482, 485 Gautam. M., 411,419 Gauthier. Y., 510. 518 Gautier. R.. 198. 199, 200, 201,207, 208 Gavalas. A.. 117. 118, 119, 123, 125, 427, 433,443, 449, 469. 474. 479. 485, 494, 545,562 Gavin. A. C.. 7, 16 Gavis. E. R., 6, 16 Gavrilina, G., 510, 516 Gawantka, V., 138, 146 Gay, N. J., 10, I8 Gaztambide, S.. 506, 514 Gearhart, J. D.. 225,230 Gebauer. E. 7. 10, 16 Gebbia, M., 357,361,363 Gebre-Medhin, S., 313,323, 326, 413, 41& 467,482, 489 Gebuhr, T., 357,364 Gecz, J., 376, 393 Gee, H.. 266, 274 Gehring, Wi J., 533,535, 536 Geiger, B., 408. 418 Geiger, H.. 194. 207 Geisendorf. S., 532, 537 Geiser. A., 226. 230 Gelbart, Vr 343,360 Gemza. D. L., 526, 527, 535 Genbacev, O., 173, 174, 176 Gendron-Maguire, M., 136. 149, 224, 232, 427, 432, 433, 443, 449, 473. 474. 477,485, 494 Generoso, W: M., 22, 34

A u t h o r Index

Geng, L., 4t5, 4 I Z 4]8 Geng, X.. 579, 589 Genin. A., 347, 364, 366 Geoffroy. V., 288, 289, 292 Georgakopaoutis, D., 343, 350. 351,352, 359 George, E. L., 222, 229, 340, 352, 361 George. E V~, 373. 390 George, S.. .44. 50, 227, 231 Georges, M.. 264, 2 74 Georges-Labouesse, E.. 94, 100. 183, 187, 188, t89, 572. 5 83,585, 586

George-Weinstein, M., 263, 264, 274 Georgiev, O., 317,325 Georgopoulos, K., 9 l, 103, 204, 2i0 Georgopoulou, A., 204, 2t0 Geraedts. J. R. 174, 179 Gerald, W:, 400. 418 Gerber. A. N., 356, 359 Gerber. H. R. 223,229. 29 t, 292 Gerety, S. S.. 218, 225. 229 Gerhart, J.. 40, 41,44, 50, 263. 264, 274 Gerken, S. C.. 412. 417 Gerlach, L. M., 550. 562 Germain. L.. 3 t 7, 324 German, M. S., 321,325, 328, 329, 451. 474, 475,494, 509. 515 German. R., 453,483 Germiller. J. A., 550, 562 Germino, E J., 414. 419 Germino. G. G., 414, 415,419, 420 Gerrard, B., 287. 292, 470, 485, 486 Gershon. M. D., 244, 251 Gertenstein, M., 168, 169. 172, 173. i74, 174, 178, 203, 20Z 209, 223,228, 349, 355, 367 Gertner, J. M., 443,497 Gertner, R., 286, 294 Gerton, G. L., 7, 16 Gertz, B. J.. 512, 515 Gerwe. E. A., 84, 9Z 435,469, 476. 481 Gerwins, R, 504, 515 Geschind, D. H., 247, 251 Geske, R., 442, 443, 495 Gespach, C., 271. 277 Gessler, M.. 347, 363, 415, 417 Gesteland, K., 38, 52 Ghahremani, M., 399. 417 Ghatas, I., 79, 89, 99 Ghatpande, A., 343, 347, 361 Ghatpande, S., 343,347, 361 Ghattas, i., 79, 89, 102, 422, 437. 450. 451,471,472, 473, 474, 484, 489, 493 Gherardi, E., 317, 328, 404, 419, 420 Ghosh, A.. 86, 92, 100, 102, 103 Ghosh, S., 318, 323

6 t9

Ghysetinck, N., 91, ]Ol, 341,346, 347, 352, 362, 469, 484, 485, 525,536, 545,562 Giancotti, E G., 410, 417 Gibbons, R. J., 376. 390 Gibson, M., 334, 361 Giguere, "~L,167, t 7 Z ] 79, 346, 363 Gitard i-Hebenstreit, R, t 19. 125, 135,147. 546, 564 Gilbert, D. J., 168, 176, 41 I, 418 Gilbert, T., 403, 4t8, 420 Gilbert, W., 525, 537 Gilbride, K., 3 t4, 328 Gilgenkrantz, H., 342, 368 Gitl, G. N., t35, 146 GilIemans, N., 343, 369 Gillies, S., 287, 292, 470, 486 Gilmore, E. C., 94, t00 Gilmour, K. C., 288, 294, 442, 443, 453, 493 Giniger, E., i 85, ]89 Ginsbu~, M., 183, 189 Ginzbe~, R. D., 560, 563 Giocanazzi, S., 376, 393 Giraldez, K, 422, 466, 494, 496, 542, 543, 545,546, 559, 562, 564, 565

Girbal-Neuhauser, E., 581,588 Girgis-Gabardo, A., 260, 270, 271,272, 276 Giros, B., 51 t, 513 Giros, R., 34t, 352, 362 Giroux, S., I71,173, 176, 318, 324 Gisselbrecht, S., 340, 36I Gitelman, I., t 35, t46 Gitelman, S. E., 280, 292 Gitlin, J. D., 3 t3, 325 Giton, Y., 84, t00 Gittenberger-de Groot, A. C, 219, 226, 229, 232 Gittenberger-de Groot, A. C., 333, 34 I, 360, 366, 453, 467, 494

Gittes, G. K., 319, 321,324, 452, 491 Giuli, G., 381,390 Glaser, T., 375,392, 415,416, 41Z 526, 527, 528, 535 Glasser, S. W., 313, 314, 329 Gleeson, J. G., 94, 100 Gleiberman, A. S., 478,496, 504, 505, 506, 507, 508, 509, 514, 515, 518

Glickman, S. E., 388, 393 Glimcher. L. H., 527, 530, 53 i, 536 Glimcher, M. J., -442, 485 Gliniak, B. C., 173, 179 Glinka, A., 44, 50. 325, 338, 361, 362 Globerson, A., 196, 20Z 245, 251 Glomski, C. A., 2 t 9, 220, 232 G!otzer, J., 185, 189 Glowacki, J., 285, 293, 442, 443,488 Glynn, M., 287, 292, 579, 586 Gnarra, J. R., 173, 176, 227, 229

6

2

0

A

u

Gocza, E.. 42, 5I, 158, I60, ]7Z I78 Goda, H., 5 t I, 515 Goda. R, 5t0, 516 Goddard, A., 402, 420, 579, 589 Goddard, J. M.. 466, 490, 545,561 Goddetl. M. A., t93,207 Godfraind, C.. 166. ] 76, 321. 325 Godfrey, R. 443,485, 508.515 Godin. I.. 185. 187, 188. 189. i 95.196. 199. 200. 207. 208. 209. 220. 229 Godin. R. E.. 171.175. 408.417 Godsave. S. E. 263.274 Goebbels. S.. 92. 104 GoeddeI. D. V.. 223. 230. 341. 370 Goedecke. S.. 317.328, 404, 419 Goering, L.. 342. 360 Goetinck. R, 588 Goff, D. J., 308.328 Goff. J. R. 246. 251 Goff. S. C.. 204. 207 Goff. S. R. 205.208 Goffinet. A. M.. 94. 100 Gofflot. F.. 132. 146 Gob. K. L.. 173, 176 Goins, T. L.. 237. 249 Gold. L. I.. 467.493 Golden, J. A., 79. 82.86. 100. 117. 122 Golden, K., 336. 337.366, 370 Goldfarb. M.. 166. 175. 313.314. 315,317. 324, 325 Goldhamer, D. J.. 258, 260, 261. 263.267. 274. 275. 277 Golding, J. R, 424, 425, 485 Goldman. D.. 132. 146. 148, 422. 427.496, 527.535 Goldman. S. A.. 95. 100 Goldowitz. D., 94. 104 Goldstein, A. M.. 287, 292, 339. 361, 470. 486 Goldstein. E. S.. 341. 358 Goldstein, M. M., 347,362 Goldstein. R. S., 131. 146 Goldstein. S. A.. 443.482 Goldstein, S. L., 314, 326, 410. 418 Golosow, N.. 319, 325 Gotuboff. L., 5I I, 515 Gomez, A. E, 40. 43, 50, 477. 484 Gomez, R. A.. 412,420 Gomez-Pardo, E., 373. 374. 385.393. 398. 420, 475.496. 544, 565 Gomez-Skarmeta, J. L.. 550. 561 Gomperts, M.. 186, 187, 189, 579. 584 Goncharov, T., 341,369 Gong, Q., 84, 100 Gonzalez, F. J.. 311.312. 326, 474. 488 Gonzatez, G. A., 286. 292 Gonzalez-Ramos, M., 453.486 Gonzalez-Sanchez, A...~.~/,""".540,"341, 36t, 364, 365

t

h

o

r

Index

Good, A., 322, 326 Good, D. J., 5 i2, 515 Goodall, H., 26, 34 Goodard, A., 12 t, 123 Gooday, D., 87, 94, 98 GoodelI. M. A., 193, 206, 208, 270, 271,275, 3I 8, 325 Goodfellow, R N., 28 t, 282, 292, 293, 377, 380, 381. 384, 386, 387, 388. 390. 39I. 392, 393 Goodman. D. S.. 468. 469.495 Goodman. L.. 402.420 Goodrich, E. S., 421. 442. 449, 456.471. 485 Goodrich. L. V.. 78.80, 100. 121. 123, 287. 293, 470, 485, 579, 586 Goodship: J., 352. 354, 366 Goodyear, R. 379, 391 Goodyear: R.. 555.562 Goossens, M.; 508, 516 Gorczyka. M. G.. 334. 361 Gordaze, R R.. 122, 122 Gordon. D. E. 509. 51 O, 515 Gordon. J. I.. 309.310. 325, 329 Gordon. J. S., 581. 586 Gordon-Thomson. C.. 337, 361 Goridis. C.. 239. 250, 435,484 Goring, D. R., 528, 536 Gorivodsky, M., 167, 171. t 74, t 76, 312, 314, 315, 323 Gorlin, R. J., 292, 294, 442. 485 Goronowitsch. N.. 423.485 Gorry, R, 443.451,453. 469, 474, 485, 489, 490, 545,563 Gortz, J.. 578, 583,586 Gosden, C., 375,391, 399, 419 Gossett, L. A.. 342, 361 Gossler. A., 132. 134. 135, 136, 137. 138. 139, t40, 144, 145, 146, 552. 553,555,558, 559, 563, 564 Goszczynski, B., 165.177, 316, 325 Gotay, J., 226, 227. 233 Gothe, S.. 510, 515 Goto. J.-I.. 338, 368 Goto. K., 531,536 Goto, S., 91,101 Goto, T., 92, 105 Gotoh, O., 525, 535 Gotoh, S., 506, 516, 549, 564 Gotsch. U., 226. 228 G6tt. R. 316, 323 Gotthardt, M., 94, 105 Gottschalk, M., 311. 329 G6tz, M.. 83, 85, 87.9 I, 94, 98, 100, 105 Gould, A., 1 t 7, 118, t 19, 123, 124, 544, 563 Gould, D. B.. 453.479, 488 Gould, E., 237, 247. 248. 250 Gould, S. J.. 442,485 Goulding, M., 260, 263, 264, 266, 268, 274, 275, 4t0, 418 Goumans, M. J., 222, 229, 467, 484

Author Index

621

Goumnerov, B., 227, 232 Gourdie, R. G., 219, 228, 231, 334, 339, 360, 368 Gourdji, D., 510, 5t8 Gove, C., 343, 361 Gowen, J. ~:. 453, 488 Grabet, L. B., 7.19 Grabowski. C. T., 161, ] 78 Gradwohl. G.. I36, 147, 166, 176, 310, 32I, 325, 435,484, 526,536 Graham. A., 4,.,.. "~ 425,428. 4_~2. ~" 434, 435,481, 6,4. ~' 4~,~. 485, 490, 497, 543.562 Graham. C. F.. 26, 33 Graham. H.. 572, 583, 585 Graham. J. M., 470, 487 Grahovac. M. J., 11.12, 17 Grail, D., 194. 208, 453, 486 Grainger, R. M., 435,469. 476, 484, 530, 536, 542, 56;,? Grainger, R. M., Jr., 529, 538 Grammatopoulos, G. A.. 433,485 Granadino, B., 525,535 Granato, M., 546, 557, 564, 565 Grange, D. K., 292. 294 Granholm, A.-C.. 402. 419 Grant, D. B.. 506. 514 Grant. J. H., 468,485 Grant, J. Wi, 356, 366 Grant, R, 435,498 Grant. S. R., 343,365 Grapin-Botton, A., 426, 427,433,483 Grass. S., 258, 2 74 Grau, E.. 40, 42, 51, I65, t7Z 303. 327 Graves, J. A., 386, 387, 388, 390, 39I, 392, 393 Graveson, A., 422, 484 Graw, J.. 527, 532, 537 Gray, C., 402, 420 Gray, N. K., 10, tl, 15, 16 Gray, S. "gv:,312, 328 Grayson, D. R., 316, 324 Grayzel, D., 357,358 Graziadei, R R, 77, 79. 84, 100, 105 Graziano. M. S. A.. 237, 250 Greaves, M., 194. 207 Greco. T. L.. 137, 146, 511, 512. 5]8 Green. E. D., 453.486 Green, H., 582. 583,585 Green. J. B. A., 167, 179 Green, M. C., 479, 485 Greenbaum, L. E., 318, 324 Greenberg, M. E.. 86, 100 Greenberger. J. S.. 246. 251 Greene, E. C., 442, 485 Greene, R., 424, "442, 443,482 Greenfield. A., 28 I. 295, 377. 390, 39I Greenhaw, G. A., 286, 292 ~

Greenlund, A. C., 287. 293 Greenspan, D., 305, 329, 453, 467, 496 Greenwood, A. L., 237, 250 Greer, I. A., 174, 177 Greer, J. M., 580, 584 Greewald, J., 354, 367 Gregg, B. C., t28, ]48 Gregory, M. C., 412, 417 Gregory, "v~ K., 42 I, 473, 485, 493 Grenier, G., 585 Grenier. J. K., 471,493 Grepin, C.. 342, 343, 361 Gretz. N., 415, 4t9 Greve, J. M., t4, t6 Gridley, T., 135, 136, 137, 138, 146, t48, 149, 224, 226, 227, 229, 230, 232, 423, 427, 432, 433, 443, 449, 473, 474, 477, 485, 48Z 494, 546, 553, 555, 563, 565 Griffin, K. J., 167, 176, 334, 361 Griffit,hs, B. L., 377,388, 393 Griffiths, D., 416, 420 Grigofiadis, A. E., 442, ~3.485, 497 Grigorieva, E., 260, 267, 2 75 Grigoriou, M., 88, 89, 10], 402, 420, 432,485 Grigoriuo, M., 432, 497 Grim, M., 258, 264, 266, 274 Grimatdi, R A., 27 I, 277 Grimes, R, 522, 527, 535, 549, 562 Grimmond, S., 390 Grinberg, A., 83, 87, 9I, t03, 106, t73~ 176, 17Z 376, 389, 402, 419, 432, 498. 508, 5t7. 521,522, 527, 537 Grindtey, J. C., 84, 85, 100, 3 t t, 3 t 2, 313, 314, 315,323, 325, 435, 451,470, 475, 476, 485, 509, 513, 521,522, 527, 530, 536 Grinspan, J. B., 82.86, 100 Gripp, K. W., 468, 485 Gritsman, K., 45, 47, 50, 51, 78, 100, 338, 353,354, 356, 357, 36I, 370 Gritti, A., 235, 242, 244, 246, 250 Grobet, I., 264, 274 Grobstein, C., 319, 325, 404, 4] 7 Groffen, J., 314, 325, 434, 467, 487 Grogg, K. M., 227,231 Grompe, M., 246, 250, 3 i 8, 326, 327 Grondona, J. M., 341,346, 347, 352, 362, 525,536 Gronenborn, A. M., 377, 393 Groome, A., 528, 533, 535 Gros, D., 348, 360 Gros, R, 79, 99, 266, 27Z 475,484 Grose, R., 574, 583, 584 GroskopK J. C., t 62, 176 Gross, C. G., 237, 250 Gross. M. K., 160, 175, 183, t90, 260, 266, 274 Gross. R. E., 239, 250

62 2

A

u

t

Grosschedl, R.. 83, 86, 91, 92. t00, 172, I75, -440, 471, 488, 49Z 579, 583,585, 58Z 589 Grosskortenhaus. A.. 336. 367 Grosveld. F.. 165, 17Z 195.196, 199.200. 202. 207, 208, 210, 343.369 Grosveld. G.. 201. 204. 209, 220. 231 Groudine. M., 169, 175, t 77 Grounds, M. D., 269, 27 I. 274 Grove, E. A., 82, 83, 84, 86, 87, 100, 10t, 105 Groves, A. K.. 238, 239. 24 t, 251, 252. 543, 546. 562 Groves. N.. 345. 348. 366 Grow. M. W.. 334. 348.361 Grubber, J. M., 467.488 Grubel. G.. 408.419 Gmber. R J.. 344. 352. 361 Grun. F.. 525.536 Grund. C.. 570, 586, 587 Griineberg, H., 132. 137. 146, 441,442, 453,456. 475.479, 485 Grunfeld. J. R. 375,389 Gruning, W~. 415.418. 579. 585 Grunwald. D. J.. 214. 230. 557.564 Grunz, H.. 44. 52. 338. 366 Gmsby. M. J.. 527. 530.53 I. 536 Gruss. R. 79.81.83.85.87.91.92. 94. 95, 9Z 100, 102, 103. 105, 113.115. 125. 129. 137. 140. 145, 14Z 160. 173.178, 179. 183. 190, 258. 260. 263, 264. 265. 266. 270. 272. 272. 273, 276, 311.321,327. 329. 373.374, 385.393, 398.417, 420, 432.451. 453,473.475.477. 48I, 482, 484, 48Z 490, 495. 496, 498, 519. 521,522, 525,527, 530. 532. 533. 535. 536, 53Z 538, 544, 547, 550. 561, 564. 565 Grussendorf, E. I., 570. 584 Gruters. A.. 508, 516 Grutzner. F.. 388. 392 Gu, D., 318, 322. 325, 326 Gu. H., 14. 16, 319. 325 Gu. Q.. 579. 589 Gu, S., 382. 389 Gu, T.-L., 201. 204. 209, 210. 220, 231. 233 Gu. Y.. 87. 101. 136. 145, 341,362 Gu. Z., 47, 50 Gualandris, A., 224, 232, 504. 517 Gualdi. R.. 316, 317, 325 Gubbay, J., 377, 390, 391 Gubler, M. C., 375, 389 Gubler, R, 10, 18, t 9 Gudas, L. J., 469, 470, 484 Guenet, J. L.. 386, 390, 476. 477.496 Guerrier. D., 381. 382, 389. 390 Gui, Y.-H., 340. 364 Guichet. A., 185. 189 Guille, M., 343,361 Guillemot, F., 79, 83, 88, 89, 90, 95, 98, 99, t00, 105, 136,

h

o

r

Index

146, t47. t66. t68, 174, 174, 176, 178, 239, 250, 310, 321,325. 435.484, 490, 526, 527,536, 538, 555.566 Guioli, S., 281,292, 378, 380, 38 t, 382, 387, 388, 389, 390, 39t, 392, 393 Gukovsky, I., 506, 508, 509, 5t6, 518 Gulisano, M., 79, 83, 100, 104, 290, 294, 376, 393, 422, 427, 428, 433,450, 47i, 472, 473,478, 48I, 48Z 495 Gull, K., 15, 15 Gullberg, D., 409, 419, 574, 589 Gultig, K.. 558, 563 Gumbiner. B. M.. 408.417. 420 Gumeringer, C. L., 341,352, 368 Gummer. A. W~, 558, 563 Gundersen. K.. 269. 274 Giines. C.. 317,325 Gunther. T.. 130. 132. 140. 148, 264, 265,277. 423, 492 Guo. H.. 12. 17 Guo, L.. 343. 347, 361 Guo, Q., 122, 122, 226, 227, 229, 336, 352, 359 Guo. W.. 378, 379, 389, 393 Guo. Y.-J., 432, 498 Gupta, R., 31, 33, 47.50. 451,476, 483, 508, 5t4 Gupta. S., 318, 324 Gurdon, J. B.. 203, 209, 263,264, 274, 275, 303. 330 Guris, D. L., 361 Gurley, C. M., 268. 269. 274 Gurney, A.. 121. 123 Gurtner, G. C.. 162. 170. 176 Gussoni, E., 193,206, 20Z 246, 250, 270, 271,274 Gutch, M. J., 91, t 00 Guthrie, S., 427, 485, 545,563 Gutierrez-Hartmann, A.. 505, 5t7 Gutierrez-Ramos. J. C., 205,206 Guttmacher. A. E., 227, 229, 231

H Haaf, T., 9, 1Z 388, 392 Haar. J. E., 317, 32 7 Haas, O. A., 281. 293, 381,391 Haas. T. L.. 9o3 229 Haase, I., 589 Habal. M.. 292. 294 Habara-Ohkubo, A., 336. 365 Habener, J. E, 322, 328 Haber, D. A., 374, 375, 392, 400, 4t5, 4 I Z 418 Habets. R E. M., 341,345, 347, 348, 350, 359 Habib, R., 416, 419 Hackenmiller, R., 286. 292 Hacker. A., 377. 378, 380, 381,384, 386, 387, 390, 392, 393 Hackett, B. R, 313,323, 325 Hackney, C. M., 558, 563 Hacohen, N., 466, 485, 488, 49I

Author Index

Hadchouet, M., 166, t 78. 263,264, 273 Haddon, C. M., 547, 562 Hadjantonakis, A.-K., 3, 4, 23, 35, 38, 51, 132, t47. 166, 174, 174, t 79, 347,352, 360 Hadrys. T., 549, 562 Hadzic, D. B., 347. 349. 361 Haendel, M. A., 170, 176 Haenig, B., 137. 147 Haenlin. M., 343.360. 361 Ha.fen, E., 470, 484 Haffen, K.. 308,325, 326 Haffner-Kraus, R., 167, 171. ] 74, 176, 312, 314, 3 t 5, 323 Haffter, R, 78, 103, 351,354, 355,359, 546, 565 Hafner, M., 415, 419 Haftek, M., 581. 588 Hagan, B., 530, 532, 535 Hagan, D. M., 387, 390 Hage, M., 183.190 Hagedorn, L., 236, 250 Hagenbuchle, O., 321. 324, 326 Haghighat, A.. 11. t6 Hagman, J., 9t, 100 Hagoort, J., 343, 345,348. 366 Hagopian-Donaldson, S.. 79. 106, 529, 538 Hah, C. S., 347. 362 Hahn, H., 287, 292, 470, 485, 486, 579, 586 Hahn, S., 468, 495 Haines. C. E. 84, 9Z 435, 469. 476, 48t Haines, L., 266. 276 Haitjema, T., 227, 231 Hajihosseini, M., 312, 324, 505,514, 546, 562 Hajra, A.. 202, 208 Hake, L.E., 7,17 Halasz, B., 504, 516

Halata, Z., 258, 264. 266, 274 Hal& J., 310, 321,325 Halder, G., 533,535, 536 Hale, L. R, 227, 231 Halevy, O., 341. 361 Halford, M. M., 453,486 Hall, B. K.. 279, 292, 421,422, 423, 424, 426, 427,432, 442, 443.449, 465, 47 t, 482, 483, 484, 486, 489, 497 Hall, D. E., 262. 277 Hall. E L., 314, 329 Hall, J., 470, 486 Hall, M.. 84, 106, 132, 146, 469, 497 Hall, S. E.. 387, 393 Hall. S. H., 452, 487 Halladay, A. K., 91,106 Hallais, M. E. 194, 201,209 Hallas, G., 173, 177 Halliday, A. L., 87, 100 Hallmann, R.. 219, 232 Hatlonet, M., 78, 79, 8t, t00, 113, 123, 522, 527,536

623

Halmekyto, M., 228, 230 Halmesmaki, E., 162, t 79 Halpern, M. E., 78, t04, 339, 34t, 349, 35 t, 364, 370 Hamada, H., 47, 50, 5I, 89, 95, 99, 102, 183, 190, 244, 25I, 338, 352, 354, 356, 357, 365, 367. 368, 370, 506, 5t6, 5 25, 535 Hamada, Y., 136, I46, 316, 317, 325 Hamburger, V., 40, 50, 568, 572, 586 Hamersma, H., 292. 294 Hamilton, D.. 15, 18 Hamilton, H., 568, 572, 586 Hamilton, R. L., 161, t 75, ! 78 Hammer, G. D., 379, 38t, 390, 392 Hammer, R. E., 94, 105, 432, 465,478, 483, 498. 511,516 Hammerschmidt, M., 287, 294, 336, 363, 442, 443, 470, 486, 496, 546, 565 Hammitt, D. G., 22, 35 Hammond, K. L., 530, 533,536 Hammond, V. E., 160, 167. 175 Hamosh, A., 347, 360 Hampt, A., 8, 10, t8 Han, J., 228, 229, 246, 249, 426, 482 Han, M. J., 3, 4 Han, Y., 34t, 362 Hanada, K.-I., 244, 251 Hanafusa. T., 321,327 Hanahan. D., 223, 232, 28 I, 291,294, 320, 321,323, 324, 580, 586 Hanai, A., 557, 564, 565 Hanakawa, Y., 588 Hanamure, Y., 473, 498 Hanaoka, K., 260, 276, 318, 328 Hanauer. A., 506, 517 Hancock. S., t32, 145 Handyside, A. H., t58, t76, 180 Hanken, J., 139, 148, 421,422, 423,432, 441,457,482, 486

Hankin, M. H., 527, 535 Hankinson, O., 172, 173, 17Z 225,230 Hanks, M. C., i09, t 15, 123 Hanley, K., 580, 58 I, 585, 586 Hanley, N. A., 387, 390 Hannon, G. J., 341, 36t, 483 Hannon, K., 258, 274 Hansen, C. S., 44, 50 Hansen, J., 57 I, 586 Hansen, L. A., 169, 179 Hansen, S. E., 322, 328 Hanset, R., 264, 274 Hanson, I. M., 451,475,486, 527, 528, 530, 533,536 Hanson, R. D., 376, 390 Hantsoo, L., 35 I, 364 Happle, R., 443, 486 Haqq, C. M., 381,390

624

Hara. M., 375.384. 392 Hara. T.. 376, 392 Hara. Y., 311, 3 i 2, 326, 474, 488 Harada, A., 38, 51 Harada, T., 531,538 Harbison. M. D.. ~ 3 , 4 9 7 Hardcastle. Z.. 86. 100. 121. 123. 437.439. 451. 486. 496 Hardin. J. D.. 205.208 Harding, G. W.. 286. 292, 558. 561 Harding, J. D., 32t, 328 Hardman. M. J., 568.582, 583, 586, 587 Hardman. R. 404. 420 Hardy. M. H.. 573.574. 575.576. 585. 586. 589 Hardy. R. R.. 245.250 Hargett, L. K.. 85. 100. 132. 149. 305.329. 453.467.479. 496, 498. 506. 516 Hargrave. M. R.. 281. 294 Harkes. I. C.. 343.369 Harland. R. M.. 40.41.44.48.49, 50. 51.52, 78.98. 141. 147. 238. 252. 260. 264. 265.275. 304. 323, 338.362, 468.482, 498. 550. 561, 579.587 Harlap, S., 22, 34 Harley. 'v: R., 282. 293, 377. 380. 381. 384. 387.390. 391, 392 Harlow. E.. 483 Harpal. K.. 43.50. 51. 133. 135. 145. 149, 158. 165. 175. 203.207. 223.228, 303,304. 316. 317.324 Harper. J. W.. 287.294, 528. 532, 538 Harper, S.. 570. 587 Harris. E., 109. 114, 115. 123. 124, 126, 228. 233. 428. 491 Harris. M. J.. 423.486, 487 Harris, R C.. 415.420 Harris, R E., 500, 518 Harris, R. C., 169, 179 Harris. ~\ A., 77, 99, 113. 125, 522. 525.533.538 Harrison. D. E.. 245.250 Harrison. D. J.. 416.4I 9 Harrison. K. A., 319. 325 Harrison, R. W. 475,481 Harrison, S. M.. 135. 145 Harrison. W.. 352. 354. 366. 380. 389 Harry, J. L.. 387. 392, 393 Harsh, G.~ 512, 516 Hart. C. E., 467,495 Hart, C. R, 443, 474, 490 Hart, K. C.. 453,485 Hartenstein. A. Y., 136, 146 Hartenstein, V.. 136, 146, 205.209, 367 Hartigan, D. J.. 78.79, 85.86, 88. 104 Hartigan-O'Connor. D. J.. 80. 82, 88.98, 321,329 Hartley, L.. 31, 34, 203,209, 3 t 7. 325, 333, 334, 343, 345, 348, 349. 352, 356. 364 Hartley. R., 575,586 Hartmann, D. J., 573,587

A u t h o r Index

Harv%5 R. B., 31, 34 Ha_rve~z R. R, 40, 44, 50, 203, 209, 317, 325, 333, 334, 338, 339, 34 I, 343, 3-44, 345, 347, 348, 349, 350, 351,352, 353, 355, 356, 357, 358, 359, 362, 364, 366, 36Z 369 Hasegawa, A., 1 1, i 2. t7 Hasegawa, G., 195, 208 Hasel, K. W.. 511. 514 Hashiguchi, H.. 338. 354. 356, 367, 368 Hashimoto. C.. 579. 587 Hashimoto, G., 313, 315, 327 Hashimoto, K., 188, 189.. 512, 518, 588 Hashimoto, N., 7. 16, 376, 391 Hashimoto. O.. 577, 586 Hashimoto. R., 557. 564 Hassan, B. A., 555.558. 559, 56I Hassan, M.. 507,514 Hasson, T., 556, 565 Hastie, N. D.. 302, 327, 341,365, 374. 375,380. 389, 391, 393, 399. 400. 416. 416, 417. 419. 420, 451. 475,486, 527. 528. 530. 536, 537, 538 Hasty, R. 110. 123. 260, 2 74 Hata. N., 135, 137, 138, 148 Hatanaka, Y., 84, 98, 100 Hatano. O.. 375. 376, 380, 390, 392 Hatch, G.. 89. I06, 450, 471,498 Hatcher. C. J.. 347. 362 Hatini. \(. 81.84. 86, 100, 101, 408.417, 479,486, 522, 525,527. 536 Hatta, K.. 528. 536 Hatta, T.. 547. 564 Hatten, M. E., 94, t00, 106, t22, 122, 453, 49t Hatton, K. S.. i0, 16 Hattori. N., 170, 176 Hatva, E., 162, t 79 Hatzistavrou, T., 334, 358 Haud. O.. 2.4 Haudenschild, C.. 220. 229 Haugen, B. R., 509, 510. 515 Hauri, H. R, 308. 325 Hauschka, S. D., 140, t4& 257,262, 263.264, 269, 276, 277 Hawes. N. L.. 527, 535 Hawkes, R., 94, I01 Hawkes, S. G., 39] Hawkins, J. E., Jr., 443, 482 Hawkins, J. R.. 377, 388, 390, 393 Hawkins, M. G., 165, 175 Haworth. I. S., 452, 453.474, 487 Hay. M. E, 454, 486 Hayakawa. K., 245.250 Hayasaka, M., 260, 276 Hayashi, D., 336, 365 Hayashi, H., 14. 18 Hayashi, S., I69, 175, 442, 498, 53 t, 536

A u t h o r Index

Hayashi. T., 414. 418 Hayashi. Y., 3I 1. 329, 577, 586 Hayashida, C. Y., 509. 517 Hayashiz~, Y., t t, 12, 18, 51 t. 516 Haydar, T. F.. 87, 100, 101 Hayes. N. L.. 245. 249 Hayflick, A. J., 512. 516 Haynes, J. I. II, 532, 535 Hayward. D.. 136. I48, 262. 276 Hayward. R., 453,494 Havward, R. D.. 45~, " " 466, 474. 9498 He. B., 402, 419 He, C.-Z., 344. 362 He, T. C., 86, I00. 579, 586 He, X., 89. 100, 506, 5t6 Headon. D. J., 44 I. 486. 491, 497 Heaty, L., 199, 200, 20t, 210 Heaney, A. R. 504. 515 Heaney. S.. 290. 294. ~ 8. 399 420, 4o7,450. 451,466, 474. 495, 527, 530, 533, 53& 550, 565 Heanue, T. A.. 268. 274, 399, 4 t 7 Heasman, J., 14. ]6, 183, 184, 185, 186, 187, 188, 189, t 90, 303, 323, 330 Heath, J. K., 167.174, 313. 326, 467, 482 Heath. M. J., 86. 102 Heaton, M. B., 435, 49I Heavey, B.. 204, 209 Hebert, J. L., 313,323 Hebert, J. M., 578,583, 586 HebrinL D., 287, 294 Hebrok, M., 319, 320. 325, 326 Hecht, E, 506, 5 l 7 Hecht. J. T., 286, 292 Heckscher. E., 45, 47, 50, 5t, 78, t00, 338, 36t Hedstrand, H., 3 t 3, 323, 467, 482 Heemskerk, J., 78, 104, 14 i, 148 Hefti. F., 91, 98, 402, 420 Hehr. A., 433,492 Held, R J., 165, 180, 316, 330 Heikinheimo, M.. 165, 176, 179, 333.342, 362, 466. 486 Heikkila, M., 384, 385, 393 Heilig. R., 398, 416, 550, 561 Heimann, G.. 509, 517 Heimfeld. S., 192, 209, 244, 248, 252 Heimrich, B., 92, 104 Heine, U. I., 314, 325 Heintz, N.. 94, 106 Heinz, R. 311,324 Heisenberg, C. R, 78, 102, 546, 565 Heisterkamp, N., 314, 325, 434, 467, 487 Heitzler, P., 343, 360, 361 Hetbling, R M., 225, 229 Held, M., 281,293. 294, 380, 38 t, 391, 393 Heldin, C. H., 226, 230, 468, 486

625

HeIgren, M., 559, 561 Heller, N.. 546, 562 Heller, R. 579, 585 Hetter, R. S., 310. 321,325 Hellmich, H. L., 402, 417 HelIstedt, R, 375,392 Hellstrom, M., 173, 178, 313, 323, 326, 414, 41& 467, 482 Helman, L. J., 268, 275 Hetmbold. E. A ~ 7 . ~ ~1 Helminen, H., 284, 293 Helms, J. A., 223, 232, 28 t. 291,294, 469, 470. 486 Helwig, U.. 263, 272 Hemati, N., 271,276 HembeNer, M., 17 I, t 76 Hemmati. H. D. ,,4~, 245.251 Hemmati-Brivanlou, A., 303,325, 533, 535, 585 Hemming, E, 504, 514 Henderson, C. E., 402, 420 Henderson, D. J., 334, 350, 362, 365, 475, 483 Henderson, J. A., 469, 470. 475, 486 Henderson, N., 15, 18 Henderson, R., 287, 292, 312, 313, 3 t 4, 315, 323 Hendricks, M., 251 Hendrickson. W. A., 408, 419 Henegar, J. R., 237, 249 Heng, H. H., t21. 124, 451,453,470. 47t, 491 Heninger, G. R., 247, 249 Henion, E D., 243, 244, 250 Henke, E S., 219, 232 HenkeI, G. Vvl, 168. 179 Henkmeyer, M.. 135, t49 Henn, W., 288, 293 Hennings, H., 580, 586, 587 Henricksen, K., 291,292 Henrique, D., 134, t35, 138, 139, 145, 146, 14Z 553, 559, 561, 577, 585 Her~', G. L., t 66, 176, 303,325 Henr?; J. J., 530, 534, 536, 537, 542, 562 Henry, M. D., i6t. 179 Hentges, K., 79, 86, 100, 243, 250, 379, 384, 39t Hentges, S., 504, 51 I, 515 Hentsch, B., 317, 325 Hentschel, H., 395, 417 Her, H., 530, 533, 538 Herbert, J. M., 225, 226, 229 Herken, R., 574, 583, 584 HermeNng, H., 86, t00. 579, 586 Hermesz, E., 45, 50, 508, 515 Hermiston, M. L., 309, 3 t 0, 325 Hernmndez-Verdun, D., 170, t 76 Herregodts, R, 502, 515 Herrera, D. G.. 106 Herrera, R L., 321,328 Herrick, T. M., 94, ]01

6 26 Herring, S., 427,452,486 Herrman, J., 506, 5 i 7 Herrmann, B. G., 45, 47, 50.. t32, I33, 135, t37, 140, 146, 14Z 149, t 67, t 79, 393, 468, 483 Herrup, K., 94. I00, 1 t2, 115, 123, 124, t69, ] 79, 318. 326, 428. 491 Herskowitz, I., 158.176 Hertte. M. D., 572. 586, 589 Hertwig, R. 544. 562 Hertz. R.. 164. 176 Herva. R.. 412.418 Herz. J.. 94. 100. 105 Herzenberg, L. A.. 157. i80, 194. 207, 245,250 Herzig, A., 170. 179 Herzlinger. D.. 400. 40 I. 405. 408, 417. 418, 419, 571. 588 Herzog, V~, 577, 583.584 Hess. H.. 160. 175, 178 Hess. J. L.. 376. 390. 393 Hetherington, C. M.. 84. 101, 451,475.486, 527. 528.530. 536 Heuchel. R.. 317.325 Heutink. E. 227.23I Hevner. R.. 83.84. 85.90. 92.95.98, 102. 103. 104 Hewick. R. M.. 467. 498 Hewson. J.. 236, 252 Hextall. R J.. 377,390 Heyberger, S.. 506. 517 Heyer. B. S.. 12, 16 Heyer. J.. 43.50, 353. 357. 365 Heyman, H. C.. 260. 268, 272, 468,482 Heyman, I., 425. 485 Heyman, R. A.. 511,513 Heymann. S.. 266. 274 Heymer. J.. 83.98, 470. 482 Heynen. A.. 11.19 Heyworth. C.. 194. 207 Hiatt. K.. 196. 202. 210 Hibbs. M. L.. 453.486 Hibi, M.. 171,173. 176 Hibino, H.. 220, 231 Hicklin. D. J., "~97 233 Hicks. C., 92. 103. 136. I49 Hidaka, K.. 262. 268. 274 Hidaka. M.. 217, 230 Hidalgo-Sanchez. M.. 1! 2, 123 Hidenori. I.. 588 Hiebert. S. W., 201,204, 209, 220. 231 Hieda. Y., 436, 486 Hiemisch, H.. 42.51 Hiesberger, T.. 94. 100. I05 Higashinakagawa. T., 376. 391 Higgins, K. M., 78, 80, 100, 121,123. 579. 583.588 Higgs, D. D., 202, 207 Higgs, D. R., 376. 390

A u t h o r Index Hilberg, E, 318, 325 Hildebrand, J. D., 79, t00 Hildebrand, M., '449, 47I, 486 Hilding, D. A., 560, 563 Hill. C. S., 342, 362 Hill. D. R. 50 Hill, J.. 468. 486 Hill. R. E.. 84. 85, 87.94. 98, 100, 105, 302, 32Z 435, 451, 453.468. 474. 475.476. 485. 486, 489, 491, 52I, 522, 527. 528. 530. 533.536 Hill. R. J.. 165.180. 316, 330 Hill. S., 349. 355.358 Hillan, K. J.. 203,207. 223, 229 Hilton, D., 40, 44, 50, 323, 338, 358 Himeno. M.. 288, 293 Himes. D.. 559. 563 Hindmarsh, R C.. 31, 33, 47, 50, 376. 389, 451,476, 483, 508,514 Hinds. J. ~:, 237.250 Hinterberger. T. J.. 258. 274 Hinuma. S.. 258. 260. 276 Hioki, T.. 321. 326 Hirabayashi, T., 341. 352, 370 Hirakow. R.. 334, 362 Hirano. S., 91. t01. 421,475,488, 492, 525, 538 Hirano, T., 171,173, 176 Hirashima, M.. 228, 233 Hirata. K.. 84. 98 Hirata. M., 341. 352. 370 Hirato, J.. 512, 518 Hiratsuka, S., 217. 230 Hirchenhain. J.. 410, 418 Hiroi. H.. 504. 516 Hiroi. Y.. 336. 343.365, 368 Hirokawa. N.. 38, 51 Hirokawa. Y., 381,392 Hiromi. Y.. 466. 485, 488 Hirose. J., 350, 362 Hirose, M., 350, 362 Hirose, S., 169, 175 Hirose, Y., 525, 536 Hirota, A., 45 l, 475,476. 492 Hirota, H., 341,362 Hirsch. B., 382. 387, 388, 392 Hirsch. J. C.. 356, 362 Hirsch. M. R.. 239, 250 Hirschi. K. K., 226. 230 Hirsinger, E., 136, 140, 14I, 142, 146, 147, 262, 263, 264, 266. 273, 274, 276 Hirth. K. R. 217, 220, 229 Hirvonen, H., 528, 538 His. W:, 21 t, 230 Hitchcock, R E, 53 I, 536 Hixson, D. C., 318, 324

Author Index

627

Hla, T., 227,230, 231 Hnadkiski. B.. 574, 588 Ho, C., 22, 34, 214, 231, 285, 293, 442, 443, 489 Ho. I. C., 527, 530, 53 I, 536 Ho. J.. 203, 209, 217. 232 Ho. R. K.. I4, i7, 118. 125. 339, 34t. 349,370 Ho. S. Y.. 352, 354, 367 Hobson. J. R. 9"~9 .:._7.23] Hobson, K.. 236, 240. 241,250 Hochedlinger, K., 87, 104 Hocking, A. M.. 405,418 Hockley. A. D.. 453,466. 474. 498 Hodges, M., 174, 174 Hodgetts, C.. 552, 553. 555. 558, 559, 564 HodgMn, J., 382, 387. 392 Hodgkinson, C. A., 525,536 Hodgman, R., 7, 14. 19 Hodgson, G., 194, 208 Hodivala-Ditke. K. M 9"~3 230, 574. 585 Hoefen, R S.. 196. 198.210 Hoeffler, J. R, 226. 230, 511,515 Hoepker, M, 525,536 Hoffarth. R. M.. 94, 101 Hoffer. B. J., 402.419 Hoffman. J. I., 333. 356, 362 Hoffman, S., 134, 148, 350. 370 Hoffmann. i., 408. 417 Hofmann, C., 408.418, 45 I, 467.490 Hofmann, U.. 574, 588 Hofstetter, W.. .443, 485 Hogan, B. L., 40, 42, 43, 49, 52, 82, 84, 85, 86. 99, I00, 101, 105, I06, 132, I35, i48, 149, t61,176, t83, t90, 203.210, 987. ~92, 302. 305 309, 3 t'~ 313.314. 315 319. 320, 321,326, 327, 329, 402, 404, 418, 439, 451, 453. 467. 470, 475,479, 485, 486, 488, 490, 494, 495, 496, 49Z 498, 506, 516, 527. 528, 530, 536 Hogan, B. L. M.. t88, 189, 190, 238. 246. 251, 303, 308, 31I. 312. 313, 314, 3t5,323, 325, 326, 328, 336, 338, 355.363, 370, 403,418, 504, 505, 507, 508, 510. o't8.' 570, 573,589 Hogan. R, 469, 498 Hoht, D.. 582, 583.584, 587 Hokfelt, T., 559. 564 Holash, J., "~94 230 Holbrook, K. A.. 568, 573, 575, 580, 583,584, 586, 588 Holdener, B. C., 31, 34 Holder, N., 119, 123, 342, 36Z 468, 486, 52t, 522, 537 Holland, L. Z., 475,486 Holland, N. D., 475,486 Holland, R W., 266, 277, 478, 498 Holland, R W. J., 305.323 Hollander, G., 195,210 Hotlemann, T., 78, 79, 81,100, 521,522, 525, 527, 533, 535, 536 .,-.

..,

.

?

.

.;-~

.

.

,~

Hollenberg, S., 257, 258, 277 Hollenbe~, S. M., 349, 362 Holleran, W. M., 582, 584 Holley, M. C.. ~.~o, 563 Hotlidav, M.. 85, 101 Holtiday, R., 6, I6 Holloway, J. M., 502, 5t8 Holtweg, G., 570, 584 Holmberg, C., 412, 418. 419 Holmberg, E.. 287, 292, 470, 486, 497 Holmes, D. IF., 572, 583.585 Holmes. G., 375, 39t Holmes, R. L., 504, 514 Holmgren, R., 470, 487. 497 Hotmyard, D., t 70, 173, ] 74 Holst, R L., 402, 417 Holstein, A. E, 386, 393 Holt. C. E., 264, 274 Holthofer, H., 4 t 2, 417 Holtmann, H., 34 t, 369 Holtzer, H., 140, 144, 263, 274 Holtzer, S., 263, 274 Holtzman, D. M., 91, 99... 101 Holzman, L. B 4t v 4 t 7 Hom. D. B., 343, 369 Homayouni, G., 94, 99 Homburger, S. A., 547, 562 Homgren, R., 121. 123 Honda. S., ~75. a 6..~84. 392 Honjo, T., t36, 137, I4Z 320, 32I, 323, 509, 5t4 Hood, L., 347, 364 Hoodless. R A., 3 I, 35, 164, 165, 17_5, t79, 303, 316, 329, 468, 497 Hoogerbrugge, J. ~:, 385,389 Hooghe-Peters, E., 502, 515 Hooper, J. E., 470, 481 Hooper, M. L., 416, 4]9 Hooshmand, E, 506, 509, 5t4, 556, 558, 562 Hoover, E, 527,535 Hopson, A., 442, -443, 449, 456, 470, 481 Horcher, R G., 314, 329 Horigome, N., 421,488 Homer, J. "~:, 532, 536 Homer, M. A., 316, 325 Homer, N. A., 165, 176 Homing, S. E., 376, 393 Horowitz, M. C., t 73, t79 Horsburgh, G., 84, 101, 451,475, 486, 527, 528, 530, 536 H/Srstadius, S., 434, 486 Horton, D. B., 6, 17 Horton, H. E, 318, 328 Horton, S., 89, 101 Horton, W. A., 286, 292, 294 Horvath, E.. 512, 515 .~

" 5

~

6

2

8

A

u

t

Horvitz, H. R., 158, 176 Hor~vitz, G. A.. 504, 515 Hos "ldng, B. M., 342, 343, 359 Hosoda, K.. 432, 465,478, 483 Hosoda, T., 336, 365 Hosoda, Y.. 258. 274 Hosti~:a, S. L., 412, 417 Hou, H., Jr., 415,420 Hou. Z.-H.. 251 Houart. C.. 22.34, 77.78. 10] Houghton, D. C., 416, 419 Houghton, L.. 335. 343.3-44. 345.346. 347.370 Houliston. E.. 23.34 Housman. D.. 34 I. 352. 363. 374. 375.391. 392, 399. 400. 415.416. 417. 418. 419 Houssaint, E., 317,325 Houston, D. ~:, 303. 330 Houweling. A. C.. 341. 345.347. 348. 350. 359 Houzelstein. D.. 267, 274 Hovens. C. M.. 453.486 Howard. K.. 186. 187, 188. 189. 190 Howard. L., 201,204,209. 220, 231 Howard. M.. 349. 362, 549. 553. 565 Howard. T. D.. 453.486 Howell. B. W.. 94, 100, I01. 104 Howells. N., 318.325 Howes. G.. 203.207 Howles. R N., 434. 453.467, 483, 493 Hoyer, J. R., 415,419 Hoyer, R E.. 373,390 Hoyes. A. D.. 570. 586 Hoyner, A. L.. 102, 471,486 Hrab~ de Angelis, M.. 31.35, 132. 136, 137. 139. 140, 145, 146. 292, 292, 320. 32 i, 323, 551,552, 553, 555,557. 558. 559. 560. 563, 564 Hromas. R., 316. 325 Hrstka. R. E, 161. 179. 527. 532, 537 Hseih. C.-M., 347.359 Hsiao, J.. 580. 585 Hsieh, J. J., 136. 148, 262, 276 Hsieh-Li. H. M., 79. 82. 10I Hsu. D. R.. 44. 50, 338. 362 Hsu. L. C.. 525.538 Hsu, S. C.. 579. 587 Hsu, Y.-R R, 7.19 Hu. B., 399, 419, 530, 533, 537 Hu, D.. 469, 470, 486 Hu, G., 267, 272, 450, 47 !, 472. 473.498 Hu, M., 194, 207 Hu, N., 318, 326 Hu, Q. L., 217, 232 Hu, S., 402, 417 Hu, T., 347, 366 Hu, Y., 437, 453,486, 498, 580, 581,583.586

h

o

r

Index

Hu, Z., 402, 41Z 579, 583,588 Huang, B., 380, 390 Huang, H. R, 310, 321,322, 327 Huang, K., 400, 418 Huane. L.. )v5 230 Huang, R., 13 t, 146, 266, 277 Huang, S., 376, 389, 432. 498 Huang, S.-R. 83, 87.91. 103, 106, I30, 131,132, 145, 402, 419, 508. 517. 521. 527,537 Huang, W., 281,282, 285,292, 293 Huang, X.. 204, 210, 220, 233 Huang, Z.. 527, 536 Huarte. J.. 10. 14. 18, 19. 321,328 Huber. G. C.. 25, 34 Huber, M., 582, 583.587 Hubner, K., t 83, t 90 Hudson, A. J., 27 I, 275 Hudson. B. G., 412. 417 Hudson. C.. 166. 176, 303.325 Hudson. J. B.. 336, 370 Huebner, K.. 290, 294. 450, 47 t. 472, 473, 495 Huetz. E. 165, 174, 303,323 Hug, B.. 214. 230 Huggins. G. S., 341. 343. 347. 352, 362, 368 Hughes, G. J., 3";1,326 Hughes, S. M.. 265.268. 269. 272, 274, 276 Huh. C. G.. 94, I03, 226, 230 Huh. N., 580. 587 Huh, N.-H., 580, 588 Huh. S., 8I. 86, 101, 408.4tZ 522. 527, 536 Huhtaniemi. I.. 511. 516 Hui. C. C., 79, 85, 101, 103, 115. 120, 121,123, t24, 125, 310, 312, 3t8~326, 327, 439, 451,453,470, 47t, 486, 48K 491, 505,517, 522, 537 Huisman. T., 506, 517 Hukriede. N. A.. 136, 145 Hulander, M.. 479. 487 Hume, C. R.. 45, 52 Hummler. E., 314. 324 Humphrey. J. S., 227, 229 Humphries, C., 269, 273 Humphries, R. K., 242. 252 Hungerford, J. E.. 226. 230 Hunsicker, R, 31, 34 Hunt, C. X/:, 166. 176 Hunt, D. E, 226, 230 Hunt, J. S., 173, 179 Hunt, R, 422, 425,426, 427,433, 449, 467, 474, 482, 487, 571,586 Hunter, R J.. 170, 174, 176 Hunzelman. J., 285,287, 293, 294, 442, 488 Huot, C., 5 t 2, 514 Huot, J.. 171,173. 176 Hurtin, R J., 580, 586

A u t h o r Index

629

Hurston. E., 318. 324 Hurt, C. R.. 260. 267, 277 Husslein, R, 174, t 77 Hustad, C. M., 342, 365 Hustert, E., 281,294, 380, 393 Huszar, D., 560, 561 Hutcheson, A. M., 527, 530, 531,537 Hutchins, G. M.. 3t2, 329 Huth. J. R.. 377, 393 Hutson. M. R.. 550, 562 Huttner. A., 510. 518 Huxham. I. M., 161, 176 Huyhn, A., 194. 207 Huylebroeck, D.. 222, 226, 227, 229. 336. 352, 359 Huynh, T., 353, 357. 364 Hwang, J. Y., 580, 581,583,587 Hwang, S. G., 318,324 Hwang, S.-Y.. 8, 10, I2, t3, 16. t Z 18 Hyink, D. R, 412, 414, 417, 419 Hymer, W. C., 511.517 Hynes, A.M., 85, 106 Hynes, M., t0t, 121, t23, 579, 589 Hynes, M. A., 110, 121,126 Hynes, R. O., 170, 173, 176, 180, 183, 187, I88, 189, 204. 206, 219, 222, 223,228, 229, 230, 233, 340, 352, 361, 366. 424, 48Z 571,572. 574. 581,583, 585, 589 Hynes, T. O., 223, 230 Hyslop, R S. G.. 347. 352,360

tkeo. K., 533, 536 Ikeya, M., 260. 264, 275, 428, 487 tkura, T., 465, 492 Ikuta, K., t 92, 194. 196, 20Z 20R 245,250 Ikuta, T., 580, 587 Itgren, E. B., 166, 176 Illmensee, K., 570, 586 Imai, H., 437,438,487 Imai, K., 263,272, 45 t, 475,493, 498 Imai, T.. 376, 380, 390 Imaizumi, K., 398,419 Imakado, S., 581,586 Imaki, J., 53 i, 538 Imamoto, A., 157, 180, 361 Imanaka, T., 308, 329 Im.hof. B. A., 20I, 209 Inada, M., 288, 293, 442, "443, 453, 488 Inadera, H., 27 t, 277 Inagaki, T., 336, 344, 362 Incardona, J. R, 78, 99, 10t Ingenhoes, R., 174, t 77 Ingham, R W., 78, t0t, 264, 265, 269, 272, 273, 275, 276, 470, 497, 534, 536 Ingraham, H. A., 89, 100, 376, 379, 380, 38i, 384, 390, 391, 392, 502, 509, 5]5, 5]8 Inoue, H., 32 t. 325 Inoue, K., 5 t I, 515 Inoue, S., 504, 516 Inoue, T., 77, 101, 340, 352, 354, 363, 367, 370, 408, 417.

I

Ion, A., 376, 391 Iozzo, R. V., 570, 572, 583,584, 585 Ip, H. S., 165, 17Z 303, 32Z 342, 366 Ip, N. Y., 559, 561 Irintchev, A., 27 I, 275 Ifion, U., 336, 367 Iruela-Afispe, M. L., 219, 226, 230, 233 Irvine, K. D., 136, 146, 555, 563 Irvine, S. M., 47t, 493 Irving, C., t I 0, t 12, t 15, 123, 428, 449, 487 Isaac, A., 354, 355, 356, 362, 366 Isaacs, H. V., i 67, 176 Isaacs, I., 240, 244, 252 Isacson, O., 89, 99 Iscove, N. N., t 93,207 Iseki, S., 422, 423,424, 435,437, 451,452, 475,476, 478,

Iangaki, T., 38.52 Iannaccone, R, 470. 497 Iavarone, A., 474, 489 Ibafiez, C. E, 402, 420 Ibraghimov-Beskrovnaya, 0., t 6t, ] 79 Ibra.him, S. N., 512, 515 Icardo. J. M.. 333, 351,352, 354, 355, 356, 359, 362, 366 Ichikawa. I., 403, 4i8 Igarashi, R, 313,328, 413,419 Iida, H., 376, 392 Iida, K., 47 i. 479, 487 Iimura, T., 451,475, 476. 492 Ikawa, M., 136, 146, 581. 583. 589 Ikawa, Y., 7.16 Ikeda, H., 502, 511,515 Ikeda, K., 506, 516, 549, 564 Ikeda, M., 47, 51, 347, ~" "9 _ . 360 Ikeda, T., 27 I, 2 75 Ikeda, Y., 375,376, 379, 380, 381,384, 390, 391, 392, 510, 515,516

Ikematsu, S., 350, 370 Ikemura, T., 226, 232 Ikenaka, K., 94, I03

418

487. 490, 492

Isenmann, S., 173, 175, 260, 266, 272, 3t7, 323, 404, 4t7 Ish-Horowicz, D., t34, 135, 138, 139, 146.. 147, 553, 559, 56t, 577,585 !shibashi, M., 136. 146, 147, 310, 321,325, 526, 527, 538 Ishida, Y., 537 Ishida-Yamamoto, A., 58 t, 582, 583, 587 Ishii, S., 121,123

63 0

A

u

t

Ishii. Y., 308, 325 Ishikawa. H.. 512,515 Ishikawa. T.. 172, t 73. t76, 465,489 Ishimam. Y.. 47.50, 354. 370 Ishino. K.. 580. 58,8" Isner. J. M.. 220. 228 Israel. A.. 136. 146 Israel. M. A.. 174. t 77 IsseIbacher. K. J., 339. 368 Itami. S., 452. 496, 580. 58 I. 583. 589 Itasaki. N.. 117, 118, I23, 355,362 Iten. L. E.. 548. 550.551.56I Itie, A.. 468,495, 5t0, 514 Ito, C. Y.. 415,417 Ito, M., 6. 19. 376, 378, 379,389. 393 Ito, T.. 171,178, 314, 327 Ito, Y.. 220. 228,229. 231, 246. 249, 288. 293. 318. 325 Itoh, H.. 228,233, 354. 370 Itoh, M., 11, 12, I8, 171, I73. 176 Itoh, N.,311 31 "~ 313.314,315,321 323,324,325.32Z 328, 505.517 Itskovitz-Eldor, J., 303,329 Ittmann. M., 505,517 Ivanova, N., 194. 206, 209 Ivanyi, E., 42, 51, 158, 160, ] 77. 178 Dins, K. J.. 469.498 Ivkovic. S.. 91.10] Iwamoto, M.. 289. 294 Iwana. Y.. 504, 517 Iwasaki. T., 512, 518 Iwata, T., 286, 293 Iyer, N. V., 225.230 Izac, B.. 194, 207 Izawa, M., 1 I. 12, 18 Izpisua-Belmonte. J. C., 39, 51, 338, 351,353, 354. 355. 356. 357, 359, 364, 36Z 433,478, 489, 496, 507, 508, 518, 580. 581,583.587 Izumo. S.. 219. 232, 334, 34 I, 343, 345.347. 348. 349. 350, 351. 352.359, 361,363, 36& 369

J Jaber. M., 34I, 352. 362, 51 I, 513 Jabs. E. "~u 452. 453,474, 486, 487 Jacenko. 0.. r JSckte, H., 316, 329, 469, 483 Jacks. T.. 318, 32.L 483, 528, 537 Jackson. B. W:, 570, 586 Jackson, C. E., 227. 231, 453.487 Jackson, D. I.. 377, 390 Jackson. I. J.. 31.34, 560. 56I. 565 Jackson. K. A.. 193.206. 208. 270.271. 275 Jackson, M. R.. 12, 17 Jacob, M. P., 585

h

o

r

Index

Jacobs, H., 170, 179 Jacobs, H. C.. 312. 316, 323, 324 Jacobs, J., -440, 498, 577, 589 Jacobs. M. L.. 349. 369 Jacobs. Y., 443.449. 476. 479. 482, 487. 495 Jacobsen. S. E.. 1 1 . 1 7 Jacobson, A. G., 335, 337, 339. 362, 36Z 422, 423,435, 48Z 492, 542, 563 Jacobson. K., 23.33, 166. 167, ] 7_5, t 78 Jacobson, L., 510, 5 t 2, 516 Jacobson, R. D., 530, 536 Jacquemin, R. t 66. t76, 32 t, 325 Jadjantonakis, A.-K., 357,368 Jaenisch, R.. 79, 104, 258, 260. 273, 276, 3t4, 326, 34t, 352. 363. 375,391, 400. 410. 418, 427. 443,467, 469, 484, 489. 491, 495, 529. 537. 559. 560. 561, 562, 573, 574. 583,584, 585, 587 Jaffredo. T., 199, 200. 201, 20Z 208, ! 987 Jagla, K.. 260. 266, 273, 27.4. 276 Jaglarz, M., 186, 187, 188. t89 Jahoda, C. A.. 585 Jain. M. K., 347, 359 Jain. R. J.. 218. 223.230 Jain. R. K., 225. 229 Jaing, T. X.. 577, 589 Jaiswal. R. K.. 206. 209 Jalanko. H.. 412. 419 Jalic. M.. 416, 420 Jalife, J.. 334, 348. 367 James. B., 185. 189 James. C. D.. 287. 294 James. R.. 160, 167. 174, 175, 509, 510, 5t5 Jameson, J. L.. 6, ] 9, 376, 378, 379, 389, 393, 500, 518 Jami, J., 320. 324 Jamrich, M., 521,522. 527, -"'~ o_, 536, 53 7 Jan. L. Y.. 136. 145, I85, t89, 257, 276, 334, 349, 358, 365. 553.563 Jan. Y. N.. 87.9Z 136. 145, 185, 189, 334, 349, 358, 365, 553.561,563 Janis. L. S., 91,101 Jankovski, A.. 95, 10I Janne. J., 228, 230 Janney, M. J., 27 I, 276 Janse, M. J.. 348. 360 Jantapour, M. J., 174, 177 Janumpalli, S., 247. 251 Japon, M. A., 502. 515 Jarnik, M., 582, 583,587 Jarriault. S.. 136, 146 Jar~-Guichard, T.. 348, 360 Jarvis, L. A., 466, 491 Jaskoll. T.. 436. 487 Jat, R S., 404, 420 Jaubert, E, 375, 387, 389, 392

A u t h o r Index

Jaw, T. J., 91,103 Jaworowski, A., 161, 179 Jaye, M., 453,487 Jean, D.. 5v'~._,536 Jeannotte. L., 171, 173. ] 76, 312. 323 Jeffreys, R. V., 505, 513 Jeffs. E, 264. 277 Jegou, B., 382, 389 Jeltsch. M.. 218, 223.230 Jen, W., 138. 146 Jenkins, N. A., 78.79. 8t, 85, 99, 100. 103, 168, 169, 173, ~ 345 , 175. 176, ] 79, 1%, 204, 208, 32 I, 328, 34I , 34J., 348. 349, 9.~_. ~-9 . 359.. 360. . 365. . 366, . 41 . i . 418, 443. 467. 476. 482, 485, 48& 508, 515, 521,525, 533, 536, 53Z 538, 556, 565 Jenkins, R. B., 287, 294 Jenkinson, E. J., 166, 177 Jenkinson, J. V~/'., 442. 487 Jensen, A. M., 86, 101 Jensen, J., 166, t 76, 310. 321. 325, 327 Jensen, R J., 570, 589 Jensen, U. B., 570, 586 Jepeal, L., 268, 273, 528, 535 Jerabek, L., 244, 252 JernvalI. J.. 44 I, 48Z 493, 497 Jerome. L. A., 353. 357, 362 Jeske, Y. Vv:, 377. 391 Jessee, J., 1 I. 18 Jessell, T. M., 3, 4, 3 I, 34, 43, 46, 47, 48, 50, 52, 78, 79, 80, 8 I, 82, 85, 87, 88, 98, 99, t01, I02, t04, 105, 106, 108, 109, 120, 121,122, t22, t23, 124, 125, 1411;14& 165. ] 79, 250, 316, 319. 320, 321,323, 326, 329, 397, 420, 423,474, 489, 494, 504, 505,506, 514 Jetton, T. L., 309. 319. 320, 327, 506, 516 Ji, Q., 453, 476, 493 Ji, X., 53 t, 536 Jiang, E, 165.177, 303. 327, 341,343,352, 368 Jiang, R., 423,487, 553,555,563 Jiang, 1". X., 574, 577, 578, 579, 585, 586, 587 Jiang, X.. 228, 229. 230, 246, 249, 250, 334, 348, 362, 426, 482 Jiang, Y., 339, 342, 346, 347, 362, 363, 581,586 Jiang, Y.-J., 139. 146, 546, 565 Jiang, Z., 90, 95. 106 Jiangming, L., 346, 363 Jimenez, M. J.. 289; 293 Jimenez, R., 388, 391 Jin, D., 416, 420 Jin, O., 31, 33, 42, 43, 46, 49, 51, 1t6, 122, 323, 45t, 478, 481, 521,527, 535 Jin, R, 105 Jing, S., 402, 417 Jing, X., 502, 515 Jishage. K., 95, I02, 506, 516

63 !

Job, C., 3 t 0, 327 Jobe. S. M., 343, 363 Jobert, A. S., 285,293 Jochum, W., 87, 104, t 73, 179 Johansem E-E., 342, 369 Johansen, K. M., 136, ]49 Johansen, T., 546, 563 Johansen, u A., 452, 487 Johansson. C. B., 236, 237, 244, 245,246, 248, 249, 250. 318, 324 Johansson, S., 574, 589 John, R., 3 t t ~..4 Johnson, D., 11, I2, 18 Johnson, D. K., 3I, 34 Johnson, D. R., 453, 471,487 Johnson, D. "vV,227, 231 Johnson, G. R., 195, 20 t. 20& 314, 317,325, 328 Johnson, J., 8, 17 Johnson, J. A., 168; t77 Johnson, J. D., 321,328 Johnson, J. E., 84, 88, 89, !0t, 102, 168, t 76, I77, 239, 250, 428, 487 Johnson, K. R., t 0, 12, 16, 94, 105, 550, 563 Johnson, M. C., 356, 357, 358, 366 Johnson. M. D., 467, 493 Johnson. M. H., 5, 17, 23, 26, 34, 156, 158, t 66, 175, 177, 178, 180 Johnson, M. L., 442, 487 Johnson, R., 453, 486, 54t, 542, 546, 552, 555, 558, 559, 564, 580, 581,583, 586 Johnson, R. L., 136, t37, 138, t41, t45, ]46, 262, 264, 265, 275, 280, 287, 293, 305,328, 354, 355,356, 364, 45 t, 470, 485, 487, 489, 490, 494, 57 I, 579, 586 Johnson, R. S., 225. 232, 442, 487 Johnston, J. G., 94, 101 Johnston, M. C., 423, 468, 48Z 497 Jollie, M., 42i, 487 Jollie, W. R, 161,177 Joty, J. S., I t6, 125 Jones, B., 453, 494 Jones. B M.. :~.x 493 Jones, C. J., 251, 415, 417 Jones, C. M., 40, 43, 49 Jones, D. H., t 56, 177 Jones, E. G., 84, 106, 322, 326 Jones, E. M., 318, 326 Jones, J. E., 553,562 Jones, J. M., 303,329 Jones, K. R., 91,101, 549, 559, 562 Jones, K. T.~ 8, 16 Jones, M. C., 338, 362 Jones, M. J., 2, 4, 17t, t73, 174 Jones, R E, 224, 231, 232 Jones, R H., 570, 587 9

.

.

4

"~'~

63 2

A u t h o r Index

Jones. R. C., 410. 4t8 Jones. R. O.. 195,208, 314, 326 Jones. S., 45 l, 453.47t, 498, 506, 518 Jones. W.. 416. 420 Jones. Y. Z.. 172, 173. 174. 341 357 358 Jones-Seaton, A., 2I, 34 Jonsson. J.. 319. 320. 322. 325 Jorcano. J. L.. 574. 583,584 Jordan. C. T.. 192. 208 Jordan. S. A.. 3 I. 34 Jordan. T.. 451. 475.486. 527. 528. 530. 536. 538 Jorgenson. R.. 507.515 Josephson. R.. 89. 101 Josso. N., 372, 374. 387.391, 392 Jost. A.. 372. .~ ~,-v ,4. 391. 507.515 Jostes. B.. 475. 487 Jouanneau, J.. 223.230 Joubin. K.. 41.45.51 Joukov. V.. 162. 179, 218. 223,230 Joulin. V.. 382. 389 Joutel. A .. 227. 230 Jouve. C. 134. 135. 138, 139. 142. 146, 262. _6.~. 264, ~74 Jowett. A. K.. 437.439. 490. 497 Joy, K. C.. 12. I7 Joyner, A. L.. 79.84. 85.88. 100. 101. 102, 103, 106. 109. It0, 111.1 I2. 1 I3, 114. 1 i5, 1 t6, 120. 121,123, ]24, 125, 126. 168. t76, 228.230, 233. 239.250, 428,451. 453.470, 471. 487, 489. 491, 493, 497 Juhasz. M.. 91, 97 Jullien. D.. 9. 14. 15 Jiinemann. G.. 443.486 Jung, D.. 173. 175 Jung, H. S.. 441. 493. 574. 577.578. 579. 585. 586. 587. 588 Jung. J.. 316. 317. 320, 324. 325 Junien. C.. 416, 419 Juppner. H.. 285.293. 294. 442. 443. 489 Jurata. L. W.. 135, 146 Jurecic. V.. 353. 357, 364 Jiirgens. G., 316. 329, 469, 483 Juriloff. D. M.. 423.486, 487 Juriscova. A., 13, 17 Jussila. L., 218. 223,229 Just, W:. 388, 391 Justice, M. J.. 292, 293 Justus. M.. 85. 103 ,

~,~

~,

K Kaartinen, V., 314, 325. 434, 467. 487 Kabl. J. M., 165. 177 Kablar, B.. 258. 260. 261,262. 267, 271. 275 Kachar, B.. 557, 56 I Kachinsky, A. M., t32, 148, 263, 276

Kadesch, T., 257, 2 75 Kadler, K. E., 572, 583, 585 Kadokawa, Y., 136, 146 Kaesmer, K. H., 40, 42, 51, 135, 146, 165, 17Z 303, 3 t 0, 325, 327. 479, 487 Kageyama, R., 136, 146; ]4Z 3 t0, 32t, 325, 526, 527, 538, 555.566 Kahane, N.. 13 I, 132, 140, 141. ]44, 145, I46, 255, 256, 273,275 Kahn, A., 262. 268. 276. 342. 368 Kaibuchi, K.. 95.105 Kaipainen. A., 162, 179, 218. 223, 230, 580, 589 Kaitila. I.. 286, 292, 453,481 Kakeya, T., 511,515 Kakinuma, H.. 471,479. 487 Kakizuka. A.. 581. 588 Kakoi, H., 557. 563 Kaku. M.. 443.453. 487 Kalamaras, J.. 321,328, 329, 451,470, 475, 494, 497 Kalantr;y, S.. 2, 4 Kalatzis. V.. 398.416, 550. 561, 563 Kalb. J. M., 316. 325 Kalb. R. G.. 225.231 Kalcheim, C., 131,132, 140, 141,144, 145, 146, 255,256, 273, 275, 422, 423,424, 489 Kalen, M., 414, 418 Kalka, C., 220, 228 Kalla, K. A., 89, 95. 104, 506, 509, 510, 514, 5t6, 517 K/illen, B., 75, 98 Kalman. E. 345, 356, 369 Kalnins, V. I.. 527, 535 Kaltenbach, J. A., 557, 563 Kalter. H.. 442, 487 Kalvakolanu. D. "v:. 343,365 Kalyani. A. J., 236. 240, 241. 250, 251 Kamachi. Y., 283.293, 381,391, 529, 530. 53 I, 534, 536, 538, 565 Kamada, N., 432, 450, 465,489 Kamada. S.. 399, 419 Kamada, T., 557,565 Kamino, K., 343, 3612 Kamisa~o. M.. ~-6, 358 Kamiya, A., 318,325 Kamps, M. R, 478, 479, 488, 489 Kan, M.. 287, 293 Kanai. Y.. 38.51, 380, 391 Kananen. K., 511. 516 Kanatani, N.. 289, 294 Kanatsu. M., 203,208 Kandel. E. R., 250 Kane. D. A., 546. 565 Kanekar, S., 526, 527, 535 Kaneko, K. J., 5, 9, 10. 17 Kaneko, S., 525,535

Author Index

Kaneko, Y., 189, i89, 202, 208 Kang, I.-S., 354, 365 Kang, S., 470, 487 Kania, A., 31, 34, 43, 46. 47, 48, 52, t04, 397.420 Kantor. A. B., 245,250 Kanzawa, N., 334, 368 Kanzler, B.. 468, 488, 57 I. 572, 574, 585, 587 Kao, K. R., 335,362 Kapiloff. M., 510, 515 Kaplan, D. H., 287, 293 Kaplan, D. R., 9 t, 101 Kaplan; E S., 271,276 Kaplan, M. S., 237, 250 Kappel, A.. 228, 230 Kappen. C., 312. D_5 "7 Kapur, R. R, 78, 99, i01, 465.498 Karagogeos, D., 553.562 Karaplis, A. C.. 285, 293, 442, 443.488, 489 Karashima, T.. 581. 585 Karasuyama, H., 87, 101 Karavanov. A., 104, 508.5t7 Karavanova, I. D., 405,417, 439, 497. 577, 578, 583,589 Karels, B., 385, 389 Kargul, G. J., I 1, 12, t7 Karin. M.. 87, 104, 453,486, 504, 509, 5 t0, 513, 514, 516, 580, 581,583,586 Karis, A., 343, 369 Karl, J., 373,391 Karlsson, L.. 313, 326, 414, 418 Karlsson, S., 203, 20Z 226, 229. 230, 467, 484 Karlstrom, H.. 246, 249, 318, 324 Karlstrom, R. O., 85, 101, 505,515, 532, 534, 536 Karolyi, J., 14, 15 Karp, S. J., 287, 293, 442, 488 Karpen, S. J., 316, 324 Karperien, M., 285, 293, 442, 443, 489 Karr, D.. 286, 292 Karram, K.. 88, 98 Karsenty, G., 280, 288. 289, 292, 293, 294, 408, 418,, 451, 467. 490 Kartasova, T., 581,582, 583, 587, 588 Kartori, Y.. 506, 516 Kas. E., 9. 14, 15 Kasahara, H., 343,347,361, 363 Kasai, N., 511,516 Kashiwagi, M., 580. 58Z 588 Kashtan. C. E., 412, 416, 418, 419 Kaspar, R, 156, 179 Kass, D. A.. 343, 350, 351,352,359 Kastner, R, 9I, I01, 341,346. 347, 352, 362, 368, 451,468. 469, 470, 484, 489, 490, 525,536, 557, 562 Kastschenko, N.. 4_~, 488 Kastury, K., 290. 294, 450, 471. 472, 473,495 Katagiri, C., 198, 208

633

Katagiri, T., 27 t, 275 Katahira, T., t 12, i t4, i i 5, ]23 Kataoka, H., 200, 209 Kataoka, Y., 47 t, 479, 487 Katayama, A., 553, 562 Kathiriya, I. S., 349, 360 Kato, A.. 525, 538 Kato, K., 263,274, 275, 557, 565 Kato, N., 471,479, 487 Kato, S., 505, 517, 525,535 Katoh, M., 22, 34 Katoh-Fukui, Y., 353, 354, 355,356, 363, 376, 39t Katori, Y., 549, 564 Ka~ev, S., 336, 368 Katsura, Y., 200, 209 Katz, A., t 94, 207 Katz, B. Z., 408, 418 Katz, L. C., 94, 102 Katz, R. W., 509, 514 Kau, C. L., 198, 208 Kaufman, M. H., t27, 128, 131, I46, 195, 196, i99, 208, 333, 362, 395, 418. 451,475,488, 493, 500, 504, 515, 541,542, 563, 568, 57 I, 574, 579, 58 i, 587 Kaufmann, E., 165, t 77, 453~ 479, 488 Kaur, R, 570, 589 Kaur, S., 79, I05 Kavanaugh-McHugh, A., 357, 358 Kawabata, M., 356. 367 Kawabe, K., 379, 391 Kawabe, S., 580, 58Z 588 Kawachi, Y., 582, 583,587 Kawai, T., 452, 496, 580, 581,583, 589 Kawaichi, M., 136, 137, t4 7 Kawakami, A., 43, 50, 88, 99 Kawakami, K., 399, 419 Kawamoto, H., 200, 209 Kawamura, K., 95, 102, 471,479, 48Z 504, 506, 515, 516 Kawano, H., 95, 102, 506, 516 Kawasaki, T., 223,230 Kawase, E., 188, 189, 226, 232 Kawase, T., 506, 516, 549, 564 Kawashima, H., 350, 362 Kawasoko, S., 443, 453, 487 Kawata, T., 443,453, 487 Kawate, T., 222, 231 KawaucN, H., 547, 564 Kawauchi, S., 525, 527, 530, 531,536 Kayden, H. J., 16I, I75 Kazanietz, M. G., 585 Kazaro,,~ A., 200, 207 Kaznowsld, C. E., 92, 102 Kazuyuki, O., t 83, t 90 Kearne, M., 220, 228 Kedinger, M., 308, 310, 324, 325, 326

6

3

4

A

u

Keene. D. R., 572, 573,583,584, 587 Keeney, D.. 453,476, 493 Kehrl, J. H., 319.325 Keighren, M., 23, 34 Keijser, A. G. M.. 34 I, 345, 347, 348, 350. 359 Keiper-Hrynko. N.. 6. ] 7 Keithley, E. M., 556. 558. 562 Ketcher. J.. 76, 77, 82. 83. I03 Ketemen. M.. 195,208 Keller, G., I95,200, 203. 207. 209, 510. 518 Keller. G. A., 223,229 Kelley. C. M., 198. 203. 208. 210 Kelley. M. W.. 553.555.557. 562. 563, 564. 565 Kelly. D.. 166. 179 Kelly, R.. 140. 14I. 145. 148. 258. 263. 264. 273. 277. 341. 343. 344. 345, 356. 361, 362 Kelly, T.. 507.515 Kelly, Wl A.. 534. 535 Kelsey. A.. 375.391 Kelsh. R. N.. 546. 565 Kelvin. D. J.. 342. 361 Kemler. R.. 15.16. 408. 409.41 O. 418, 419. 420 Kemp. C.. 31.33. 44. 48.49. 50. 78.98. 304. 323. 338. 358 Kemper, O. C.. 341. 369 Kempermann. G.. 237. 250 Kendall. S. K.. 511. 512.515. 518 Kennedy, M. 195.200...0/. 209 Kennedy, T. E.. 510. 514 KennerdelI. J. R., 14, 1 5 . 1 7 Kenny. D. A.. 135. 146 Kenny-Mobbs. T.. 140. 146 Kent. J.. 380. 387. 391 Kenyon, K. L., 532, 536 KerS.nen. S., 441. 487. 497 Kere. J.. 441.48& 493 Kern. M. J.. 433.476, 477.478.489 Kern, S. E.. 468.495 Kerr. R. 341. 359 Kerr. W G.. 157. 180 Kerrebrock. A. \~. 136. 137. 147 Kertesz. N.. 40. 49, 323 Keshet. E.. 193. 204, 208, 225,229 Keshvara. L.. 94. 99 Kessel. M.. 39.41, 46, 51, 78.81. 103, 129. 147. 427. 488, 546, 564 Kessler. D. S.. 45.52. 239. 250, 303.325 Kessler, J. A.. 239. 251 Kestevan, S.. 348, 349. 352, 353.357.358 Kesteven. H. L.. "442, 488 Kestila, M., 412, 4 t 8, 419 Ketting, R. E, 14. t8 KettiewetI, J. R.. 382. 387. 388, 391.392 Kettunen, R. 441.48Z 493 Kettunen, R J., 452, 488, 494

t

h

o

r

Index

Keutet, J., 28 I, 294, 380, 393 Keverne, E. B., 435, 488 Keynes, R.. t t9. ]23, 225,229, 422, 425, 428, 490 Keynes. R. J., t 34, ]47, t48 Khan, K. M.. 547, 564 Khan. M. K., i 93, 206, 20Z 246, 250, 270, 271,274 Khavari, R A., 578. 579, 580, 583,585, 588 Khoo, R. 3, 4, 422, 427, 482 Khoo, V~. 341,370 Khumna, T. S., 94, 104 Kid& S., 136, 147 Kidder. G. M., 5. ]7. 156, ]77 Kido. M.. 38.5] Kidwell. J. F.. 453.488 Kieboom. D.. 192, 210 Kieboom, K., 376, 389, 453,481 Kieckens, L., 203.20Z 223.228 Kiecker, C.. 338.362 Kiefer. R. 545.563 Kier. A. B.. 204, 208, 318, 327, 453,483 Kiernan, A. E., 546, 548, 551,552, 557, 56t, 563, 565 Kiessling. A. A.. 14, 18 Kikuchi. K., 560, 563 Kikuchi. T., 506.516, 549. 564 Kikuchi, Y.. 303.326 Kikuyama, S., 504, 515 Kil. J., 558, 563 Kilby, M. D., 174, ] 79 Kilkenny, C.. 402. 420 Killary. A. M., 267.2 77 Kilner. R J.. 351,363 Kilpatrick, T. J., 86, t0] Kim, C. H., 469, 470, 486 Kim, C.-Y., 354. 365 Kim, E.. 375.390, 415,418 Kim, H. J., 452. 488 Kim. I. S.. 288. 293 Kim. J.-H., 342, 360 Kim. J. I., 527, 530, 53 I, 536 Kim, J.-S., 173, 17Z 348, 363, 379. 391 Kim. M.-S.. 347, 362 Kim. R C.. 310. 326 Kim, S.-H.. 40. 44. 51, 243,249. 338, 359 Kim, S. K., 319, 320, 325, 326 Kim, Y., 334, 343, 361, 363, 474, 488 Kim. Y. H.. 217, 220, 229 Kimata, K., 284, 294, 350, 370, 442, 443,493, 497 Kimberling, W: J., 414, 418 Kimble. J.. 10, 19, 165, 176, 316, 325, 326, 340, 358, 471, 493 Kimble, R. B., 292, 292 Kimelman. D., -44. 45.51, I67, 176, 334, 361, 579, 584 Kimmel, C. B., 118, 125, 167, 176, 465, 491 Kimmet, R. A.. 109, 123, 228, 230

Author Index Kimmel. S. G.. 310, 326 Kimura. C., 31.34. 46, 5t, ! 14, 124, 451,478, 488, 490, 521. 527,537 Kimura. S.. 81.82. 88, 89, 90, 91. 105, t94. 208, 311,312. 326, 32Z 474, 488, 504, 505~ :~0 - "J, 508.510, . . . . 518 Kirnura. T.. 288. 293, ~ 3 , 4 9 2 Kimura. Y.. 8.17. 408. 418 Kina, T., 192, 194, 207, 245,250 Kindblom. J. M.. 510, 5 t 5 Kinder. S. J., 3, 4. 38. 50, 51. 132. 147. 397, 420 King. A.. 267,274 King, C. Y., 38I. 390 King, J. A.. 82. 101, 315,326 King, M . L. , 6. t 7. ~~3~ , . 2~;" DO Kin~.,. T.. "'~, 356. 363 Kingbury, B. E. 421,488 Kingdom, J. C. R, 174, 177 Kingsley, D. M.. 82.10t, 315.326, 467,488, 496 Kinloch, R. A.. 15, 18 Kinnon. C., 201,208 Kinoshita. C., 581. 582, 583.587 Kinoshita, N., 338. 363 Kinoshita. T., 318. 325, 581,583.589 Kintner. C. R., 138. t46, 226, 232, 408, 409, 410, 417': 418 Kinzler. K. Wi, 86, 100, 579. 586, 587 Kioschis. R, 327 Kioussi. C., 353. 354. 355, 364, 398, 420, 433,478, 489, 496, 500, 509. 515 Kioussis. D.. 31,34, 31 I, 3t6, 327 Kiraly, K.. 284, 293 Kirby, C.. 22. 34, I57.177 Kirby, M. L.. 14, 17. 334, 348, 357,363, 45t, 473.495 Kirchhoff, S.. 173, 177 Kirillova. I.. 318. 329 Kirsch. i R.. 5 t "~ 515 Kirschner, M. W, 338, 363 Kishida, T.. 227. 229 Kishimoto. T.. 204. 209, 220, 232, 288, 293, 3t 8, 325, 341, 352, 370, 442, 443,453, 488 Kishimoto; Y., 336. 363 Kispert, A., 45, 47.50, 135, 137. t40, 14I, 147, 336, 338, 355,359, 363, 384, 385,393, 405. 406, 407, 418, 419, 468. 483 Kissa, K.. 9, 14. 15 Kisslinger. J. A., 136, 148 Kist, R.. 380, 392 Kitagawa, M.. 286. 293, 294 Kitaguchi, T., 357, 363 Kitajewski, J., 140. 141, 14Z 226, 230, 232, 262, 264, 275 Kitajima, K.. 317,327 Kitajima, S., 340, 352, 363, 367 Kitamoto, Y.. 413,418 Kitamura. K.. ~,"'" 354, 355. 356. 363, 557, 563 Kitamura. N., 174, 179, 404, 420

63 5 Kitamura, T.. I86, 190 Kitamura, Y.. 288 293. ~ 2 , ~'_~, " ~.~, 488. 555.565 Kitanowski, F., 451,453, 474, 489 Kitchen, J. R., 1 I, 12, ] 7 Kitos, R, 200, 20 t, 209 Kitsukawa, T., 223,230 Kittappa, R., 200, 209 Kizumoto, M., 200, 209 Klagsbrun, M., I56, ] 78, 223, 230, 232 Klaine, M., 200, 208, 209 Klamt, B., 347, 363 Klausing, K., 504, 510, 516 K!ausner, R. D., t 73, 176, 227,229 Klein, G., 411,418 Klein, R S., 405,418 Klein. R.. 91,105, 119, t23, 171, I73, i74, 266,275, 559, 564 Klein, R. D., 402, 4t8 Klein, ~ H., 204, 209, 260, 261,262, 274, 276, 277, 289, 294, 527, 535, 536, 556~ 558, 565 Kleinman. H. K., 415,420, 424, 442, 443, 482 Ktewe-Nebenius, D., 23.34, 160, 166, 178 Ktima, M., 473,488 Ktine. A. D.. 4~... 486 Kline, D., 8, ] 7 Klingensmith, J., 40, 42, 43, 45, 48, 49, 50, 5t, 52, 78.98, 217, 229, 304, 323 Ktisak, I.. 452, 453, 474, 487 Kloc, M., I, 4, 8, t6, ]7 Ktoos, J., 423, 493 Kloter, U.. 533, 535 Kluk, M.. 227,230 . ~M.W., 4.~.~, Klvmkowsky. "~'~ 482 Knapp, L. T., 507, 508, 511,514 Kneppers, S. L., 285, 293 Knezevic, V., 40. 51 Kn6chet, W., 165, 17Z 453, 479, 488 Knoedtseder, M., 94, 104 Knoepfler, R S., 478, 479, 488, 489 Knoetgen, H., 41, 46, 51, 427, 488 Knofler, M., 174, t 77, 32 t, 326 Knoll, J. H., 288, 293 Knowles, B. B., 6, 8, 10, 1 i, i2, i3, t5, 16, 17, t8 Knowles, H. J., 313, 323 Knudsen, K. A., 264, 274, 340, 364, 366 Knudson, C. M., 196, 198.2t0 KniiseI, B., 9t, 10t Knust, E., t 36, t45, 149 Ko, K.. 219, 229, 231 Ko, M. S. H., I 1. t2, 17 Koban, M. U., 343,366 Kobayashi, A., 397, 420 Kobayashi, S., 186, ! 90 Kobelt, A., 281,293, 381. 391

636 Koblar. S. A.. 173. 179 Kobler. J. B.. 353.357. 365 Koch. A. W.. 408.4]8 Koch, C. J., 225.229 Koch. R J.. 582. 583,587 Koch. V< J.. 341,352, 362, 363 Kochanowski. H.. 12, 16 Kodama, T., 432,450, 465.489 Koder. A.. 172, 173, 174. 34I, 357. 358 Kodjabachian. L.. 40, 5I Koehler. G.. 205,206 Koehler. M.. 388, 392 Koentgen, K, 31,34, 345, 348, 366 Koentges, G.. 422,425.485 Koester. S.. 264. 268, 275 Koetgen. F.. 348. 349. 352. 353. 357.358 Kofron. M.. 14, 16 Koga, Y.. 312. 328. 505.517 Kohan. R., 543.563 Kohl, L., 15, 15 Kohn. G.. 174, 178 Kohtz. J. D.. 78, 81, 99, 101, 110. 121. 123, 124 Koishi, K., 269, 274 Koiussis, D., 46. 47, 48, 51 Koivisto, T., 314, 327 Koji, T.. 504. 516 Kojima. I., 321. 324, 327 Kojima, M.. 317.32 7 Kokaia, M., 247, 249 Kokaia, Z.. 247. 249 Kola, I.. 22.34 Kolatsi-Joannou. M.. 404, 420 Kollar, E. J.. 421,438, 439. 48], 488, 491 Kollet, O., 341. 369 Kollufi, S. K., 346, 364 Kolquist, K. A., 400, 4t8 Kolski. C., 506. 517 Komaki, M.. 271. 275 Kominami. R., 537 Komiya. T., 258. 272 Komori, T.. 204, 209, 220, 232. 288, 289, 293, 294, 442, 443,453,488 Komura. I.. 343.368 Komuro, I.. 334, 336, 343,347, 361, 363, 365 Komuves, L., 580, 581,582, 583,585. 586, 588 Kon, Y., 51 t, 516 Kondaiah. R, 467, 49] Kondo, M., 194, 208., 241,250 Kondo, S., 317, 32Z 353, 354. 355.356. 363 Kondo, T.. 241,246. 250, 305,310. 326 Kondoh, H., 47.49, 5I, 52, 120. 121. 125, 283. 293, 318. 328, 357, 365, 38 I. 391, 522, 527. 528. 529. 530. 531, 532, 533. 534, 536, 53Z 538, 565 Konieczny, S. K, 258, 274

Author Index Konno, H., I 1, 12, 18 Kontgen, E. 156, 160, 167, t 75, 179, 203, 209 K6ntges, G., 266, 27Z 422, 423, 424, 425, 428, 488 Kontos, C. D., 225, 230 Kooh. R J.. 136, t45 Koopman, R, 281,293, 295, 377. 380, 381,387, 389, 390, 391, 392 Kopan, R., 136, I46, I4Z 14& 262, 263, 275 Kopen, G. C., 246, 250 Kopf, G. S., 7.8. 13. I6, IZ t9 Kopf. M., 205,206 Kopp, J. B., 408,420 Kordower, J. H., 247, 251 Korematsu. K., 91, IOI Korfhagen, T. F., 313, 314. 329 Korhonan, J., 228. 230 Korhonen. J.. 218, 230 Kornberg, T. B., 470, 481 Korniszewski. L., 380, 392 Korsmeyer. S. J., 376, 390, 393 Korving, J., 183, 190, 451,496, 549, 565 Korving, J. R W: E M., 336. 363 Korzh, "v:, 78, I04, 141,148, 546. 563 Kos, L., 402, 4I 7 Kosaki, K., 47, 51, 357. 363 Kosaki. R., 47, 51, 357, 363 Koseki, H., 135, 137. 138, 140, 145, 14Z 148, 398, 41Z 471,475,479, 483, 48Z 492 Koshiba, K., 354, 370 Koshiba-Takeuchi, K., 525,536 Koshida, S., 41, 51 Kostakopoulou, K., 266. 273 Kostamarova, A. A., 22, 34 Kostas, S. A., 14, 16, 341,360 Koster, M., 266, 274 Koster. R., 533. 537 Kostetskaia, E.. 346. 347, 363 Kostetskii, I.. 346, 347.363 Kotanides, H., 227, 233 Kotch, L. E., 225, 230 Kotecha, S., 40, 44, 50, 323, 336, 338,339, 342, 355, 35& 368 Kothapalli, R., 47, 51 Kothax?; R., 23, 35, 169, 179, 258, 264, 266, 274 Koudrova, M., 266, 274 Koutsourakis, M., 165, 177 Kouzarides, T., 342, 368 Kovacs, C. S., 285.294 Kovacs, K.. 511,512, 513, 515, 518 Kovari, I. A., 412. 417 Kowalsld. J., 223.229, 291,292 Kowk, G.. 281. 292, 380, 390 Koyama, E., 423, 424, 451,475, 476, 478, 490 Koyama, H.. _o, 35

Author i

n

d

e

Koyama, Y., 531,538 Kozak, C. A., 284, 294, 442, 443,497, 521,525,535, 537 Kozak, K. R., t 72, 173, ] 7Z 225,230 Kozian, D., 264, 277 Kozu. T., 202, 208 Krahl. T., 318, 322. 326 Krakowski, M. L., 318, 322, 326 Kral, A., 415.417 Kramer. S.. 466. 485, 488 Krane. S., 583.587 Kraner. S. D.. 243,249 Krantz, I. D., 347,364, 366 Krapp, A., 321,326 Krasnow, M. A., 311. 327. 466, 485, 488, 491 Krastel, K., 258, 260, 26 I, 262, 267, 275 Kratochwil, K., -440. 44 I, 451, 47 t, 475, 481, 488, 493 Krause, D.. 194, 207 Krause. M., 257, 27Z 341,360 Krause, W: L.. 453.495 Krauss, S., 31, 33, 47, 50, 78, 101, 451. 476, 483, 508, 5t4, 546. 563 Kraut, N., 169, t75. 177 Kraybill, A. L., 415,417 Krebs, C., 174, 177 Krebs. L. T., 226. 230 Kreiborg, S., 292, 294 Kreidberg, J., 183, 187, 188, 189 Kreidbe~, J. A., 94, 98, 3t4, 326, 341,352, 363, 365, 375, 376, 389, 391, 392, 400, 4!0, 418, 572, 574, 58.5 Kremser, T., 334, 363 Kress, C.. 166, 178 Krezel, W., 91,101, ]04, 525, 536 Krieg, R A., 334, 339, 347, 348, 359, 360, 361, 369 Krieger, J., 346. 358 Krieglstein, K., 314, 324 Kriegstein, A. R., 86, ]02 Krimpenfort, R, 583,589 Kritzik, M. R., 318, 322, 326 Kriz, R. W., 467, 498 Kromer, L. E, 9 I, lO1 Ir H. M., 285,286, 287, 292, 293, 294, 442, 443, 470, 488, 489, 497 Kronenberg, M. S., 452, 484 Kros, C. J., 553, 563 Kroumpouzou, E., 357, 358 Krude, H., 31 i, 326, 508, 516 Kruger, O.. 173, 177 Krull, C. E., 426, 427, 494 KrumlauL R., 45, 52, 117, 1t8, 1t9,123, 124, 125, 1!42, 147, 422, 424, 425,426. 427,428, 433, 449, 453, 468, 469, 474, 479, 484, 485, 487, 488, 490. 492, 494, 496, 544, 545,562, 563, 571,586 Krushel, L. A., 94, 101 Krust, A., 469, 489, 491, 494

x

6

3

7

Krylova, I., 379, 390 Kuan, C. Y., 87, 100, lOl Kuang, WiJ., 223,230 Kubalak, S. W., 341,344, 346, 352, 359, 36], 363, 367 Kubiak, J. Z., 7, 18 Kubo, H., 14, ]7 Kubo, T., 555,565 Kucera, J., 559, 562 Kuch, C., 2 ~ , 277 Kucheflapati, R., 43, 50, 353. 357,358, 365, 415,420 Kudlow, J. E., 51 t, 516 Kudoh, S., 336, 343, 365. 368 Kuehn, M. R., 338, 352, 354, 355, 356, 359, 363, 364, 370 Kugter, J. D., 357, 358 Kuht, M., 337, 363 Kuhlman, J., 401,419, 57t, 588 Kuhn, H., 421,442, 457, 488 Kuhn, H. G., 237, 250 Kuida, K., 87, t01 Kulbacki, R., 205, 206 Kutesa, R M., 424, 488 Kutesh, D. A., 570, 589 Kulikowski, R. R., 355,364 Kuliszewski, M., 173, 174, 175, 313, 329 Kulkarni, A. B., 94, 100, 103, 203,207, 226, 229, 230, 467, 484 Kulkarni, S., 171, t 75 Kumada, M., 432, 465.478, 483 Kumanogoh, A., 34t, 352, 370 Kumar, A., 338, 363 Kumar, S., 3 t 8, 328 Kuma_r, T., 44, 51, 441,467,468, 490, 491, 500, 510, 5t6 Kume, R., t37, 147 Kume, T., 402, 404, 4I& 453,479, 488 Kun, J., 28 i, 294 Kunath, T., 23, 35, t66, 167, 179, 357, 368 Kundu, R. K., 428, 452, 489 Kunisada, T., 442, 498 KunkeI, L. M., 193, 206, 207, 246, 250, 270, 271,274 Kuno, J., 95, 102, t22, 122, I73,179, 220, 222, 231, 404, 420, 506, 516 Kuno, T., 2 I7, 230 Kuo, A., 286, 292, 453,484 Kuo, C. J., t 64, 177 Kuo, C. T., 165, 177, 316, 326, 342, 343, 352, 363 Kuo, E C., 203, 209, 5 t0, 518 Kuo, H.-C., 342, 343, 352, 363 Kuo, T. S., 91,103 Kuo, W. L., 347, 364 Kupper, T. S., 570, 587. 588 Kupperman, E., 340, 341,363 Kupriyanov, S., 168, t 79 Kurant, E., 9 t, 103

638

A u t h o r Index

Kuratani, S., 31.34, 46, 51, 83, I06, 1 I4, 124, 305,307, 328, 9 374, 392, 403.418, 42 t, 422, 424, 426, 435,437. 442.451. 457. 475,476, 478. 488, 490, 492, 496, 521, 52 9 537 Kurihara. H., 349, 369, 432, 450, 45t, 465.473,489, 496 Kurihara. Y.. 349. 369. 43 "~ 450, 451 465.473.489, 496 Kurisu. K., 289, 294 Kurkinen. M.. 161.176 Kurland. S.. 467, 482 Kuroiwa. A., 41.47.51, 78. t04, 1 !7. 124, 427.485 Kuroki. T.. 580. 587. 588 Kurosumi. M.. 511. 515 Kuroume. T.. 506. 518 Kurzrock. E. A.. 301. 326 Kusakabe. M.. 11. 12.18. 557.565 Kusumi. K.. 136, 137. I47 Kutsche. L. M.. 350. 369 Kuttenn. F.. 375.389 Ki.ittner. F.. 316. 329. 469.483 Kutunumu. N.. 581. 589 Kuwaki. T.. 432.450. 465,478.483. 489 Kuwana. E.. 76. 77.82, 83.85.99. 103 Kuzmin. I.. 227. 229 Kwan. C. T.. 118. 119. 124. 544. 563 Kwan. K. M.. 31.34. 43.46. 47.48.52. 104. 397. 420 Kwee. k.. 162. 170. 177. 219. 230 Kwok. C.. 380. 381~ 391 Kwon, Y. T.. 94. 98. 101 Kwong. R D.. 408.419 9

~...,

.,

9

L Labastie. M. C.. 201,208 LaBonne. C.. 422. 423.481, 489 Labosky. R A.. 82. 106. 203.210, 305.309.319. 320. 327, 329, 439. 453.467.468. 488, 496, 498. 506. 516 Labouesse. M.. 14, 15.16. 165.176, 316. 325 Labow. M. A.. 162. 170. 177, 219. 230 Lacerda. D. A.. 442. -443,489 Lachman. R. S.. 286. 294 Lacktis. J. W.. 355.364 Lacombe. D.. 398, 416, 550. 56t Lacroix. B.. 308, 325 Lacy, E.. ~.. "~ 4 Lad& A. N., 334, 335, 336. 337, 363, 370 Ladher, R.. 422. 484 Lagace. G.. 512, 514 Lagasse. E.. 246. 250, 318, 326 Laget, M.-R. 470. 481 Lah. M., 40, 44. 50,. 323, 338. 358 Lahn, B. T., 387, 391 Lahtinen. I.. 228.230 Lai. A . E . 9.18 Lai. C. C., 318, 326

Lai, E., 78, 81, 82, 84, 85, 86, 99, 100, I0t, I02, ]05, t06, 164, t 65, 177, t 79, 24 I, 243, 244, 251, 252, 408, 417. 479, 486, 498, 522, 525,527, 536 Lajeunie, E., 452, 453, 484, 489 Lake, R. J., t36, I44, 263,272, 555, 56] Lakkaraju, S., 427, 433,443, 449, 474, 479, 494 Lakso, M.. 218. 223. 230 Lala. D. S.. 375,376. 390, 391, 510, 5i6 Lallemand. Y.. 31, 33, 46, 48, 49, 114, I22, 322, 428, 45 t, 478. 481, 521. 527, 535 Lalli. E.. 378. 379. 384.39]. _ 9.~ Lalor. R A.. 194. 207 Lam. C. ~i. 579,589 Lam. G.. 15.17 Lam. R, 268, 2 73 LaMantia. A. S., 84. 9Z 106. 435, 469, 476. 48t, 483, 497 LaMantia. C.. 169. 179 Lamb. A. N.. 380, 390 Lamb. L. C., 467, 494 Lambie, E. J.. 316, 326 Lamerdin, J., 412. 418 Lamers, J. M. J.. 343, 345,348, 366 Lamers. W. H., 173. 177. 334. 341,343,344, 345, 347, 348. 350. 35 t. 352. 356. 359, 360, 36], 363, 365, 366, 369 Lammer. E. J., 468. 497 Lammert, E., 321. 326 Lamolet, B., 478, 489, 507, 5 t 6 Lamond. A. I., 400, 417 Lamperti, E. D., 94, 100 Lampron, C., 469, 489. 491 Lampugnani, M. G.. 226. 229 Lan, Y., 423,487, 553,555,563 Lance-Jones, C., 269, 277 Lanct6t. C.. 478. 489, 507, 508. 510, 514, 516, 5 t 8 Lander. E. S.. 136. 137. 147. 380. 392 Landis, C. A.. 512, 516 Landis, S., 239, 249 Landolt, A. M., 505, 5t3 Landry, C., 321I. 326 Landry, J.. 171, 173, 176 Lane, A. H., 381,382, 393 Lane, J., 416, 417 Lane. N.. 9. 18 Lane. R Wi, 442. 490 Lane, Vv: S.. 251 Lanford. E J.. 553.555,563 Lang, H.. 547.563 Lang, R, 357. 358 Lang, R. A.. 219. 229., 230, 23], 533,535 Langegger, M., 41 t, 418 Langeland, J. A., 478. 496 Langenbach, R., 156, 177 Langeveld, A., 165, 177

Author Index

639

Langitle, B. L., 341,361 Langille, R. M.. 422. 4,..~. "~'~ 424..9-443.449.47I. . . . . . 489 Langley, E., 342, 368 Lan~imm, C. J., 236. 249 Lankes, "~:, ~.~,"'"~ ~'-6 , 361 Lansdorp, R M., 242, 245, 251, 252 Lanske. B.. 285, 287, 293, 294, 442. 443.470, 489, 497 Lapi, R. 311. 326 Lapic, S.. 581,588 Lapidot, T.. 341,369 Lapointe, E. 264, 266. 273, 277 Lapointe, L., 167. ] 79 Lapvetelainen, T., 284. 293 Lardetli, M.. 135. I49 Larder. R., 390 Largman. C.. 242, 252 Larkin, S. B., 262. 277. 469, 49] Laroia, G., 1I. 17 Larouche, L., 171.173, ] 76 Larson, C., 415,418 Larsson. E., 413, 418, 467. 489 Larsson. L. I., 32 I. 327 Larsson. S. H., 39t, 400, 4t7 Larsson-B tomberg, L.. 402, 419 Lame, L., 409, 410. 418, 419 Lash, J. W., 263. 274, 333. 336. 340, 364 Lasko, R, 182, 185. 186, t90 Lasky, L. A., t93, 210 Lasorella. A., 474, 489 Lassar, A. B., 140, 141,142, ]47, t48, 257,258, 262, 263. 264, 268, 273, 274, 275. 276, 277. 282. 293, 328, 334, 335. 336. 337. 339, 341, 36t, 365, 367, 369, 399, 417 Latham, K. E., 5, 9, 10. 11, t2, t3, I6, 17, ]8. i9, 168;, ]78 Latit, F., 227,229 Latinkic, B., 336, 339, 342, 368 Lattanzi. L., 271,273 Lau, A. L., 44, 51, 440, 484 Lau, C. K.. 84, t02 Lau, K. K., 165. t 77. 316, 325 Lau, Y. E, 378. 390 Laub, F., 581,588 Laufer, E., t 41,146, 262, 264, 275, 470, 487, 494 Lau~hner, E.. "~'~5 230 Laurent, M., 579, 587 Laurie. G. W., 415,420 Lauster. R., 577. 583, 584 Lavdas. A. A., 88, 89, 101 Laverriere, A. C., 316, 326 Lavker, R. M., 570, 578, 585, 587.. 589 Law'. D. J., 357, 364 Lawitts. J. A., 228, 232 Lawler, A. M., 129. ]47, 225,230, 260, 264, 275, 353, 356, 367 Lawlor, R, 553,563 ,~ .

.

.

.

*--,,

,

Lawrence, H. J., 242, 252 Lawrence, R A., 550, 563 Lawsh6, A., 466, 486 Lawson, K. A., 38, 39, 44, 45, 47, 51, 52, 158, i T Z t83, ]90, 303, 3 ~ , 326, 333, 336, 338, 363 L~ne, M. D., 347. 359 Layton, x,,~M., 352, 354, 363 Lazarus, J. H., 3 t I. 324 Laszzarini, R. A., 170, 173, 174, t 74, 178 Lazzaro, D., 85, t0'3, 189, 190, 31 i, 326, 478, 48t, 508, 513 Le, T., 379, 389 Le, T. C., 415,420 Leardkamolkarn, Vi, 412, 417 Le Bail, O., 136, 146 Lebeche, D., 313, 315,326, 327 Leberquier, C., 308, 310, 324 Lebtanc-Straceski, J., 357, 358 Lecerf, E, 27 I, 2 77 Lechteider, R. J., 339, 368 Lechner, M. S., 399, 402, 41Z 420 Ledan, E., 7, 18 Ledbetter, D. H., 94, 103 Leder, R, 40, 52, 167, 17 I. 173, 179, 286, 292, 338, 368, 453,484 Ledermann, B., 321,326 Le Douarin, G. H., 250 Le Douarin, N. M., 39. 40, 50, 77, 84, 85, 98, 113, t23, 128, t40, 141,142, 145, 147, t48. 196, 200, 201,207. 236, 237, 241,244, 249, 250, 256, 261,263, 264, 266, 27 I, 273, 276, 277, 316, 3 t 7, 320, 326, 422, 423, 424, 426, 427, 433, 435,483, 489, 502, 507, 514, 569, 571, 572, 585, 587 Leduque, R, 320, 324 Lee, C. S., 141,147 Lee, D., 506, 517, 532, 53Z 549, 564 Lee, E., 43, 50, 78, 80, 83, 85, 86, 87, 9 l, 9& t03, t06, 12I, 123, 141,145, 260, 265,273, 3t9, 323, 439, 451, 470, 482, 508, 5 t Z 52I, 527, 535, 537 Lee, E. J., 402, 4i9, 432. 498, 508, 517 Lee, E. Y., 318, 326 Lee, H.-G., 2, 4 Lee, H.-H., 336, 363 Lee, H.-J., 354, 365 Lee, J., 314, 318, 326, 330, 579, 585 Lee, J. C., 16I, ]79 Lee, J. E., 92, 102, 12 I, ]24, 321,328 Lee, J. J., 470, 489 Lee, J.-S., 12, tZ 341,360 Lee, K., 285, 287, 294, 442, 443, 465,470, 489, 491, 497 Lee, K. E, 94, 9Z 285,293, 294, 469, 489, 559, 562 Lee, K.-E, 580, 58 t, 583,587 Lee, K. H., 42, 51, 349, 355,358 Lee, K.-H., 336, 363

640

A u t h o r Index

Lee, K. J.. 45, 52, 78, 10t, i20, I21,122, 122, I24, 343, 366 Lee. M.-E., 347. 359 Lee. M. H.. 483, 528, 537 Lee, M. J.. 227. 230, 314, 315.329 Lee. M.-J., "'~7 231 Lee, M. O., 525,537 Lee, R. K., 3-44. 368 Lee. R. Y.. 346, 363 Lee. S. B.. 400. 418 Lee, S. H.. 219. 226, 230. 233. 580. 587 Lee. S.J. 129, 14Z ~,~'7 ~ 9 260. 264. 275. 353 356. 367 Lee. S. L.. 510, 516 Lee. S. M.. 82. 84. 86. 101, 104. 428.48Z 489 Lee. S. M. K.. 116, 117. 124 Lee, W.-H.. 243.244. 249. 318. 326 Lee. Y.. 343.363 Leeming, G. L., 437, 490 Lefebvre, V., 281. 282, 283, 284, 289, 292, 293. 294. 295 Leffell. D. J.. 287, 292, 470, 486, 579. 586 Lefkowitz, R. J.. 341. 352. 362 Legeai-Mallet. L.. 286. 294. 453.494 Leger, J.. 507. 514 Leger, S.. 337. 367 Legouy, E.. 5, 9. 14. 15. 19 Legrand. J. F.. 408.419 Lehman, A. L.. 6. 17 Lehmann. M. S., 408. 419 Lehmann. R., 14. 19, 185.186. 188. 189, 190 Lehner. C. F., 170, 175, 179 Lehrer, M. S., 570, 589 Lehtonen, E., 559, 564 Lei, H., 47.50, 376, 393 Lei. L.. 312. 319. 326 Leibel. R. I.. 443.497 Leibovici. M., 398, 416, 550, 561 Leibowitz. R. T.. 247.251 Leiden. J. M.. 165, 177, 179, 316, 326, 341,342, 343,347, 352. 362, 363, 366, 368 Leimeister, C., 347, 363 Leinwand, L. A., 269, 275 Leinwand, M., 314, 315,329 Leiser, R., 174, 177 Leiter, A. B.. 310, 321,322, 327 Leitgeb, S., 570, 587 Leitges, M.. 137, 147 Le Lievre, C. S., 426. 489 Lelievre-Pegorier. M., 403,418 Lemai~e, F. R. 166. 176, 32 I, 326 Lemaire, L., 39.40, 51 Lemaire, R, 263,264. 274, 303, 324, 330 Lemarchandel, V., 200, 208 Lembo, G., 341. 363 Le Merrer, M., 285. 286, 293, 294, 452. 453,484, 489, 494 9

s

.

.

Le Meur, M., 4(), 46, 47, 52, 478, 494, 583,586 LeMeur. M., t14, 125, 310, 312, 321,325, 32Z 435,443, 469, 474, 484, 489, 490, 49], 544, 545,563 Lemieux, M.. 312, 323 Lemischka. I. R.. t 92, t 94, 200, 205, 206, 208, 209 LeMouellic, H., 571,587 Lendahl, U., 135, 149, 236, 237, 244, 245, 246, 248, 249, 250, 318. 320. 321. 323, 324 Lenhard, T.. 291,292 Lenkkeri. U.. 412, 418, 419 Lentz. S. I.. 411, 4]8 Leon. Y., 466, 494, 545,564 Leonardi, A., -442, 484 Leonardo. E., 91. 102 Leong. L. M.. 335,337. 339, 355.365, 366 Leopold. R L.. 579. 588 Lepori, N. G.. 356, 363 Leptin, M., 336, 367 Lerman, M. I., 227, 229, 231 Lerner. C. R. 245. 250 Lerone. M.. 346. 358 Leroy. J. G.. 281,293, 381.39t Leroy, R, 469, 484, 489, 557,562 Lesot, H.. 455.489, 493 Lessey, B. A., 156. 175 Letarte. M., 227, 23] Lettice. g. A.. 451. 453,474, 489. 530. 533,536 Lettieri. K., 104, 508, 517 Leung, D. W., 223,230 Leung, K. K., 380, 389 Leung, S. W., 225, 230 Leveen, R, 313,323, 413.418, 467, 482, 489 Levenberg, S.. 408. 418 Levi. G., 290. 292, 450, 453,472, 473,481, 5 I0, 518, 546, 549, 561, 564 Levi. T., 339, 343, 347. 357, 358, 359 Levin, L. S.. 442. 485, 507. 515 Levin, M. E., 94, 106, 356, 363, 364, 473,489 Levitt, R, 84, i 0 t kevorse, J. M., 451,476, 493 Levy, D. E., 286, 292, 294 Lew, D., 504, 5 t O, 516 Lewandoski, M., 15.17, 31,35, 84. 106, 114. t 15, 124, 125, 337, 340, 352, 365, 36& 453. 466, 489, 496. 497 Lewin, M., 47, 51, 357, 363 Lewis, A. E, 204, 210, 220, 233 Lewis, J., 139, 146, 466, 491, 545,546, 547, 549, 552, 553, 555, 558. 559, 561, 562, 563, 564, 565 Lewis. K. E.. 534. 536 Lewis, P., 441,483 Lewis. P. M., 312, 328, 577, 578, 583,589 Lewis. R. S.. 8. 16 Lewis, S., 509.510, 515 Lewis, W. H., 26, 34, 4 t 5, 4 IZ 529, 536

Author Index

Leyns, L., 40, 44, 49, 51, 52, 141,144, 323, 338,366, 473, 477,482 Leys, A., 398, 419 Leysens, N. J., 478, 495, 507, 517 Li, C., 167, 171,173, 179, 226. 227, 233, 286, 292, 293, 411.418

Li, C. W., 542. 550, 563 Li. D. Y., 226. 230, 232, 556, 558, 565 Li. E., 47, 50, 94, 98, 353, 356, 357, 366, 468, 469, 489, 492

Li. E. 225, 23I, 375,392 Li, H., 79, 82, 90. 94, 95, 10I, 104, 105, 106, 162, 170, 176, 319, 320, 321,323, 326, 339, 36& 502. 507, 508, 509, 516, 517. 521,530, 536 Li, J., 317, 326, 577,578,583,589 Li, L., 228, 230, 347, 364, 58Z 589 Li, N., 356, 366 Li, R, 89, 95, 104, 240, 250, 283, 284, 289, 293, 294, 506, 517

Li, Q., 343, 357, 364, 574, 580, 581,583, 587, 588 Li, R., 203,209, 334, 343, 345,348, 349, 352, 356, 364 Li, S., 3, 4, 78, 81, 82, 86, 99, 260, 276, 509, 516 Li, S. C., 81, 86, 101, 522, 527, 536 Li. S. W.. 284, 293 Li. T.. 220, 228, 527. 530; 53 t, 535, 536 Li, W., 239, 250 Li, X., 290, 292, 452, 453,474, 487 Li, X. E, 174, !79 Li, Y., 9t, IOI, 172, 173, 177, 424, 442, 443,489, 493 Li. Y.-X.. 14, 17 Li, Z., 266, 277 Lia, M., 43, 50, 353.357, 365 Liang, I., 235, 244, 251 Liang, J. O., 78, 104, 351,364 Liang, L.-E, 14, 16 Liang, M. Y., 527, 535 Liang, Y., t 1, 12, IZ 556, 564 Liao, E. C., 214, 230 Liao, W., 214, 222, 230, 231 Liao, X., 245,249, 312, 328 Liberatore, C. M., 342, 347, 364, 367 Licht, J. D., 375,392 Licht, R, 388, 393 Lichter, R, 479, 487 Lichti, U.. 169, 179 Lichtlen, R, 317. 325 Lichtler, A. C., 452. 484 Lidral, A. C., 292, 294 Lieber, T.. 136, 147 Liebhaber, S. A., 443,493 Liegeois, N. J., 532, 536 Liem, K. D., Jr., 423,489 Liem. K. E, 102 Liem, K. E. Jr., 121,124

641

Lien, C.-L., 342, 364 Lieschke, G. J., 194, 208 Liggitt, D., 173, 179, 322, 328 Lighffoot, R, 453,483 Lightman, S. L., 506, 514 Ligon, K. L., 135, ]45, 267, 273 Liguon, G., 338, 352, 370 Lilly, B., 262, 273, 341,364, 476, 483 Lim, C., 509, 5] 7 Lim, D. A., 236, 237, 244, 245,248, 249 Lim, D. J., 551,552, 557, 563 Lira, H., 156, 177 Lim, L., 82, 102, 321,328 Lim, M. K., t 1, 12, 17 Limon, J., 83, 100 Lin, B., 346, 364 Lin, C. H., 271,275 Lin, C. R., 353, 354, 355,364, 433,478, 489, 508, 509, 512 516

Lin, D. M., 435, 489 Lin, H., 91,100, 204, 208. 341,343, 352, 368 Lin, R, 227, 23t Lin, Q., t 65, 168, t 75, 177, 3 t 6, 32Z 340, 34 i, 342, 343, 346, 347, 349, 350, 352, 361, 364, 365, 368, 451,473, 49_5, 580, 583, 588 Lin, S., 135, 147 Lin, S.-C., 508, 516 Lin, V., 258, 276 Lin, V~:,450, 470, 473, 481, 546, 561 Linask, K. K., 333,336, 340, 356, 364, 369 Linchan, W: M., 227, 231 LindahI, M., 403, 419 Lindaht, R, 173, 178, 313, 323, 326, 4 t 4. 41& 467, 482 Lindberg, M., 580, 586 Linder, G., 577, 583, 584 Linders, K., 376, 393 Lindout, D., 288, 293 Lindroth, J. R., 341,369 Lindsay, E. A., 353, 357, 364 Lindsay, R. M., 91, 9Z 98, 434, 489, 549, 561 Lindsay, S., 387, 390 Lindsetl, C., 136, 137, 148, 149, 262, 276, 553,555, 563, 565

Lindvall, O., 247, 249 Line, S., 284, 294, 442, 443, 497 Linehan, W. M., 173, 176, 227, 229 Ling, G., 574, 588 Ling, K. W., 380, 389 Linney, E., 469, 483 Linnoila, R., 313, 323 Lints, T. J., 78, 79, 80, 81, 85, 99, 120, 123, 317,325, 333, 334, 339, 356, 364, 369 Linzer. D. I., 162, 176 Lipkin, S. M., 510, 516

6

4

2

A

u

t

Lira, S. A., 402, 419 Lis. ~,~ I"., 158. ] 78 Lissauer, M. E., 322. 328 Litingtung, Y., 43.50, 78, 80, 85, 86, .98, t 2 I, I23, 141. 145. 260. 265,273, 312, 319, 323, 326, 439, 45t, 470. 482, 521,535 Litovsky. S. H.. 219, 232. 341. 343.352.360. 369 Little. C. D.. 223.226. 229. 230 Little. M.. 375.391 Little, M. H., 416, 4t9 Little. R. 174. 174 Littlepage. L. E.. 7, t 7 Littlewood Evans, A., 557.563 Litvin. J.. 337. 340, 341. 364. 365 Liu, A.. 84. 102. 110. 112. 115. 116. 124. 428. 453.489 Liu. C. H.. 227.230. 23I Liu. F.. 353. 354. 355.364. 433.478. 489. 496 Liu, F. C.. 507. 508. 518 Liu. J.. 312. 327 Liu. J. K.. 79.88.89.99. 102. 103. 121. 124. 290.292. 435. 450. 451 453.471 47 "~ 473 474. 477. 478. 484, 489. 549. 562 Liu. M.. 92. 102. 559. 563 Liu. R. 43.45.51. 202. 204. 208. 210. 220. 226. 227. 233 Liu. Q.. 531. 536 Liu. R.. 14. 17 Liu. S.. 79. 89. 102, 473.474. 489 Liu. W H.. 31.34 Liu. X.. 219. 233, 573. 583.584, 587 Liu. Y.. 168. 178. 227. 231. 289. 294, 428. 452.484. 489 Liu, Z., 116. 126 Ljungberg, R, 412.419 Llacer. R., 341. 360 Lloyd, R.. 504. 511. 512.5t5, 516. 518 Lo, C. W,, 157.17Z 357. 369 Lo, D. C., 94. 102. 106 Lo. J., 225.232 Lo. L.. 236. 239, 242, 243,250. 251 Lo, L.-C.. 79. 88.89. 102. 103. 168, 176. 177. 239. 250 Lo, R C. H.. 334, 364 Lo. S. H.. 572. 586 Lobel. R.. 240, 243,251 Locascio. A.. 355, 359. 423.482 Lock, L. F.. 188. I89 Lockwood. ~: L.. 34 t, 358 Lodish. M. B., 343, 352.360 Logan. C.. 116, I25 Logan, M.. 337.339. 343,347, 354. 359. 364 Logeat, E. 136, 146 Loh, Y. R, 502, 5t4 Lohikangas, L.. 574, 589 Lohnes. D.. 3I "~ 327, 451 453 468. 469. 489. 490 Lohning, C., 415.417 Lohr, J., 349, 355, 356, 358, 364 /-,

.

,

,

h

o

r

Index

Lois, C., 90. 95, 102 ~.~7. 251 Lonai, R. 167, 171,174, 176, 3 t2, 314, 315,323, 34i, 369, 467, 492 Long, C.R., 8, ]6 Long, E. 286, 293 Long, J. E., 79, 99, 290, 292, 435,450, 45 I, 453.471,472, 473.477. 478. 484, 549. 562 Long. Q., 89. 106. 450. 47 I. 498 Longaker, M. T.. 452, 49t Longenecker, G.. 94, t03 Longhi, M. R, 227, 231 Longley, M. A., 582, 583, 587 Longo, K. A.. 27 t, 276 Loomis. C. A.. 109, 115, 123, 228, 230 Loosli. E, 521,525. 531. 533, 537 Lows da Silva, S.. 506, 513 Lopez. J. M.. 289, 293 Lopez. M.. 353, 357. 365 Lopez. S.. 268. 276 Lopez-Fernandez, J., 510.516 Ldpez-Mascaraque, L.. 84, 90, 95, 99, 102, ]05 Lopez-Otin, C., 289. 293 Lopez-Rios. J., 522, 525,535, 537 L6ra. J.. 316. 320, 324 Loring. J. F.. 443,493, 559. 562 Loring. J. M., 341. 352, 363. 375.391, 400, 418 Losken. H. W.. 433.492 ,~:_~. 489. 49t Losos. K.. 84.102, 110. I15.116.124, 428,~-" LoTurco. J. J., 86, 102. 239. 250 Lou. L. J.. 312. 323 Lough, J.. 317.330, 335,336, 337. 342, 360, 364, 368, 370 Loughan, S.. 227, 231 Loughna, S.. ,_--,'~9231 Louis. J.-C., 402, 417 Loune, E.. 511,516 Lourenco, D. M., 509. 517 Louvet, S.. 7.18 Lovell-Badge, R., 372, 373,377.378. 379, 380. 38t, 383, 384, 386. 387, 388, 389, 390, 391, 392, 393, 527, 529, 530. 531. 534, 536, 537 Lovicu. F. J.. 532. 537 Low. M. J.. 502, 510. 513, 515 Lowe. C.. 47 I. 493 Lowe, L. A.. 338, 352. 354. 355, 356. 357, 359, 36:4, 370 Lowe, S. W:, 341,370 Lowell. S., 570. 586 Lowenheim. H., 558, 563 Lowenstein. D. H., 92. 102, 247, 248, 251 Lowik. C. W.. 285.293 Lowry. O. H.. 269, 274 Lowy. D. R.. 171. 173, 178 Lu. B.. 269. 275, 338. 359 Lu. J.. 342. 365, 370 Lu, J.-R., 342, 343, 353, 364, 365

Author Index

643

Lu, L., _0.~, 207, 223 229, 506.51Z 549. 564 Lu, M. E. 354, 355. 364, 433, 451,476, 477, 478, 489 Lu, M. M.. i65 17Z ,.6o, 274, o0o. 316. 37'6 327, 042, o4a, o3z, .~)a, o)7, 363, 365, 366 Lu, N., 468, 49t, 500. 516 Lu, Q., 478, 489, 580, 58 t, 583, 587 Lu, W., 415.417, 418 Lu, Y.. 171, t73, t78 Lucas, R C., 271,276 Lucero, M. T.. 240, 250 Ludgate, M., 31 t, 324 Ludlow, J. W., 173. 174. 176 Lufkin, T.. 290, 292, 312, 32Z 443.451,453,469, 474, 485, 489, 490, 497, 5.44, 545,549, 563, 565 Lui, H., 284. 293 Lumsden, A., 116, 119, t23, I25, 260, 263, 264. 265. 266, 273, 274, 422, 423,424, 425,427, 428, 433, -443,466, 468.469. 474, 485, 488, 490, 491, 493, 544, 545, 546. 562, 563, 564, 565 Lumsden. A G.. 422, 4o~., "~ 4.~8, " 439. 454, 490 Lumsden, A. G. S.. 438,490 Lun, Y., 136, 137, 138, t45 Lund, L. R.. 29 i, 292 Lundh-Rozell, B., 470. 497 Lundrigan, B. L.. 377. 387.393 Lunsden, A., 119. 122, 123 Lunsford, L., 186. 188, 190 Luo, G., 408, 418, 451,467, 490 Luo, J.. 167, 177 Luo, K., 268, 275 Luo, L., 12, 1Z 556, 558, 562 Luo. W.. 428, 452, 453,474, 487, 489 Luo. X .~ 5. 376. 390. 391, 510. 515, 516 Luoh, S. M., 579, 589 Lupo, G., 525,535 Lupu. F., 226, 229 Luria, A., 8.18 Luria, V.. 341,369 Lusis, A. J.. 508. 516 Luskin, M. B.. 95, I00, 237,251 Luteijn, Y., 199, 200, 201,202, 204, 207 Lutgens, E.. 226, 229 Luton. D., 200. 20t. 209, 220, 232 Lutz. Y.. 343, 361 LuValle, R. 443,487 Luyten, E R, 408, 420 Luz. A., 285,293, 294, 442, 443. 488, 489 Lyapunova, E.. 388, 391 Lye, S. J.. 173, 174. 175 Lyght, M., 226, 230 Lyman. S. D., 188, 189, 193, 204, 208 Lymboussaki, A., 162, t 79, 218, 223, 229, 580, 589 Lynch, J., 343,347.36t Lynn, A., 479, 482 9 '~

~

,

,

_

.

.~

~ 7

.

.

.

.

.

~

Lyon, M. E, 6, 1Z 39t, 45i, 475, 486, 527,528, 530, 536 Lyon, M. G., 84. t01 Lyonnet, S., 357, 364 Lyons, G., 132, 14Z t 70, t 76, 258, 262, 267, 269, 272, 273, 275, 276, 333, 341,342, 349, 360, 364, 365, 473, 483 Lyons, I., 203, 209, 3 t 7, 325, 333,334, 343. 345, 348, 349, 352, 356, 364 Lyons, J.. 512, 516 Lyons, K. M., 45, 47, 50, 82, 99, 105, 238, 25I, 308, 32 t, 326, 338, 355,359, 408, 417. 45I, 467, 468, 483, 484, 490, 495 Lyons, R. H., 556, 564 Lyons, "~i E., 559, 562 Lysakowski, A., 555, 558, 559, 561, 563

M Ma, L., 290, 294, 341,363, 437,450, 45 I, 452, 453, 466, 474, 487, 495 Ma, N., 453, 486 Ma, Q., 83, 89, 95, 99, 102, 435,490, 526, 538, 559, 563 Ma, X., 201. 204, 207 Maas, R.. 268, 273, 290, 294, 398, 399, 420, 437, aA0, 44 I, 450, 45 t4 453,466, 474, 475,481, 482, 483, 493, 495, 527, 528. 530, 532, 533, 535, 537, 538, 550, 565 Maatta, A., 58I, 585 MaNe, R C., 239, 250, 251 Mabry, M., 313,323 Macara, L. M., 174. t 77 MacArthur, C., 115, 1 t 6, t23 MacArthur, C. A., t65, 179, 466, 486 MacArthur, C. T. R. 116, 124 MacAuley, A., 163. 169, t 70, 177 Macchia, R E., 311,324, 326, 478,481, 508, 513 Macchia, V, 478, 481, 508, 513 Macdonald, R.. 78, 102, 521,522, 537 MacDonald, R. J., 321. 328 MacDougald, O. A., 27t, 276 MacGregor. G., 183, 190 Macias, D., 219, 228, 23t Mackarehtschian, K., 84, 102, 205, 208 Mackay, A. M., 206, 209 Mackem, S., 40, 45, 50, 5t, 508, 515 MacKenzie, A. L., 437, 441,490 Mackenzie, S. L., 425, 428, 432, 484, 490 Mackool, R. J., 452, 491 MacLetlan, R R, 342, 364 MacLetlan, W. R., 341,364 MacNaughton, K. A., 415, 417 MacNeit, t., 192, 194, 207. 245, 250 MacNeitl, C., 316, 326 Maconochie, M., 117, I t 8, 124, 449, 468, 479, 484, 492 MacPhee, D. J., 156, 177

644 Madan, A., 225, 231 Madden. S. L., 400. 418 Madel. R.. 314, 328 Madison, K. C.. 581,589 Madri, J. A., 223.229 Madsen, O. D.. t66~ 176, .310, 321,325, 327 Maeda, M.. 341.352,363, 375,391, 40.0, 418 Maeda, T.. 92, 102, 290, 294, 437. 450, 45t, 466. 474. 495 Maeda, Y.. 415. 420 Maekawa. A., 531. 538 Maekawa. T.. 121. 123 ~' 359, 4.~.. Maemura, 9K.. MT. "~'~ 450. 465. 489 Maeno, M.. 198. 208 Magdaleno, S. M.. 3 I5.326 Magenheim, J.. 164. 176 Maggio-Price. L.. 468.485 Maggs. A. M.. 269. 274 Maghnie, M., 508, 509. 514, 516 Magin, T. M.. 570. 572. 583.587 Magli. M. C.. 206. 206. 246. 249 Magner. M.. 220. 228 Magnuson. M. A., 309.319. 320. 327. 453.476. 493 Magnuson. T.. 3 I, 34, 169. 179 Magram, J.. 228, 232 Magre, S., 372, 387, 392 Maguire. C. T.. 343.350. 35 I. 352. 359 Maguire, M.. 226. 230 Mah. S., 408. 409. 410. 417 Mah, S. R. 410. 418 Mahadevaiah. S. K., 377. 386. 389 Mahan, M. A., 194. 206. 209 Mahboubi. K.. 225. 231 Maheshwari. H.. 511,513 Mahlapuu, M., 527. 530, 532. 535 Mahmood, R.. 116, 125, 545,546. 563 Mahon, J. A., 470, 484 Mahon. K. A.. 45.50, 94. 104, 173. 177, 402. 417. 450. 471. 494. 502. 504. 505.507. 508. 509. 510. 511. 512. 514. 515. 517. 518. 521. 522. 527. 537 Mahony. M., 556. 565 Maida. J. M., 117, 122 Mailhos. A.. 79. t03. 52I. 533.537 Mailutha. K. G.. 312, 32 7 Maina, F., 266, 2 75 Maioli. M., 337, 338,369 Maire, R. 262, 268, 276 Maisonpierre, R C., 224, 225.227, 230, 231. 232 Majesky, M., 342, 359 Majumdar, A., 395.417 Majumder, K., 352. 354, 366 Majumder, S.. 5, 9. 17 Majzoub, J. A., 510. 5t2. 516, 581. 586 Mak, T. W., 136, 137, 139, 145, 147. 341,370, 468. 495, 509, 514

A u t h o r Index Makarenkova, H., 2 t9, 229, 574, 577, 588 M~,K.,5tl,516 MN:i, R. A., 168~ 179 Maki, Y., 79, 99 Makino, T.. t 67, t80 MaIach, S., 405,407, 416, 417 Malapert, R, 142, 146, 262, 263, 264, 274 Matas, S., 91. 102 Malcolm, S., 433.452. 453,466, 482, 492, 493, 494 Maleszewski, M., 8. 17 Maliakal, J., 416. 420 Maticki, J., 546, 563 Mallamaci, A., 40, 46, 52, 79, 92, 102, 104, 290, 294, 450, 471,472, 473, 478.495 Mallo, M., 427, 432,433,443,449, 468,469, 473, 474, 477, 485, 488, 490, 494 Malpel, S., 312, 315,326 Maltais, L. J.. 311. 323 Maltepe, E.. 172. 173, 174. 174, 225,231 Malter. H. E.. 23.27.35 Maltosy, A. G.. 568.588 MaMahon. A. R. 84, 101, 246. 249 Mamuya, W S.. 219, 229 Manabe, M., 581,587 Manaia. A.. 200, 208 Manasek. D. M. D., 352. 354, 363 Manasek, E J.. 354. 355.364 Manasek. M. D.. 352, 354, 363 Mancilla, A., 423, 490 Mandel, G., 243. 249 Mandel, J., 283, 284, 289, 294 Mangalam, H. J., 509, 515 Mangelsdorf~ D. J., 468, 490 Mangia. F., 10, 16 Mango, S. E., 165, 176, 316, 325, 326 Maniatis, T., 136, 145, 226, 233 Manivel. J. C.. 416, 419 Mankoo. B. S.. 260. 267.275 Manley, N. R., 305, 311,327. 545,561 Mann, J. R., 443,482 Mann, R. S., 91, t03, 478, 490 Mann, S. J., 574, 587 Mannens, M., 174, 174, 375,391 Mannikko, M., 412. 418 Manning, S., 2, 4 Mannisto, A., 511,516 Manova. K., 2, 4, 164, 175, 303, 323 Manseau. L., 185. 189, 190 Mansour, S.. 281. 292, 380, 38 I, 390, 391, 466, 490, 545, 563 Mansouri. A.. 83. 103, 135, 1_~ "~7, 147, 260, 270, 272, 276, 311.321. 327, 329, 432, 451,473, 475, 477, 490, 498 Mansukhani. A., 286, 294 Mantero, M. R, 290, 292

A u t h o r Index

Mantero, S., 450, 453, 472, 473, 481, 549, 561 Manzanares, M., 1i8, 119, 124, 453,490, 544, 563 Mao, J. I., 453, 487 Mar, J. H., 262, 274, 277 Marasso, M. I., 580, 583,587 Marazzi, G., 226, 232 Marceau. N., 3 t 7, 324 Marcetle, C., 141, I47, 200, 207. 262. 263.264, 265,275, 435,475, 48t. 496 Marchac. D.. 453,489 Marchionni. M. A., 94, 97, 240, 244. 252 Marchuk. D. A.. 227, 229, 23t, 232 Marcil, A., 510, 518 Marco, S., 9t, 98 Marcos, E., 508, 516 Marcos, M. A., 196, 207 Marcotti, W., 553,563 Marcus, R., 81, 86, 101 Marcus, R. C., 85, 102, 522, 527, 536 Mardon, G., 268, 274, 399, 4 t Z 530, 533,535 Marekov, L. N.. 570, 58 I, 582, 584, 588 Margolis, B., 94, 105 Mari~ni, M., 525,535 Mariano, A., 31 t, 324, 478, 481, 508, 513 Marigo, V., 105, 265,275, 470, 490 Marfn, E. 9 i. ]00, 428.490 Marin, O., 81, 82, 85, 88, 89, 90. 9 t, 102, 105 Marin, X., 435, 484 Marinkovich, M. R, 572, 587 Marino, B., 356, 357,359, 365 Marino, S., 317, 325 Marin-Padilta, M., 20t, 204, 209, 210, 220, 23t, 232, 233 Marion, C. D.. 44. 50 Marital, J., 502, 5t5 Mark, M., 94, 100, 1 t7, 123, t72. 173, 179, 305, 3t2, 32Z 329, 341,346, 347, 352, 357, 362, 368, 369, 427, 433, 443,449, 451,453,468, 469, 474, 479, 484, 485, 489, 490, 491, 494, 525, 536, 544, 545, 562, 563 Mark, T., 15, 18 Markakis, E. A., 236. 251 Markel, D. S., 227, 231 Marker, R C., 82. IOI, 3 !5,326, 467, 488 Markert. C. L., 7, 17 Markham, B. E., 343,363, 365 Markkula, M., 42, 51, 158, i77, 5t 1,516 Marklin, R. J., 354, 365 Markov, L. N., 581,582, 587 Markova, N. G., 580, 583,587 Markovich, D., 323 Markowitz, G., 415, 420 Marks, S. C., Jr., 442, 490 Markwald, R. R.. 334, 336, 342, 344, 345,347, 348, 350, 35 I. 352, 357, 365, 368, 369, 370 Markwell, R. R.. 357, 369

645

Martton, R, 202. 208 Maro, B., 7, 18 Maron, B. J., 357, 367 Marone, M., 188, t90 Maroteaux, R. 286, 294, 453,494 Maroto, M., I41, t42, t48. 262. 264, 268, 275, 276 Marovitz, "#: E, 547, 552, 564 Marquardt, T., 5 t9, 530, 533,535 Marques, S., 3 t, 33, 44, 50, 338, 358 Mars, W. M., 246, 25t Marshak, D. R., 206, 209 Marshall, C. J., 201,208 Marshall, D. M., 582, 587 Marshall, H., 1I7, 1 t8, 119, I24, 125, 422, 425,427,449, 468, 479, 484, 485, 48Z 490, 492, 544, 563 Marshall, "vi S., 303, 329 Marshall Graves, J. A., 376, 392 Marshall-Graves, J. A., 387,392 Marsh-Armstrong, N., 525,537 Marsushima, J., 557, 565 Martens, E., 268, 2 75 MLrtensson, I. L., 47, 50, 45 t, 476, 483, 508,514 Marth, J. D., 14, t6 Martf, E., 470, 490 Martidate, M. Q., 471,493 Martin, C., 196, 200, 20 i, 206, 20Z 208, 219, 229 Martin, D. I. K., 356, 357,360, 369 Martin, D. M., 557, 561 Martin, G. R., 3, 4, 15, t Z 31, 35, 45, 50, 79, 84, 85, 86, 98, 99, 102, 105, 106, 1I3, 1 i4, 115, 116, 123, 124, 125, 132, ]45, I46, t66, 178, 317, 321,324, 328, 337, 340, 352, 360, 365, 36& 415,420, 422, 428, 432, 440, 449, 450, 451,453,466, 475,483, 489, 490, 491, 492, 494, 496, 497, 578, 583,586 Martin, G. V., 555, 565 Martin. J. E, ">6v ~73, 333. 342. 354, 355. 360, 364, 365, 433, 451,476, 477,478, 489, 490, 496, 549, 565 Martin, J. S., 203, 207. 226, 229, 467, 484 Martin, L. J., 94, 103, 264, 274 Martin, R, 547, 564 Martineau, J., 373, 383, 39t Mart/nez, C., 91,102 Martfnez, S., 77, 78, 79, 84, 85, 86, 88, 89, 95, 99, 102, t03, 104, 106, 1t3, 1I4, 1t6, 123, t24, t25, 422, 428, 453,490, 494,497 . . . . . Martinez-Arias, A., 136, 145 Martinez Barbera, J. R, 31, 34 Martinez-Barbera, J.-R, 31, 33, 46, 47, 48, 50, 51, 311, 316, 32Z 451,476, 483, 490, 508, 514 Martinez-De Velasco, J., 559, 562 Martinsen, B. J., 490 Maruniak, J. A., 237, 249 Maruthainar, K., 118, 124 Marvin, M. J., 334, 335, 336, 337, 339, 365

646 Marxer. A., 308, 325 Marziati, G.. 189. 190 Masai, I., 78, 102 Mascara, T.. -~3,495 Maseki. N.. 202, 208 Mashima. H., 32 I, 327 Masiakowski. R "~99 v99 Mason. A. J., 512. 516 Mason. A. K.. 512.5]6 Mason. C. A.. 85. I02 Mason. I.. 84, 102, 110, 112, 115. 116. 123. 125. 428. 449. 487. 545. 546. 563. 565 Mason. J. O.. 87.94. 105 Mason. S.. 11, 12. 17 Massabanda, J.. 264, 274 Massaddeq, N.. 109. 122 Massagu6. J., 47.5I, 468. 485 Massalas, J. S.. 87.91. 103, 508. 517. 521,527.537 Masson, N.. 512. 514 Masson. R., 162. 179 Mastick, G. S.. 85, t02 Masuda. H., 220. 228 Masui. T.. 303.327 Masui. Y.. 7.17 Masuno, M.. 398. 419 Masuya. H.. 83.102 Mata de Urquiza, A.. 91. 105. 120. 125 Mathers. R H., 521. 522.527, 537 Mathews, L. S.. 511. 513 Mathieu. M.. 506. 517 Mathis. J. M.. 506. 516 Matise. M. R, 85. 102, 103, 121. 124, 125, 505,517 Matsubara, Y., 525, 537 Matsubasa, T., 528. 536 Matsuda. A.. 531. 538 Matsuda. H.. 531. 538 Matsuda. Y.. 223. 230 Matsui. D.. 312. 328, 505.517 Matsui. T.. 318,325 Matsui. Y.. 188. 189. 190. 246, 251 Matsuk. M. M.. 44. 51, 440, 484 Matsuki, M., 58 t, 582, 583,587 Matsumoto. K., 10. 17, 466. 496, 525,536, 571,586 Matsumoto, M., 94, t06 Matsumoto. N., 581,588 Matsunami, H.. 340. 366. 408.417. 418 Matsunashi, T.. 204. 209, 220. 232 Matsuno. K.. 553, 561 Matsuo. H.. 466. 496 Matsuo, I., 31, 34, 46, 49, 517 83.84. 105. 106, 1 t 4, 124, t25, 374, 392, 403,418, 421. 451. 457. 478. 488, 490, 496, 521. 527. 537 Matsuo. N., 423.424, 451. 475,476. 478, 490 Matsuo. T.. 423, 424. 45 I, 475, 476, 478, 490

A u t h o r Index Matsushima-Hibiya, Y., 531,538 Matsushita, S., 303, 329 Matsuzaki, E, 95, 105 Matsuzaki, T., 557,565 Mattei, M. G., 85, 9 t, 100, 103, 3i I, 324, 372, 387, 392 Matthews. A. N., 550, 563 Matthews. K. L., 437, 450, 497 Matthews. L., 352, 354. 366 Matthyssen, A., 167, 177 Mattiacci, M., 263,274 Mattot. V.; 226, 229 Mattsson. R., 167, 178 Matzuk. M M. 1"~9 122, "~'~6 997 ;~9 336.357 359, 441,467, 468, 490, 491, 500, 510, 5t6 Mauch. T. J.. 397, 418 Mauer, S. M., 416, 419 Mauger. A., 572, 573.587 Maulik, C., 292, 294 Maurer, M., 574, 588 Maurer. R. A., 508, 517 Mauro, T., 580, 582, 583, 585, 58Z 588 Maury, M.. 31, 33, 46, 48.49, 1 i4. ]22, 322, 428, 45 t, 478, 481.521, 527,535 Mavilio. F., 206. 207, 246, 249, 27 I, 273, 274, 443,449, 478, 482 Max-Audit, L., 200, 208 Maxeiner, S., 173, t77 Maxson, R.. 290. 294, 428, 437, 450, 45 t, 452, 466, 474, 484, 489, 494, 495 Maxwell. R, 2257 229 May, G.. 199, 2007 201,210 May, L. L., 260, 267, 275 May, N. R., 957 I02, 141. 147, 506, 516 May, R. T., 248. 251 May. S. R.. 48, 49, 78, 98. 304. 323 Mayen. A. E.. 193, 207 Mayer, B.. 31, 35 Mayer, E. L., 203. 209 Mayer, M.. 583. 585 Mayer. Vv:. 9, 1Z 18 Mayer-Proschet, M., 240, 241,251 Mayo, K. E., 443,485, 508, 511,515, 516 Mayo, M. L.. 428. 491 Mayor, R., 423,490 Mayordomo, R., 541. 564 Mazan. S.. 31.33, 44. 46, 48, 49, 52, t 14, 122, 322, 428, 451,478, 481, 508, 513, 52 I, 527, 535 Mazzalupo, S.. 570. 587 Mbamalu, G.. 171.175 Mbiene. J. R. 552. 564 McAllister. A. K.. 94, 102 McAllister, D., 317. 330 McAllister, K. A., 227, 231 McAndrew, J., 51 I, 516

Author I

n

d

e

McAvey, B. A., 7. t(5 McAvoy, J. \~,~.532, 537 McBurney, M. W.. 236, 245, 251 McCabe, E. R., 378, 379, 387, 389, 393 McCaffery, R, 345. 346, 347,366, 469, 482, 492, 498, 525, 537 McCaffe~', R. J., 346. 367 McCall, A. E.. 1~ t_,~ McCm,'thy, J. G.. 452. 491 McCarthy, K. J.. 511,516 McCarthy, M., 102, 241, 2 ~ , 251 McCarthy, M. M., 500, 516 McCarthy, M. T., 427,484 McCarty, M.. 172, 173, 180 McCaw. R S.. 257, 2 75, 2 76 McClain, J., 223, 224. 227. 231, 232, 559, 561 McClearn, D., 49I McClive. R J., 382. 388, 393 McClure, M. H., 316. 324 McComb. D. J.. 512, 515 McComb. R. D.. 166. 179 McConnell, S. K.. 84, 87.92. 94, 98, 102, ]06 McCormick, E, 86, 105, 5t2, 516. 579, 589 McCormick, M. K., 227, 231 McCormick, R. J., 525. 538 McCoy, M. J., 162, 170, I76 McCready, R, 412, 418 McCurrach, M. E.. 341,370 McDade, J. R., 347, 349, 36I McDermott, M., 509, 510, 515 McDevitt, D. S., 534, 538 McDonald, D. M., 223.224. 227,232 McDonald, L.. 348, 349, 352, 353, 357,358 McDonough, B., 357, 358 McDowall, S. G., 381,39] McDowell, T. L., 376. 390 McElreavey, K.. 375, 376, 382, 387, 389, 390, 39t, 392 McEvilIy, R. J., 89, 95, I04, 506, 5tZ 556, 558, 562 McEwen, B. S., 247, 249, 250 McEwen, D. G., 286, 292, 558, 561 McFadden, D. G., 168, i 75, 342, 344, 347, 349, 352, 359, 361, 365, 366 McFadden. K. A., 82. 86, 100 McGaughey, R. Vv:, 13, 16, 27, 33, 34 McGhee, J. D.. 165, 175, t 77. 316, 325 McGrew, M. J., 134. 138, 139, 147 McHugh. K. R, 223. 230 Mclnnes. L., 341,365 Mclnnes, R. R.. 527. 535 McIntosh, I., 286, 292, 453, 48], 495 McIntosh, T. J., 582, 587 McIntyre, J. Ii, I36, 137, t46, 553, 563 McKay, I. J., 466, 49], 545. 546, 564 McKay, R. D., 88, 89, 98. t0t, 235. 247, 249, 251

x

6

4

7

McKearn, J. R, 192, 208 McKeehan, W\ L., 287.293 McKendry, R.... 579, 587 McKenna, H. J.. 173, 179 McKercher, S. R., 168, ] 79 McKinnon, W. C., 227, 23I McKinsey, T. A.. 269. 277, 342, ~+~, 353.364, 365, 369. 370 McLain, K., 204, 208, 3t8, 327 McLm,en, A., 26, 34, t83, 189, 189, 382, 384, 389, 391 McLaughlin, J., 10, t 2. 13, 18 McLaughlin, K. A., 339, 367 McLoon, S. C., 88, t05 McMahon, A. R, 40, 44, 48, 49, 51, 78, 79, 82, 85. 86, 98, 99, 10I, 102, I04, 105, 106, 112, i16, I17, t20, t21, 123, 124, t25, t32, t33, 135. t37, 140, 14I, t45, t4Z 148, I49, I66, t67, 175, 228. 229. 230, 232, 238, 246, 249, 250, 260, 262, 264, 265 275, 287,292, 293, 294, 3C.~, 308, 310, 312, 315, 3t7. 323, 324, 328, 329, 334, 336, 348, 362, 363, 384, 385.386, 389, 392, 393, 405, 406, 407,418, 4t9, 423, 426. 428, 439, 440, -441,442, 443, 45 t, 453,468, 470, 47t, 482, 483, 484, 486, 48Z 488, 489, 49~ 491, 496, 49& 504. 505, 506, 518, 529, 538, 545,565, 577, 578, 583,584, 585, 589 McMahon, G., 217, 220, 229 McMahon. J. A.. 23, 33, 78.85. 99, t0I. I~z, I~7, I4t I45, ]4Z 148, 166, I67, 175, 260, 264,265,275, 304, 317, 323, 329, 428,468, 470, 482, 485, 491, 50,4, 505, 506, 518, 577, 578, 583, 584, 589 McNamara, J. O., 247, 25i McNiff, J. M., 579, 584 McNoe, L. A., 398, 419 McPherron, A. C., 129, I47, 260, 264, 275 McQuinn, T., 342, 357, 359, 369 Mead, R E., 198, 203, 208, 210 Mecklenberg, L., 574, 588 Medberry, S. L., t 5, 17 Medlock, E. S., 317, 327 Medvinsky, A., 194, 195, 196, 199, 200, 207, 208, 220, 231, 317, 327 Meegdes, B. H., t 74, t 77 Meehan, M. A., 452, 492 Meehan, R. R., 302, 327 Meeson. A. R, 219, 231 Megeney, L. A., 258, 260, 27 t, 275 Mehler~ M. E, 239, 250, 251 Mehlmann, L., 8, ] 7 Mehrara, B. J., 452, 491 Mei, W. Y., 579, 589 Mei, Y. L., 402, 417 Meier, R, 79, 104, 529, 537 Meier, S., 424, 428. 491 Meijers, J. H., 385, 389 Meijers-Heijboer, H., 528, 538

648 Meijlink, E. 45 t. 476, 477. 478, 491, 496, 549, 565 Meindl. A.. 378.392 Meinecke, R. 468. 485 Meiners. L. C., 94. 99 Meinhardt, G.. I74. 177 Meinhardt, H.. 138. 139. 147 Meininger. C. J., 189.190 Meins. M., 334. 365 Meisler. M. H., 428, 475,497 Melancon, E.. 265.269. 273 Melega, W.. 239.241. 251 Mellitzer. G.. 1 t9, t24, 126. 225, 231, 233 Mello. C.. 14, 16, 185,9190. 240, ~4.~. " " 249,. 251. . . .~5 :~ Mellon. E L.. 504,509.510. 512.513. 516, 517. 518 Mellon. S. H.. 510. 513 Melmed. S.,510.512.513. 518 Melnick, M.. 436. 487 Meloy, D.. 31.34. 46.47, 48, 51, 311.316. 327 Melton. D. A.. 3.4. 166. 176, 303. 304, 308. 310. 319. 320, 321. 325. 326, 328. 329. 405.418 Melton. D. W.. 570. 587 Meltzer. E S.. 347,366 Mendell. J. R., ,./1 "~" ,_~73 Mendelsohn, C., 3 I2, 326, 32Z 403, 418, 45 I, 453. 468. 469. 489, 490. 491 Mendelsohn, M.. 122. I24 Mendez, R.. 7. 10. 11.1Z 18 Mendis. D.. 11.19 Mendosa, E. S., 543. 564 Meneses. J. J.. 38.39.45.47.51.79. 83.87.89.90. 91.92. 95.97, 98. 99, 103, 290. 292. 303,304. 321. 326. 329, 333,363, 410. 418, 422, 435.437. 450. 451,453,470, 471. 472. 473.477. 478, 481,484, 493, 549. 562. 570. 589 Meng, A., 135, 147 Meng, X., 218, 223,230, 403, 419 Menissier. E. 264. 274 Mennerich. D.. 266. 275 Meno. C.. 47, 51, 354, 357. 365, 370 Menon. G. K.. 580. 581. 582. 584, 585. 587 Mentink. M. M., 219. 232, 333, 360 Menzel, E. 286. 292 Meo, T.. 386. 390 Meraz, M. A., 287, 293 Mercader, N., 9 t. 102 Mercer, B., 228, 230, 269, 27Z 342, 364. 365, 369 Mercer, E. H., 109, 114, 126, 228,233 Merchant-Larios, H.. 382, 384. 387. 391. 392. 393 Mercier. Y.. 9.15. 18 Mercola. M.. 219. 229, 334. 335,336. 337. 338. 339. 340. 356. 363, 366, 36Z 473.489 Mercurio, S.. 92. 102 Meredith. A., 89, 10I Meredith, M. M., 25, 34

A u t h o r Index Meredith, M. R., 158, 176 Merentes-Diaz, E., t 60. ] 78 Mericksay, M., 266, 27Z 335,337, 340, 358 Merker. H. J.. 570, 584 Merlet-Benichou, C., 403,418, 420 Merlie, J. R, 4I 1,4t9 Merlino. G., 268. 275 Merlo, G. R.. 290, 292, 450. 453, 472, 473,481, 549, 561 Mermer, S.. 573,584 Mershcer, S.. 353, 357,365 Mertelsmann, R., 288, 293 Mertim S., 381, 39t Mertineit, C.. 382, 393 Meskenaite. V., 453,486 Mesnard, D.. 27, 33 Mesquita, S. E. 346. 358 Messaddeq, N., 94, I00. 341. 346. 347, 352, 362, 366, 469, 485, 583.586 Messdeq, N.. 341,352. 368 Metcalf. D., 192, 193, 194, 195, 196, 198, 203, 208, 209 Metsaranta, M., 573.588 Metsiiranta. M., 424, 442, 443,494 Mett. I. L., 341,369 Mettei. M. G., 40, 47, 5t Metzger, D.. 109. 122 Metzger. R. J., 311,327 Meyer. A. N.. 453,485 Meyer, B. I.. 547,564 Meyer, D., 349. 365 Meyer. J.. 281. 293, 294, 380, 381. 391, 393 Meyer, M., 204, 207. 317. 324 Meyer, T., 225, 230 Meyerowitz, E. M., I I, 17 Meyers, E. N.. 31.35, 1 t4, 1 I5, 124, 337, 340, 352, 365, 368, 466, 489, 496 Meyers, G., 453, 487 Meza. U.. 511. 514 Mezey, E., 206, 207 Mi. T., 193, 206, 208, 270, 271,275 Mi, Y., 227, 23I Miano, J. M., 228, 230, 342, 365 Miao, H. Q., 223,232 Mic. E A., 525,537 Michael, D., 580, 586 Michalak, M., 343, 347, 361 Michaliszyn, E., 510, 514 Michalopoulos, G. K., 318, 327 Michaud. J.. 95.9Z 102, 121,124, 125, 451. 453,470, 47t, 491, 493, 506. 513, 516 Michel, E., 281,293, 381,391 Michel, R. N., 269, 27Z 342, 369 Michels, A., 91.98 Michelson, A. M., 136, I45, 165, 178, 316, 32& 340, 361 Michte, C., 263, 274

Author i

n

d

e

Michon, A., 185.189 Miettinen, R, 314, 32Z 465, 491 Mignatti, R, 224. 232, 504, 517 Mikawa, T., 219, 228.23t, 334, 347. 362, 365, 368 Mikkota. I.. 52 I, 522, 537 Mikoshiba. K., 94. t03, I04, 106, 122, t22, 357,363, 423, 492 Milbrandt, J.. 510, 516 Milenkovic. L., 78.80. I00, 121,123, 470, 485 Miles, C., 416, 419 Milhalek, R. M., 251 Milla. R J., 305,328 Millan, F. A., 467.49t Miltauer, B., 227, 23I Millen, K. J., 115. 122, 124, 428, 453, 491 Miller. A. R. 506, 508. 509, 5t8 Miller, C. L., 245, 25t, 385, 39I Miller. C. R. 322, 328 Miller, C. T., 465.49i Miller. D., 475, 485 Miller. G.. 470. 486, 579, 586 Miller, I., 188, I89 Miller, J. B., 132, 148, 263, 269, 275, 276 Miller, J. D., 204, 210. 220, 233 Miller, J. R., 337. 363, 405,418 Miller, S. A., 355.365 Miller, T. A., 204, 208, 318, 327 Miller, W., 12, 16, 438, 49I Millet. S., 1i4, 115, 124, 428, 491 Millonig, J. H., 122, i24, 453, 491 Mills, A. A., 571, 581,583,587 Min. H., 312, 327 Min. J.-Y., 354, 365 Mina, M., 439, 491 Minamino, N., 466, 496 Minasi, M. G., 246, 250 Mincheva. A.. 479. 487 Min Deng, J., 3 I, 34, 328 Mine, T., 32 I, 327 Miner. C., 466, 494, 545,564 Miner, J. H., 260, 276, 411. 412, 418, 419 Ming, J. E.. 470. 49I Minichiello. L., 559, 564 Minkoff, R.. 469, 470, 486 Minnerath, S. R.. 94. l O0 Minoo, R, 312, 314, 327 Minowa, O., 122, 122, 173.179, 217, 230, 404, 420, 506, 516, 549, 564 Minowada. G.. t 15. 116. 123, 466, 491 Minshull, J., 338, 363 Mintz, B., 245, 249 Mione, M., 79, 88, 89, 90, 94, 95, 97, 99, 450, 471,484 Miralles, E, 321,327 Miranda. B., 313, 3t5,327

x

6

4

9

Miranda, M.. 9, t 0, / 7, / 9 Mirtsos, C., 468, 495 Mishina, Y., 336, 352, 365, 385, 39I Miska. E. A., 342, 368 Missate, C., 51 I, 5]3, 516 Missero, C., 311,326 Mitchell, K., 341,370 Mitchell, R J., 79, 104, 529, 537 Mitra, J., 7, L/7 Mitsiadis, T., 509, 518 Mitsialis, S. A., 31 t, 3 t2, 323 Mitsock, L. M., 467,498 Mitulia, B., 28 i, 293, 38I, 39I Miura, H., 353, 354, 355, 356, 363 Miura, N., 47 t, 479, 48Z 491 Miwa, H., 528, 538 Miyagawa, J., 321,327 Miyagawa, K., 391, 4t6, 419 Miyagawa-Tomita, S., 340, 352, 353, 354, 355, 356, 363, 367 Miyake, H., 506, 518 Miyake, S., 288, 293 Miyake, T., 442, 486 Miyama, S., 92, I05 Miyamoto, K., 466, 496 Miyamoto, M., 84, 98, I00 Miyamoto, N., 83, 106, 374, 392, 403, 418 Miyasaka, M., 350, 362 Miyashita-Lin, E. M., 83, 84, 85, 86, 92. 95, 100, ] 02, 104 Miyata, K., 10, I7 Miyata, T., 92, 94, I02, t03, 106 Miyazaki, J., t 60, 178, 340, 352, 36Z 443,492 Miyazaki, S., 8, t6 Miyazaki, T., 408, 418 Miyazaki, Y., 403, 4t8 Miyazono, K., 226, 230, 231, 356, 36Z 468, 486 Miyoshi, H., 202, 208 Miyoshi, I., 511,516 Mizuno, K., 580, 587 Mizuno, M., 521,537 Mizuno, N., 529, 534, 537 Mizuno, S., 388, 392 Mizuno, T., 41, 5t, 303, 308, 324, 329, 343,368 Mizutani, Y., 140, 145, 147 Mizutani-Koseki, Y., 47i, 479, 487 Mjaatvedt, C. H., 334, 344, 347, 348, 350, 351,352, 357, 365, 370 Mo, R., t21, ]23, 124, 310, 312, 326, 327. 439, 451,453, 470. 471,486, 491 Moak, J. R, 357, 367 Mobley, W C., 91.10! Mochida, K., 47.51, 338, 354, 356, 357, 365, 367, 368 Mochii, M., 237, 252, 525, 529, 534, 537 Mochizuki, T., 352, 354, 365, 4t4, 418

6

5

0

A

u

t

Modolell, j., 550, 555,561 Moegetin, A., 452. 498 Moens, C. B., 118, 125, 34 l, 352, 365 Moens, G., 223, 230 Moers, A.. 508. 5t6 Mohandas. N., 44", ~ .~ 493 Mohanty, N., 14. t 7 Mohiaddin, R. H.. 35 t. 363 Mohler. J.. 78.99. 141. 145. 470. 484 M6hle-Steinlein. U.. 442. 443.497 Mohr. S.. 341. 369 Mohri. T.. 8.16 Mohun, T.. 10. 15, 40, 44, 50, 337. 338, 339. 345. 348, 358. 366 . . 275. "" .~.~. "" 342. 355. 359, Mohun. T.J. . ."~6"~ i ~)..~.~6. ...... _~9. 36I. 365, 368 Moisan. M. R. 375.390 Molinar-Rode. R., 313. 327 Molkentin. J. D.. 165.177. 257. 262. 275. 316. 327, 334. 340. 341. 342. 343. 347.352.360. 361. 365. 367 Moll. I.. 570. 587 Moll. R.. 570. 587 Mollard. R., 314, 327 Molnair, Z.. 92, I02 Molotko'~~.A.. 525,537 Mombaerts. R. 435,491 Momma, S., 236, 237, 244. 245.248, 250 Momose. T.. 525.536 Monaco, L., 376. 393 Monaghan, A. R, 40, 42, 44. 50, 51, 165, 177, 303, 314, 324, 325, 327. 479. 487 Monaghan, R. 528.538 Moncada. S.. 315, 324 Moniot, B.. 381,390 Monkley, S. J.. 172, 173.177. 336. 337,365 Monod. C.. 9, 14. I5 Monreal. A. W.. 44.1. 491 Monsoro-Burq, A.-H.. 140, 141. 147 Montesano, R. 223. 230, ..,-1 ~ 8 Montezin, M., 58 I, 588 Montgomery'. C.. 442. 443.495 Montgome~', M.. 341,364 Montgomery. M. K.. 14. 16. 17 Montgomery. M. O., 337. 340. 341. 365 Montgome~,. R. A., 347, 360 Monti-Graziadei, A. G.. 77, 79.84, I00 Montminy, M. R., 286. 292, 293 Montonen. O., 441. 488 Montrocher. C., 585 Monzen. K.. 336. 343. 365, 368 Moody, S. A., 435.491, 532, 536 Mookerjee, B., 225,231 Moon, R. T.. 44, 45.51, 337, 363, 405,418, 579, 584 Mooney, J. R., 577, 587

h

o

r

Index

Moons, D., 165, 17Z 316, 325 Moons, L., 203, 20Z 223, 225, 226, 22& 229 Moorby, C., 404, 420 Moore, A. T., 533, 535 Moore, A. ~:. 334, 341,349, 365 Moore. B I.~... 147 Moore, C. C.. 380, 381,384, 392 Moore, G. D.. 8, 17 Moore. K. A.. 194. 205. 206. 208, 209 Moore, K. J., 525.536 Moore. K. L., 568, 587 Moore. L.. 185, 186. 188. 190, 568, 582, 583, 586 Moore. M. A., 195, I96. 198, 20I, 208, 317, 325 Moore, M. W:, 203, 20Z 223, 229, 402, 418, 420 Moore. R.. t 5, 16 Moore, R. L.. 20t. 208 Moore. \u A.. 194, 207 Moore, W. J., 42 I, -442, 449, 456. 457, 47i, 491 Moorman. A. E M.. 334. 341. 343,344, 345.347. 348, 350. 351. 352. 355. 356. 359. 360, 361, 362, 363, 365, 366. 369 Moorman. M. A., 206. 209 Moos, J., 8. 17 Moos. M. C.. Jr., 14, 16 Mora, M., 246. 250 Morais da Silva. S.. 380, 38 I, 384, 387,392 Moran-Rivard, L.. 260, 266, 274 Morasso. M. I.. 173, 177 Morassutti, D. J.. 235,236, 242, 244, 245,250, 251 Morata, G.. 91,102 Moreau. A., 478, 489 Moreau. E.. 403.418, 420 Morell, R., 475.481 Morell, R. J.. 556. 564 Moreno, T. A., 423,481 Moreno-Mendoza, N.. 382, 387, 391, 392 Moreno-Rodriguez, R. A.. 3-44. 345, 348, 360, 365 Morgan, B. A.. 305,328, 575,576, 577, 578, 579, 58Z 588 Morgan, D., 352, 354, 366, 441,488 Morgan. E. R, 452, 492 Morgan, J. I., 313, 327 Morgan. M. J.. 351. 356, 360 Morgan. R. A.. 14. 16 Morgan. W. C.. 137. 147 Morgenbesser, S. D., 528,537 Moil, C., 136, I37, 14Z I73, t79, 341,352, 370, 404, 420 Moil, K., 237, 252, 435, 491 Mori. M.. 512. 518, 528, 536 Morikawa. Y., 318,325 Morimitsu, T.. 537 Morin, E J.. 86, 100. 579, 586 Morin. S.. 342, 343.366 Morishima. M.. 353. 357. 364 Morishima, Y., 58 I, 582, 583. 587

Author I

n

d

e

Morita, M., 527, 530. 53 I. 536 Morita, T.. 41 "~,.,418 Moriuchi, T., I 1, 12, I8 Moriwaki, K., 83, 102 Moriyama, H., 195.208 Moriyama, K., 94, I04. 502, 508, 509, 5t 7.. 557, 564 Morkin. E.. 343.360 Morley, G. E., 334. 348, 367 Moro, J. A., 547, 564 Morohashi. K.. 375. 376. 378. 379, 380, 384, 390, 39t, 392 Morozova. L. M., 26. 27.33 Morrell, C.. 1t, 12. 17 Morrice, D. R.. 534, 536 Morris, J. E, 400, 418, 419 Morris. J. H., 260, 274, 276, 277 Morris, N. R, 442, "443. 489 Morris, R. J.. 570, 587, 589 Morrisey, E. E., 165, 177, 303, 316.326, 32Z 342, 343. 352. 363, 366 Morrish, T. A.. 349. 358 Morrison, A., 117, 118. 119, 124, 544, 552, 553, 555, 558, 559. 563, 564 Morrison, A. D.. 469.49] Morrison, M., 118, t23 Morrison, S. J.. 235. 236, 238, 239, 240, 24 t, 243, 244, 245,248.249, 251 Morriss-Kay, G. M., 132, I46, 199, 200, 201,210, 424, 437. 452, 468, 469, 484, 485, 48Z 49i, 494, 496, 545, 546, 557, 562, 563, 564 Morris-Wiman, J., 443. 482 Morrov< B. E., 353, 357, 365 Morrow. E. M., 527.535 Morrow, K. E., 378, 390 Morshead. C. M., 235, 236, 244. 245, 25I Morsli. H., 478,491, 541. 542, 550, 551,552, 555,558, 559, 564, 579 Mort-Hopkins, T., 453,483 Morton, C. C., 346, 358 Morton, S., 43, 50 Mortmd, M. T., 510, 513 Mosca. J. D., 206. 209 Mosca. R. S.. 356. 362 Moser. C.. 378, 393 Moses. H. L., 467. 493 Moshnyakov, M., 559. 564, 565 Mosinger. B. J., 508, 517 Moss. J. B., 345.346, 347,366 Moss, M. L., 452, 492 Mossman, H., I4, 16 Mostachfi, H., 173, 174, 175 Motegi, M., 51 I, 514 Motegi, Y., 353, 354, 355, 356. 363 Motoike, T., 222, 231 Motoyama, J.. 121,123, I24, 317, 321,327

x

6

5

1

Motro, B., t 93, 204, 208 Motta, R, 7, 19 Motte, J., 94, 99 Mouly, V., 344, 362 Mourton, T., 169, ] 79 Moury, J. D., 423, 492 Mouse Genome Database, 516 Moussa, S. M.. 5 I2, 515 Moutard, M. L., 94, 99 Mucenski, M. L., 204, 208, 318, 327 Muchamore, I., 422, 425,427, 468, 485, 48Z 490 Mueller, C., 316, 326 Muenke, M., 78, I02, 27t, 276, 357,359, 433, 452, 453, 468, 470, 48I, 485, 491, 492 Muglia, L. J., 58 l, 586 Muhr, J., 78. 79, 80, 81, 85, 99, t08, 120, t23, I24 Mujtaba, T., 240, 241,250, 251 Mukai, M., 186, 190 Mukai, T., 262, 268, 274 Mukherjee, R, 196, 202, 2I 0 Muller. A. M., 194, 195, I96, t99, 200, 20Z 208, 220, 231 Muller, B. K., 525, 535 Muller, C. V~(,346, 358 Muller, E, 338, 362 Miitler, K.-H., 3 t 7,325 Miiller, M., 134, 147 Mailer, R.. 89, lOI Muller. T. S., 131, t46, 266, 2 73, 353, 356. 36t Muller, U., 410, 41& 452, 453, 474, 48Z 557. 563 Muller, W., 34t, 362 Muller, W. J.. t 71, 175 Muller-Rover, S., 574, 578, 588 Mulligan, G., 483 Mulligan, R. C., t 93. 205, 206, 20Z 208, 246, 250, 270, 27I, 274, 285, 293, 442, 443, 488, 489 Mutliken, J. B., 227, 232, 288, 292, 293, 294, 452, 453, 474, 487 Multins. M. C., 546, 565 Mulnard. J. G., 27, 34 Mumby, M. C., 94, I O0 Mummew, C. L., 222, 229 Mundlos, C., 288, 293, 294 Mundlos, S., 281,288, 293, 442, 443,453,492, 493 Mundschau, G., 574, 583, 588 Muneoka, K., 3, 4 Munnich, A.. 286, 294, 452, 453, 484, 494 Munoz, E. E, 3t 4, 325 Munoz, E, 44 t, 488 Munoz-Chapuli, R., 219, 228, 231 Miinsterberg, A. E., 140, t4I, I4Z 262, 263, 264, 268, 275, 377, 381,390. 392 Muragaki, Y., 427, 443,484, 492 Murakami, M., 5 t2, 518 Murakami, S., 287,293

652

A u t h o r Index

Muramatsu. H., 350, 370 Muramatsu, M.. 11, 12. 18. t83. 190. 504. 516 Muramatsu. T.. 350. 370 Murase. N., 246, 25t Murata, T., 49.51 Murillo-Ferroi. N. L., 337,359 Murohara. T.. 220, 228 Murone. M., 579, 589 Murphy. D., 502, 5t5 Murphy. M. ~:. 382. 392 Murphy. R, 468, 491 Murray. D.. 163. 175 Murray. J. C.. 354. 370. 478. 495, 507.517. 527. 532. 537 Murray. M. J.. 467.495 Murray, R, 200, 208 Murre, C.. 257.275, 276 Murrell. J.. 227.23I Murtaugh. L. C., 282. 293 Muscat. G. E., 281. 293. 381. 392 Muscat. G. E. O.. 342, 343.359 Muscatelli, F.. 378,392. 393 Musci. T. S.. 117. 125. 443.474. 483. 544. 561 Muskavitch. M. A.. 136. 145 Mustonen. T.. 218.223.229. 230. 441. 493 Muthukumar. S., 553.562 Mutoh. H.. 310. 321. 322. 327 Muzio. L.. 92, 102 MvKinley. M.. 31, 34 Myat, A.. 553. 559, 561 Myer, D., 357. 366 Myers, R. M.. 287.293. 579. 586 Myglia. L. J.. 510. 512.516 Myokai, E, 423,424, 451,475,476. 478. 490

N Na. E.. 102, 24 l, 244. 251 Na. S.. 87. 101 N ~ . A. M., 510, 5t6 Nabeshima, Y.. 258. 260. 272. 274, 276 Nachtigal, M. W., 376. 381. 391. 392 Nadal-Ginard, B., 262, 277 Nadeau. J., 263,272, 313, 314, 315.324 Nagai, K., 378. 390 Nagai, R., 432. 450, 465,489 Nagai, T., 122, 122, 357. 363. 423,492 Nagakubo, D.. 350, 362 Nagamine, C. M.. 378,390. 392 Nagano. M.. 376. 393 Nagao, H., 435.491 Nagaoka, S., 11, 12, t8 Nagase, H.. 223.231 Nagashima, K., 506. 518 Nagata. A., 95, 102, 506, 516 Nagata, E., 94, 106

Nagayama, Y., 504, 5t 6 Nagayoshi, M.. 7, t6 Nagorcka, B. N., 577. 587 Nagy, A., 3, 4, 23, 35, 38, 42, 51, t32, 14Z 158, t60, 164, 166, i68, 172, 173, 174, 174, 175, 176, ]7Z 178, I79, 203, 20Z 223, 228, 302, 324, 357, 368 Nags~ L., 469, 484 Nagy. R.. 42.51 Naito, M., 195, 196, 208, 209, 228, 233 Nait-Oumesmar, B., 170, 174, 174, 178 Nakafuku. M.. 95. 105, 120, 12I, ]23, 125, 451,475.476, 492. 522. 537 Nakagata. N.. 10. 17 Nakagawa. M.. 343. 347, 366 Nakagawa, O., 343,347, 366 Nakagawa, S., 408. 418 Nakagawa, Y., 84. 102 Nakahara, K., 526, 527, 538 Nakahara, Y., 353,354. 355, 356, 363, 376, 391 Nakahata. T.. 341. 352, 370 Nakai, S.. 95, 102, 506, 516, 549, 564 Nakajima. K., 94. 103, 104. 106 Nakajima. O.. 527. 530. 531. 536 Nakamura, A.. 186. 190, 355,364 Nakamura. H.. 112. 114. 115, 116. I22, t23. I24, 355,362, 525.536 Nakamura, K. ~4_, .~47. 361 Nakamura, M., 577, 583, 584 Nakamura, S., 77.95, 101, 105, 473,498 Nakamura, T., 476, 482, 577, 586 Nakamura, Y., 479, 482 Nakanischi, S., 136, 147 Nakanishi, S., I36, 146, 237, 252, 526, 527, 538 Nakanishi, Y.. 436, 486 Nakano, H.. 14, 17 Nakano, T., 49, 51, 136. 137. 147. 509,514 Nakao, K.. 228. 233 Nakaoka. T.. 336. 365 Nakase, T., 555,565 Nakashima. K., 318. 325 Nakashima, M., 85, 103, 12t. 125, 505,517 Nakata, K., 289, 294, 357, 363, 443,492 Nakatake. Y., 315, 324 Nakatsu. M. N., 260, 266, 274 Nakatsu, Y., 6, 17 Nakatsuji, N., 188. 189, 226, 232, 352, 354, 365 Nakauchi. H.. 2.44. 251 Nakayama, A., 525,536 Nakayama, H.. 168. 169, 170, t74, I78, 189, 189 Namioka. S.. 511,516 Nanda, I., 388, 392 Nanobashvili, A.. 247. 249 Napoli, J. L.. 469. 497 Narasimhaswamy. S., 342, 366 Narc?; E, 286, 294

Author I

n

d

e

x

Narimatsu, M., I7t, t73. 176 Narita, N., 45,50, 160. 164, 165. 175, i78, 179, 323, 341, 343, 352, 366 Nammiya, S., 581,588 Narvaez, V., 372, 378, 379, 387.39.3 Nascone, N., 334, 335, 336, 337. 340, 366 Nash, A., 40. 44, 50.. 323. 338, 356' NasM, M. C., 167. 17i. t73, I79, 286,293, 453,492 Nathans, J., 527, 535, 556, 558, 565 Nations. M.. 509. 517 Nau, R, 580. 585 Nava, V. E., 227.23] Navankasattusas. S., 345,367 Navaratnam, V., 333.362 Navascues, J., 547, 56I Nave, K.-A., 92, 104 Nawata, H.. 379, 391 Naya, E J., 92, 102. 104, 269, 277, 310, 321,322, 327, 342, 369 Nayeem. S. M., 345.346, 347. 366 Nayernia, K., 386. 393 Naylor, S., "440, 494 Nebreda, A. R., 171. 173. I74 Neel, "v~. 136, 145 Neeman, M., 225,229 Nef, S., 386, 392 Neff, C. D., 415.419 T f f . L. A.. 285. 294 Ne Negus, K., 287, 292, 470, 486 Nehls, M. C., 160, 167, 178 Neidhardt, L., 135, 137, t47 Neihrs. C., 2, 4 Neilson, E. G., 4 t 5, 419 Neilson, K. M.. 453,492 Nelkin, B., 313, 323 Nellen, D., 577, 587 Nelson, A. R., 509, 515 Nelson, C. E., 305, 328 Nelson, D. L., 357.361 Nelson, M. C., 172, 173, t 74, 34 I, 357, 358 Nemali, M. R., 3 t 8. 328 Nemer. G., 342, 343,359, 361 Nemer, M., 342, 343, 352, 359, 360, 361, 363, 366, 36 7 382. 393 Nemes, Z., 581,582, 587, 588 Nemeske~, A., 504, 516 Nemeth, A., 504, 516 Nessmann, C., 387,392 Nestler, E. J., 247,249 Netchine, I.. 508, 516 Neub/.iser, A., 131, I46, 263,272, 321,32& 440, 44t, 451, 466, 475,491, 492, 493, 494 Neufeld. G., 223, 232 Neuhaus, J., 219, 231 Neuhauser-Klaus, A., 522, 527, 535, 549, 562

6

5

3

Neuhauss, S. C., 395, 41Z 546, 563 Neumann, H. R, 4 t5,420 Neumann, J., 453, 483 Neumann, R E., t t 7, 125 Neve, R. L., 415, 4i7, 469, 498 Neville, C. M., 335, 343, 3 ~" , 345, 346, 347, 370 New', D., 110, 125 Newbury-Enob, R. A., 357, 364 Newgreen, D. E, 453,486 Newman, C., 340, 358 Newton, J. M., 6, 17 Newton, V. E., 525,538, 560, 564 Ne~, C., 102, 24I. 244, 25t, 265, 266, 276 Nezanov, N., 570, 589 Ng, L. J., 281,293, 380, 381,389, 392, 5 I0, 515 Ng, M., 341,370 Ngai, J., 435,489 Nghiem, M., 318, 327 Ngo-Muller, V., 3, 4 Nguyen-Luu, D., 550, 562 Nguyenphuc, Q., 355,364 Niaudet, R, 375, 389 NichogiannopouIou, A., 204, 210 Nichols, A., 321,328 Nichols, D. H., 424, 425,437, 492 Nichols, J., 23, 34, i 58, 160, 166, ] 78 Nickel, D. D.7, 235,242, 244, 250 Nickolls, G. H., 282, 294 Nicol, R. L., 342, 364 Nicolas, J.-F., 9, 16 Nicolis, S., 390 N ~ico11. . C. . . S., . 500, 5!3 Nicolson, T., 557, 564 Nieburgs, A., 502, 5t4 Niederlander, C., 2, 4 Niederreit.her, K., 341,346, 347, 352, 366, 469, 492, 545, 564, 573, 588 Niehrs, C., 44, 50, 51, 78, 103, 138, 146, 325, 32Z 338, 361, 362 Niessen, C. M., 408, 420. 583,589 Nieto, M. A., 135, 14Z 354, 355,359, 367, 423, 428, 482, 490, 492, 495, 546, 564 Nieuwkoop, R, t08, 124, 185, 190 Nigam, S. K., 415,417 Nigam, V., 342, 343,367 Nigro, V., 40, 46, 52, 79, 10.4, 478, 495 Nihei, H., 352, 354, 365 Nihonmatsu, I., 84, 98 Nii, A., 525, 538 Nijhof, W., 245, 249 Nijja~ S., 343, 36t Nijweide, R, 285, 293 Niki, M., 220, 231 Nikolics, K., 5 t2, 516 Nikovits, J. W., 343, 369

6

5

4

A

u

Nikovits, W.. Jr.. 3.~. _4.~, .~44..~4~, .~46, ~4/, 370 Nilson, J. H., 376, 39t, 5 t0, 515 Niisson, A.-S., 402, 420 Nilsson, E., 246, 249, 3 t 8.324 Nilsson. M.. 467.482 Nilsson. S. K.. 194. 208 Nimpf, J.. 94, 105 Ning, G.. 317. 326 NinE. ,_ Y.. 380. 390 Ninomiya. C.. 292,294 Ninomiva. Y.. 376. 393, 4,._. "~ 424. 425.435 . 4.~7.438 " . 442. 443.451. 453.475.476. 487, 489. 492, 493, 572. 588 Nirenberg. M., 334. 363, 474. 488 Nishi. M.. 95.102, 506. 516 Nishi, S.. 531. 538 Nishida. K.. 171. 173. 176 Nishiguchi, S.. 527,530. 531. 537 Nishigushi, S.. 91. 102 Nishihara. E.. 504. 5t6 Nishikawa. S., 172.173. 176, 188. 190. 196. 200. 209. 228. 233. 442. 498

Nishikawa. S.-I.. 173. 178. 188. 190. 200. 203.208. 209 Nishikawa. T.. 570. 584 Nishikori. T.. 547. 564 Nishina, H., 318.327 Nishino. Y.. 338, 354. 356. 368 Nishi-Takeshima. M.. 506. 516, 549. 564 Nishiyama, T.. 79.99 Nishizaki, Y.. 121,125. 522. 537 Nishizawa, K.. 290, 294, 437.450, 451. 466, 474. 495 Nishizawa, M.. 527, 530. 531,536 Nissinen, M.. 412, 418 Niswander. L.. 166. 178. 401,419. 571. 577.585. 588 Niswender. K. D.. 453.476. 493 Niveleau, A.. 9, 17 Niwa, H., 23.34, 160, 166, 178 Niwa. O.. 376. 392, 393 Noakes. R G.. 411. 419 Noben-Trauth. K., 556, 564 Noble, M., 240, 24 t, 251, 404, 420 Noda, M., 525, 538 Noda, T., 122, 122, 173, ]79, 217. 220. 230, 231, 404, 420, 506. 516, 549. 564 Nodasaka. Y.. 557.565 Noden. D. M.. 254. 276. 345.366, 421. 422, 424. 425.426. 428, 433.434, 435.449. 483, 491, 492. 497. 558. 562 Noebels, J. L., 92, 102 Nofziger. D., 136, 148, 262, 276 Nogawa, H.. 17 I. t 78, 3 t 4, 32 7 Noguchi, K.. 376. 39t Noji, S., 47, 50, 116, t23, 354. 357. 365, 370, 423,424, 451. 471,475, 476, 478, 479. 487, 490, 492 Notan, R M.. 560, 561 Noten, A. A., 452, 492 Noll, M., 398, 419, 470, 481

t

h

o

r

Index

Nomura, M., 47, 50, 356, 366, 375,376, 390, 392, 393, 468, 492 Nomura, S., 288, 293, 3 i O, 327, "442, -443,453,488, 555, 565

Nonaka. I.. 260. 276 Nonaka. K.. 282. 294, 465,492 Nonaka, S., 38, 51 Nonchev, S., 117. 118, 1 t 9, 124, 453, 468, 490, 492, 5.44, 563

Noramly, S.. 575,576, 577. 578, 579, 587.. 588 Nordborg, C.. 237. 247, 248. 249 Nordheim. A.. 342, 358 Nordlund. M. L., 34 t, 352, 359 Nordqvist, K., 373, 383. 391 Norlin. S.. 78, 104, 141. 148, 504, 505,506, 514 Norman, D. J.. 506. 508. 509. 518 Nornes, H. O.. 398, 417 Noms; D. R, 354. 367. 386, 387. 390 Norris, W. E., 40. 52, 265,276 North, "12,201,204, 209, 220, 23t Northcutt. R. G., 421. 427,433,457,485, 492 Norton. C. R.. 136. 149. 226. 230, 423, 487 Norton. J. D.. 10, 17 Norwood. V. F., 412, 420 Noseda, M.. 474. 489 Nothias. E, 84, 103 Nothias. J.-Y., 5.9, 10, 17. 19 Nothwang. H., 388. 392 Nougues. J.. 271,273 Novacek, M. J., 442, 457,492 Nova_k, J., 527, 535 Novikoff, R M., 318, 324 Novitch. B. G., 341,361 Novoselov. V., 338. 363 Nowak. S. J., 351,364 Nowakowski, R. S., 87, 92, 105, 245, 249 Nowling, T., 166. 179 Nowotschin, S., 354, 355,359 Nuckolls, G. H., 465,492 Nunes, E D., 550, 561 Nusse, R., 45, 51, 52, 82, 106, 405, 420, 577, 579, 588 Nusslein-Volhard, C., 351,355,359, 546, 557, 564, 565

Nutt. S. L.. 204, 209 Nuyens, D., 226. 229 Nye, J. S.. 89, 100, 136. 14Z 262, 263.275

O Oakey, R. J.. 6. 17 Oates, A. C.. 14, 17, 214, 230 Oba, K., 379, 391 Obara-Ishihara, T., 401,419, 571,588 Obinata, T., 258, 274 O' Brien. M. A., 341,358

Author i

n

d

e

O'Brien, M. J., 26, 34 O' Brien, T. X., 343.366 Ochi, T., 288, 293 Ochikubo, E, 557. 563 O' Connell, M. L., 10, 19 O'Connetl, S. M., 89, 95, 104, 353. 354, 355, 364, 433. 478. 489, 496. 504. 505, 506. 507, 508, 509, 514, 517, 518. 556, 558, 562 O'Connor. D. S., 225,231 Oda. H.. 321. 326, 432. 450. 465.489 Oda. M., 167, t80 Oda. S.. 8, 16 Oda, T., 347, 366 Oda, Y., 580. 583, 588 Odenthal, J., 546, 565 Odland. G. E. 568. 583,586 Ody, C.. 201,209 Oelgeschlager, M.. 342, 358 Oellig, C.. 558, 564 Oesterle. E. C.. 558. 565 O' Farrell, R H., 170. 175 Ofer~ L., 166, 178 Offield, M. E. 309, 319, 320. 327 Ogasawara, M., 475.492 Ogasawara. N., 557. 565 Ogawa, M.. 94. 103. ]06. t96, 209, 228, 233, 442, 498 Ogino, H., 527, 530, 53 t, 536, 537 Ogle, R. C., 452,492 O' Gorman, S., "443. 449, 479, 495 O'Guin, W M., 581,587 Ogunrinu. G.. 353. ."~-"7 , , 364 Ogura, K., 525,536 Ogura, M., 506, 516, 549, 564 Ogura, T., 525,536, 571,586 Ogura, Y., 173, 178 Oh, B., 8, t0, 12, 13, 16, t7, 18 Oh, M. H.. 354, 3 6 5 Oh, S. H., 546, 564 Oh, S. R. 353, 356. 357. 366 Oh, S. R., 92, 103 Ohba. M.. 580. 58Z 588 Ohfuji, Y.. 47.51 Ohgane, J., 167, 180 Ohishi, S., 47, 51, 356, 367 Ohkawara, T., 525,538 Ohki. M., 202, 208 Ohlsson, C., 510. 515 Ohlsson, R.. 167. 173, t 78 Ohmori, T., 289. 294 Ohmoto, T., 247,249 Ohnishi, H., 321,327 Ohnishi. S., 581,588 Ohno. S., 580, 587 Ohsaki. K.. 537 Ohshima, H., 290. 294, 437, 450, 451,466, 474, 495

x

6

5

5

Ohshima, Z, 94, 100, 103 Ohsugi, M., 410, 418 Ohto, H., 399, 4t9 Ohtsuka, T., 136, 147 Ohuchi, H., 47.50, 116, ]23, 3 I2, 328, 353, 354, 355,356, 363, 370, 423,424, 451.47 I, 475,476, 478. 479. 487, 490, 492, 505, 517 Ohyama, K., 313, 329 Oikawa, T., 313, 314, 323 Oishi, C., 53 I, 538 Oiski, S., 357, 365 Ojeda, J. L.. 4 t5.419 Oka, C., 136, t37, I47 Oka, S., 379, 39t Oka, T., 336, 343, 365, 368 Okabe, M., 136, 1461, 466, 488, 581,583, 589 Okabe, S., 88, 89, 98, 101 Okada, H., 220, 231 Okada, T. S., 529, 531,534, 536, 537 Okada, Y., 38, 5] Okafuji, T., t 12, I14, 115, 116, 123, 124 Okaiima, K., 347, 366 Okamoto, K., 183, 190, 442, 443, 453,488 Okamom, R., 288, 293 Okamura, A., 91, t01 Okamura, H., 442, 498 Okamura, R. M., 440. 497. 583, 589 Okamura, Y., 79, 99 O'Kane, S., 434, 496 Okazaki, K., 7, t6 Okazaki, S.. 136, 137, 147 Okazaki, Y., t t, 12, ]8 Okazawa, H., 183, t 90 O'Keefe, S. J., t4, 18 Okkema, R G., 165, 177, 316, 325 Okuda, A., 183, 190 Okuda, T., 49, 52, 201,204, 209, 220, 231 Olanow, C. W., 247, 251 OIdenettel, I., 85, 103 Otdmixon, E. H., 224, 227, 232 Oldridge, M., 453,466, 474, 494, 498 O'Leary, D. D., 84, 102, 103, 119, 125, 525, 537 O'Leary, D. D. M., 92, 94, 98 Olek, A., 9, ]8 Oliner, J. D., 400, 418 Oliphant, A. R., 412, 4I 7 Oliver, G., 79, 103, 218,219,233, 268,274, 317, 32I, 329, 399, 417. 451,475,495, 52 i, 527, 530, 532, 533, 534, 537. 538.. 571,585 Oliver, J. A., 405, 407, 4]6, 417 Olivo, J. C., 85,103, 443, 474, 490, 545,563 Olney, A. H., 470, 487 Olsen, A., 412, 4t8 Olsen, B. R., 227, 232, 280, 281,288, 292, 293, 294, 424, 427,442, 443, 453,484, 487, 489, 492, 493

656 Olson, E. N.. I35, 136. 137, t38, t45. 148, t65, 168, 175, 17Z 228, 230, 250, 257. 258, 260, 261,262, 267, 269, 273, 274, 275, 276, 27Z 287, 289, 292, 294, 316, 32Z 3 o 334, 336, 340, 34t, 342, 343, 044, 346, 347, 049. 350, 352, 353, 355, 358, 359, 360, 361, 364, 365, 366, 367, 36& 369, 370, 45I, 465,473.476, 477. 483. 490. 495. 496 Olson. L., 509. 518 Olsson. M.. 88.89.98. 103. 110. 125. 241. 244. 249 Ottz. E. M., I06 Otwin, B. B.. 27 I. 273 O'Malley, B. W.. Jr., 556. 558. 565 Omi. M.. 3.4 Omori, A., 84, 100 Omura, T.. 375,376, 380. 384. 390. 392 O'Neil. J.. 525,537 Ong. A. C., 4 t 5.420 Ong. E. S.. 172. 173. 174. 341. 357. 358 Ong, L.. 580. 585 Ong. S.-H.. 171.173. I78 Onichtchouk. D.. 338.361 Onigata, K.. 506. 518 Ono. K.. ~ 3 . 4 9 2 Ono. T.. 525.537 Ontell. M.. 269. 275 Onuchic. L. F.. 414. 419 Onuma. Y.. 338. 368 Oosterveen, T. C.. 476, 477. 478, 491 Oostra. B. A.. 227. 231 Oostuyse. B.. 226. 229 Opperman, L. A.. 452. 492 Opthof, T.. 348. 360 Orban. R C.. 14. 16 Orci. L.. 321. 328 Ordahl. C. R. t28, 130. 131. I32. 140, 141. I45. 147. 149, 219, 229, 254, 255,256. 258. 261. 262, 263. 264. 266. 268.271,273, 274. 276. 277, 334. 360 Oreal, E.. 372. 387.392 Orellana. S. A., 415.419 Orkin, R. W.. 577. 578. 579, 587 Orkin. S. H., 202, 203.204, 207, 209, 210. 219. 232. 271. 276, 318. 328, 341. 343.352, 360. 369. 415.417, 510. 518 Orlic, D.. 196. 202, 210 Ormsby. I., 453. 467. 494 Ornitz, D. M., 116, 126, t 67, 17 I. 173, t 79, 286. 292, 293, 313.314. 315,324, 453.492, 558. 561 Ornov. A.. 174. 178 Oro, A. E., 579, 585 Oropeza, V., 578, 587 O'Rourke, D. M., 376, 390 Orr-Urtreger, A.. 467, 492 Orr-Weaver. T. L.. 169. 175 Ortega, S.. 505, 517

A u t h o r Index Ortiz de Luna, R. I., 453,486 Osawa, K., 525, 535 Osawa, M., 244, 251 Osawa, Y., 376, 393 Osborn, J. W., 437, 492 Osborne, L., 246, 250, 318, 326 Osborne, N.. 340. 34 t, 363 O'Shea. K. S 2,..~. );~9 Oshima, K., 403, 4t8 Oshima, R. G., 168, 179, 570, 589 Oshima, T., 506, 516, 549, 564 Oshimura, M., 478, 488 Oskarsson, M., 7, ]8 Oster, A., 32 t, 327 Ostrer. H., 387, 390 O' Sullivan, M. G., 382, 392 Osumi, N.. 77.95, 101, 105, 451,475, 476, 492, 525, 538 Osumi-Yamashita, N.. 422. 423.424, 425,435,437,438. 451. 453.475.476. 478, 48Z 490, 492, 493, 527, 530, 535, 572. 588 Oswald. J., 9. I8 Otani, H., 547, 557, 564 Oto, A. E.. 574, 577, 579, 583, 588 O'Toole, B. A., 453,483 Ott. M. O., 132, 147. 165, ] 74, 258, 276, 303, 323 Otten, B., 509, 5 t 7 Otto, F., 288, 293, 294, 442, 443,453, 493 Ottolenghi, C., 382, 392 Ou, C.N.,318,327 Oudejans, C., 174, 174 Oudhof, H. A., 452. 493 Oulad-Abelghani. M., 40, 47.51 Overbeek, R A., 349, 352, 354, 355, 364, 365, 366, 369, 380, 389, 441,486, 49I, 49Z 527, 530, 53 i, 532, 537 Overton, G. C., 194, 206. 209 OvertruL K., 318, 327 Ovitt, C., 183.190, 31 t, 324, 442, 443, 497 Ow, D. W., 15, I7 Owen, D. A. J., 26, 33 Owen, M. J., 288, 289, 293, 294, 442, 443, 453, 493 Owens. D. F.. 86. 102 Oxford. J. T.. 442. 443.489 Ozaki, H.. 399, 419 Ozaki, K., 321,327 Ozato, K., 183, I90 Ozawa, M., 408, 41 & 419 Ozcelik. C.. 353, 356, 36I

P Pabst. O.. 310. 327 Pace. J. M., 424, 442. 443,493 Pachnis, V.. 88, 89, 101, 260, 267, 275, 402, 403,419, 420, 432, 485, 497

Author Index

Pacifico, F., 338, 360 Packard, D. S., Jr., 134, ]47 Packer, A. I., 312, 32 7 Pagan, S., 364 Paganessi, L., 337,354. 364 Pagan-WestphaI, S. M.. 337,354, 364 Page, D. C., 387, 39] Pai, C. Y., 91. 103 Paige, C. J., 193.20Z 318, 327 Pajusola, K.. 218. 223.229 Pakarinen, L., 452, 494 Pakzaban, R. 89, 99 Palacios. D., 5 I0. 516 Paldi, A., 320, 324 Paleari, L., 290, 292, 450. 453,472, 473,481, 549, 56t Palfrey H. C., 9 t, 105 Palgi, J.. 559. 564. 565 Palis, J., 195,209 Palko, M. E., 559, 562 Pallister, R D.. 506. 5] 7 Pallon, J., 580, 586 Palmeirim, I.. 134, 135, 138, 139, 146, t47 Palmer. D. A.. 348, 358 Palmer. M.. 219,231 Palmer. M. S.. 377, 388, 393 Palmer. R., 400. 4] 8 Palmer. S. 34t _~4~.347, 348, o~ , 359, 366 Palmer. S. J., 377, 378, 383,386, 389, 392 Palmer. T. D.. 236, 237.241,244, 251, 252 Palmes, C., 318,328 Palmieri, S. L., 160. 178 Palmiter. R. D., 318, 328, 374, 385,389, 511. 513 Palomino, M. A., 344. 348, 360 Palomino, T.. 5 t 0. 517 Pan, D.. 136, 147 Pan, L., 559, 561 Pan, S. S., 188, 190 Pandita. R. J., 353,357,365 Panelli, S., 171, 173.174 Pang, K., 105 Panganiban. G.. 471,493 Panic, S., 307, 329 Panitz, F., 52 I, 522, 525,533, 535 Pannell, R., 203,210 Pannese, M., 290, 294, 450, 471,472, 473,495 Pant, H. C., 94, t03 Paolucci. E., 206, 20Z 246, 249, 271,274 Paonessa, R D., 11, t2, ] 7 Papadapoulos, N., 224, 231 Papadimitriou, J. C., 200, 207 Papageorge, A. G., 171.173, 178 . . " ~ 7 , t45, 157, 166, 175, Papaioannou, V.E. 132. 133.. . 135. 1~ 176, 178, 353, 357, 362, 442, 487 Papalopulu, N., 86, 100

657

Papaloucas, A., 570, 584 Papapetropoulos, A., 225, 231 PapapouIos, S. E., 285,293 Papayannopoulou, T., t 73, 179 Papiernik-Berkhauer, E., 22, 33 Papkoff. J., 140, 141, 14& 263,264, 277 Paquette, A. J., 243. 249, 252, 263, 264, 266, 274 Parada, L. F., 193,204, 20& 34t, 352, 359, 365, 386~ 392 Paradies, N. E., 550, 563 Paradis, G., 193, 207 Paradis, R, 342, 343, 359 Parameswaran, M., 38.52, 333, 366 Parati. E. A., 235,242, 244, 250 Pardanaud, L., t 99, 200, 201,209 Pardini, C., 83, 92. 100, 1.02 Parent, J. M., 247, 248, 25 ] Pavia, B. C., 156, t77. I78 Park, B. K.. 89, 106, 450, 471,498 Park, H. L., 85, I02, t03, t21,124, 125, 505, 517 Park, J. H., 4 t5, 420 Park, L., 292, 294 Park, M., 266, 27Z 337, 341,360, 363, 366 Park, W. Y., 313, 3 t 5, 32 7 Parker, J., 465,491 Parker, J. D., 510, 513 Parker, K. L., 375, 376, 379, 380, 38 t, 384, 389, 390, 391, 392, 510~ 5t5, 516 Parker, S. B., 287, 294 Parks, D. R., I94, 207 Parks, J., 500, 504, 506, 509, 5t7 Parlow, A. E, 506, 509, 512,513, 5t5, 516 Parmacek, M. S., I65, 17Z 303, 3 t 6. 326, 327, 342, 343, 352, 363, 366 Parmar, M., 9 t, t05 Parnavelas, J. G., 88, 89, 10t Parr, B. A., 385, 386, 392 Parravicini, E., 405,407, 4 t6, 4i 7 Pmrrington, E R., 442, 493 Parrington, J., 9, 18 Parrott, J. N., 10, t8 Parslow, T. G., -440, 49Z 583, 589 Parsons, L. M., 333, 334, 343,345, 348, 349, 352, 356, 364 Parsons, S. M., 260, 267, 277 Partanen, J., 224, 231 Pmrtington, G., 343,361 Parton, L. A., 10, t 9 Papdnen, M., 580, 589 Pasantes, J., 281,294, 380, 393 Pask, A., 376, 386, 387, 392 Pasko, D., 530, 536 Pasquali-Ronchetti, I., 588 Passarelli, R. W., 452, 492 Pastor, F., 547, 564 Pastorcic, M., 416, 420

6 58 Pastore, C.. 220, 228 Pasyk. K. A.. 227. 232 PS.szty, C., 443,493 Patan. S.. 224, 232 Patapoutian. A.. 260. 276 Patek. C. E., 416. 419 PateI. B.. I2. I3. ]8 Patel, K.. 264. 266. 272, 354, 355,366, 574. 577.588 Patel, S., 224. 232, 504, 517 PateI. Y.. 23.33, 166, t 67.175. 178 Paterson. A. J.. 511.5t6 Paterson. B. M.. 341. 364 Patient. R.. 165. 177. 198. 200. 207, 342. 343.361. 367 Paton, I. R.. 388. 392, 534. 536 Patrene, D. K., 246. 251 Patten. B. M.. 354. 366 Patterson. C.. 421. 493 Patterson. K. D.. 347. 348. 359. 360 Patterson. L. T.. 408.409.410, 417 Patton. B. L.. 411. 418 Pauken. C. M.. 13.18 Paukku. T.. 511. 516 Paul. J.. 317. 327 Paulding, C.. 400. 418 Paulin. D.. 266. 277 Paus. R.. 574. 577.578. 583.588, 589 Pause, A.. 227. 229 Pavlova. A.. 415.417. 418 Paw. B. H.. 214. 230 Pawling, J.. 203.207. 223.228 Pawlowski, S. A.. 434. 467. 493 Pawson. A. J., 171. 175 Pawson. T.. t71. 173. 178 Paydar. S.. 474. 494 Payne, C.. 303,330 Payne, R. M.. 356. 366 Paynton, B. V.. 11.18 Payrieras. N.. 338. 366 Pay san. J.. 84. I03 Paznekas. W. A.. 453.486 Pearce. J. J.. 40. 44.52. 160. 166. 167. 168. 178. 179. 303. 327, 376. 390 Pearlman. A. L.. 94. 104. 237,251 Pearse. A. G.. 320. 327 Pease. W.. 357.367 Peault. B.. 192, 194. 200, 201. 207, 208, 209. 220. 232, 245,250 Pedersen. A. C., 29 t. 292 Pedersen, E. E., 310, 321,325 Pedersen, R. A., 26. 32. 35, 3 8 . 3 9 . 4 5 . 4 7 . 5 I , 79.83.87. 89. 90, 91, 92.95, 97. 98, 99, 103. 158. 177. 179, 290. 292, 303.30-4. 321. 326, 329. 333.336. 338.363, 410. 418. 422, 435,437. 450, 451,453,470, 471. 472. 473, 477, 478. 481,484, 493, 549, 562, 570, 589

A u t h o r Index Peeters, L. L.. t74, 177 Peeters. M. C. E.. 196, t 99, 200. 201. 202, 207 Pei. Y. E, 519,537 Peifer. M., 443,478, 493, 575,588 Peissel, B.. 415.418 Pekna, M.. 313.323, 414. 418, 467, 482 Pekny, M.. 313.323, 413, 4t4. 4t& 467, 482, 489 Pelet. A.. 286, 294, 453.494 Petlas, T. C., 188, 190 Pellegrini, M.. 83, ]03 Pelletier. J.. 341. 352. 363. 374. 375. 379. 391, 392, 399, 400. 415.416.41Z 418, 419 Pelliniemi. L. J.. 51 I. 516 Peltarri, A.. 284, 293 Pelto-Huit,cko, M.. 527, 530. 532. 535 Pelton. K. M.. 308. 326 Pelton. R. W.. 308.326, 467, 493 Peltonen. L., 412. 418 Pena. R. 510. 517 Peng, J., 217, 227, 229 Penman-Splitt. M.. 352. 354. 357,361, 366 Penner. J.. 570, 583.584 Pennisi. D. J.. 172. 173. I77. 336. 337, 365 Penny. G.. 40.44.52 Pennypacker. J. R. 424, 442. 443,482, 493 Penttila, T. L., 580, 589 Penzes. R. 469, 497 Pepicelli. C. V.. 312. 328, 407,419 Pepling, M. E., 6, 7, 18 Pera, E. M.. 78.81. 103, 546. 564 Perantoni, A. O., 405,407, 4 ! Z 419 Percival, A. C., 320, 328 Perea-Gomez, A., 41.42.43, 44. 47, 52 Pereira, A.. 346. 358 Pereira. E A., 92, 106, 334. 343, 345, 346, 352, 366, 559, 563 Perens. E.. 222.23] Perez. A., 577, 578. 579, 587 Perez. L.. 17I, 173. 174 Perez-Miguelsanz, J.. 527. 535 Perez-Moreno. M. A., 355.359, 423,482 P6rez-Navarro. E.. 91, 98 Perez-Pomares, J. M., 219. 228, 231 Perfilieva, E., 237, 247. 248, 249 Pericak-Vance, M. A., 227,231 Perkett, E. A.. 467, 493 Perkins, A., 318, 328, 34 I, 352, 359 Perkins, D.. 325, 470, 485 Perkins. G.. 341 .~_. 367 Perl. A.-K.. 311. 322. 324, 328 Perlmann. T.. 91. 105, I20, 125, 509. 518 Permutt. M. A.. 321. 325 Pernasetti, F.. 509. 517 Perreau, J., 205,207

Author I

n

d

e

Perrimon, N., 45, 52 Perrin-Schmitt. E, 452, 453, 482, 484 Perris. R., 423,424, 484, 493 Perron, M., 522, 525, 533, 538 Perry, M., 262, 276 Persico. M. G., 162, t79, 338. 352, 360, 370 Persing, J. A., 452. 492 Persson, H., 9 t, 98 Pertz, O.. 408.4t8 Pesce, M.. 86. 103, i 88, 189, ] 89, 190 Peschon. J. J., 318. 329 Pestell. R.. 86, 104, 579. 588 Peter, W.. 160, 178 Peterka, M., 454. 455,489, 493 Peterkova. R.. 454, 455,489, 493 Peteropoulos, H.. 342. 368 Peters. C., 577,583, 584 Peters D., 305,329 Peters D. J.. 414, 418 Peters, D. M.. 453,467, 496 Peters. G.. 545.565 Peters. H.. 290. 292. 294, 398, 399, 420. 437, 440. 44 t, 450, 451. 466, 474. 475,492, 493, 495, 498, 527, 530, 533, 538. 550. 565 Peters. K. G.. 214, 225,227, 230. 231, 312, 314. 328, 330 Peters, M. A.. 525,537 Peterson. A., 79, 84, 86, 92, 9& t00, 236. 243, 250, 252 Peterson, B. E., 246, 25] Peterson, C. A., 268, 269, 274 Peterson, C. L., i 66, 179 Peterson, D. A., 237, 241,247, 248, 249, 252 Petit. C., 398. 416, 550. 561. 563 Petkovich. M., 469, 470, 484. 525,537 Petrenko, O., 200. 209 Pettersson. K., 167, 178 Pettersson. R. E, 558, 564 Pettit, J.. 185, I90, 243, 252 Peverali. G., 382. 387. 388. 390 Pevny, L. H., 204, 209, 260, 267. 275, 377, 392, 53I, 537 Pexieder, T., 341,366 Pey, R., 415,419 Peyrol, S., 585 Pfaff. D. W.. 435,495 Pfaff. M.. 574, 589 Pfaff. S. L.. 104, 319, .~_0. 321 323, 325. 504. 505.507. 508, 510, 517, 518 Pfziffle, R., 509, 517 Pfeifer, D., 380, 392 Pfeiffer. R. A., 281,293, 381,391 Pham. T. D., 244. 251 Phan, H., 185,189, 190 Phelps, A. L.. 357, 369 Phelps, C., 511. 517 Phelps, D. E., 398, 4]9

x

6

5

9

Philbrick, g( M., 285,294 Philips, R. J. S., 44t, 493 Philipsen, S., 343,369 Phillips, H. S., 402, 4]8, 420, 512, 516 Phillips, R E., 467,495 Phillips, R. I., 194, 206, 209 Phillips, R. J., 443,486 Phillips, S. J., 31 t, 330 Philp, N., 356, 369 Philpott, A., 522, 525, 533,538 Philpott, M., 578, 588 Phinnes~ D. G., 246, 250 Phippard, D., 506. 5] 7. 549, 564 Phyllis, R. Wl, 334, 361 Piatigorsky, J., 532, 535, 538 Picard, J. J., 32I, 326 Picard, J. Y., 372, 374, 387, 391, 392 Piccoli, D. A.. 347, 364, 366 Piccolo, S., 3 i, 33, 40, 44, 50, 5t, 52, 338, 358, 366 Pichel, J. G., 374, 390, 402, 4 ] 9 Pickel, J., 89, 101 Pictet, R. L., 319, 321,328 Pieau, C. 83, 104..~7,.. 387 :~9) Piedra, E., 35I, 355, 356, 359 Piedrm M. E., 349, 354, 360, 366 PiehI, E, 559, 564 Pieter, T., 81, I00, 521,522, 525,527, 533,535, 536 Pierani, A., 80, 82, 88, 9& 120, 121,125 Pierides, A.. 414, 418 Pierpont, M. E. M.. 346, 347, 35& 364 Pietryga, D. W., 204, 208, 318, 327 Pignoni, E, 399, 419, 530, 533,537 Pike, B. L., 347, 366 Pilia, G., 357, 361 Pinard, J. M., 94, 99 Pinchera, A., 311,326 Pineau. T., 311, 312, 326, 474, 488 Pinto, S., 251 Pintucci, G., 224, 232, 504, 517 Pintus, G., 338, 369 Piotrowska, K., 1, 4 Piper, D., 240, 250 Pirottin, D., 264, 2 74 Pirro, A., 285, 293, 294, 442, 443, 489, 574, 583,584 Pirro, M. T., 31 t, 326 Pirvota, U., 558, 559, 564, 565 Pisano, M. M., 528, 537 Pischetola, M., 31 I, 330 Pispa, J., 44t, 493 Piston, D. Vr 82, 86, 99 Pitera, J. E., 305,328 Pittenger, M. E, 206, 209 Pitts, S. L., 322, 328, 512, 516 Pitts-Meek, S., 12I, 123

660 Piussan, C., 506, 517 Pixley. S. K.. 82. t05 Plaas. A. H.. 350. 362 Placzek, M., 78, 79. 80, 8I, 85.99, I04, 120. 123, 14t, t48, 5"~ , 537 ~:,1 Ptasterk, R. H. A., 14, 18 Plate, K. H., 227, 23I Platt, J. B., 423,493 Platt, K. A.. 85. 102. 103. 121. 123, 124, 125, 470. 487. 493, 505.517 Playford. R. J., 167, 174. 305,323 Pleasure, S. J., 83, 90, 92. 102, 103 Plehn-Dujowich, D.. 79, i06. 529. 538 Ploder, L., 527, 535 Ploemacher, R. E., 192. 193,207. 210, 343.369 Plowden, J.. 357.358 Plowman, G. D., 314, 328 Plum, A., 173. 177 Plump, A. S.. 164. 175, 303.323 Poea, S.. 271,277 Poelmann. R. E.. 219. 226, 229. 232. 316. 326, 333,341. 360, 366 Pohl, T., 85,103 Poirier, E, 183, 190 Pokrywka, N.. 185, 190 Potani. R, 183, 189 Polanski. Z.. 7, 18 Polarlds. R, 575.588 Poli, V.. 318,324 Polish, J. A.. 510, 5t6 Poljak, L.. 442. 484 Polk, C. E.. 343,368 Pollard. J., 173, 178 Pollefeyt, S., 203.20Z 223,228 Poller, N., 138, 146 Pollock, R. A., 402, 420 Polonskaia. O.. 91. 101 Pompa, J. L. D. I., 318.327 Poncelet, D.. 264. 274 Ponsot. G.. 94. 99 Pontiggia. A.. 377. 392 Pontoglio, M., 166, 178 Ponzetto, C.. 266, 271. 273, 275 Poole. M. D., 453, 466. 474. 498 Popp, R. A.. 204, 208 Popperl, H., 45, 52, 117, t 18, 124, 449, 479. 484 Porcher, C., 203, 209 Porras, A., 171. 173, 174 Porteous, D. J., 399, 416, 419 Porteous. M. E.. 227.231 Porter, E. 508. 517 Porter. E D., 87, 91, t03, 173. 176, 376, 389, 512, 515, 521,527.537

Author Index Porter, J. A., 78, 98, ]0], 120, ]25. 141, t45, 287, 292, 470, 483, 489. 493 Porter. T. E., 51 t, 518 Porteu, A., 262, 268, 276 Porteus, M. H., 79, 88, 89, 98, 103 Portillo, E, 355,359, 423. 482 Posadino, A. M.. 338, 369 Post. L. C.. 3 t 3,328 Post. M.. 121. 124, I73. 174. 175, 312. 313.327, 328, 329, 385.389, 413,419 Postiglione, M. R. 95.9Z 290. 292. 450, 453,472. 473, 478.48I. 491, 506, 513, 549, 550, 55 I, 552, 561, 564 PostIethwait, J. H.. 2t4, 230, 231 Posttethwaite, M., 91, t02 Postmus, J., 174, 174 Pote. J.. 581,586 Potten, C. S.. 570, 587 Potter. S. S., 79, 82, 83, 94, 95, 101, i04, I05, 204, 208, 318.32Z 352. 354. 355,364, 368, 433,476, 477, 478. 489, 502, 507. 508, 509, 516, 517 Poueymirou. W: T., 166, 175, 292. 292 Poulat, E, 381. 390 Poulin, G., 509, 517 Poulsen, K. T., 402, 420 Pournin, S., 546, 565 Pourqui6, O.. 134. 135, 136, 138, 139, 140, 141, I42, 146, 147, 262, 263,264, 266, 273, 274. 276, 347. 366 Poustka. A., 32 7. 415, 417 Powell, B. C., 578, 588 Powell, T. J., 217, 220, 229 Powell-Braxton, L., 9 I, 98. 203, 20Z 223, 229. 341,363 Pownall. M. E.. 167. 176. 263,276 Powrie. J. K., 507. 514 Prakkal, P. E. 568. 588 Pramparo, T., 353, 357, 364 Pratt, H. R, 158, t 78 Pratt. S. J.. 214. 230 Pratt. T., 87.94. 105 Prawitt. D.. 379.39t Prescott. A. R., 527, 530, 53 t, 537 Presley, R., 79, 99, 290, 292, 422, 432, 435, 437, 442, 450, 451,453. 456, 47i, 472, 473, 477,478, 484, 493, 549, 562 Pressman, C., 287,292, 354, 355,364, 451,470, 486, 489, 579,586 Pretsch. W\, 522, 527, 535, 549, 562 Prezioso, V. R.. 164. 165, 175, t 77, 179, 303, 316, 323, 329 Price. B. M. J., 167, 179, 203, 207 Price. D. J.. 85.87.94. 9& t05 Price. J., 83, 85.91. 105, 237: 239, 252, 341,352, 368 Price. M.. 85, 103, 311,326, 450, 484 Price. S. D., 555.558, 559, 561, 563 Price, S. M., 338, 353,354, 356, 357, 370

A u t h o r Index

66 t

Prideaux, E. M., I58, 177 Prideaux, V., 23, 35, 42, 51, 169, 179 Priess, J. R., 165, 180, 185, 190, 240, 243, 251, 252, 316, 330

Prigent, M.. 200, 209 Primig, M., 263,264. 273 Prim F., 572, 574, 585 Prince. V. E., 118, t25, 427, 493 Prinz. M. R, 341,363 Pritchard, J., 506. 514 Pritchard-Jones, K., 374, 375,389, 399, 416, 4t9 Probst, E J., 556. 557, 561, 564 Prockop, D. J., 246, 250, 284, 293 Proctor, J., 128, t 37, 146 Proesmans, W., 398, 4t9 Proetzel, G., 434, 467,493 Proia, R. L., 227, 231 Prokscha, A., 336, 370 Prosser, J.. 45 i, 475,486, 527, 528, 530, 536 P~byla, A. E.. 321,328 P~,wes, R., M_. 360, 369 Puech, A., 353, 357,365 Puelles, L., 75, 76, 77, 78, 79, 82, 83, 84, 85, 86, 88, 89, 92, 98, 99, 103, t04, 422, 428.450, 471,484, 490, 494 Puissant. E, 27, 34 Pulleyn, L. J.. 433. 453.466, 474, 492, 494, 498 Punnett. H. H., 380. 38 I, 391 Purcell, K., 186, 190 Purcell, R, 532, 538 Purdie, L. A., 451,453.474, 489 Puri, M. C., 224, 231 Piischel, A. "v~:,475,493 Putaala, H., 412, 418 Puzis, R., 258, 263,274 Pytowski, B., 227, 233

O Qi. H., 136. 144, I48 Qi, M.. 347, 364 Qi, P., 94, 104 Qian, E, 414. 419 Qian, X., 171, I73. 178 Qiao, J., 405.4t7 Qiao, w , 286, 292 Qin, M., 15, 17 Qin, Y., 224, 228, 232, 380, 389 Qiu, M.. 78, 83.85, 87, 89, 90, 9t, 95, 9Z 103, 422, 437, 450, 451,470, 471,472, 481, 493, 496 Qiu, Y., 310, 321,322, 32Z 334, 343,345, 346, 352, 366 Qu, S., 451,453, 476, 493 Quadrelli, R., 346, 358 Quaegebeur, A., 511, 517

Quaggin, S. E., 3t3, 32& 413,419 Quarmby, J., 185, i89 Quere, R, 201,209 Quinlan, E., 11, 19 Quinlan, G. A., 3.4, 30, 35, 38, 39, 40, 41,47, 51, 52, 132, 147

Quinlan, R. A., 527, 530, 531,537 Quinn, A. G., 287, 293, 579, 586 Quinn, J. C., 451,475,493 Quintana, D. G., 25t Quintana-Murci, L.. 382, 392 Quintin, S., 165, t 76, 316, 325 Quo, R., 440, 497, 583, 589 Quondamatteo, E, 574, 583, 584

R Raabe, M., 161, 178 Raats, J., I85, t90 Rabbitts, T. H., 203, 210 Rabejac, D., 556, 562 Rabl, W., 378, 392 Racer, R A., 388, 393 Radcliffe, J., 505, 513 Radeke, M. J., 9 I, 98 Radice, G. L., 340, 366 Radich, J., 316, 325 Radovick, S., 509, 514, 517 Radtke, E, 223,229 Radziejewski, C., 224, 227, 231 Raeburn, A., 357, 364 Raft, M., 24 t, 246, 250, 34 t, 359 Raffel, C., 287, 294 Raffin, M., 337, 339, 366, 367 Rafter?', L. A., 226, 23t Raghavan, S., 574, 583, 588 Rago, C., 86, 100 Ragsdale, C. W., 83, 87, 100, 105 Rahal, J. O., 443, 485, 508, 515 Rajewsky, K., 14, 16, 341,362 RakJc, R, 84, 87, 92, 94, 9Z 98, 99, 100, 101, t03, 136, 148, 248, 251 Ralphs, J. R., 468, 497 Ram, R T., 12, 19 Ramachandran, B., t 88, 190 Ramain, R, 343,360, 361 Ramakrishna, N., 405, 417 Rarnalho-Santos, M., 308, 310, 328 Raman, S., 376, 390 Rambhatla. L.. 19 13.18 Ramesa_r, R., 292, 294 Ramirez, A., 574, 583, 584 Ramirez, E, 442, 443,489, 573, 581,583,584, 588

6

6

2

A

u

Ramirez-Weben R-A.. 470.48I Ramdn y Cajfil, S.. 248. 251 Ramos. C., 525.535 Ramsdetl. A.. _.~4. ~44, 350. _~. t. 357..~6A 473,493 Rand. E. B., 136. 149, 347.364 Rand. M D I36, 144, I48. 263. ;72. 555,561 Raneanavakulu. G. 334, 341 _6/ Ranganayakulu, R., 34t. 364 Rangelt. L.. 223.229 Rangini. Z.. 395.417. 546. 563 Rankin. C. T.. 353. 356. 367 Rao. C. V. 510. 518 Rao. M. S., 236. 239. 240. 241. 250. 251,252, 318.328 Rao. Y.. 90. 95, 106, 136. 149, 521. 536 Rapaport, D. H.. 556, 558. 562 Raphael. Y.. 556. 557.561. 564 Rapola. J.. 22.34 Rapolee. D. A.. 23.33 Raposo do Amaral, C. M., 292. 294 Rappolee, D. A.. 166, 167. 175, 178 Rasberry. C.. 378. 387. 390 Rashbass. R. 88.99, 132. 133. 144. 149, 528.537 Rassner. U.. 581. 582. 585. 586 Rassoulzadegan. M.. 391 Rastan. S. _~,6. 378. 386. 387.390. 393 Ratcliffe. E. 225.229, 415.420 Rathbun. G.. I06 Ratner. N.. 341. 352.359 Ratty. A. K.. 502, 515 Rau. gi. 388. 391 Rauch. M., 382, 389 Rauchman, M., 532. 537. 538 Raulet. D. H.. 205.208 Rausa. F.. 321. 328 Rauscher. R J. III.400. 418. 419 Rauskolb, C.. 443.478.493 Rauvala. H.. 218. 223.230 Raven. C. R. 423.493 Raven. H. C.. 473.493 Ravi. R.. 225.231 Rawls. A.. 136. 137. 138. 145. 148. 250. 260. 262. 267. 273. 276. 277. 287.294 Rawson, E. J., 509, 516 Ray. D. W., 512. 518 Ray. J.. 241. 252 Ray, M.. 309, 319. 320, 327 Rayburn, H., 170, 180, 204, 206. 219, 223.228, 229, 230, 233. 340. 366, 571.581,583,589 Raymond, C. S.. 382, 387, 388. 391, 392 Raynaud, A.. 139, 148 Raz, Y.. 557. 564 Read, A. R, 525.538, 560. 564 Readhead. C., 512. 5 t3 Reardon, ~\. 433, 453.492, 493, 494 .

"~7

t

h

o

r

Index

R6aume, A. G., 135, 136, 137 145, t48 Rebagliati, M. R., 78, 103 Rebay, I., i36, t45 Rebel. "V. !., 245, 251 Reber. M.. 165, 174, 303~ 323 Rebrikov, D., 34I, 369 Recan, D., 378. 392 Reda, D., 169, ] 79, 349, 367 Reddi. A. H., 467, 494 Reddy, J. C.. 375.3 92 Reddy. E H.. 247. 251 Redmon. J.. 452. 492 Redmond. L.. 92. ]03 Reed, K. J.. 382, 388, 393 Reed. R., 263, 274 Reeders. S. T.. 412. 415.417 Reeve, W. J.. 158. 178 Reeves. A.. 237. 250 Reeves. R. H.. 509, 514 Refetoff. S., 31 t, 329 Regan. C. L.. 136. 145 Reginato. A. M.. 280. 294 Rehorn. K.-R. 165. 178. 316. 328 Reichardt. L. F.. 91.10I. 239, 249. 402, 410, 418, 549, 562 Reichenberger, E.. 292. 294 Reichmann. V., 336. 367 Reid. C. B.. 94. t05. 235, 2-44, 251 Reid. L. M.. 314. 324 Reid. S. W.. 173. 179, 341. 352, 359 Reifers, R, 115. t25, 337. 367 Reik, W., 9, 18 Reimold, A. M.. 318. 328 Reiner. C., 271. 273 Reiner. O.. 94. 103, 104 Reintjes, M.. 452. 492 Reissman. E.. 238. 251 Reiter. J. F.. 303.326, 342. 367 Reiter. R., 478,495, 507,517. 527, 532, 537 Reith, A.. 193.204. 208 Reitsma. M., 246. 250. 318. 326 Reitz. R. 322. 328 Relaix. E, 268,276 Remak, R., 131,148 Ren. C. J.. 224. 232, 504. 5 t 7 Renard. J.-R. 5.9. 14, 15, 18, 19 Renaud, D., 250 Renault, B., 357, 358 Renfree. M. B.. 372. 376, 386, 387,389, 392, 393 Renier, D.. 452. 453,484, 489 Renkawitz-Pohl. R., 334. 363 Rennels. E. G., 512, 515 Rennke. H., 314. 326, 410. 418 Renshaw, B. R., 173, 179 Rentschler, S., 334, 348, 367

Author Index

Repaske, D. R., 506, 515 Rep6rant, J., 83, t04 Represa, J., 466, 494, 545,547, 5591 564 Resau, J. H., 405, 417 Reshef. R., i4t. 142, 148, 262. 264, 268. 274, 275, 276, 399, 417 Resnick. J. L., 7, 19, t 88, 189, 189.. t 90 Retaux, S.. t I3. 125 Reutebuch. J.. 468. 485 Reuter, R., 165, 178, 186, ] 90, 316, 328 Revest. J.. 546, 562 Revest, J.-M.. 312~ 324, 505, 5]4 Rex, M., 308. 325 Rey, S.. 303.324 Reynaud-Deonauth, S., 10. 15, 342, 368 Reyne, Y., 271,273 Reynolds, B. A., 86, t03, 206, 206, 236, 245,246, 248, 249, 251, 252 Reynolds, D. M., 414. 415,418. 420 Reynolds, K.. 89, 101 Rhee. J., 341.36I Rhee, J. T.. 344. 345.370 Rhee-Morris, L.. 292, 294 Rhim, J. A.. 318. 328 Rhinn. M., 3 I. 33, 40. 44. 46, 47, 49, 50, 52, 1I4, 125, 323, 338, 358, 451,478,481. 494, 521. 527, 535 Rhodes. S. J.. 258, 274, 509, 517 Rhodin, J. A., 519, 537 Ricci. S., 94, 99 Rice, D. R, 452, 488. 494 Rice. D. S., 94, 99, t03, 104, 510, 516 Richard~ S.. 117, I 18, 124 Richard-Parpaillon, L.. 116, t25 Richards. A.. 188, t90 Richards, J., 527,530, 531,537 Richards. Wi G., 15, l& 226, 232 Richardson, C. D., 222, 231 Richardson. G.. 555,562 Richardson, J. A., 94, 105. 260, 261,262. 269, 273, 276, 277. 342, 343.344, 347, 353.364, 365, 366, 432, 465, 478, 483, 498, 532, 537 Richardson, M. K., t39, 148, 236, 249, 252 Richarson. J. A., 89, 101 Richieri-Costa. A., 468, 485 Richter, J. D.. 7, 10. 1t, 14. 16, t Z t8, 19 Richtsmeier. J. T., 442, 494 Rickles. R. J., 10, 14, 18, ] 9 Rickmann, M., 134, 148 RidalI, A. L., 288, 289, 292 Riddle, R. D., 117, t22, 141, I46, 262, 264, 275, 470, 48Z 494 Ridgeway, A. G., 342, 368 Ridgway, E. C., 504. 509, 5t0. 513, 515 RieckhoK G. E., 91,103

663

Riedl, A. E., 547, 562 Rieff, H. I., 94. 104, 343,369 Riethmacher, D., I70, 173, ]75, 179, 260,266,272, 317, 323, 404, 417 Riethmacher-Sonnenberg, E., 170, 173, 179 Rietze, R. L., 206, 206, 246, 249 Rifkin, D. B., 224, 232, 504, 517 Rigby, R Vv\ J., t 83, 190, 258, 260. 267, 275, 276, 475,496 Riggs, S., 357, 358 Rijli, E M., 117, 118. 119, t23, 125, 427, 433.443. 449, 45 t, 468, 469. 474, 479, 485, 490, 494, 545,562 Riley, B. B., 543, 557, 564 Riley, J. K., 287,293 Riley, R, 168, t 69, t78, 179, 349, 350, 352, 355, 367 Riley, R. R, 343, 349, 360, 367 Rimoin, D. L., 286, 294 Ring, B. D., 312, 327 Ring, B. Z., 527, 530, 53 t, 537 Ring, M., 91,103 Rinkwitz-Brandt, S., 85, 103, 549, 550, 562, 564 Rintata, M., 424, 442, 443,494 Rio, C., 94, 104 Ripoche, M. A., .~_0. 324 Riquet, J.. 264, 274 Risau, W., 203, 20Z 2t 1,212, 215, 217, 219, 220, 222, 223, 224. 226, 227, 228, 22& 229, 230, 231, 232 RisIing, M., 236, 237, 244, 245, 248, 250 Ristoratore, E, t 16, 125 Ritchie, H. E., "443, 453, 495 Rittenhouse, E., 442, 443, 494 Rivas-Ptata, K., 239, 249 Rivera, A. J., 27 I, 277 Rivera-P6rez, J. A., 3, 4, 40, 43, 50, 422, 427, 432, 473, 477, 482, 484, 494 Rivolta, M. N., 553,563 Rizzino, A., 166, 179 Robanus-Maandag, E., 376, 393 Robb, D., 185, 190 Robb. L., 31 34, 203. 209, 334, ~4_~..~ ~, ~48. o49. 352. 353, 356, 357, 358, 364 Robbins, E. S., 224, 232, 504, 517 Robbins, J., 343, 365 Roberson, M. S., 508, 517 Robert, B., 267, 274, 4i2, 414, 419, 476, 477, 496 Robert, C., 570, 588 Roberts, A. B., 226, 230, 314, 32L 339, 368, 467,468, 469, 494, 495 Roberts, A. W:, t 94, 208 Roberts, D. J., 305, 308, 328, 364 Roberts, H. M., t 83, 189 Roberts, J. M., 558,563 Roberts, R. C.. 451,475,494 Roberts, S., 44, 50 Roberts, u J., 467, 494, 509, 513

664

Author Index

Robertson, E. J., 30, 31, 33, 35, 37, 42. 43.44. 45, 46, 47, 49. 50, 5I, 78, 82. 98, 99, ]05, 133, 135, I37, 145, 148, 160. t 71,175, 204, 209, ~.,8, 251..~u_ 321 326, . . . . _~_~8,.339..~4. "" 355 . 357. 358. 359, 367. 368. 329, _~.6. 408.417. 422. 426. 427.451. 467. 468. 481,483. 484. 490, 495, 497. 532. 538 Robertson. K. E.. 534, 536 Robertson. S.. 195,209 Robey, E.. t 36, 148 Robin, N. H.. 433.492 Robins, E, 579, 585 Robinson. G.. 173.177. 450. 47 I. 494 Robinson. I. C.. 31 ,~3 4" 50. 451 476. 483. 500. 506. 509,514 Robinson. I. C. A. F.. 508.514 Robinson. N. A.. 581. 588 Robinson. V.. 119. 126. 225.233. 423.424. 494 Robison. W. G.. Jr.. 528. 533.535 Robitaille, L., 342, 343.366 Robl. J. M., 8. 16 Robson. L. G.. 266. 269,273, 276 Robson. S.. 357.364. 387. 390 Rocancourt. D.. 258. 260, 263.268. 277 Rocco. M. V.. 415.419 Rochette-Egly, C.. 469, 485, 489 Rockman, H. A.. 341. 352. 362. 363 Rockwell. K., 203, 209 Rockwell. R . _,,7. "~'~ 233 Rodaway. A.. 342. 367 Roder. J. C., 42.5I Roderick. T. H.. 527. 535 Rodig, S. J., 287.293 Rodrigo. I.. 355.359, 423,482 Rodriguez, T., 3 t. 34. 46, 47, 48.51.311,316, 324. 327, 339, 348. 360, 476, 490 Rodriguez de Cordoba. S., 522.525.535, 537 Rodriguez-Esteban. C., 354. 367. 478. 496 Rodriguez-Gallardo. L.. 541. 564 Rodriguez-Mallon, A.. 311. 324 Rodriguez-Rey, J. C.. 349. 354. 360, 366 Rodriquez-Estaban, C.. 507. 508. 518 Roehl. H.. 471,493 Roeten. B. A., 183, t90, 222, 229 Roelen, B. A. J., 336. 363 Roelink, H., 43, 50, 78. 99, t01, 102, 104, 120, 121,124. 125,914t 148, 4,.~, ~" 489 Roeser. T., 39, 51 Roessler. E.. 357, 359, 468, 470, 485, 491 Roessner, A.. 443,486 Rogers, A., 374. 375.392 Rogers, G.. 578. 588 Rogers. I.. 22. 34 Rogers, J. M.. 161, 178 Rogers, K. E., 12, 17 .

Rogic, D.. 416, 420 Rohdewohtd, H., 160, ] 78 Rohovsky, S. A., 226. 230 Rohrer. H.. 238. 251, 252, 434, 489 Roisen, K. 2 3 5 . 2 4 2 . 2 ~ , 250 Rojas. J.. 292, 292 Roiink, A. G., 204, 209 Roller, M. L.. 507, 509. 5]5 Rollh~iuser-ter Horst. J.. 423.494 Roman, B. L., 1222, 231 Roman& R., 557. 564 Romeo, G.. 346. 358 Romeo, R H., 200, 20 t, 208 Romer. A. S., 421,494 Rones, M. S., 3""J. 339. 366, 367 Rong. R M.. 140. 148 Rongo. C., 185.190 Ronicke, V., 228, 230 R6nning, O.. 424. 442. 443. 494 Roomans. G. M., 580, 586 Roop, D. R., 467, 468.490, 491, 571,580, 581,582, 583, 584, 587. 588 Ros. M. A.. 349, 351. 354. 355. 356. 359, 360. 366 Rosa. E M., 303.328, 338.366 Rose. E. A., 415. 417 Rosen, E. D., 289, 294 Rosen. V.. 271,275, 467, 498 Rosenberg, R. 409. 419 Rosenberg, R. D.. 219, 228, 229 Rosenblatt. M., 203,209, 387, 392, 510, 518 Rosenfeld. M. G., 85, 89, 91, 95, 97, 99, 100, 104, 105, 353, 354. 355,364, 367, 433, 478, 489, 496, 502, 504, 505.506, 507, 508, 509, 510, 51t,514, 515, 516, 517, 518, 556. 558,562 Rosenfeldt, H. M.. 227, 231 Rosenquist, G.. 344. 367 Rosenquist. G. C., 303,328 Rosenquist. T.. 95, 102, 506, 516, 578, 583,586 Rosenthal, A.. 85, lO1, 106, 110, 121. 123, I26, 402, 418, 420 Rosenthal, N. S., 31, 34, 335,339, 343, 344, 345, 346, 347, 350, 35 t, 352, 359, 366, 36Z 370 Rosewell, I., 288, 294, 3 t2, 324, 442,443, 453,493, 505, 514, 546, 562 Rosner, M. H.. 183, 190 Ross, A., 85, 100, 39t, 528, 537 Ross. F. R. 223,230 Ross. J.. 341. 352. 362, 363 Ross. M. E.. 94, 100 t Ross. R. S.. 345.367, 467, 495 Ross. S. A.. 346, 367 Ross. S. E., 271. 276 Rossant, J., 23.26. 31.33, 35, 40, 42, 43, 44, 46, 48, 49, 50, 51, 52, 78, 98, 1 t0, 112, 1 t5, 116, 121,122, 123, 125,

Author I

n

d

e

t33, 135, 136, I37. 139. I45, 148, ]49, 157, 158, 160. 165, 166, 167. 168, 169. 174. I74. ]75, i76, t77. 178, 179, t82, 183, 189, 193, 203,204, 20& 209, 2 t 7, 223, 224, 228, 229, 231, 232, 303, 304, 313, 316, 3 t 7, 323, 324, 32& 34t, 352, 357, 365, 36& 413, 4]9, 45t, 468. 478, 481,483, 49_5, 509, 514, 521,527,535 Rossel, M.. 119. 125, 545,564 Rosselli-Austin, L.. 237, 252 Rossier, C., 479. 482 Roth. W.. 577.583,584 Rothbacher. U.. 579. 587 Rothman. A. L., 287, 293. 579, 586 Rothman, J. H.. 165.180, 316, 330 Rothman. T. R. 244. 251 Rothnagel, J. A., 580. 581,584, 586 Rothstein. J. L.. 11, I2, t& I9 Rottapel, M.. t 89, 190 Rouaud, T.. 267, 274, 335. 337, 340. 358 Roume, J., 285,293 Rousseau, E, 286. 294, 453,494 Rousseau. G. G., 166, 176, 321,326 Rousseau. S., t 71.173, 176 Roussel, M., 556, 558. 561 Roux, I. L.. 553.559, 561 Rowe. D. ~:, 452, 484 Rowe, L. B.. 11. 12. I7 Rowitch, D. H., 112, 125, 228, 229, 230, 246, 249, 250, 334, 336. 348, 362, 363, 407, 418, 419, 426, 482 Rowley, J. D., 202, 209 Rowlitch, D. H., 86. 104 Rowning, B., 44, 50 Royer-Pokora, B.. 528, 538 Rozet, J. M.. 286, 294, 453.494 Rubel, E. W\, 558, 563, 565 Ruben, R. J., 542, 550, 552. 558, 563, 564 Rubenstein, J. L., 110, 114, 1 t 6. 121, ]24, t25, I26, 290, 292, 321. 329, 4,.,. "~v 477, _ 9428, 4"" "- 437, -449, ~_~,434, 4.~, 450. 451,453,466. 470, 471,472, 473,474, 475,477, 478, 481, 484, 489, 490, 493, 494, 495, 496. 497, 498, 549. 562 Rubenstein, J. L. R., 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85.86, 87. 88.89, 90, 91, 92, 94, 95, 9Z 98, 99, 100, 102, 103, 104, t05. t06, 443,449, 472, 479, 484, 495 Ruberte. E., 443. 469, 470, 474, 48"4, 490, 491, 494, 545, 557,562, 563 Rubin. E. M., 443.493 Rubin, G. M., 136, 147 Rubinstein, A. L., 78, 104, 351. 364 Rubinstein, M., 502, 515 Rubinstein. S.. 8, 18 Rubock. M. J., 479,482 Ruch, J. V., 454, 455,489, 493 Ruddle, E H., 157, 175, 376. 390. 450, 471,496 Ruderman, J. u 7. 17

x

6

6

5

Rudnick. A.. _~..I 325 Rudnicki, M. A., 258, 260, 261. 262, 267, 269, 270, 27 !, ~'7~ 273, 774, 275, 276 Rueda. J., 55 t, 557, 563 Rueger, D., 238, 251 Ruffins, S., 423,426, 494 Rugendorft\ A., 136, 146, 367 Rugh, R., 3 t 9.32& 442, 494, 500, 517 Ruhin, B., 426, 427, 433, 483 Ruili, L.. 317, 325 Ruiz, I., 579, 585 Ruiz i AItaba, A., 78, 83, 84, 86, 99, 103, !04, t2!. t24, 125, t41, t48, I65, I79, 316, 329 Ruiz-Lozano, R, 172, 173, 174, 34t, 342, 343, 352, 357, 35& 363, 367 Ruland, S. L., t61, t75 RuIleyn, L. J., 453, 493 Rumyantsev. R R, 341,367 Running, M. R, 11, 17 Ruohala-Baker. H., 185, t89 Ruosta,hti, E., 410, 4] 7 Ruotsalainen, V., 412, 418, 419 Rupp, R., 257, 277 Ruppersberg, J. R, 557, 564 Ruppert, S., t 83, 190 Rusch, A., 557, 564 Rusconi, J. C., t 36, t48 Rush, A. J., 247, 249 Rush, M. G., 287,292, 312, 314, 3 t 5,323 Russ, A. R, 160, 167. 178 Russell, A., 167, t74 Russell, E. S., 194, 195, t 99, 209 Russell, L. B., 467, 48& 525, 538 Russell, R. G.. 353, 357, 365 Rustin, R, 34 t, 352, 368 Rfither, U., 83, 85, 98, 103, 105, 342, 358, 442, 443, 470, 482, 497 Rutishauser. J., 506, 517 Rutland. R, 433,453,466, 474, 492, 493, 494, 498 Rutledge, J. C., 22, 34 Rutter, W. J., 168, 169, 175, 3 t 9, 32 I, 324, 32& 329, 349, 360, 509, 515 Ruvinsky, I., 339, 341,349, 370 Ryan, A., 541,542, 552, 555, 558, 559, 564, 579, 589 Ryan, A. E, 556, 558, 562 Ryan, A. K., 89, 91, 95, 104, 354, 367, 478, 496, 506, 507, 508, 509, 516, 5t7, 5l& 556, 558, 562 Ryan, A. M.. ,._'~')3,229. 9291 ,292 Ryan, G., 204, 210, 220, 233, 400, 419 Ryan, H. E., 225,232 Ryan, K., 160, 167, t78 Ryan, S., 4 t 6, 420 Ryans, A. M., 402, 418 Ryazanov, A., 15, ]8

666

A u t h o r Index

Ryder. E. F.. 527,535 Ryoo. H. D.. 9t. I03 Rzhetskv. A 5 v~, 538

S S~iS.m~inen, A. M., 424, ~ 2 . ~ 3 . 494 Saarialho-Kere. U.. ~ 1. 488 Saarma, M., 402,403.4t9, 420, 559. 564. 565 Sabapathy. K.. 87. 104 Sabbagh. W.. 354, 367 Sabin. E, 200, 209 Sabin, F. R.. 211,232 Sab!itzky, F.. 10. t 7 Sabourin. L. A.. 260. 270. v'71,,.272. ,.'~76 Sachs. M.. 266. 276 Sack, R. A., 510. 516 Sadaghion. B.. 425.494 Sadeghi-Nedjad. A.. 507.517 Sadikot. A. F.. 86. 104 Sadl, V.. 118. I24 Sadler, T. W.. 110. I25 Sadovsky, Y., 510. 516 Safar, E, 236. 251 Saffitz. J. E.. 165. 179 Saffman. E.. 182. 185. 190 Saga. Y.. 135. 137. 138. 146. 148, 340. 352. 363. 367. 512. 518

Sagai, T., 83, 102 Sagata. N.. 7. 16. 18 Saha. M.. 347. 360 Sahai, E.. .~4~.. " "~ 369 Sahenk. Z.. 271. 273 Sahly. I.. 398.416, 550. 561. 563 Sa_hni. M., 286. 294 Saiardi, A.. 51 I. 517 Said. J.. 510. 518 Saijoh. Y., 47.51. 352. 354. 356. 357.365. 367 Sainio. K.. 375.392, 403.419 Saito, M.. 341,352. 370 Saito. T., 88. 102, I68, 177. 239. 250 Saitou, M.. 581. 588 Sakagami, M.. 555.565 Sakai, L. Y.. 572, 587 Sakai, M.. 183, 190, 53 I, 538 Sakai, N., 45 I. 475,493 Sakai, R., 17I, 175 Sakai. T.. 136. 137. 147, 511,515 Sakai, Y.. 338. 354. 356. 368 Sakano. S.. 282. 295 Sakse!a. E.. 405, 419 Sakuma. R., 338.354. 356, 367, 368 Sakuma. S .~_0. 370 Sakuta. H., 525,538

Salama, R. H., 350, 370 Salamon-Arnon, J., t 74, 178 Salas-Cortes, L., 387, 390, 392 Salazar-Grueso, E., 402, 420 Saldivar, J. R., 426, 427,494 Saientijn, L., 452, 492 Salinas. R C., 45.52, 268. 269, 274 Salminen. M.. 262. 268, 276. 547. 564 Satomon, D. S., 338, 367 Saltiel. A. R.. 162. 176 Salunga, R. C., 12, 17 Satvatoni, A., 509. 514 Salzberg, A.. 91. 103 Samad. T. A.. 91. 101, 104 Samadani. U.. 321. 328 Samanta Roy. D. R., 87. 101 Samoylina, N. L.. t 96, 199. 200, 208, 220. 231 Sampath, K.. 78. 104. 354, 355,364 Sampath, T. K.. 78.81.99, 408, 416, 420, 467. 494 Samper. E.. 509. 514 Samson. D.. 398.416, 550. 561 Samuelson. L. C.. 51 1. 512, 5]5 Sanak, M.. 286, 294 Sanbo, M., 223, 230 Sanchez. A.. 388. 391 Sanchez, M. R. 402. 419 Sfinchez. S.. 354. 367, 423.495 Sanchez de Vega, M. J., 333. 352, 354, 362 Sanchez-Gomez, C., 344, 348, 355,360 Sanchez-Pacheco, A., 510, 5 t 7 Sandell. L. J., 282. 295 Sander. 88. 104 Sander. M., 32 I, 328, 451. 474, 475,494 Sandgren, E. R. 318.328 Sandilands, A.. 527,530, 53 t, 537 Sands, A. T., 287,294 Sandulache. R., 522. 527, 535. 549. 562 Sanes. J. R.. 237.249, 251, 411. 412, 418, 419 Sanford, L. R. 453, 467, 494, 550, 563 Sangha. R. K., 174, 179 Sangiorgi, F., 428, 452, 489 Sanicola. M., 403,420 ,~. 589 Sanjo, H.. 452. 496, 580. 581, 8.~, San Jose. I.. 559, 564 Sanne, J. L., 469, 483 Sano. Y.. 11.12. I7 Sans. A.. 552. 564 Santanna. C.. 292, 294 Santella. L., 23.34 Santerre. R. F., 258.274 Santoro. S. A.. 314. 329 Santos, E., 171. I73. 178 Santos, V., 428, 49t Santschi, L., 239, 250

A u t h o r Index

Sanvito, F.. 321,328 Sanyanusin, R, 398. 419 Sanz, R., 525,535 Sapin, V., 557,564 Sapir, T.. 94, 104 Saplakog!u, U.. 357, 359 Sarapura, V. D., 509, 515 Sarasa. M.. 337, 359 Sargent, M. G., 355, 356. 362, 423,492 Sareent. T. D. 17_ t77, 580. 583.587 Sariola, H., 341,352. 363, 375, 391, 392, 400, 402, 403, 418, 419, 420, 453,467, 494 Saris, J. J., 4i4. 418 Sarkar, D. K., 504, 511. 513, 5t5 Sarkar, L.. 440. 494 Sarvetnick, N., 318. 322, 325, 326, 328 Sasai, Y., 44, 52, 338. 359 Sasaki. E, 504. 517 Sasaki. H., 40, 41, 42.43, 47, 50, 52, t20, t2!, 123, t25, 135. 148, 303.328. 338. 355,370, 376, 393, 470, 477, 479, 484. 490, 494, 522. 537 Sasaki. K.. 204. 209, 220, 232, 288, 293, 442, 443, 453, 488 Sasaki, N., 1 I, 12, 18. 511. 516 Sasaki, T., 318.32Z 574, 583. 584 Sasse. J. 317..,30..~.~7. 370 Sassevitle, R.. 86. 104 Sassone-Corsi, R. 379, 384, 391, 393 Sassoon, D.. 226, 232, 258, 262, 267, 269, 273, 274. 275, 276. 277, 334. MS. 367. 385 391. 476.4c.~ Satake, M., 220, 231 Sater, A. K.. 335. ~ . 339 362, 367 Satin. J.. 343.344. 35 t, 367 Sato, B., 466, 496 Sato, K., 84, 100, 3I 1,330 Sato. M., 288, 293, 442. 443,453, 488 Sato. N., 579, 588 Sato. S., 266, 277, 399, 419 Sato. T., 1i2. 114, 1t5. 123, 312,328, 505,517, 525,535 Sato. T. N., 222. 223, 224. 226, 227. 228. 231, 232 Sato. Y.. 571,586 Satoh, N.. 266. 27Z 475,492 Satoh. T., 512, 518 Satokata, I., 290. 294, 437. 440. 450, 45 t, 453. 466, 474, 482, 495 Sauer, B.. 15, 18 Sauer. H., 402, 418 Saueressig, H., 410, 418 Saugier, R, 286, 294 Saulnier, D. M.. 225.229 Sauls, A. D., t t. 12. 17 Saumhueter, S., 193.210 Saunders, G. E, 45I, 475. 486, 527, 528, 530, 536, 538 Saunders. J. C., 506. 517. 549, 564

667

Saunders, T. L., 6, I9, 378, 379, 393, +43, 482, 507, 509, 51 i, 512, 5t4, 515, 556, 564 Sauvageau, G., 242, 252 Savary, R.. 265,272 Sawada, Y., 588 Sawai, S.~ 318, 328 Sawchenko, R E.. 89. 95, 104, 506, 508, 5t 1, 5t3, 516, 517 SaweI. L., 579, 586 Sawyer, D., 3 t4, 324 Sawyer, H., 214, 230 Saxen, L., 22, 34, 375,392, 405.419 Saxton, T. M., I7I, t73, 175, t78 Sayama, K., 588 Sayre, E.. 94, 103 Scaat, M., 266, 276 Scalera, L., 338, 360 Scambler, R J., 353, 357,364, 365, 367 Scandrett, J. M., 165, 176, 333, 342, 362 Scapoli, L., 382, 392 Scartett, L. M., 507, 508, 500. 5 t 1, 5]4, 515 Scarpa, A., 27 I, 2 73 Scarpetli, D. G., 318, 328 Schach, U., 546, 565 Schaefer, J. J., 534, 537 Schafer, K., 260, 266, 275, 276 Schaffner, W., 3 t 7, 325 Schaible, K., 183, 187, 188, t89 Schatler, S. A., 3, 4 Schalling, M., 313,323, 374, 375,392, 467, 482 Scharer, E., 582, 583,587 Scharfmann, R., 321,32 7 Schartt. M., 388, 392 Schatteman, G., 220, 228 Schauer. G., 47, 51, 357, 363 Schechter. J., 512. 517 Schecterson, L. C., 558, 559, 564, 565 Schedl, A., 88, 99, 34 i, 365, 380, 393, 528, 537 Schedt, T., 242, 252 Scheel, D. W., 321,328 Scheele, J., 478, 489 Scheffer, I., 94, t00 Schelt, U., 433, 492 Schelling, M. E., 223, 232 Scherer, G., 281,293, 380, 381,390, 39i, 392, 393 Scherson, T. Y., 423, 425, 495 Schier. A. E, 45, 47, 50. 5t, 52, 78, t00, 338, 353, 354. 355,356, 357, 359, 361, 368, 370, 395, 41Z 428, 468, 495, 505,515, 534, 536, 546, 563 Schieren, G., 415, 419 Schilling, T. F., 422, 433, 465,491, 495, 497 Schimmang, T., 543, 545,546, 559, 564, 565 Schimmenti, L. A., 398, 419 Schindeler. A., 345, 348, 366 Schinke, M., 219, 232, 34 t, 343, 352, 369

668 Schinko, I., 560, 565 Schinzel, A., 281,293, 381.39t, 433,492 Schipani, E., 285. 286, 287, 292, 293, 294, 442. 488 Schlaeger. T: M.. 228. 232 Schlange, T., 340, 367 Schleinitz. M., 343,369 Schler. A. E, 85. 101 Schlessinger, D.. 357. 361, 44 1. 488 SchlondorfE J., 42, 5t, 479, 487 .-, ,.) SchmahI. J.. _~8~..392 Schmahl. W.. 94. t04, 263.272 Schmid. M.. 388. 392 Schmid. W., 314. 324 Schmidt. C.. 45.52, 130. 131. 132. 145, 146. 317. 328. 404. 419 Schmidt. H.. 281. 293. 381.39I Schmidt, J. V.. 225.231 Schmidt. L., _~7,~'~229 Schmitt. E. M.. 318. 325 Schmitt, J. R. 350. 353. 357.359 Schmitz. M. L.. 281. 293. 381. 391. 393 Schmoll. M.. 264. 277 Schnabel. C. A.. 449. 479, 487 Schnabel. D.. 508. 516 Schnapp, B. J.. 6. I8 Schne~elsber~... .. R N.. 258. 260. 276. 4_,.'~v484 Schneider, C.. 238. 252 Schneider. M. D.. 341. 342. 364 Schneider, R. -441. 497 Schneider, R. J.. 11, 17 Schneider. V. A.. 336. 337. 338. 339. 340. 367 Schneider-Maunoury. S.. 118. 125, 510. 518. 546. 564. 565 Schnurch. H.. 226. 228 Schoderbek, V~( E.. 508. 517 Schoeberlein. A., 264. 274 Schoeller. H.. 183.184. 185,189 Schoenherr. C. J.. 243.252 Schoenwo!f, G. C.. 38, 40. 50, 52, 132, 148, 317,328, 333, 334, 335, 336. 344. 361. 362, 368, 370, 397. 418, 422, 427. 433,496, 497 Schoter, H.. 23.34, t 60. 166. 175. t 78, 183, 190, 311,324 Schonemann, M. D.. 89. 95. 104, 506, 517 Schopp. J.. 441. 497 Schorderet-Slatkine. S., 7, 16 Schorle. H., 79, 104, 353, 357,365, 529. 537 Schorpp-Kistner, M., 173, 179 Schott, J.-J., 343, 345. ~48, .~" 9, 350, 351 352. 357. 359. 367 Schowing, J.. 432. 495 Schreck. R., 217. 220, 229 Schreiber. G., 16 I, 179 Schreiber. J., 170, 173, 179 Schreiber. M., t73, t79 Schreiber, R. D.. 287. 293

A u t h o r Index Schreiner. C. M., 2 ~ , 208, 318, 327 Schroder, R., 570, 587 Schroeter, E. H., 136, I46, 14,, 148 Schubert, C., 240, 243, 251, 252 Schubert. E R.. 140, 145, 263, 2 ~ , 265, 266, 273, 277 Schuchardt, A., 402. 419 Schuchmann. M.. 34 t, 369 Schuger, L., 314. 328 Schughart, K., t30, 132, 140, I4& 264. 265, 27Z 397, 420, 423. 492, 522, 527. 535, 549. 562 Schuh. A. C.. 203 209. "~17. "~'~'~ ~3") Schuhbaur. B.. 341,346. 347,352. 366, 469, 492, 545, 564 Schulte. D.. 525.537 Schulte. T.. 547. 564 Schulte-Merker. S., 336. 363 Schultheiss. T. M., 263,274, 328, 335. 336, 339, 367 Schultz. G. A., 5.9.18. 19, 310, 325 Schultz, J. R., 89. t06, 450, 47I, 498 Schultz. M.. 432. 473.477.498 Schultz. R. A 334. o4.. 361 Schultz. R. M., 5 . 7 . 8 . 9 , 11. I2, 13, 14, I5, I6, I Z l& 19 Schulz. R. A.. 262.276, 334. 34 t, 363, 364, 367 Schumacher. C. A., o" ~ 4 ''~o. 345,348, 366 Schuster. G.. 352. 354. 366 Schutz, G.. 40, 42, 51, 165, 17Z 303,314, 324, 32Z 479, 487 Schwab. M. H., 92. t04 Schwanzel-Fukuda, M.. 435.495 Schwartz. C., 470, 481 Schwartz, J. H., 250 Schwartz, K.. 343,366 Schwartz. L.. 43.50. 133. 145. 158, 165, I75, 203, 209, 217. 224, 231. 232. 303.304, 3 t 3, 316, 317, 324, 328, 413.419, 468.495 Schwartz. R. J., 336, 342, 343, 344. 359, 360, 364, 366, 367, 369. 370 Schwarz. J.. 342, 350. 352. 364 Schwarz. M., 115. 125, 3t 1,329 Schwarz, S. M., 27, 34 Schweickert, A., 35 I, 353, 354, 355, 356, 359, 36t Schweppe, R. E., 505,517 Schwind. J. L.. 433.495, 500. 517 Schwitzgebel, V. M.. 321,328 Scofield. R. M.. 27 I. 273 Scott. A., 318, 328. 453,487 Scott, B. W., 247.252 Scott, I. C.. 169. t 70, 178, 179, 349, 367 Scott. M. E, 78.80, 86, ]00, 106, t21, t23, 287, 293, 294, 470, 485, 574, 577, 579, 583, 585, 588 Scott. W. J., Jr., 204, 20& 318, 327 Scotting, E J., 308.325 Scully, K. M., 502, 514 Scully, S., 226, 232, 312. 327 Seale, R, 260, 269, 270, 271,272, 276

Author Index

Searcy, R. D., 342, 346, 367 Searcy-Schrick, R. D.. 347. 364 Seasholtz, A. E, 502, 5 t 1. 513 Sebbag, M., 58 t, 588 Sechrist. J.. 4~.~. "~'~ 425,426, 4 ~'" ~ . 47~_, 48t, 494, 495, 496 Seckt, J. R., 506. 514 Seeburg, R H., 51 ~.. "~ 5t6 Seed. J.. 269, 276 Seegmiller, R. E., 424, 442, 443,493. 495 Sefton, M.. 354. 367. 423, 495 Segal, Y.. 415, 417 Seghezzi, G.. 224, 232, 504. 517 Segil, N., 556, 558, 561 Segr~, G. V., 285,287. 293, 294, 442, "443, 470, 489, 497 Segr6, J. A.. 271,274, 570. 572, 58I, 583,586, 588 Seibel, W., 161. ] 79 Seidah, N. G.. 136, ]46 Seidman, C. E., 339. 343. 345,346, 347, 348, 349, 350, 35I, 352, 353,357, 358, 359. 367 Seidman. J. G., 339, 343,345.346, 347, 348, 349, 350, 351,352. 353.357,358, 359, 367 Seifert, E., 316. 329 Seifert, K. J.. 382, 387. 392 Seifert. R. A., 467,495 Seike, M.. 94. 103 Seitanidou, T., 118. 124, 125, 468. 492, 546, 564, 565 Seitz, C. S., 580, 583,588 Seker. M.. 573,584 Seki, T., 582, 584 Sekimoto, T., 305,307, 328 Sekine, K., 312, 328, 505.517 Selby, R B., 288, 294, 442, 443,453, 493, 495 Seldin. M. F.. 172, 173.176, 476, 498 Seleiro, E. A., 45, 52 Self, T.. 556, 565 Selin. L. K., 415,418 Sellars, S., 292, 294 Selleck, M. A., 39, 40, 41, 52, t2I, 123, 125, 423, 484, 495 Seller. M. J., 525,535 Sellheyer. K., 468, 49] Semb. H.. 322, 324, 409, 419 Semba, I.. 282. 294 Semenova, M. L., 199, 208 Semenza, G. L., 225,230, 231, 232 "~ . 370, . 478. . . "~ ~ , 537 Semina. E. V.. ~54, 495, 507. 517, 527, 5~.. Senba, E., 318, 325 Senft, E., 522, 527, 535, 549, 562 Sengel, R, 568, 57 t, 572, 573, 574. 58Z 588 Senior, B.. 507, 517 Senior, R. M 2_a, 232, 281 29i 294 Seno, M., 321,327 Seo, J.-W.. 352, 354. 365, 367 Sepulveda, J. L., 342. 343, 367 Sepulveda, V. N., 342, 366

669

Serbedzija, G. N., 79, 85, t02, 106, 237, 252, 339, 36& 424, 425,495, 529, 538 Serrano, A., 9 i, 102 Serre. G.. 58 t, 588 Serreri, L., 443, 449, 479, 495 Serup, R, 80, 82, 88, 9& 310, 32t, 325, 327 Sesay, A. K., 9, 18 Sessa, V~[C., 225,231 Sestan, N.; 136, 148 Setalo, G., 504, 516 Seth. R K., 580, 585 Seth, R., 3I 4, 329 Seto, M., 376, 390 Seufert, D. W., 395, 420 Seung, K. J., 82, 101 Seung, K. S., 3t5,326 Seuntjens, E., 5 t t, 517 Seydoux, G.. 185, 190, 243, 249, 252 Sha, W. C., 3 t 8, 323 Shabanowitz, J.. 226, 230 Shackleford, G. M., 466, 486 Shafritz, A. B., 27 t, 276 Shafritz, D. A., 318, 324 Shah, N. M., 235,238, 239, 240, 2-44. 25], 252 Shah, S. B.. 45, 52 Shahar, S., 22, 34 Shahinian, A., 341,370 Shalaby, E, 203,209, 217, 223,232 Sham, M. H., 1t7, 1t8, It9, 124, 380,389, 422, 427,433, 468, 48Z 490, 492, 544, 563 Shamim, H., i 16, I25, 546, 565 Shamu, C. E., 382, 387, 392 Sham Z., 388. 392 Shani. M.. 258, _6~, 267, 274 Shanle?, S., 287, 292, 470, 486 ShanmugaIingam, S., 78, 86, 104 Shannon, J. M., 313, 324 Shapiro, B. M., 27, 33 Shapiro, D. N., 268. 273 Shapiro, L., 408, 419 Shaeiro, M. D., 335, 343, 344, 345.346, 347, 366, 370 Shapiro, R. L., 224, 232, 504, 517 Shapiro, S. D., 223, 232, 28 I, 291,294 Shapiro, S. S.. 303, 329 Sharara, R., t 1, t2, 17 Sharkey, A., 357, 358 Sharkis, S., 194. 207 '~ 7 Sharma, A..: ~2_. 328 Sharma, K., 104, 504, 505, 507, 508, 510, 5 t Z 518 Sharon-Friiing, R. 5~_, 537 Sharp, R A., 14, 19 S h ~ , A., 162, t70, 176 Sharpe, A. H., 201. 204, 210, 220. 232, 233 Sharpe, M., 317, 328, 404, 419

6 70 Sharpe, R T., 121. 123, 422, 432, 434, 437, 438, 439, .440, 441,450, 451,467, 47t. 472, 478, 484, 485, 486, 490, 493, 494. 495, 496, 497 Shashikant, C. S., 376, 390 Shatz, C. J.. 92.9Z ]02 Shaw. G.. 372. 386. 389. 393 Shaw. J. R. 22.34. 451. 475.488 Shawber. C.. 136. 148. 262. 276 Shawlot. W.. 31.34, 40. 41.42.43.44, 46. 47.48.52, 104, 114, ]25, 32& 374. 392, 397, 420, 478. 494 Shawver, L. K. "~17. ~ 0 ,,~.7 229. 231 Shea, M. J.. 3t. 34, 4 3 . 4 5 . 5 I , 132. 137, 148 Sheehan. K. C., 287. 293 SheldahI. L. C.. 337.363 Sheldon. M.. 94, 99, 104 Shelton, J. M.. 94. 105. 269. 273. 277, 342. 369 Shen. H. M.. 162. 170. 177, 219. 230 Shen. L.. 78.99. 141. 145. 336. 363. 384. 389. 402, 407. 418, 419. 470. 484 Shen. M M.. 31.33. 40. 45.50. 5~ ~.6 ~. 272. 338 353. 354. _~6. 35 J, 359, 360. 368, 370. _~8_. 387. ?92 428. 450. 468. 471. 472. 473.495. 498 Shen, W. H.. 376. 380. 381. 384. 390. 39]. 392 Shen, Y.. 94. I03 Sheng. G.. 546. 565 Sheng. H.. 502.508. 509. 517 Sheng. H. Z.. 83.94. 104. 106, 402. 419, 500, 504. 505. 507. 508.510. 517. 518 Shepard. A.. 416. 420 Shepherd. K.. 314. 326. 410. 418 Sheppard. A. M.. 94. 104 Sher. A. E.. 442. 473.495. 542. 552. 553.565 Sherbon. B., 471. 493 Sherman, M. I., t 56, ] 74 Sherr, C. J., 170, 179 Sheth. A. N.. 87. 104 Shevchenko. V. I.. 9. 18 Shi. H.. 15. I8 Shi. L.. 84, 88.89.90. 92.97. ]04, 450. 470. 481 Shi. X. M.. 121. 124. 451. 453.470.471. 491 Shi. Y.. 336. 368 Shiang. R.. 286. 292, 294. 453.495 Shiba. H.. 376. 393 Shibata. Y.. 376. 392 Shibusawa. N.. 512, 518 Shibuya. m.. 217, 230 Shiere. F., 507.514 Shigetani. Y., 475,492 Shih, J., 40, 52 Shih, M. S.. 416, 420 Shiino. M., 5 t2, 515 Shikayama, T.. 379, 39t Shilo. B.-Z.. 340, 358, 370 Shim, E. Y., 243,249

A u t h o r Index Shim, H. H.: 398, 419 Shimamura, K., 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 88, 89. 92, 98, 102, t03, t04, t06, 110, 121, t25, 126, 408, 418, 422, 427, 428, 494, 495 SNmasaki, S., 577, 586 Shimazaki. T.. 89. 9I. 104 Shimizu. H.. 570. 583.584 Shimizu. K.. 202. 208 Shimizu, Y., 288, 293, -442. 443. 453.488 Shimon, I., 510, 518 Shimono, A., 47, 49, 51, 52, 318. 328, 357, 365 Shimouchi, K.. 58t. 588 Shin. J. J.. 556, 565 Shin. I". H., 243.249 Shinanura. K., 83. 103 Shinmei. Y.. 531. 538 Shinoda. K.. 376. 393 Shinomura. T.. 350, 370 Shinpock. S. G.. 204. 208 Shintani. T.. 525.538 Shinya, M.. 41, 51, 78, I04 Shinya. T.. 427.484 Shioda. T., 339. 368 Shioi. T.. 343. 363 Shiojima, I.. 336, 343.365, 368 Shiojiri, N.. 317. 328 Shiota, K.. 136. 137, 146, ]47. 167~ I73, 179. 180, 341. 352. 370, 404, 420 Shipley. J. M.. 223,232, 281,291,294 Shipley. M. T., 84. I00 Shirakata. Y.. 588 Shirakawa. H.. 8.16 Shiratori, H., 338, 354, 356, 368 Shirayoshi, Y., 226, 232, 352, 354, 365 Shiroishi. T., 83, 102, 376, 391 Shivdasani. R. A., 203.209 Shmitt, D., 58 I. 588 Shoji. H.. 577.586 Shoji, R.. 557. 565 Shope. C.. 559, 563 Shore. E. M.. 271. 276 Shores, E. W.. 442, 484 Shors, T. J.. 237,250 Short, R. V., 372, 392, 393 Shou. J., 555.566 Shou. W.. 44.5I Shown. E. R. 378,390 Shtein. G. I., 169, 180 Shtutman, M., 86. 104, 579. 588 Shu. H.-B., 341,370 Shugar, J. M., 552, 564 Shuler. C.. 314. 325, 434, 465.467, 48Z 491 Shultz, L. D., 195,208, "442, 498 Shum, L., 282, 294, 314, 329, 428, 465,491, 492

Author I

n

d

e

x

Shutter. J. R., 226, 230, 232 Sibitia, M., 169, ] 79 Siciliano, M. J., 202, 208 S ideras-Haddad, E.. 580. 585, 587 Sidman, C., 194, 207 Sidman, R. L., 92, 94. 97, i03 Siebel, K. E.. 193,207 Siebenlist, U., -442, 484 S ieber. B.-A.. 402. 420 Sieber-Blum, M., 236. 237. 249, 252 Sieget-Bartelt. J.. 478. 495. 507.5t7 Sigala, S.. 51 I. 516 Siggers, R. 390 Si~rist. . . K.. . 165. . 177. . . 303.316. . . .326. 3;~Z _~4z. "~ "~ .~4~, "~ ~ _352. 363 Siiberbach. G. M., 357, 358, 367 S ilberg, D., 310, 325 Silengo, M., 346. 358 Sillence, D. O.. -443. 453, 495 Silos-Santiago, I.. 91.99, 402.4t9, 559, 562 Silve. C.. 285.293 Silver. J.. 542, 559, 561 Silver, L. M.. 132. !45, 339. 34t, 349, 370 Silver. M., 220. ,._~8, 5"".~.535 Simak. E.. 264. 274 Simard. A. R.. 269. _. P77. .54,. " "~ 369 Simcha, I., 86, 104, 579, 588 Simeone, A., 31.33. 40, 46, 48, 49, 52, 79, 83, 84, 95, 97, 103, 104, 112, 114, 122, I23, 124, 266, 275, 290, 292, 294. 311 322, 330..~.~8. 360, 428. 450~ 451 453.47 I 472, 473,478. 481, 491, 495, 508. 513, 52 i, 527, 535, 549, 550. 55 I. 552. 56t, 564 Simmons, D. M.. 89, 100, 502, 506. 509, 514, 515, 5t6, 518 Simmons, W. W.. 219, 228 Simon, M., 58t, 588 Simon. M. C.. t 65, 172. 173, 174, 174, t 79, 204, 209, 225, 231, 286. 292 Simon. R.. I 0. 18 Simon-Assmann. R M., 308, 325, 326 Simonet, W. S., 312. 327 Simonetti. D. V~[,206. 209 Simpson, B. B.. 47, 50 Simpson, E. H., 31, 34, 378, 387, 390 Simpson, K.. 379. 390 Simpson, R. 136. 148, 343, 360. 36I Simpson, T. H., 557, 563 Sinclair, A. H., 379, 382, 387. 388. 393 Sinclair, A. M., 192. 209, .~77, J80, .~8,, 388, 391, 393 Sinclair, R. A., t94. 208 Sineone, A., 506, 5 t3 Singal, A., 341,369 Singh, G., 79. t05 Singley, C. T., 424, 495 ,

.

' ~

~

~

,

~

~

"7

..

,

6

7

1

Singson, R., 504, 515 Sinickas, V., 194, 208 Sinning, A. R., 342, 368 Siracusa, G., 86, 103, t 88, 190 Siracusa, L. D., 157, 178 Sirard, C., 468, 495 Sisi, R, 568, 582, 586 Sissman. N. J., 34i, 354, 355,368 Siwik, E., 34 t, 369 Sjodin, A., 409. 4] 9 Skaer. H., 171, ] 79 Skandalakis, J. E., 312, 328 Skapek, S. X., 341,361 Skeath. J. B., 340, 36t Skerjanc, I. C.. 342, 368 Skoldenberg, E., 504, 515 Skolnick, M. H., 412, 4t7 Skoultchi, A. t., 353, 357, 365 Skowronski, J.. I t, 12, 18 Skromne, I., 45, 48, 50, 52, 427, 484 Slack. J. M., 27.34, 167. 176. ,6_~, 274, .~20. 328 Sladek, E M., 164, t 77, 179 Sladek, N. E., 525, 53 7 Sladek, R.. 167. ] 77 Slaney, S. E, 453,466, 474. 494, 498 Slav~n, H. C., 282, 294, 314, 329, 428, 465, 49t, 492 Slepecky, N. B., 557, 558, 565 Slieker, W. A. T., 192, 210 Sloviter. R. S., 247, 251 Slusarski, D., 121,123, 470, 48Z 497 Small, K., 95, 101, 507. 516 Small, K. M., 82, 105 Small, K. W.. 478, 495, 507, 517 Smalley. M.. 440, 494 Smalls, O.. 357, 358 Smart, i. H., 87, 104, 572, 588 Smets, G., 502, 515 Smeyne, R., 313, 327, 559, 562 Smidt, M. R, 506, 513 Smiga, S. M., 76, 77, 82, 83, 84, 92, 98, 103 Smith, A., 23, 34, 160, 166, 178, 423,424, 494 Smith. A. G., 160, 178 Smith. A. J.. 203. 210 Smith. B., 34I, 352. 362 Smith. C. A., 379, 382, 387, 388, 393 Smith C. K. D., 258, 274 Smith C. K. II. 271. 276 Smith, D., 397,418 Smith, D. E., 135, 148 Smith, D. M., 308. 309, 328, 329, 337, 354, 364 Smith, E. A., 286, 292 Smith, G. H., 226, 230 Smith, J. C., 40, 49,. 167, ] 79, 203, 20Z 209, 303,323, 329, 337, 338, 362, 368

6 72

A u t h o r Index

Smith, J. M., 467. 494 Smith. K.. 223. 227. 232 Smith. L. J.. 25.26.34. 390. 432. 496 Smith. L. T.. 573.583.584. 588 Smith. M J. ~ t7.388. 393 Smith, M. M.. 433.436, 495. 497 Smith, R. 251 Smith, R., 26. 34, 84, 102 Smith. R. A.. 219. 220, 232 Smith. S., 314. 315.329, 330 Smith. S. M.. 34I . 342.35"~. 367, 368 Smith. T. H.. 132. 148, 263.276 Smith. T. W.. 219. 228 Smith, V. V., 305,328 Smith, W. C., 44.50, 52. 238.252 Smithers. L.. 139. 146 Smith-Fern~indez, A.. 83. 104 Smits. R. 283. 284. 289. 294 Smyth, I.. 287,292. 470. 486 Snead. M. L., 428. 452. 489 Snell. W: J.. 8. 18 Snider. L.. 169, 177. 258. 277 Snider. \~ D.. 411. 418 Snoeck. I.. 94. 99 Snow, M. H. L.. 128. 148, 183.188. I89. 190 Snyder. E. Y., 86, 104. 527. 538 Snyder-Keller. A., 91.98 Soares. M. B.. 390 Soares, V. C.. 78.81. 106, 243.252, 408.417, 479. 498 Sobe; T., 341. 369, 556. 565 Sobrier. M. L.. 508. 516 Sockanathan, S.. 88. 105. 377. 386. 387. 390. 53 I. 536. 537 Softer. D.. 341. 369 Sofroniew, M.. 376. 393 Sohal. G. S.. 426. 495 Sohocki. M.. 408.418, 451,467.490 Soker. S.. 223.232 Sokol. S. Y.. 415.418. 579,585 Solloway. M. J.. 82.86. 105. 135. 148. 336. 368, 426. 467. *

~

3

"v

.

495

Solnica-Krezel. L.. 78. 104. 395.41Z 546. 563 Solomin. L., 509, 518 Solomko, A. R. 26, 27, 33 Solter, D.. 6. 8.9, 10. 1I. 12. 13.16. 17. 18 Solum, K., 443, 487 Solursh, M., 424, 473,495, 498 Somerset, D. A., 174. 179 Somlo. S., 414. 415,418, 419, 420 Sommer, A., 292, 294 Sommer, L., 102, 236. 239, 250. 251. 252, 320. 321,323, 526. 538 Sommer. E, 316, 323 Sonenberg, N.. 11.16 Soneoka, Y., 193, 206, 207, 246, 250., 270. 271,274

Song, D. L., 113, 125 Song, H., 588 Song, K., 267,277 Song. Y.. 437,498 Sonksen, R H.. 507, 514 Sonnenberg, A.. 572. 583.585, 589 Sonnenberg-Riethmacher, E., 260, 266, 273 Sood, S., 453,495 Sorensen, L. K.. 226, 230, 232 Soria. J. M.. 84. I05 Soriano, R. 79.94. 100, 10I, 157. 173, 178, 180, t83~ 190, 228.229. 230, 246. 249, 250, 334, 341,348, 352, 359, 362, 413,414, 418, 419, 426, 442, 443, 467,482, 495 Sornson, M. W., 506, 508,509, 5t 8 Sorrentino, V., 189. t90 Sosa-Pineda. B., 317.321,329, 451,475,495. 532, 537 Sotelo. C.. 95.101, 113. 122 Souabni. A.. 311. 326, 533,535 Soudais, C., 165, 177, t 79, 316, 326, 342, 343, 352, 363 Souleyreau-Therville, N., 375,382. 389, 392 Soulez, M.. 342, 368 Soults. J.. 357,358 Southard, J. L., 137. 149, 311,323 Southee. D.. 384, 391 Southwell, B. R., 161, 179 Souza. R, 313,329 Sowden, J. C., 334. 365 Spada. E. 9. 18 Spagnuolo, R., 226, 229 Spana, E. E, 532, 537 Spangmde, G. J., 192, 209, 244, 248, 252 Spano, R, 511,516 Sparkes, R., 452, 453,474. 487 Sparks. A. B., 86. 100, 579. 586 Sparrow, D. B.. 335,336, 337. 339, 342, 345.348, 355, 365, 366, 368

Speck, N. A.. 196. 199. 200, 201,202, 204. 207, 209, 210, 220, 231, 232, 233 Speicher, S. A.. 136, 149 Spemann, H.. 40, 48.52, 427,495, 538 Spence, A.. 42.52, 110, 125, 158, 178 Spence-Dene, B.. 312, 324 Spencer, J. B., 136, 137, 147 Spencer-Dene, B., 505,514, 546, 562 Sperbeck. S.. z.~_. 537 Spicer, D. B., 341,361 Spiegel, S.. 227, 23t Spiegelrnan. B. M.. 289, 294, 442. 487 Spiegelman, M.. -442, 443,494 Spindle, A. I.. 158.179 Spinner, N. B., 347. 364, 366 Spirina. O. M.. 510. 518 Spiro, A. C., 88, 98 Spitz, E, 262, 268, 276

Author Index

Splitt. M., 357, 359 Spohr, G.. 1O. 15, ~4,_ 368 Spooner. B. S., 319, 329 Sp/Srle, R., t30, t32. 140. 148. 264, 265. 277, 423,492, 522. 527, 535, 549,562 Sporn, M. B., 226. 230. 314. 325, 467, 468, 469, 494, 495 Spotila, J. R., ".~8,. v 393 Spotita, L. D.. 387,393 Spradling, A. C.. 6. 7.18 Sprawson. N.. 425.490 Sproat, G.. 1 t 8, t 23 Sretavan. D. "%~.85. 102. 506, 514 Srivasta, A. K., ~ 1,488 Srivastava, D.. t 68, 175, 341,342, 343,344, 346, 347, 349, 350, 352, 355,358. 360, 361,365, 366, 368, 369, 370, ' 495. 496 451 , 465. 497.~. St. Jacques, B., 78, 86, 99. 104. 141,145, 287, 293, 294, 442, 443,470, 484, 488, 496, 577,578, 583, 589 St. JohmR L., 412. 4t4. 417. 419 St. Jore, B., 353, 357. 365 St. Onge, L.. 321,329 St. Vil, D., 307. 329 Staccini, L., 27 I. 277 Stacey, A., 443.495 Stacker, S. A., 162, 174, 453,486 Stackhouse, T.. 227, 229 Stacy, Z, 201,204. 209, 210, 220. 231, 232, 233 Staeheli, E, 388, 392 Stahl, J., 5 t 1,512, 518 Stahle-Backdaht, M., 287, 292, 470, 497, 579, 586 Staines. W. A., 236, 245, 251 Stainier. D. Y., 1t5, t25, 213,214. 222, 230, 23!, 232. 303, 323, 326, 340. 341,342, 363, 36Z 546, 563 . . 337. . . 339..~" . "4 I.. .344. . . ~4~, " ' " M9. 361, 9 Stainier. D. Y. R... 334, 367. 368, 370

Stall, A. M., i 94, 20Z 245.250 Stalsberg, H., 34 I, 344, 355. 368 Stamp, G. W., 288, 294. 442, 443, 453,493 Stanford. W. L.. 203. 209, 2 i 7, 230, 232 Stanley, E., 31, 34, 40, "44, 50. 194, 208, 323, 338, 348, 349. 352, 353, 357. 358 Stanton. B. R., 341. 352, 365 Stappenbeck, T. S., 310, 329 Stark. K. L., i32. 137, 148, 226, 230, 232, 260, 276, 317, 329. 407. 419 Stark. M. R., 141,147. 262. 263. 264, 265,275, 435,475, 481, 496

Start, E., 202, 209 Starzinski-Powitz, A., 264, 271,275, 277 Stauber, C., 504, 510, 512, 516, 517 Staudt, L. M., i 83, 190 Stauffer. T. R, 225, 230 Stavlijenic, A., 416, 420 Stavnezer. E.. 268, 273

6 73

Stayton, C. L., 376, 393 Stead, R. H.. 258, 260, 276 Stebbins-Boaz, B., 10, I 1, 18 Steding, G., 386, 393 Steel, F. L. D., 442, 493 Steel, K. R, 546, 55 I, 552, 556, 557, 560, 561, 563, 565 Steele, K., 44, 50 Steele-Perkins, Vi, 400, 4] 9 Stefana, B., 512, 518 Stefanadis, J. G., t 61, 178 Stefaneanu, L., 5t l, 512, 513, 518 Stein, R, 9. t 4, I8 Stein, R. Wi, 309, 319, 320, 327 Steinbeisser, H.. 323, 354, _ ~ 359. 477. 482, 483 Steinbeisser. K. ~:, 40, 50 Steiner, D. Y., 22, 34 Steiner, K., 50 Steiner, K. A., 30, 31, 35, 39, 40, 4 I, 46, 52 Steinert, R M., 570, 580, 58 I, 582, 584, 586, 58Z 588 Steingrimsson, E., 168, 169, t73, 175, 179, 349, 36~ 525, .

3

" ~

.

.

.

.

536, 538

Steininger, Z L., 91, t05 Steinmetz, H., 341,363 Stemple, D. L., 235, 248, 252, 395, 41Z 546, 563 Stennard, E R. K., 203, 209 Stensas, L. J., 94, 103 Stephens, R. J., 313,324 Stephenson, E., 185, ]90 Stem, C. D., 2, 4, 39, 40, 41, 45, 48, 50, 51, 52, 75, 99, 134. t 38, 147, 148, 303, 329, 356, 364, 387, 393, 427, 484, 546, 565 Stem, H. M., i40, i48, 262, 263, 264, 277 Sternberg, E. A., 342, 361 Sternberg, N., 15, t8 Sternfeld, D. C., 204, 208 Sternglanz, R., 349,362 Stevanovic, M., 281,292, 380, 386, 387, 390 Steven, A. C., 582, 583, 587 Stevens, K. A.. 165, t 74, 303,323 Stevens, L. C., t37, 149, 532, 538 Stevens, M. E., 443,493 Stevenson, B. J., 32 t, 324 Stevenson, L., 31, 33, 46, 49, 451,478, 481, 521,527, 535

Stewart, A. E, 262, 277 Stewart, C. L., 43, 50, 156. 162, 170, 175, t7Z 179, 219, 230

Stewart, M. E., 582, 587 Stewm,t, R., 44, 50 Stewart, T. A., 322, 32& 512, 516 Stifani, S., t 68, 178 Stocco, D. M., 379, 393 Stock, D. Vv\, 450, 47 t, 496 Stock, J. L., 79, 105

674

A u t h o r Index

Stockdale, E E., t40. 144. 254, 263, 268, 269, 273. 275, 277, 335. 343.344. 345,346. 347. 369, 370 Stockinger, W.. 94. 105 Stoenaiuolo. A.. 478.49_5 Stoetzei. C.. 453,482 Stoller, J.. 334, 361 Stone. D. M.. 121, ]23, 402. 420 Stone, J. S.. 558.565 Stoner. C. M.. 469. 470. 484 Storey, K. G.. 40. 52 Storm. E. E.. 467. 496 Stornaiuolo. A.. 40.46.52. 79.83. I04. 206. 207. 246. 249. 271. 274. 290. 294. 450. 471. 472.473.495 Stott, D., 166. 176, 185, 189, 303, 325 Stott. N. S.. 577. 589 Stout. R. P.. 84. 105 Stoykova, A., 8 3 . 8 5 . 8 7 . 9 I. 94. 100. ]05. 451. 475.490 Strachan. T.. 357. 364. 387. 390 Strachen. T.. 352. 354. 366 Stradler, J.. 453. 488 Strahle. U.. 338.362, 366 Strain. A. J.. 174. ] 79 Strand. F. L.. 501. 518 Strates. B. S.. 271. 277 Strauss. A. W., 356. 357. 358, 366 Strawn. L. M., 227.23 t Street, S. L.. 441. 491 Strehle. M 35" 356. 361 Streit, A.. 44, 52 Streit, W. J.. 206, 209 Streuli. M.. 188. ]89 Strickland. C. D.. 193. 206. 207, 246. 250, 270, 271. 274 Strickland, K. R. 271. 275 Strickland. S., 10. 14. 15.18, 19 Striegel, J. E.. 416. 419 Strom. T. M., 378,392. 393 Strome. S.. 185.190 Strong, C. F.. 136, I46 Strong. D.. 284, 294, 442. 443.497 Strong, L. C.. 528. 538 Stroobant, R W.. 240, 244, 252 Stroschein, S. L., 268, 275 Strouboulis. J.. 195, 196, t 99, 200. 208 Strubin, M.. 32 I. 324 Struhl. G., 136, t48, 550. 563. 577. 587 Strunk. K. E.. ,_60, "~ " 265. _~72, _~76 Strunski, V.. 506. 5I 7 Struwe. M.. 130, 132, 140, 148. 264, 265.277 Smart, E. T.. 398, 420, 432, 473.477. 498 Studer, M.. 1 t7, 118. 119, I24, t25. 427.469, 485, 545, 562 Sti.ihmer. T.. 89. 106, 450.471. 498 Stutz, A., 7. 10, 16, 18 Su, B.. 172, t73, 180 ..

"~.

Su. G., 3t2, 327 Su. H., 346, 353, 357,364 Su, M. S., 87, I0t Su, W. C., 286, 294 Subbarao, V., 318, 328 Subbarayan, V.. 34I . . 368, . . 469, 492, . . 346. . . 347. ~""~ . , . 366, 545,564 Subramanian, S. V,. 262, 277 Sucov. H. M "~8 )79 230, 246. 249, 250, 334. 341 346. 348, 352. 362, 363, 36Z 368, 369, 426, 469, 482, 489 Suda. T.. 271. 275 Suda. Y.. 83.84. 105. I06. 114. 125, 451. 478. 496 Sudbeck. R. 281. 293, 381. 390. 39]. 393 Sudo, T.. 442, 498 Suehiro, A., 353,354. 355,356. 363 Suematsu, S.. 341. 352. 370 Suffolk. R., 31,34 Suga. Y.. 582. 583.587 Sugai. S.. 581. 588 Sugaya, K.. 226, 232 Sugi, Y., 335,336. 337, 364, 368 Sugihara. A.. 531. 538 Sugihara, M.. 354. 370 Sugimoto. A.. 165. ]80, 316. 330 Sugimoto. K., 189. 189 Sugimoto. T., 506. 5] 6, 549. 564 Sugimura, K., 473,498 Sugino. H.. 577. 586 Sugitani, Y.. 506. 516, 549. 564 Sugiyama, S., t I2, 1 I4, 115, ]23 Sugiyama. T., 471,479, 487 Suh. H., .~)o, 354. 355, 36I 4.._~,478. 485 Suh, Y. _4_,. 249. 354. 365 Suhonen. J. A.. 241. 252 Sukhatme. V. R. 400, 418 Sulik. K. K.. 38. 52, 341,352, 362, 468, 497 Sullivan. A., 220, 228, 246. 251 Sullivan, M. J., 398, 4 t 9 Sumida, H., 355, 362 Summerton. J.. 14. 18 Sumoy, L., 579, 584 Sun, B. I., 470. 489 Sun. E. S.. 136. 1."7 ~.147 Sun, L., 204, 210, 217, 220. 229, 588 Sun. Q.-Y.. 8. 18 Sun. T.. 11.12. ]7 Sun. T.-T.. 570. 585, 58Z 589 Sun. X.. 31.35. 337, 340. 368, 466, 491, 496 Sun. Y. H.. 91. 103 Sunada, Y., 161. t 79 Sundberg, J. R, 226. 230, 423, 487 Sundin. O.. 346. 36& 5"~9 5o0. 5o,.. 536, 538 Sunthornthepvarakul, T., 3 t 1,329 Suoqiang, Z., 558, 564

Author I

n

d

e

Supp, D. M.. 9~,., " ""~ ~>,. " "A 355,364, 368 Surani, M. A.. 158, 176, 258. 264. 266, 274 Surani, M. A. H., 26, 35 Suri. C., 223. 224. 227. 231,232 Susasurya, L.. 185, 190 Sussel. L., 80. 8 t, 82, 83, 88, 89, 90, 91, 9& 103, 105, 321, 328. 329 Sussman. D. J.. 45, 52 Sutcliff. K. S.. 312, 329 Sutcliffe. J. G.. 5I 1. 514 Sutcliffe, M. J.. 377, 386, 389 Suter, U.. 236. 250 Sutherland. D. J.. 226,231, 466, 485 Sutherland. H. E. 353.357. 364 Sutter, C. H.. 225, 231 Suvanto. R, 403,419 Suzuki, A., 336, ~:~.. "-'~ 365 Suzuki, H., 23, 35, 432, 450, 465.489 Suzuki. M.. 478.488, 506, 516, 549, 564 Suzuki, N.. I83. 190, 305, 329. 453, 467. 496 Suzuki, R.. 353,354, 355.356, 363, 525. 538 Suzu'ld, S. T., 91. 101 Suzuki, T.. 226. 232 Svensson, E. C., 341,343. 352, 368 Svensson. K., 167, 178 Svensson, M.. 574. 583,584 Svoboda, M. E.. 532, 535 Svoboda, R, 14, I8 Swain. A., 372, 378, 379, 380. 384, 387. 390. 39t, 392, 393 Swalla. B. J., 471,493 S wann, K.. 9. 18 Swanson. B. J., 170. 176 Swanson, G. J., 547, 549, 553, 564, 565 Swanson, L. "vV.89, 97. 100, 502, 506, 509, 5 t t, 514, 515, 516, 518 Swaroop, A., 528, 531,532, 536, 53Z 538 Swartz, M., 218. 223,230 Swartzendruber. D. C., 581. 589 Swat, W., 203,209 Sweeney, T. M., 452, 492 Sweeney. V~ E., 415.419 Sweet. H. O., 94, 105 Swerdlow, S. H., 204, 208, 318. 327 Swiatek. R J.. t36. 137. 148, 546, 553, 565 Swiergiel, J. J., 303,329 Swirnoff. A. H., 510, 516 Swolin, B., 413,418, 467, 489 Symbas, P.. 312, 328 Syrop, C. H., 22, 35 Syu, L. J.. 162, 176 Szabo, J., 452. 453,481 Szabo, K.. 504. 518 Szendo, R I.. 336. 370

x

6

7

5

Szeto. D. E, 353,354, 355, 364, 433, 478, 489, 496, 504, 505, 5f~, 507. 508, 509, 514, 518 Szollosi, D., 1, 4, 8, 16 Szonvi. E., 5 lV 516 Szucsik, J. C., 79, 82, I01, 105

T Tabata, K., 58 I, 582, 583,587 Tabata, M. J., ~ t, 493 Tabibzadeh, S., 47, 51 Tabin, C. J., 3, 4, 14 I, 146, 262, 264, 265, 268, 274, 275, 280, 285,287, 293, 294, 305, 308, 309, 328, 329, 337, 338, 339, 343,347, 354, 356, 357,359, 364, 399, 4t7.. 470, 487, 490, 494, 497, 571,586 Tada, M., 303,323, 329, 337, 368 Tadaka, N.. 31, 34 Tadic, T., 452, 484 Tadolini. B .. _~~."8.. " 369 Taga, T., 318,325, 341,352, 370, 405,407.416 Tagher, R H., 341,358 Tagishita, N.. 3 I2, 328 Tagle, D. A., 247, 25i Tago, K., 399, 419 Tahta, S. A., 349, 369 Taigen, T., 342, 365 Tainsky, M., 57t, 572, 580. 584 Taipale, J., 218, 223. 229 Taira, M., 83, 106, 374, 390 Tajbakhsh, S., 13 I, 140, t41,144, 145, I4& 258,260, 262, 263,264, 267, 268. 273, 274, 275, 277 Tajima, N., 305, 307, 328 Takabatake. T., 52 t, 53 7 Takada, K., 289, 294 Takada, R., 470, 490 Takada, S., 40, 44, 51, 132, 133, t35, 137, 141, 14Z 148, 149, 260, 264, 265, 275, 423,428, 487, 492 Takaesu, N., 45, 47, 50, 338, 355,359, 468, 483 Takagi, A., 340, 352, 363. 367 Takagi, C., 529, 53 7 Takagi, S., 528, 536 Takahara, K., 305,329, 453,467, 496 Takahashi, A., t 0, 17 Takahashi, E., 511,516 Taka~hashi. I., 282, 294, 465,492 Takahashi, J., 236, 237, 24 I, 244, 251, 252 Takahashi. K., 195, 196, 208, 209, 282, 294, 465,492 Takahashi. M., 241, 2"44, 252, 402, 420, 581,589 Takahashi, N., 271,275, 525. 536, 537 Takahashi. S., 338, 36& 5t 1,515, 527, 530, 531,536 Taka~hashi, T., 87, 92, 105, 220, 228, 52 t, 529, 534, 537 Takahashi, Y., t 0, 17, 308, 329, 571,586 Takai, S., 352, 354, 365 Takakura, N., 173.178

67 6 Takakusu. A...~'~"-tz, o76," ~80." 390, 392 Takano, H.. 220. 231 Takano, K., 338, 368 Takano. T.. 308,329 Takano, Y.. 290. 294. 437,450. 451,466. 474. 495 Takaoka, K.. 555.565 Takasaka, T., 506.516, 549. 564 Takasashi. Y.. 142. 149 Takashima, S., 223.232 Takauji, R.. 90. 105 Takayama, K.. 376. 380. 390 Takayama, S.. 308. 324 Takebayashi, K.. 555,565 Takebayashi-Suzuki, K.. 334. 368 Takeda, H., 41.47.5I. 78. 104, 532. 534. 536 Takeda. J.. 581. 583.589 Takeda. K.. 452.496, 531. 538, 580. 581. 583.589 Takeda. M.. 466. 496 Takeda. N.. 7. 16. 46.51, 83. 106. 114. 124. 451. 478. 488. 490. 521. 527. 537 Takeda. S.. 38.51. 289. 294 Takeichi. M. 226. _3~..~40. 366, 408.409.4] 7. 4 ]8. 419. 528.536. 538 Takemura. T.. 555. 565 Takenawa, J.. ! 89. 189 Takeshima. K.. 521, 53 7 Taketo. M. M.. 135. 137. 138, 148. 172. 173. 176. 512. 518 Taketo. T.. 384, 393 Takeuchi. J. K.. 525.536 Takeuchi. O.. 452. 496, 580. 581. 583, 589 Takeuchi. S.. 511. 515 Takeuchi. T.. 317.32 7 Takiguchi-Hayashi. K.. 84. 98. 100 Takimoto. E.. 336. 343.365. 368 Ta3cke. C., 138, 148 Takuma, N.. 504. 505,507.508, 5t0. 518 Talansky, B. E.. 23.27.34. 35 Talarico. D., 162. ] 79 Talbot. W. S., 45, 47, 50, 5I, 78, 85, ]00, IOt. 338. 353, 354, 356. 357.36I, 370, 505.515, 534. 536 Taljedal. I. B.. 322. 324 Talmadge, R. J.. 268. 269, 277 Talreja. D. R.. 553.563 Tam, R L., 333, 335,368 Tam. R R, 37.38, 39, 40, 41, 45, 46, 47.49.50, 51.52, 129. 142, 146, 281,293, 333.366. 380. 381. 389, 392, 397. 420. 422. 424. 425.427. 428, 482, 491, 496 Tam, R R L.. 2. 3, 4, 30, 31.35, 40. 43.44. 49. 50. 128. 130. 131. I32. 133. 135. 139. ]46, 147. 148, 182. 183, 190 Tamaddon, H.. 334, 348, 367 Tamai. K. T.. 379. 384. 39t Tamai, S., 506. 518 Tamai, Y., 172. 173, 176

A u t h o r Index Tamamaki, N., 90, 105 Tamara-Lis, W.. 166. 178 Tamemoto, H., 7, I6 Tamir, R., 402, 417 Tampanaru-Sarmesiu, A.. 5 i2, 513 Tamura, K., 354, 367 Tan. S. S., 47, 52, 132, t 34, t4& 310, 32Z 424. 425,428, 437,496 Tan. X.. 318. 328 Tanabe, Y.. 43.52, 78, 104, I20, 125, 141,148 Tanaka, A., 466. 496 Tanaka. H., 116, 123 Tan'&a, K., 479, 491 Tanaka. M.. t 68. ] 78, 219, 232, 341. 343, 345, 348, 349, 350. 351,352, 359, 368, 369 Tanaka. S.. 23.35, 166. 167. 179, 180. 357, 368 Tanaka. T., 11. I2. I7. 341. 352. 370, 531. 538, 581. 588 Tanaka. Y.. 38, 51, 12I. 123 Tanapat, R, 237.247.248. 250 Tanegashima, K.. 338, 368 Taneja. M.. 322. 328 Tang, C.. 217.220, 229 Tang, H., 135. 147 Tang, J., 5!2. 514 Tang. K., 509. 518 Tang. M. J.. 403.420 Tang. T. K.. 7.19 Tang. T. S.. 8.18 Tang. Z., 165, 177, 290, 294, 303, 32Z 342, 366, 428, 437, 450.45 !. 452. 466, 474. 489, 494, 495 Tani, H., 570, 589 Tani. K.. 220, 231 Tani. Y.. 204. 209, 220. 232 Taniguchi, M., 471. 479. 487 Taniguchi, S., 423,424, 45 t, 475, 476, 478, 490 Tanna.hill. D., 225,229 Tanne. K.. -443.453.487 Tanswell. A. K., 313. 329 Tao, H., 47.50 Tao, Q. H.. 579, 589 Tao, W., 78.81, 84, 86, IO0, t05, 106, 165, t 7 Z 243, 252, 479.498. 525.536 Tapia-Ramirez, J.. 243,249 Tapscott, S. J.. 169. 17Z 257, 258, 260, 261,262, 264, 265, 267. 268. 269, 272, 274, 275, 27Z 356, 359 Tara, D., 361 Tarcsa, E., 570, 584 Tarkows~. A. K.. 22.35 Tarle. S. A.. 202, 208 Tarpley, J. E., 312. 327 Tarutani. M., 581, 583,589 Tashiro, K., 350. 362 Tassabehji, M., 525.538 Tassi, V., 311,326

A u t h o r index

677

Tata, E, 305, 307,323 Tatla, T., 467, 484 Taub, M., 415, 420 Taub, R., 27 t, 276, 318, 324 Tavazoie. S. E, 94. 105 Tavernarakis, N.. 15, I8 Tavian. M. 194. 200. 20I 209. 370 ~.3~ Tavormina. R L., 286, 294 Tay, J., 7, 1t. 14, I9 Taya, C.. 87. 101, 3z~.. ""~ 354, 365 Taya. Y., 434. 496 Taylor. B. A.. 3 t 1,330, 424, 442, -443, 493, 527, 535 Taylor D. G.. 226, 230 Taylor. G., 570, 589 Taylor. J. M., 268, 269, 274 Taylor M. V., 262, 275 Taylor, N. R.. 432. 498 Tchervenkov, C. I., 349, 369 Teboul, L., 27 I. 277 Teesalu. T., 162. 179 Tefft. D.. 3 t 1. 329 Tefft. J. D., 314. 315,329 Teichmann, G., 228, 232 Teichmann. U., 4I, 51, 427, 488 Teillet, M. A.. 39, 40. 50, 140. 14Z 148. 250, 263,264, 276, 277, 320, 326 Teitelbaum, S., 223.230 Teitelman, G., 320. 323 Teixeira, J., 385.393 Telford, N. A.. 5, 9, 19 Telvi, L.. 376, 391 Temeles, G. L., i 2, 19 Temme. A.. 173. 175 Temple, S., 235,236, 237, 245,249, 252 Tempst, R, 405,407, 416, 417 ten Berge, D., 451,476, 477, 496, 549, 565 ten Cate, A. R.. 437, 439, 496 ten Dijke. E, 338. 363, 468, 486 ten Dujike, R. 226, 230 Tenenhaus. C., 243, 252 Ten Have-Opbroek, A. A. W.. 312, 329 Tennenbaum, T., 169, 179, 589 Tepass, U., t36, 146, 205,209 Terada. M., 581,588 Terada. N.. 452, 496, 531,538, 580, 581,583, 589 Terasaki. M., 8, 17 Terhorst, C., 195.210 te Riele, H., 376. 393 Ternet, M., 313.328 Terrett, j. A., 357, 364 Tessarollo, L., 7, 19, 173, 179, 559, 562 Tessier-Lavigne, M., 85, 99, t01, 102, i 40, I41, 142, 145, 14Z 263, 264, 266, 274, 276, 287, 292 Testaz, S., 424, 496 9

,

.

.

.

.

~

,.~.-,

,

.~

.a.

Tetsu, O., 86, t05, 579, 589 Tetztaff, V~. 86. 103 Teuscher, C., 424, 442, 443, 493 Tevosian, S. G., 2 i 9, 232, 341,343, 352, 369 Texido, G., 86, 99 Tezuka, T., 581; 583,589 Thai. L. J., 247,252 Thater, J., 3t9, 325 Thaller, C., 469, 470, 486 Thangada, S.. 227, 230, 318, 328 Thayer, M. J., 257, 258, 267,277 Theete, D. E, 206. 209 TheiI, T., 83, 105, 119, 125 Theiler, J., 568, 570, 571,573, 574, 579, 581,582, 583, 589 Theiler, K., I28, t31, t37, 140, 149, 54t, 542, 565 Theitl, k E., 504, 509, 510, 513, 514, 516 Thelen, H., 165, 178, 316, 328 Thelu, J., 585 Tnepot, D., 165, 175 Therkaut~ W., 185, t 90 Therkidsen. B., 291,292 Thesteff, I., 422, 439, 441,452, 456, 48Z 488, 493, 494, 496, 49Z 578, 587 Theveniau-Ruissy, M., 348, 360 Thibaud, E., 375,389 Thi6baud, C. H., 425,494 Thief', J. E, 201,207, 223, 230, 3 t 5, 324 Thiery, M., 22, 33 Thise, B., 78, 105 Thisse, B., 47, 50, 52 Thisse, C., 47, 50, 52, 78, 105, 266, 276 Thoma, B., t 73, t 79 Thomas, B. L., 437, 451,496 Thomas, H. D., 378, 390 Thomas, K. R., 117, 125, 428, 471,496 Thomas, L. J., -44.2,496 Thomas, N., 44 I, 488 Thomas, R, 31, 34, 46, 47, 48, 5I, 311, 316, 32 7 Thomas, R Q., 30, 3 t, 33, 35, 45, 46, 47, 48, 50, 52, 304, 316, 329, 338, 362, 451,476, 483, 508, 514 Thomas, T., 95, 97, 137, 147, 161, 173, t 79, 341,346, 349, 350, 352, 355, 368, 369, 451,453, 465, 473, 495, 496 Thomas. 9U..~ t 36, 149 Thomazy, V., 469, 484 Thompson, A., 408, 4t9 Thompson, D. A. W., 442, 496 Thompson, E. M., 5, 9, t 4, 15, 18, 19 Thompson, K., 79, 86, t00, 243, 250 Thompson, L. M., 286, 294, 453, 495 Thompson, R. R, 341,342, 357, 368, 369 Thompson, S. R., t 0, t 9 Thomson, J. A., 303, 329 Thorey, I. S., 570, 589 Thorn, L., 560, 565

6 78 Thornetl, A. R, 288. 294, 442, 44.> -3 493 " . 4~:. Thorners, J., 45, 50 Thorogood, R, 140, 146, 20t, 208, 305. 328, 421,422, 423, 424. 426, 432. 435, 441, "442, -449, 457. 467,468, 48t, 482, 486, 48Z 496 Thorsteindottir, S., 131, 132, 149 Thorsteinsdottir, U., 242, 252 Thrasher. A. J.. 20I, 208 Threadgill, D. W(. 169, 179 Threat, T. A.. 11, 12. ] 7 ThummeI, C. S.. 15. ] 7 Thurberg. B. L.. 343.352.360 Thurston. G.. 223.224. 227,232 Ticho. B.. 339. 341. 349, 355.358. 370 Tickle, C.. 280. 294. 435.468. 483. 497. 534, 536, 577, 578, 579. 587 Tierney. C.. 521. 536 Tilghman, S. M.. 168. 176 Tilmann. C.. 373. 383.391. 393 Tilney. L. G., 557. 565 Tilney. M. S.. 557. 565 Timmons. R M.. 183. 190. 475.496 Timpl. R.. 574. 583.584 Timpl. T.. 411. 418 Ting. S. A.. 571,585 Ting-Berreth. S.. 577. 578. 579. 585, 587. 589 Tiveron. M. C., 239. 250 Tiveron. M.-C.. 239. 242, 243.25t Tiziani. V., 292, 294 Tochinai, S.. 198.208 Todaro, G. J.. 465.484 Toftgard. R.. 287. 292. 470. 485, 486. 497 Tohyama, M.. 479. 491 Tokimasa. C., "443.453.487 Tokooya, K., 353. 357. 365 Toksoz. D.. 188.190 Tokunaga. H.. 413.418 Tole, S., 82. 83.84. 86. 87. 100, lOI, 105 Toledo. S. R, 509, 517 Toledo-Aral. J. J.. 243. 249 Toles. J. F.. 202. 209 Tolon. R. M., 510, 516 Tomac. A.C.. 40.. "~ 498 Toman, R D., 573.588 Tomarev, S., 268. 274, 399. 4IZ 532. 538 Tomasiewicz, H.. 228. 229 Tomihoara-Newberc, er C.. "~ 4 Tominaga, K.. 204. 209, 220. 232 Tominaga. S. I.. 399.419 Tomita, K.. 413,418, 526, 527,538 Tomsa, J. M., 478, 496 Ton, C. C.. 451. 475.486, 527, 528, 530, 536. 538 Tonegawa, A., 142. 149 Tong, C. X., t 15. ]23

A u t h o r Index Tonini, G., 378, 387, 389 Tonissen, K. F., 339, 369 ~ Toole, L., 4 .>.>. 485 Topilko, R, 510, 5l& 546, 564 Topley. G., 580, 585 Topouzis, S., 342, 359 Torban, E., .>,9,39] -,-7 Torchard, D.. 382, 392 Toresson. H.. 47, 50. 82.83, 9 I, 105, 120, ]25, 451. 476, 4&3, 508, 514 Torii. M.. 95. 105 T/Srnell. J.. 467.482 TorTes, M.. 91. 102. 321. 329, 373.374. 385,393, 398, 420, 422, 432.451. 473. 475,477,490, 495, 496, 49& 542, 543,544, 546, 550, 561, 565 Toshikawa. Y.. 133, 149 Tosi. M.. 386. 390 Toso. R. D., 511,516 Totorice. C. G.. 171.175 Touchman. J. W.. 556. 564 Tournier-Lasserve, E.. 227. 230 Towbin. J., 357. 359, 363 Towers. N.. 262. 275. 336. 339. 342. 355. 368 Toy. J.. 522. 538 Toyama. J., 335.358 Toyama. R.. 78. 103, 374, 390 Toyoda, Y., 47, 51 Tozawa, Y., 224. 232 Traber. R. 310. 325 Traill, T. A., 357,358 Trainor. R A.. t 19, t24, I32, t35, 148, 424, 425,426. 427, 428,453,485, 490. 496, 544, 563 Tran. B., 433,476, 477, 478, 489 Tran. C. M.. 341. 369 Tran, T.. 442. 484 Trask, B . J.. .>4 " -7 ,, 364 Trasler. J. M.. 382, 393 Traub, O., 173, 175, 177 Trazami, S., 342, 362 Treacy, M. N., 89, t00 Treanor, J. J. S.. 402. 420 Treier, M., 85, 105, 502, 504, 505, 506, 509, 514, 518 Treisman, R.. 342, 362, 369 Tremaine. R., 432, 486 Tremblay, G. B., 167, 179, 478, 489 Tremblay. J. J.. 478. 481, 508. 510, 513, 518 Tremblay. K. D.. 303,329 Tremblay. M.. 171,173, 176, 312, 323, 510, 514 Tremblay. R, 258. 263.264. 266, 272, 277., 475,482 Trembleau, A.. 510. 518 Tremml, G., 102, t2I, 124, 423, 489, 508, 517 Tribioli. C., 451,453, 474, 497 Trinh, L. A., 303,326 Trommsdorff, M., 94, t00, 105

Author Index

679

Trounson, A., 22, 34 Truck. T., 344, 345, 348.365 Trumpp, A., 86, 105, 422. 432, 449, 450, 453, 466, 475, 497 Truong, "v: B., 400, 4t8 Trupp. M.. 402. 420 Truslove, G. M., 453,497 Tryggvason, K., 412.41/: 41& 419 Trzaskos. J. M.. 156. 177 Tsai, E.. 443.482 Tsai. F.-Y.. 203,209. 510, 518 Tsai. L. H.. 94. 98, t01 Tsai. M. J.. 92. 102. t06. 310 321 .~,,2. 327. 334. ~4_~,345 346. 352. 366, 559, 563 Tsai. M.J., 92. 104 Tsai, S. F.. 204, 209 Tsai, S. Y., 92. 106, 334, 343, 345, 346, 352, 366 Tsakiris, D. A.. 223,230 Tsang, A. E, 343. 369 Tsang, S. H., 505,517 Tsang, T. E.. 3.4, 38. 43, 49, 51, t32, 14Z 397,420 Tschudi, C., 15.18 Tseng, T.-C., 7, t9 Tseu. I., 3t3,329 Tsuboi, H., 379, 391 Tsuchida, T. N.. 109. 123 Tsuchiya, K., ~ . . 354. 365 Tsuchiya, R., 376, 39] Tsuda, T., 356, 369 Tsui. L. C., 12 I, ]24, 45 I. 453, 470, 4715 49], 528. 536 Tsujimoto, Y., t36, 146 Tsujimura, T.. 452. 496, 531,538, 580, 581,583, 589 Tsukiyama, T.. 376, 392 Tsukui, T.. 354, 367 Tsuruo, Y., 79, 99 Tu, C. L., 580, 583, 588 Tuan, R. S., 280, 292 Tucker. A. S., 432,433,434, 437, 438, 439. 440, 441,450, 451,467, 478, 484, 485, 496, 497 Tucker. D. C., 412. 417 Tucker, R K.. 377.387,393, 526, 527,535 Tucker. R. ~,~, 587 Tucker, S. C.. 451. 453.476, 493 Tuerk, E. E.. 170. 173, 179 Tufro. A., 412, 420 Tufro-McReddie, A., 412, 420 Tufts, R., 34 I, 343, 352, 368 Tuil, D., 342, 368 Tully, T., 251 Tumas, D. B.. 244, 252 Tuorto, E, 48, 49, 84, 9,7, 1t4, 122, 478, 481, 49t, 508, 513, 549, 550, 551. 552, 564 Tureckov~i. J., 455,489 Turgeon, B., 509, 5 I7 .

-

.

,

" ~ ' ~

..

.

.

.

"~

~

,

Turley, H.. 415,420 Turnball, D., t t0, 123 TurnbulI, D. H., 78, 99, I09, 1i0, I23, 124, 125, 228, 230 Turner. B. M., 9, ] 9 Turner, D. C., !34, t47, 257, 27Z 334. 3"44, 350, 351,357, 365 Turner, D. L., 527, 538 Turner. E. E., 237, 250 Turner, R A., 89, t0l Turnpenny, L., 352, 354, 366 Turoto, R, 478, 48] Turpen, J. B., 196, 198, 203, 208, 2 t0 Tuschl, T., 14, 19 TybuIewicz, V. L., 285,293, 442, 443, 488 Tyler. M. S., 432, 497 Tymowska-Lalanne, Z., 390 Tyrell, J. B., 512, 5]5 Tzahor, E.. 334, 335, 337, 339, 369 Tzartos, S., 271,277 Tzetepis, D., t94, 208

U Uchida, N., 192, 194, 20Z 2411, 243, 244, 245,250, 251, )5) 408. 418 Uchida, T., 527. 530, 535 Uchida, Y., 582, 584 Uchikawa, M., 283, 293, 529, 530, 531,532, 534, 536, 538, 565 Uchiyama, M., 290, 294, 437. 450. 451,466. 474, 495 Udy, G. B., 7, 16 Ueda, E.. 581,582, 583, 587 Uehara, Y., t 73, t79, 404, 420 Ueki, Yi, 292, 294 Uemura, M., 473. 498 Ueno, E, 471,479, 487 Ueno, K., 473, 498 Ueno, N., 336, 352, 365 Ueno, T., 408, 418 Ueta, C., 289, 294 Uilenbroeck, J. T., 385,389 Uitto, J., 573, 589 Ullman-Cullere, M., 223, 230 Ullrich, A., 217,220, 227,229. 23t Ullu, E., 15, 18 Umeda, S., t 95,208 Umemoto, M.. 555,565 Umesono, K., 525,536 Umezu, H., 195, 208 Unabia, G., 5 I2, 514 Unden, A. B., 287, 292, 470, 486, 497, 579, 586 Uno, K., 525,536 Unsicker, K., 314, 324 Upadhyaya, C., t 71, t 73, t 78

680

Author Index

Uratani, Y., 84, 98, t00 Urbanek, R, 1 t5. 1t8, 125, 428. 475,497 Urist. M. R., 271,277 Umess. L. D.. 226, 230, 232 Urrutia. I.. 506. 5i4 Umshihara. H.. 226, 232 Ushio, Y., 91,101 Usman. M. I., 318. 328 Usuda, H.. 195. 208 Utset, M. F.. 174. 177 Uyttendaele. H., 226. 232

V Vaage. J.. 428. 475. 497 Vaahtokari. A.. 439.441,456. 497 Vaccarino. F. M.. 95, 97. 506. 513 Vaessin. H.. 257, 276 Vaglio, A., 346, 358 Vaidya, D. M.. 334. 348. 367 Vaigot. R. 201. 209 Vainchenker, V~L,194. 207 Vainio. S.. 336. 363, 384. 385.393. 405.406. 407.418. 419, 439. 497 Valayer, J.. 376. 391 Valdez. M. R.. 260. 26 t. 262. 276. 277 Vale. W. W.. 286, 292 Valencia. A.. 345.348,360 Valenzuela, D. M.. 292. 292 Valerius. M. T., 79, 95,101, 105, 507, 516 Valladares, A. 171 1..~. 174 Vallotton, M. B., 506, 517 Valverde, F.. 84. 90, 95.99, 102, 105 Valverde. J.. 91.98 van Bebber. F., 354. 355. 359 Van Beneden. E., 31.35 "van Blerkom. J.. 7. 15. 19, 33.33 Van Den Berghe, H.. 22.33 van den Eijnden-van Raaij, J., 47, 50, 467, 484 van den Heuvel, M.. 470, 497 Vandenhoeck, A., 203,207. 223. 228 van den Hoff. M. J. B., 343, 345,348,366 van der Harten. H. J.. 285.293 van der Kooy, D., 94. 101, 193.204. 208. 235.236. 244. 245.251 Van der Loo. J. C. M., i 92, 210 van der Lugt, N. M.. 376, 389, 390. 393, 453. 481 van der Meer. R., 5.~,476, 493 van der Neut. R.. 572. 583. 585, 589 VanderPlas-de Vries. I.. _.~.~, 360 van 9der Schoot, R J., 385,389 van der Valk. M., 376, 393 van der Veen, C., 574, 588 van der Zee, R., 220, 228 van Deursen, J., 20 I, 204, 209, 220, 231

van de Velde, H., 313, 323 Vande Vijver, V., 511,5t 7 Van De Water, T., 474, 497. 542, 549, 550, 553, 555, 559, 562, 563, 565, 566 Vande Woude, G. F.. 7, ] 8 Vandlen. R., 402. 420 Van Doren, M., 185, 186, 188, ] 90 van Eeden, E J., 546, 565 van Eeden, J. M., 351,355,359 van Ewijk, W.. 195, 2 I0 Van Exan, R. J., 432, 486, 573,576, 589 van Genderen, C., 440, 49Z 583, 589 Vanhaelst, L.. 502. 515 Van Hateren, N., 390 van Helmond. M. J., 385, 389 van Heyningen. ,V.. 88, 99. 375.391, 399, 419, 420, 451, 475,486, 527, 528, 530, 536, 537, 538 van Hinsbergh, V. W:, 218.230 Vankelecom, H., 511, 517 van Lijnschoten, G., 174. 179 van Lohuizen, M., 376, 389, 390, 393, 453,481 Van Maldergem; L.. 281,293, 381,391 van Mierop, L. H. S., 343.350, 369 van Oostrom, C. G., 425,435, 497 Van Regemorter, N.. 398, 416, 550, 561 van Rooijen, M. A., 222, 229 van Room M., 376, 393 van Schaick, H.. 506. 513 van Schooten, R., 285.293 Vansomphone, D., 58 t, 586 Van Swearingen, J., 269, 277 'van Vliet, G., 512, 514 Van Voorhis, B. J., 22, 35 van Wijk, I., 174, 174 van Wyk, J. J., 532, 535 VanZant. G.. 245. 249 Varfolomeev, E. E., 341. 369 Vario, G., 343,369 Varlet, I., 43, 44, 45, 46, 50, 354, 355, 357, 359, 468, 497 Varmuza, S., 13, 1Z 22, 23, 34, 35, 169, 179 Varnum. D. S., 137. 149, 532. 538 Vasa, S. R.. 318, 324 Vasavada. H.. 312. 323 Vasicek. R.. 174. 177 Vasilyev. V. V., 509, 517 Vass. V~: C., 171,173, 178 Vassalli, A., 10, 14, 18, 19. 467, 490 Vassalli, J. D.. 10, 14, 18, t 9, 321,328 Vassileva, G.. 121,123, 132, 137, 148, 407, 419 Vassiliauskas, D.. 138, 148 Vassilli, J. D., 7, 16 V/issin, H.. 136, 149 Vastrik, I., 580, 589 Vaz, C., 318,327 Vazquez, E., 559, 564

A u t h o r Index

Vazquez, M. E., 91, t05 Veeranna, 94, 103 Vega, Q. C., 402, 420 Veile, R., 321,325 'I~ 3 497 Veitch. E.. ~+_~_. Veitia, R., 382. 392 Vekemans, M., 79, 99, 475,484 Velasquez, T., 260, 266~ 274 Veldhuisen, B., 414. 418 Velinzon, K., 25I Velkeniers. B.. 502. 515 Vendrelt, V., 543.545,546, 565 Veniant, M. M., 161.178 "v~nnstrom, B.. 510, 515 "v~nters, S. J.. 13 I. 132. 149 Ventura, C., 337,338, 369 Venuti, J. M., 260, 274, 277 Verano, N. R., 354, 365 Verbeek, F. J.. 345, 356, 369 Verbout, A. J., 131,134. 149 Veress, B.. 246, 249, 318, 324 u A., 343.369 Verlhac, M.-H., 7, t8 Verma, I. M., 258, 27Z 580, 581,583, 587 Verma, R.. 412, 417 Vermeulen, J. L. M., 343, 345, 356, 366, 369 Vermot, J., 341,346, 347, 352, 366, 469, 492, 545, 564 Verrotti, A. C., 10, 19 Verschueren, K., 226, 227,229, 336. 352, 359 Verwoerd, C. D., 425,435, 497 Vescovi, A. L., 206, 206, 235, 242, 244, 246, 249, 250 Vesque, C., 78, 81, 91, 99, 100, 118, 124, 468, 492 Vestlund, K. N., 512, 514 Vestweber, D., 226, 228, 4 t O, 420 Vetter. M. L., 526, 527, 535 Viallet, J. R, 571,572, 574, 585, 587 Vicaner, C., 379, 391 Victor, J. C., 550, 562 Vidgen, D., 527, 535 Viebahn, C., 26, 31.35, 41, 46, 51, 354, 355, 359, 427.488 Vielmetter, E., 355, 356, 36t Vien, L., 352, 354, 366 Vigano, M. A., 183, ] 90 Viger, R. S., 382. 393 Vigh, S., 504, 516 Vigneron, J., 398, 4t6, 550, 56t Vigneron, R, 271,273 Vijh, M., 157, 178 Vikkuta. M., 227,232 Vilar, J., 403, 41& 420 Villar. J.M. ,3_," "J, 359 Villavicencio, L. G., 345, 348, 360 Vincent, A., 91, I00 Vincent, C., 141.14Z 398, 416, 550, 561 Vincent, E. B.. 342, 367

681

Vincent, M., 9. 15 Vin6s, J., 466, 482 Vintersten, K., t 7 I, 173, 174 Viragh, S., 334, 341,345,348, 356, 360. 363, 369 Viragh, S. Z., 348, 351,369 Viriot, L., 455,489 Visconti, R E., 8, 17 Visser. R, 199. 20,3, 202, 207 Visvader, J. E., 203, 210 Vitelti, E, 353, 357, 364 Vivarelli, E., 140, 14t,145, ]4& 149, 258, 263, 264, 273, 277

Vivian, J. L., 260, 277 Vivian, N., 377, 378, 387, 390, 391 Vize, R D., 395, 420 Vliemck, R., 22, 33 "vbgan, K. J., 266, 27Z 338, 354, 356, 357, 359 Vogel, H., 468, 491, 571, 581,583, 587 Vogel, K. S., 237, 252, 341,352, 359, 435,497 Vogel, W., 17t, 175, 388, 39t Vogetstein, B., 86, 100, 579, 586, 587 Vogt, T. E, 45 i, 476, 493 Volk, T., 340, 358 "vbtkel, V., t 0, 18 Vollrat.h, M. A., 555,558, 559, 561 yon Bartheld, C. S., 558, 559, 565 Voncken, J. W., 314, 325, 434, 467, 487 von Ebner, C., 129, 149 Vonesch, J. L., 109, 122, 443,451,454, 455, 468, 469, 474, 489.. 490. 493, 545, 563 von Kessler, D. R, 470, 489 Von Ohlen, T., 470, 481 yon Schack, D., 141,144 Vorbusch, B., 336, 358 Vorechovsky, I., 287, 292, 470, 485, 486, 497 Voronova, A., 257, 275 Vortkamp, A., 265. 275, 285,287, 293, 294, 442, 443,470, 48R 490. 497

Voss, A. K., 95, 9Z 137, 14Z 173, I79, 453,496 Voss, J. W., 502, 518 Vrablic, T., 38, 52 Vrmncken Peeters, M. R, 2 t 9, 232 Vriend, G., 160, 175 Vu, T. H., 2_:, 23~' 28 t 290. 291 292, 294 Vukicevic, S., 408, 416, 420 Vuorela, R, 162, t79 Vuorio, E., 424, 442, 443,494,572, 573,588, 589

W Wada, H., 266, 277 Wada, R., 227, 23] Waddin~on, C. H., 542, 565 Wagenaar, G. T. M., 343, 345, 348, 366 Wagner, D. S., 527, 535

682 Wagner, E. E. 87. 104, 169, t 73, t 79, 318, 325, 442, ~ 3 , 485, 497. 525.537 Wagner, G. C., 9 I, 106, 439, 497 Wagner, J.. 173. t76, 510,514 Wagner. M. A.. 525.53Z 555. 557. 563 Wagner. T.. 281. 293. 294, 380. 381. 391. 393 Wagner. T. E.. 509.518 Waid. D. K.. 88. 105 Wainer. B. H.. 91. 105 Wainwright, B.. 172. 173, 17Z 287.292. 336. 337. 365. 375.391, 470. 485, 486 Wai-Sum. O.. 393 Wajnrajch, M. R. "443. 497 Wakabayashi. K.. 321. 327, 512. 518 Wakamatsu. Y.. 237.252, 318. 328 Wakamiya. M.. 3.4, 3 I. 34. 43.44.45.46.47, 48.49. 51, 52. 104. 328. 422. 427. 477. 482, 494 Wakeham, A.. 136. 137. 147. 341. 370. 468.495. 509.514 Wakkach, A.. 271. 277 Waknitz. M. A.. 303. 329 Waldrip. W. R.. 3 I. 35, 468.497 Waldron. S.. 340. 341. 363 Wales. J. K.. 31, 33. 47.50, 451. 476. 483. 508.514 Walker. A. R. 378. 392. 393 Walker. M.. 471. 493 Wall. C.. I95.209 Wall. N. A.. 470. 497 Wallace. V. A.. 86. 101. 105 Wallach. D.. 341. 369 Waller. R. R.. _~ " ""~. 369 "~llin. J.. 140. 147. 475.496 Wallingford, J. B.. 395,420 Walmsley. M.. 198. 200, 20Z 343,361 Walsh. C.. 86. 94, 97. 100, 105. ,.~.'~""24~." 244. 251. ,_~5:~_ Walsh. E. C.. 115. 125, 337.367 Walsh, F. S.. 425.428. 432. 484. 490 Walsh. J.. 2.4. 556. 565 Walsh. R, 375.391 Walter. A.. 83, 105 Walter, J.. 9. 17. 18 Walter. M. A.. 390. 453.479. 488 Walterhouse. D.. 470. 497 Walther. B. T.. 319. 329 Walther. C.. 83, 85, 91, ]05, 475.487, 530, 538 Walton. D. S., 528. 535 Walz. G.. 415.418. 579. 585 Wan. J. ~i. 12, I7 Wanaka, A., 479, 491 Wandycz, A. M., 243,245,251 Wang, A., 556, 564 Dang, B., 12I, I25, I95,210 Wang, C.-C.. 40. 44. 50, 323. 338.345.348. 358. 366 Wang, D., 410. 418 Wang, D.-Z., 343, 353, 364

A u t h o r Index Wang, E. A., 105, 467, 498 Wang, E, 90, 92, 95.98 Wang, G. F.,. ~ . . 34o, . .~- -,. ~45.346. . 347..369, 70 Wang, H., 40, 52. 218, 225,229, 232, 338, 368, 450, 47 I, 472.473.498 Wang. J.. 204. 210. 313. 329. 509. 515 Wang, g.. 342. 369 Wang, M. Z.. I05 Wang, Q., 10, 19, 201,204, 209, 21 O, 220, 23 I, 232, 233, 453,492 Wang, S. L. 15. l& 247. 252. 260, 276, ,o8 . 390 Wang, S. V~. 527. 536 Wang. W.. 136. 148. 268. ~./.~. "~'~- 280. 290. 292, 294, 34 l, 352. 370. 474, 49Z 549, 565 Wang, X., 11, 12. 1Z 44. 50, 217, 220, 229, 246, 250, 318, 326, 338. 362, 381. 382. 393, 469. 497 Wang, X.-J.. 571. 581. 583.586, 587 Wang, X. Y.. 549. 562 Wang, Y.. 262. 273, 318. 326, 476. 483, 500, 516, 588 Wang, Z.. 504. 510, 515 Wang. Z. Q.. 173. 179. 442, 443, 485, 497 Wanke. E.. 235,242, 244, 250 Wanninger, F., 570, 587 Warburton, D.. 31 t. 314, 3 t5,325, 327, 329, 330, 434, 467, 487 Warchol. M. E., 553, 555, 557. 562, 563 Ward. C. J., 415,420 Ward. J. M.. 94. 103. 171. 173, 176, t78, 226, 230, 311, 312. 326. 474, 488, 504, 505. 507, 508, 510. 518, 525, 538 Ward. S.. 341. 346, 347, 352, 362, 468, 49t, 545, 564 Ward, T. A., 398, 419 Wardle. E C.. ~9,., ~09. 249 Ware. C. B., I73.179, 405,407,416 Ware. M. L., 94. 105 Waring, M. T., 547,562 Waring. R. 167, 174, 305,323 Warkany, J., 346. 369 Warman. M. L.. 227. 232, 44-2. 443, 452. 453.474, 487, 489 Warot. X., 109. 122, 310, 329 Warren. A. J.. 203, 210, 342. 343, 360 Warren, K. S.. 351. 355,359 Warren, N., 85, 87.94, 105 Warren. R D.. 156. 175 Warrior, R.. 186. 190 Warsowe. J., 12, 16 Wartiovaara, J., 403.412, 4 t 9 Wartiovaara. K.. 403.419 Washburn. L. L.. 378, o8,., 389, 390, ~9~' Wasmuth. J. J., 286, 294, 453,495 Wassarman. K. M.. 8, 15.17, 19, 84, 85, 95. 102, t06, 114, 125, 453,497 Wassarman, R M., t 4. 15, 16, 18

Author Index

Wassef, M., 83, 84, 9& I00. 104, 1 t 1, 112, t 13, 115. t 17, 122, 125 Wasser, M., 240, 250 Wassif, C., 87, 91,103, 376, 389, 508, 5t Z 52I. 527. 537 Watanabe, H.. 284. 294, 442, 443, 453, 497 Watanabe, K., 38 I. 382, 393, 506, 516, 549, 564 Watanabe, M., 338.341,347, 354, 356, 359, 36Z 368, 369 Watanabe. N., 7, 16 Watanabe. S.. 292, 294 Watanabe. T.. 422, 424. 435. 437,475.476, 492 Watanabe. Y.. 140. t41,142, 147, 263, 264, 266, 276, 277 Whtanabe. Y. G.. 5I 1,5t4 Waterman, M. R.. 375,376, 380. 384. 390, 392 Waterman, R. E., 433,497 Watkins-Chow, D. E., 85. 1061. 502. 511,512, 518 Watnick. T. J.. 414, 415, 4!9, 420 Watson. A. J., 5, 9, 19, 156, 177 Watson, M. S., 357. 358 Watt, F. M.. 242. 252, 570. 572. 573, 580. 581,585, 586. 587. 589 Wattler, S.. 160, 167.178 Wawersik, S., 532, 538 Wawrousek, E. E. 528. 533. 535 Weaver. M.. 312. 313.314, 315,329 Weaver. R. G.. 398. 419 Webb, J. F., 422, 434, 435,497 Webb, R., 342, 364 Webb, S., 334. 348, 35 t, 369 Web't~er, E. M., 318, 329 Weber, H., 415,420 Weber. R. J.. 26, 32. 35, 158, 179, 348, 349, 352, 353, 357, 358 Weber. T. J., 379, 384, 391 Webster, M. K., 452. 453,466, 485, 497 Webster, W: S., 468, 497 Wechsler-Reya, R. J., 86, 106, 287,294 Wedden. S. E., 468, 497 Wegner. J., 450. 470, 473,481, 546, 561 Wegner, M., 170, 173. t 79, 238. 252, 281,294, 502, 510, 514, 5t5 Wehnert. M.. 94, t 03 Wehr, R., 78.79, 81. t00, 103, 521,533,537 Wek L.. 342, 369 Wei, M. H.. 227. 229 Weich. N.. 205.206 Weichenhan. D., 378, 390 Weigel, D.. 167, 179, 316, 329 Weigel, N. L., 379, 390 Weil, D., 398,416, 550, 561 Weil, M.. 266. 272, 528. 538 Weiler-Guettler, H., 219, 228 Weimann, J. M.. 94, 106 Weinberg, R. A., 318. 325. 483 Weiner. R. I.. 504, 5t8

683

Weinhotd, B., 342, 358 Weinmaster, G., 92, 103, 136, 137, 148, 149, 262, 276, 553, 555,563, 565 Weinstein, B. M., 213, 222, 23], 232 Weinstein, D. C., t 64, ! 65, 175, 179, 303, 316, 323, 329 Weinstein, M., 79, 82, 101, t05. I67, t7i, 173, 179, 226, 227. 233, 313, 329 Weintraub, B. D., 509, 514, 517 Weintraub. H., 136, 147. i 69, 175, 257. 258, 262, 263, 272, 273, 274, 275, 276, 277, 349, 362 Weir, E. C., 285,294 Weir. G. C., 322,328 Weiss, A., t 70, 179 Weiss, D. Wi, 428, 475,497 Weiss. J., 500, 518 Weiss, K. M., 450, 47 t, 496 Weiss, L. W\, 568, 589 Weiss, M., 203, 209, 38 l, 390, 5 t O, 518 Weiss, S., 86, 89, 9i, 103, 104, 236, 245, 248, 251, 252 Weissenbach, J., 281,292, 380, 390, 398, 416, 550, 56t Weissman, I. L., 192, t94, 202, 207, 208, 209, 2t0, 24t, 243. 244, 245, 246, 248, 249, 250, 25], 252, 318, 326 Weiz~icker, E., ! 34, 147 Welker, R, 588 Wellauer, E K., 321,324, 326 \~tler. D., 14, 18 Weller, R A., 281,292, 380, 381,390, 39I W~tls, C., 390 Wells, D., 11, t9 Wells, J. M., 3, 4, 304, 319, 329 Welter. J. E, 581,588 Wen, D., 402, 417 Wen, L., 52 I, 536 Wendel, D. E, 226, 230 Wendling, O., 109, 122, 172, 173, t79, 305,329, 341,346, 347, 352, 357, 362, 369, 545, 5612 Weng, Y., 227, 229 Wenger, R. H., 225, 230 Weninger, W. J., 335, 355. 365 Wenink, A. C., 334, 369 Wennerberg, K., 574, 589 Wenstrom, K. D., 22, 35 Wentworth, J. M., 3 t 1,324 Werb, Z., 163. 166, 168, 169, 170, 175, ] 77, t78, 179, 223, 232, 233, 281,290, 291,292, 294, 314, 32Z 329, 349, 360, 465,491 Weremowicz, S., 346, 358 Werner, M. H., 377, 393 W~rner. S.. 312, 328, 574, 583,584 Werner-Linde, Y., 580, 586 Wernig, A., 27 i, 27_5 Wert, S. E., 312, 313, 314, 328, 329, 330 Wertz, K., 393 W~rtz, R W:, 581,589

684 Wessets. A.. _~',, 342, ~44. 345. 348. 350. 351 356, 357. 360, 363, 365, 369, 370 Wessels. N. K.. 319, 329 Wessels. R. B., 574, 589 West, J. D.. 23, 34, 45 I, 475,493 Westerfield, M., 77, 78, IOt, 265, 269, 273. 435,450, 470. 473. 475.481, 493, 498. 546, 561 Westerman. C. J.. 227. 231 Western. E S.. 379. 382. 387. 388.393 Westerveld. A.. 174. 174 Westlund. B.. 242. 252 Westoll. T. S.. 442, 493 Weston. A. R, 504, 513 " .~ .~4.~. 250. _)5~Weston. J. A. , 2_.,7 ~ ?44. _ Westphal. H.. 43, 50, 78.80, 83, 85.86. 87, 91.94. 98. 103. 10"4, 106. 121. 123, 141,145, 173, 176. 260. 265, 273, 312, 319, 323, 326. 374. 376, 389, 390. 402. 419, 432. 439, 451,470, 482, 498, 500, 502.504. 505.507. 508, 509, 510. 512, 515, 517, 518, 521. 527. 535, 537 WetseI, W. C.. 511. 513 Wharton, K. A., 136. 149 Wheatley. M.. 236, 252 \~qaeatley, S.. 281. 293. 380. 381,387, 389, 391,392 Wheeler. E. F., 558,559, 565 Whitcomb, R. W., 500, 518 White. J. M., 8, 18, 287, 293, 424, 481 White. M. C.. 505,513 White. P. A.. 241,252 White. P. M., 235, 236, 238.239, 240. 241,243. 244. 245, 248, 251 White. R. A., 118, 123 White. R. D.. 355,365 "~hite, R. I., Jr., 227, 229 Whitelaw, E., 356. 357.360, 369 V~qaitesides, J., 84, 106, 469. 497 Whitfield, L. S., 377, 387,393 Whitfield, T. T.. 546, 565 Whiting, J.. 422. 425,427, 487 Whitington. P. M.. 185, 190 Whittock. K. E.. 435.498 Whitman, M.. 226. 233, 338, 354. 356. 367. 368. 479. 482 Whitney, J. B. D., 378, 390 Whitsett. J. A.. 311.312. 313.314, 328. 329, 330 Whitten. ~( K.. 157, 175 ~,~itters, M. J., 467,498 Whittingham, D. G., 8. 16 Whittle, M. J., 174, 179 Whitworth, D. J., 281,282, 292, 380. 388. 389, 393 Whyatt, D. J.. 343. 369 Whyte, D. B.. 504. 513 Wianny, F,. 14. 19, 26, 32.35. 158. 179 Wicherle, H.. 106 Wichmann. W.. 506. 517 ~ ,., _. Wicht, HI....' ~ "~8. ~57

A u t h o r Index Wickens, M. R, t 0, 11, 15, 16, I9 Wickjng, C., 287,292, 470, 485, 486 Wickramarame, G. A., 479, 485 Wickramasinghe, D., 7, 19 Wicks, I. R. 3I, 34 Wictorin, K., 89. I03 Widetitz, R. B., 574, 577, 578, 579, 585, 586, 587, 589 Widmer. D. A., 91,106 Widmer. H. R.. 91.98 Wieand. S. J.. 91.97 Wieduwilt. M.. 465.483 Wiegand, S. J., 91, 9Z 224, 230, 23t Wiekowski, M., 9, 19 Wierda. A.. 165, 174, 303.323 Wieschaus. E.. 136. ]46. 443.478. 493 Wiese. R. J., 343.363 Wiest, W., 570. 587 Wigle. J. T., 218, 219, 233, 317.329, 527. 530. 532, 538 Wijgerde, M.. 202.2]0 Wilbertz. J.. 246. 249, 318, 324 Wilby. O. K.. 128. 137, 146 Wilcox. J. N.. 512.516 ' ~ 7 9, 387, 393 Wilcox. S . A.. _~ Wilcox, W. R.. 286, 294 Wilde, A. A. M.. 348, 360 Wilder. R J.. 166. 179 Wiles. M. V.. 434. 467, 493 Wiley, L. M.. 26.33, 35 Wilkes. A. J., 35 I, 363 Wilkes, D., 452, 466, 482 Wilkie. A. O.. 452, 453.466,474, 487, 498 Wilkin, D. J., 286. 294 Wilkins. A. S., 387, 393 Wilkinson. D. G.. 118. 119. 124, 125, I26, t33, t35, i 4 Z 149. 225.231, 233. 264, 272, 422, 423,424, 425,427, 433,487, 492, 494, 498, 525,537, 5-44, 545, 546, 563, 564. 565. 571,586 Wilks. A. E. 162. 174, 453,486 Willecke, K., 173. 175, t77. 348, 360 Willetts. K.. 3t3.323, 326, 467,482 Willhoite, A. R., 236. 251 Williams, B. A., t4t, 149, 194, 208, 255. 256, 258, 264, 266. 268.27 I, 276, 277 Williams, B. O., 528, 537 Williams, B. R, 237, 239, 252 Williams, C. J., I3, 19 Williams. C. L.. 405,407, 4 t 9 Williams. D. E.. 188. 189, 190, 193,204, 208 Williams. E. A.. 47.52 Williams. J.. 237. 252 Williams. L.. 312. 328 Williams. M.. 247.25t, 580, 585 Williams, M. C.. 311. 312, 323 Williams, M. L., 58 I. 584, 586

Author Index

Williams, N. A.. 478. 498 Williams, R., 135, 149 Williams, R. H., 319, 328 Williams, R. S., 269, 2 73, 2 76, 2 7Z 342, 369 Williams, S. C.. 432, 465,478. 483, 498 Williams, T., 79, 106, 529, 538 Williamson, K. A., 375,391 Wiltiamson, R. A., 161.179 Wilm. B.. 45 t. 475,493, 498 Wilming, L. G., 385. 389 Wilson. C. M., 312, 323 Wilson. D., 568, 584 Wilson. D. B., 45, 50. 160, 164, 165, ] 75, ] 76, 178.. ] 79,. 323, 333, 341. 342. 343,352, 362, 366, 466, 486, 504, 518

Wilson. D. I., 357. 364, 387. 390 Wilson, G. N., 339, 369 Wilson, J. D.. 373. 390 Wilson, J. G., 346, 369 Wilson. R., 336, 367 Wilson, S. W., 22, 34, 77, 78, 79, 9& IOI, t02, 106, 521, 522, 537 Wilson, V., 45, 52, 132. t33, 144, 149, 160, 167, I78, 304, 329, 407, 4I 7 Wilson Berry, L.. 242, 252 Wilson-Heiner. M., 570. 583,584 Wilson-Rawls. J., t36, 14& 260, 267, 277 Wilting, J., 130, 131,132, I40. I44, I45, I46, t4Z 255, 263, 266, 273, 277 Wilton, S., 342, 368 Wilusz, J., t 1, 16 Windle, J. J.. 504. 512. 513, 51Z 5t8 Windsor, D., 388, 392 Winer-Muram, H. T., 351,354, 369 Winfrey, V., 453,479, 488 Wingate, R. J., 119, I22 Winkler, J., 247, 252 Winkler, S., 521,525, 53 I, 533,537 Winkling, H., 378, 390 Winnier, G., 82, 106, 203. 210, 287, 292, 313, 3 t 4, 323, 439, 467, 479, 498 Winnier, G. E., 132, 149, 506, 516 Winokur, S. T., 286, 294 Winter, B., 342, 369 Winter, J.. 173, 174, 175 Winter, R. M., 453, 493, 494 Winter, S., 570, 586 Winterhager, E., 173, ] 75, t 77 Wirth, J., 281,294, 380, 393 Wirtz, E., 15, 18 Wisdom, R., 451,453,476, 493 Wise, S. R, 84, 106 Wisniewski, S., 581,586 Wistow, G., 532. 537

685

Wit, J., 509, 517 Wittbrodt, J., 116, 125, 52t, 525, 531,533, 537 Witte, D. R, 79, 82, tOI, 105, 352, 354, 368 Witte, L., 219. 227, 231, 233 Wittier, L., 4t, 5t Witzenbichler, B., 220, 228 Wlaczak, V. R., 58 t, 586 Wodarz, A., 82, 106, 405,420 Woessner, J. E, Jr., 223, 23i Woh.t C., ,-~6, ~5"~ Wohlschlegel, J. A., 25]_ Wojnowski, L., 470, 486, 579, 586 Wojtowicz, J. M., 247, 252 Wolburg, H., 2 t 9, 224, 231, 232 ~3,~lburg-Buchholz, K., 224, 232 -Wold, B. J., 239, 241,251, 258, 260, 271,273, 276, 277 Wolfes, H., 14. ] 8 Wolfman, N. M., 338, 363 Wolfsberg, T. G., 424, 481 Wotgemuth, D. J., 312, 327 Wotoshin, R, 267, 277 Wolpert, L., 356, 359, 577, 578, 579, 587 Wondisford, E E., 509, 514, 517 Wong, A., 27 I. 277 Wong, B., 347, 362 Wong, C., 528, 532, 538 ~ n g , D., t 65, 174, 303, 323 Wong, J. S., t6I, 175, t78, 400, 418 Wong, K., 90, 95,106 Wong, E C., 137, 149 Wong, V., 91, 98 Wong, Y.-M. M., 341,369 ~:oo, I., 290, 294, 437, 440, 450, 451,466, 474, 482, 495, 530, 533, 538 Woo, K., 543, 565 Wood, C. R., 217, 229 Wood, H., 91, t02, 199, 200, 201,210, 527, 530, 531,537 Wood, R. I., 511, 5t2, 515 Wood, W., t 85, I90 Wood, W. B., 243, 252 Wood, W. M., 509, 510. 515 Woodland, H. R., 166, 176, 303,325 Woods, K. L., 51 I. 518 Woolf, A. S., 404, 420 Woolf, T., 105 Worby, C. A., 402, 420 Vvbfley, D., 403,420 Worley, K. C., 378, 387, 389 Worrad, D. M., 9, 1.5. 19 Wotton, D., 468, 485 Wozney, J. M., 271,275, 467, 498 Wrana, J. L., 3 t, 35, 468, 497 Wray, G. A., 47 t, 493 Wray, S., 435,498, 502, 514

686

Author Index

Wreden, C., t 0, 19 Wright. B. D.. "'~3~.~.. __;~9 Wright. C. V., 40, 43.47. 49, 50, 78, t04, 105, 133. 137, I45. 183 190, 260. 267. 275. 309. 319. 320. "~'~ 326. 3 2 / ,- 477, 48,_" Wright, C. \( E., 336, ~J4. ~>). 363, ~64 Wright, D. D.. 478,489 Wright, E. S.. 26, .~.~' ~ 281,293._~94. 381 . 39" Wright, G. M.. I39, 148 Wright, M., 397, 4 t 8 \Vrieht. M. E., 475.498 Wri_oht. S. J.. 8.19 Wright. W. E.. 257.258.275, 276 Wroblewska. J.. 22.35 Wu. C.. 342. 364 Wu. D. K.. 469. 478.483. 49I. 54 I. 542. 546. 550, 551. 552. 555,557, 558. 559. 561, 564. 579 Wu. G., 226, 232. 414. 415.418, 420 Wu. H., 219, 233. 269. 273. 277, 342. 369, 583.587 Wu. J. E.. 314. 329 Wu. J. Y.. 90. 95, 106, I36. 149. 521. 536 Wu. L., 11. t 9, 204.210. 428,452. 489 Wu. M. T.. 227.230 Wu. N.. 133, 149 Wu. Q.. 226. 233 Wu. S. Q.. 47.51 Wu. w.. 44. 50, 90, 95. 106. _719. 229. 325, .~. "'~7. _~_~8.'"361. 362, 506. 508.509. 518 Wu, X.. 136. 148, 336. 337. 366. 370 Wuenschell. C. V~(, 88. 102, 168, 177 Wunderte. V. M.. 380. 393 Wunsch, A. M.. 342.368 Wtinsch, K.. 136, 137, 139. 145 Wurst. \~. 109. 114, 115. I22. 123, 124, 126, 228. 230, 428,479.48Z 49I. 522.527. 535. 549. 562 Wuytack. F.. 343. 345.348.366 Wylie, C. C.. 14. 16. 182. 183. 184. 185. 186. 187. 188. 189, 190, 303.323, 330 Wynne. J.. 342, 362 Wynshaw-Boris. A., 3 I. 33, 40. 45.50. 204. 210. 220. 233. 286, 292. 338, 353.357.360. 365, 453.484

X Xavier. R. J., 353,357. 365 Xavier-Neto. J., 335. 339, 343.344. 345.346. 347. 350. 351. 352.359. 366, 367, 370 Xenophontos. S. L.. 414. 418 Xiang, M.. 527, 535. 556, 558, 565 Xiao, D.. 346. 364 Xiao. H., 12, 17 Xiao, J.. 399. 419. 530. 533,537 Xie. B., 286. 294 Xie, J., 287, 293, 579, 586, 589

Xie, V~ E, 282, 295 Xing, L., 442. 484 Xu, C., 338, 352, 370 Xu, H., 343,353,364 Xu. J., 116. ]26, 453. 492, 521 5 "~7 537 Xu, K.. 8. 16 Xu, R X.. 398. 399, 420, 527, 530. 533,538, 550. 565 Xu. Q.. 119. 124. t26. 225, 23I, 233, 521,522, 537 Xu. S., I4, ]6, 17 Xu. T.. 136, 145. I48, 149 Xu. W.. 7. 16 Xu. X.. I67. 171. 173. 179. 226. "~'~" ~.,/, 233. 286. 292, 31o, " 329, 336.370 Xu, X. M., 555, 557, 563 Xu. Y., 87, 91. t03, 106, 508, 517 498 Xuan. S., 78.81. 106, z4.~. "~ " _~5). . . 479. . Xue. N.. 286. 294 Xue. X. J.. 263.277 Xue. Y.. 136. 149, 226, 230 Xue, Z. G., 263, _,/;~7" Xy, H ~.~.~..~/,365 Xvdas. S.. 328. 335.336, 339. 367 .

.

.

.

.

.

,

~ - , ~

Y Yablonka-Reuveni, Z.. 271,277 Yacoub. M. H.. 341, 351. 359, 363 Yacoubian, T. A., 94, 106 Yagi, H., 204. 209, 220. 232, 288, 293, -442, 443,453, 488, 506, 5 t 8 Yagi. T~. 223. 230 Yaginuma, H., 122. 122 Yaginuma, K., 504, 510, 516 Yagishita. N.. 505. 517 Yagyu. K.. 94. 103 Yahata. T.. 339.368 Yamaai, T.. 260, 266. 273 Yamada. E.. 580. 587 Yamada, G.. 432. 473,477,482, 49Z 498 Yamada. H.. 321, 32 7 Yamada. K., 352, 354, 365, 58 I, 582. 583,587 Yamada K. M., 408, 418 Yamada. M., 94. 106, t86, I90, 336, 370, 512, 518 Yamada. S., 338, 352, 364 Yamada. T., 534. 538 Yamada. Y.. 203.210, 284. 294, 318,329, 442, 443,453, 49Z 557.565 Yamagami. T.. 341,352, 370 Yamagata, M., 525,538 Yamagishi, H 349 05... 369, 370, 451 465.470, 496 Yamagiwa, H.. 288,293 Yamaguchi. A.. 271,275, 288, 293, 442, 443.453,488 Yamaguchi, T. R. 133. 135. I45, I4& I49, 203,209, 2 t 7, 223,232, 303. 317.324, 45t, 453,471,498, 506, 518

Author I

n

d

e

Yamamoto, H., 94, I03. 168. t79, t88, 189, 266, 277 Yamamoto, I., 262, 268, 274 Yamamoto, K., 286, 292, 335,358 Yamamoto, M., 527,530, 531,536 Yamamoto. T., 195,208, 534, 537 Yamamura. E, 196, 209 Yamamura, H... 334. ~ t..~,.. .... "~ 357 , . .344. . .347. . 348. . . 350. ~.~ 365, 370 Yamamura. K., 305,307. 328, 443.492 Yamanaka. H., 506, 5t6, 549. 564 Yamanishi, K., 588 Yamasaki. K.. 588 Yamasaki. M.. 312.328. 505,517 Yamasaki. N. 048, " "_~~ 368 " 049. Yamashita. E. 58 t, 582. 583,587 Yamashita. J.. 228. 233 Yamashita. S., 504, 516 Yamashita, T.. 94, I04, 227, 23t, 502, 508. 509, 5 t 1,516, 517 Yamazaki, T., 343.368 Yam C.. 31 .~," 330 Yam J., 167, 180 Yan. Q., 9I, 101 , ,.,.'~6,_)3).. Yam w.. 334, 361 Yah, Y. L.. 214. 23t Yam Y.-T., 3 l, 33, 40. 45.50. 338, 353, 354, 356, 357. 360, 370 Yanagimacht, R., 8, 17 Yanagisawa, H., 347,366, 432, 465. 478, 483, 498 Yanagisawa, M., 334, 368, 432. 465,478,483, 498 Yanase. T.. 379. 391 Yanazawa, M., 353. 354, 355. 356. 3613 Yancopoulos, G. D. 106, "~3 "~94, ~ 5 "~v7 ;~9 230, 231,232, 292, 292, 549, 561 Yang, A., 571, 58 I, 583, 589 Yang, B. B., 350, 370 Yang, B. L., 350, 370 Yang, D. D., 87, t0t Yang, G., 397. 418 Yang, G.-Z., 351. 363 Yang, J., 405, 407, 416, 579, 589 Yang, J. M., 530, 532, 536, 538 Yang, J. T.. 170, 172. 173, 176, 180, 204, 206, 219, 233, 571.58 I. 583, 589 Yang, L., 31, 33. 40, 45.50, 338, 360, 450, 471. 472, 473, 498 Yang, Q., 269, 273 Yang, w. R, 225, 230 Yang. X., 23, 35, 226, 227, 233, 240, 250, 266, 277 Yaniv, M.. 165, 166, t 75, 178 Yano, H., 189. 190 Yap, A. S., 408, 420 Yashiro. K.. 356. 357. 365, 367 Yassine, F., 200, 209

x

6

8

7

. .525. . . 527, . . 528, . -3 0. 531 , 5~".~4. ~ 536, 537 Yasuda.. K.. ~_ Yasuda, M., o_~ 362 Yasugi, S., 303, 308, 325, 329 Yasui, K., 473,498 Yasui, N., 288, 293 Yasui, T., 288, 293 Yasuno, H., 581,582, 583, 587 Yasuo. H.. 303, 330 Yatskievych, T. A., 334, 335,336, 337, 363, 370 5~azaki, Y., 336, 343, 349, 365, 368, 369, 451. 465,473. 489, 496 Ybot-Gonzalez, R, 475, 486 Ye, H.. 321,328 Ye, "v~(,85, 106, 110, I2t, 126 Ye, X., 479, 486 Yee, D., 169, 179 2~ger. H., 415, 4t7 Yeh, ',~:-C., 341,370 Yehia, G., 583,586 Yelandi, A., 318, 328 Yelon, D., 303, 326, 334, 339, 341,342, 349, 36I, 36Z 370 Yeo, C.-Y., 356, 367 Yeoh, T., 345,348,366 Yeom, Y.. 183, I90 Yeoman, E., 506, 514 Yi, C. H., 357, 364 Yi, ~&:,388. 393 Yienger, K., 171, 173, 178 Yin, M., 434, 467,493 Yin, Z., 334. 336, 370 Ying, C., 258. 260, 261,262, 267. 275 Yingling, J. M., 311, 3 t 3, 314, 315, 325, 329 Yip, L., 83, t00 Ytikoski, J., 558, 559, 564, 565 Yntema, C. L., 542, 543, 565 Yoda, H., 532, 534, 536 Yoder, E, 507, 515 Yoder, M. C., 196, 202, 210 Yokata, C., 338, 368 Yokayama, M., 353, 354, 355, 356, 363 Yokayama. T., 352, 354, 365 Yokoi, M., 237. 252 Yokota, Y.. 1.~,, 147 Yokoyama, M., 7, 16, 47, 51 Yokoyama, T., 47, 51, 354, 355.364 Yoneda, K., 570, 583, 584 Yoneda, Y., 341,352, 3 70 Yonei-Tamura, S., 354, 367 Yonekawa, H., 352, 354, 365 Yoneshima, H., 94, 104 Yoneshita, H., 94, 106 Yonezawa, S.. 557, 564, 565 Yoon, J. K.. 260, 276, 277 Yoon, S. K., 400, 418

688 York, J. D.. 225, 230 Yoshiba, K., 453. 482 Yoshida, A.. 525, 538 Yoshida. C.. 289. 294 Yoshida. H.. I72. 173. 176. 178. 200. 209. 442.498 Yoshida. K. 318. 325, 34 I. 352. 370. 405.407, 416, 53 I, 538 ~shida. M.. 83. I06. 305. 307. 328, 374. 392. 403.4t8 Yoshida, N.. 341. 352,370. 47t, 479. 487. 531. 538 Yoshida. O.. 189. 189 Yoshida. T.. 581. 588 Yoshida. Y.. 171. 173. ]76 Yoshie, O.. 350. 362 Yoshihara. Y., 435.491 Yoshii. H., 376. 393 Yoshikawa. K.. 452. 496. 580. 581. 583.589 Yoshikawa. Y., 133. 135, 137. 149, 557.563 Yoshiki, A.. 1 I. 12. 18. 557.565 Yoshiki. S.. 288, 293. 442. -44'3. 453.488 Yoshimoto. T.. 502.511. 515 Yoshinao_a. K.. 156. 175 Yoshinobu. K.. 305. 307. 328 Yoshioka, H.. 47.50. 354. 370, .442. 443.471. 479, 48Z 489 Yoshizaki, N.. 557. 564 Yoshizawa. T.. 312. 328. 505.517 Yost. H. J.. 47.50. 349.351. 355.356. 358. 359. 364, 473. 493 Young, I. D.. 281. 292, 357. 364. 380. 390 Young, J. K.. 23.35 Young, K. E.. 43.50, 78.80. 85, 86. 98, 121. 123, 141. 145. 260. 265.273. 319.323. 439. 451. 470. 482, 483. 493, 521. 535 Young. M. W.. 136, I45, 147 Young, R E.. 193.210 Young, R. W.. 452, 498 Young. S. G.. 161. 175. I78 Young, W. S. D.. 502. 518 Young, W. S. III, 512, 516 Younger-Shepherd, S.. I36. 145 Younossi-Hartenstein. A., 367 Youssef. E. H.. 442. 498 Yu. A. T.. 11.16 Yu, A. Y., 225.230 Yu, B. D.. 376. 390 Yu. D. B., 376, 393 Yu, G.. 79. 89, 99, 450, 471,484, 498 Yu, Q.-C., 572, 583.585. 586 Yu, R. N.. 6. I9. 378. 379, 393 Yu. T. ~,~. 247. 251 Yu, Y., 402, 417 Yuan, B., 135. t47 Yuan, H., 160, I80 Yuan, S.. 334, 335, 336, 346, 347, 363. 370

Author Index Yuasa, J., 525, 538 Yuasa, Y., 226, 232 Yudate, T:, 95, ]02, 506, 5 t 6 Yue, Y.. 91. 106 Yun. J. S.. 509. 518 Yun, K., 88, 89, 90, 94, 95, 97 Yumgi, T.. 228. 233 Yuspa. S. H.. 169, ]79, 580, 585, 586, 58Z 589 Yutani. S.. 506, 518 Yutzey. K. E.. 342,344, 345.346, 347, 364, 36Z 370

Z Zabel. B. U.. 281,288. 293, 294, 379, 380, 391, 393, 478, 495, 507, 517 Zabski. S. M.. 313.324 Zackai, E. H.. 452. 453,468, 481, 485 Zagouras, R. 136, I44 Zagzag, D.. 224. 230 Z~ikfiny, J.. 305.309, 310. 326, 330 Zakin, L.. "44.52 Zalokar. M.. 185, 190 Zalzman, M., 551,552, 557, 563 Zambrowicz, B. P., t 57, 180, t 83, 190 Zammit, P. S., 344, 345.356, 36I, 362 Zamore, P. D.. 14. 19 Zanaria. E.. 378. 379, 380. 384. 387. 389, 391. 392, 393 Zanchi. M.. 271. 273 Zanellato, A.. 511,516 Zanetti, A., ~ ;~9 Zang, Z., 239, 250 Zangen, D. H.. 322, 328 Zanger, U. M.. 375, 384, 392 Zanin. M. K. B., 350. 370 Zannini, M., 311,330 Zaphiropoulos, P. G., 287, 292, 470, 485, 486, 497, 579, 586 Zappavigna, 'v:, 443,449, 478,482 Zarbl, H., 15, 15 Zaret. K. S., 165, I74, 303,307, 3 t 6, 3 t 7, 320, 323, 324, 322 330 Zarkower, D., 382, 387, 388. 391, 392, 393 Zasloff. M. A.. 27 I. 276 Zatloukal, K.. 570. 587 Zavitz. K. H., 399, 419, 530, 533, 537 Zawel, L., 86, 100 Zazopoulos, E., 379, 393 Zbar. B.. 227, 229, 231 Zbieranowski. I., 163, 175 Zeeman. E. C., 139, 145 Zehentner, B. K., 282, 295 Zehetner. G., 378. 392 . . . E Zeinstra,. L.. 183.190, o" ~ 3 o, 363 Zeitler, R S., 507, 516

A u t h o r Index

689

Zekower, D.. 382, 387, 392 Zelenka, R S., 532, 535 Zetent, A., 469, 489, 49I, 494 Zelickson, A. S., 568, 589 Zelter, R., 86, 99 Zelter, U. 42I 442. 456. 457. 6.>, 471 48& 498 Zelzer. E., 340. 370 Zeng, Q., 225,231 Zeng. X., 314, 330 Zenner, H. R, 558, 563 Zerega, B., 290, 292, 450, 453.472, 473,481, 549, 56] Zernicka-Goetz. M., 1,4, 14, ] 9, 26, ,./.~"32, 33. 35, t 58, 179 Zerucha, T., 89, ]06, 450, 471. 498 Zeschnigk, M.. 264, 271,275, 277 Zetter. B. R.. 189, 190 Zetterstrom, R. H.. 509, 5t8 Zevnik, B., 23, 34, 160, 166, 178 Zhadanov. A. B.. 508, 517 Zhang, C. L., 342~ 365. 370, 579. 589 Zhang, D., 239, 251 Zhang, H., 82, 106, 336, 339, 370, 439, 450, 467, 471,472, 473,498 Zhang, J.. 45, 50, 78, 79, 100, 106, 186, 190, 303, 318~328, 330, 338, 361, 529. 538 Zhang, L., 217. 227, 229 Zhang, M., 347, 348, 350. 352, 370, 427,433.443, 449, 474, 485 Zhang, N., 135. 136. 137, 138, 149, 186. ]90, 555,565 Zhang, R, 285, 287, 293, 294, 453,486, 528, 532, 538, 580, 58 I, 583, 586 Zhang. Q., 334, 343, 36t Zhang, R., 288, 289, 292 Zhang, W., 240, 243,251, 260, 262, 276, 277 Zhang, X., 282, 295, 414, 419, 437, 498 Zhang, X.-K., 346, 364 Zhang, Y. 350, 370..~79. 390, 4.~., 498 Zhan~. Y. A.. 94. 106. v v6 P)9 357. 358 Zhang, Y.-H., 398, 419 Zhang, Y.-Y., 243, 244, 249 Zhang, Z., 12, 16, 281,282. 283, 284, 289, 292, 294, 380, 389. 437, 49& 530, 533, 535 Zhao. B., 34 I, 364, 367 Zhao. D.. 469,498 Zhao, G. Q., 473,476, 498 Zhao, J., 314, 3 I5,327, 329, 330 Zhao, Q., 79, 106, 281,295, 451,453, 476, 493, 498 Zhao, S., 92, ]04, 473, 498 Zhao, X., 437, 498 Zhao, Y. 83. I06. 376, 389, 4.~,~. "~ 498 Zhao. Z.. 450, 471,496 Zharhary, D.. t 96, 207 Zheng, B., 57 I, 58 I, 583,587 Zheng, C.. 94, 106 ,

,

.

.

9

.

.

.

4

"

,

Zheng, H., 347, 352, 360 Zheng, J. L., 555,556, 566 Zheng, M., 316, 317, 320, 324, 325 Zheng, R. Z., 134, ]47 Zheng, T. S., 87, 10] Zheng, Y., 243,249, 506, 516, 549, 564 Zhi, Q., 13 t, 146, 266, 277 Zhong, V~\,79, 89, 99, 164, t 79, 450, 47 I, 484 Zhou, C., 92, 106 Zhou, E, 286, 292, 453.484 Zhou, G., 334, 343, 345, 346, 352, 366 Zhou, H.. 82, 102, ?..,,4. ' ~ 227, 232 Zhou, J., 4t2, 415, 4t Z 418 Zhou, L., 90, t06, 313, 330, 527, 535, 556, 558, 565 Zhou, E, 440, 498, 577, 589 Zhou, Q., 268, 275 Zhou, R., 91,106 Zhou, S. X., 30, 35, 39, 40, 4 t, 52, 182, 183, t90, 268, 275, 380, 389 Zhou, W., 342, 369 Zhou, X., 95, 9Z 338, 355,370, 473, 498. 519, 521,522, 525, 530, 533, 535 Zhou, Y., 6, t7. t73, I74, 176 Zhou, Z., 582, 583,587 Zhu. A. J., 242, 252, 589 Zhu, D., 428, 452, 489 Zhu, J., 165, 180. 316, 330 Zhu, L., 387,393 Zhu, V~, 269, 273 Zhu, X., 3 t 7, 330, 337, 364, 370 Zhu. Y., 90, t06 Zhu, Y. Z., 286, 294 Zhu, Z., 227, 233 Zhuang, Y., I72, 173, I80 Zhurinsky, J., 86, 104, 579, 588 Zidehsarai, M. R, 509, 513 Zietler, R S., 95, 101 Zile, M., 343,346, 347, 36t, 363 Zile, M. H., 346, 347, 356, 369, 370 Ziller, C., 140, 14& 236, 249, 569, 587 Zimmer, A., 89, 101, 402,417, 470, 486, 579, 586 Zimmer, J., 281,294, 380, 393 Zimmerman, B., 452, 498 Zimmerman, L. B., 40, 44, 51, 141, I47. 238, 252, 260, 264, 265,275, 468, 498 Zimmermann, J. W., t 2, t9 Zimmermann, S., 386, 393 Zindy, E, 556, 558, 561 Zine, A.. 555, 566 Zinn, C., 558, 563 Zinyk, D. L., t09, 1 t4. 121,124, t26, 228, 233, 451,453, 470, 47t, 491 Ziomek. C. A., 23, 34, 158, 177, 178, 180 Zipursky, S. L., 399, 419, 530, 533.537

690

A u t h o r Index

Zirno_ibl. R. 1~_. 148 Ziyadeh, K N.. 4 t5,419 Zlot, C. H., 161. I78 Zock. C., 235, 236, 238, 239, 240. 24 t. 243. 244, 245, 248. 251

Zoeller, R. H., 512, 516 Zoelter, R. T.. 502, 518 Zoerkler. N.. 321. 326 Zoghbi, H. Y., 122. I22, 555,558. 559. 561 Zon, L. I., 198, 199, 203.208. 210, 2 1 3 . 2 1 4 . 230. 232, 336,363 Zonana, J.. ~ 1,488, 4 9 t Zorn, A. M., 172, 173, 176, 303,330 Zou, K., 350. 370

Zou, Y.. 342.. .343. ~"~ 36_~ . . . .~,.. Zschiesche, W., 3t 7, 328, 404, 4t 9 Zsebo, K., 185, 188, t89, ]89, ]90, 246, 2 5 t Zuber. M.E. , 5 _,_, ~ ..~_:~. s "~. . .~.~.~. . . 538 Zuffardi, O., 380, 381,391 Zulch, A., 550, 56] Zuniga, A.. 32 7 Zuo. L.. 506, 508, 509, 518 Zwartkruis. F.. 395.417, 546, 563 Zweigerdt, R., 3 ! 0. 327 Zwijsen, A., v~'~ "~'~6 "~';7 ~ 9 336, 352, 359 Zybina, E.. ,.~, v', 35. 163 , 169. 180 Zybina. T. G.. 23.35, i 63. 169. 180 Zygar. C. A., 529, 538

Subject Index

A

Angiopoietins, 224 Animal-vegetal, 27 Anterior patterning, ~ - 4 8 Anterior-posterior axis CNS DV pattern and, t 08 - t 09 mes/met domain development, identification of mutants, t t5 fate mapping, t t 3-114 gene expression, 1 t 2 - t t 3 organization, role of FGF8, 115-16 patterning models, 116 - 117 regulation, 110 - ! t 2 Otx2/Gbx2 mutants, t t 4-115 diencephalon, 84-85 epithelial cup, 301 fetus btasto~cyst, 29 conserved properties, 40 duplication, 22 EGO response, 41-42 node development, 3 8 - 4 0 organizer function, 4 2 - 4 4 polarity specification, 29-32 relationship, 2 5 - 2 6 gut tube regionalization, 305 hindbrain, t 17-119 nephric duct, 40 l prosencephalic neural plate, 78 somite, 134 telencephaton, 8 3 - 8 4 Aorta gonads mesonephroi CFU-S in, 196 description, 192 fate mapping, 202

ACTH. s e e Adrenocorticotrophic hormone Activin, 337 Activin receptor-like kinase-1,225-226 ActRIIa, 68 Acvrl 1. s e e Activin receptor-like kinase-I Adrenocorticotrophic hormone characterization. 499 deficiency, development, 509 functions, 500 AGM. s e e Aorta gonads mesonephroi Alport syndrome, 4 t "~ Alzheimer's disease, 247 Amh Miillerian duct differentiation. 385-386 production. 385 regulation, 38 t-382 AMLI expression. 201 liver hematopoiesis, 204 Amphibians, 197 Ang- I. 2 2 7 Angiogenesis arteries segregation, 225-226 cell-cell junctions, 226 clinical implications, 227 complexity, 227-228 definition. 212 pericyte recruitment. 226-227 smooth muscle cell recruitment, 226-227 vascular channel formation, 222-225 channel regression, 227 polarity, 227 vein segregation, 225-226

69t

692

Subject Index

Aorta gonads mesonephroi ( c o n t i n u e d ) hematopoietic progenitor source, t99 hematopoietic stern ceil source. 199 mesodermaI precursors, 200-20 I AP axis. s e e Anterior-posterior axis Arginine vasopressin. 95 Aristaless 3,476 ARNT. s e e Arylhydrocarbon receptor nuclear translator Artery segregation, 225-226 Arylhydrocarbon receptor nuclear translator angiogenesis. 225 labyrinth branching morphogenesis. 172-173 Asymmet~' blastocyst. 2 7 - 2 9 development early, 23-27 overview. 21 - 23 preimplantation, 1-2 enhancer location. 62-63 fertilized oocytes. 1 left-right diversity among vertebrates. 68-69 furin. 67-68 future challenges. 69-70 GlcNac-TI. 67 initial determination. 58-59 models. 55 morphological. 56-57 mutations. 58-59 node flow, 59-61 P i t x 2 on left side. 65-66 positive/negative feedback loops. 62-64 retinoic acid. 67 Sna on fight side, 65-66 TGF-/3 signals. 59-61 Zic3.67 oocytes, 7-8 AV axis. see Animal-vegetal AVE. see u n d e r Endoderm AVE see Arginine vasopressin Axin. 45

B BA. see Branchial arch B a p x t , 474 B a r x l , 437. 439 Basic helix-loop-helix factors, see also specific f a c t o r s angiogenesis, 225 crainofaciaI development. 473-474 MRFs as. 256-258 oocyte maturation. 10 retina development, 526-527 sensoo~' differentiation. 555

spongiotrophobtast development, t 68 ventricular specification, 348-349 BCC, 579 B cells, 204 BDNE see Brain-derived neurotrophic factor BF-1 neural stem cell renewal. 243 telencephalon, 81 BF-2. 408 bHLH. see Basic helix-loop-helix factors Blast colony forming cell assay. 217 description, 217 Blastocyst asymmet~~'.23, 25-29 axes, origins, 26 lineages molecular specification, 159-160 setting aside. 158 Blastomeres. 151 BL-CFC. see Blast colony forming cell Blood. see Hematopoietic system Blood islands, 211 B ME see Bone morphogenetic protein Bone cartilage replacement by, 279-280 formation Cbfa. 287-290 Dlx. 290 gelatinase B, 290-291 Ihh. 287-288 Msx, 290 process, 279-281 VEGF, 290-291 ossification description. 279 types. 280 skull development regulation calvarium formation, 451-452 molecular evidence,-442-443 neurocranium formation, 45 t skeletogenesis, 442 studies. 441-442 skull organization characterization, 456-457 chondrocranium, 457 morphology dermatocranium. 464-465 nasoethmoidal, 457-460 occipital region, 463 orbitotemporal, 460, -462 otic region, 462-463 sptanchnocranium, 463- 464

693

Subject Index units/divisions, 457 Bone marrow progenitors, 246 Bone morphogenetic protein, see also Transforming growth factor-/3 BMP2 cardiac induction. 339 lung development, 314 neuronal differentiation, 238-239 pituitary development, 505,506 skull formation. 452 BMP4 expression pattern, 183-185 hematopoietic specification, 203 inner ear development, 550 tens development, 532-533 lung development, 314 mesoderm formation, 203 myotome formation, 264 neuronal differentiation, 238-239 pituitars' development, 505, 506 skull formation, 450, 452 tooth development bud-to-cap transition, 440-441 dentition patterning, 437 initiation, 439 ureteric bud. 314 BMP7 metanephros development, 408 pancreatic expression, 321 cardiac induction, 3.~6.~ ~""7 cardiac looping, 355 crainofacial development, 467-468 expression in LPM. 64 follicle formation, 577 follicle spacing, 557-558 forebrain morphogenesis, 85-87 pallial telencephalon patterning, 82-83 prosencephalic neural plate patterning, 78-79 signaling, role of chordin, 44 somite patterning, I4 t Brain. see also specific regions early patterning, 109-110 endothelial cells, 2 t 9 Brain 2/4, 506-509 Brain-derived neurotrophic factor inner ear neurogenesis, 559 neural stem cell survival, 239 Branchial arch characterization, 422 dermatocranium, 464 fate mapping, 424-426 intra identits, establishing, 449 Branchio-oto-renal system, 550 Bm3b, 527

C Cadherins cell-cell junctions and, 226 epithelium mesenchyme conversion. 408-410 gene, effects on heart, 340 muscle differentiation, 263-264 -positive, vascular endothelial cells, 200 Caecum. see Intestines CAKUT syndrome, 403 Calcium, 8 Calcium/calmodutin-dependent protein kinases, 342 CaMKs. see Calcium/calmodulin-dependent protein kinases Campometic dysplasia characterization, 281 XY reversal and, 380 Cardiac jelly, 350 Cartilage ECM function, 280-28 t replacement, 279-280 Cartilage homeoprotein I, 476 /3-Catenin dermal tumorigenesis, 579 follicle formation, 555, 557 neural stem cell renewal, 242 CBFs. see Core-binding factors CD. see Campomelic dysplasia cdc5, t 69- t 70 Cell fate node, 38-40 Cell lineage analysis, t 5 6 - I58 Celt movement node, 38-40 Cellular retinoid acid binding proteins l, 9 t Central nervous system AP patterning description, 3 I mes/met domains centrally located organizer, t 10-112 development, 115 fate mapping, 113 - 1 t 4 gene expression defining, 112-113 organization, FGF8 role, 115- t 16 patterning models, 116- t 17 Otx2/Gbx2 mutant rote, 114-115 development, flat-top mutation, 243 development/patterning, 107- t 09 dorsal ventral patterning Gli cells role, t21 SHH regulation, 120-12 t TGF-/3 regulation, 121-t 22 skull support, 457 stem cells adult/fetal, comparison, 245 celt cycle status, 245 characterization, 235

694 Central nervous system ( c o n t i n u e d ) differentiation process. 24 t role of BMR 238-239 function in adults. 237 hematopoietic stem cells, comparison, 2 4 3 - 2 ~ lineage, 237-238 neuronal potential, 24I "~+'~ nonneural derivative potential. _4... "~ ~,-247 occurrence. 235-236 other nerve cells, association. 237 outstanding issues. ;4/-.A8" -" v response to nervous system injur3~'. 247-248 survival, role of neutotrophins. 239 in x'i~+'ofunction. 244-245 Cerberus. 338 CerL, 31 CFC. see Cripto-FRL-1-C~ptic factors CFU. see Colony forming factors CHDs. see Congenital heart defects Cholesterol sulfate. 580 Chondrocytes FGF. 286-287 PTH/PTHrP. 284-286 Chondroitin sulfate proteoglycans. 350 Chordin characterization, 44 crainofacial development. 468 function. 44 Chorioallantoic branching, 170 Chorioallantoic fusion description. 161 initiating, 170 Chromatin. 9 Chx 10, 5 27 C#ed2. 339

Cleft palate, 434 Cloche. 213 - 215

c-myb, 2f'N CNC. see Cranial neural crest CNS. see Central nervous system Collagens fibrils, dermal development, 572-573 type II genes, as chrondrocyte markers. 282. 283 type IV renal development, 412 Colon. see Intestines Colon cancer. 506 Colonization. 201. 205 Colony forming factors activity in mammals. 198-200 hematopoietic system, 193-!96 HSC proliferative potential. 205 mesodermal precursors, 200-201 Congenital heart defects, 356-357

Subject Index Core-binding factors bone formation, 287-290 liver hematopoiesis, 204 Cornified envelope precusor proteins, 581 Cortex histogenesis. 91-92 regionalization, 84 Corticotropin-releasing hormone in diencephalon, 95 expression, 512 CRABR see Cellular retinoid acid binding proteins Cranial neural crest cells olfactory-" development. 435 tooth development. 4 3 6 - 4 3 7 characterization. 423 migration. 423-424 pattern formation ectoderm. 432-433 endoderm+ 433 experimental evidence, 426-427 mesoderm, 428,432 studies, 421-422 Cranial suspensory ligament, 386 Craniofacia. see also specific regions pattern/development bHLH. 4 7 3 - 4 7 4 genes encoding BMPs, 467-468 chordin, 468 EGF, 465 endothelin. 465 FGR 4 6 5 - 4 6 6 follistatin, 468 hedgehogs, 470

Lef], 471 Noggin, 468 PDGF. 466-467 Ph, 4 6 6 - 467 RA, 4 6 8 - 4 6 9 TGF. 465,467 Writ, 471 genes regulating Alx. 4 7 6 - 4 7 7 Cart1, 4 7 6 - 4 7 7 Dix. 471-472 goosecoid, 477 Hand. 4 7 3 - 4 7 4 Hox. 4 7 3 - 4 7 4 N/oc. 447 Or.v, 477 Pbx. 478-379 Pitx, 477 Prx, 4 7 6 - 4 7 7

695

Subject Index Twist, 473-474 WH, 479 studies, ihisto~,, 421-422 Cre/Iox recognition system, 15 CREM. see Cyclic AMP responsive element modulator CRH. see Corticotropin-releasing hormone Cripto-FRL- 1-Cryptic factors, 338 C~statlins characterization. 528 lens development, 53 t CSL. see Cranial suspenso~ ligament CSPGs. see Chondroitin sulfate proteoglycans Cyclic AMP responsive element modulator. 286 Cyclin E. 170 Cytochrome P450, 526

D Dach. 268 Dccvl

conservation, 387 Leydig cells, 384 sex determination...-/"-8-380 Delta-like homolog I inner ear neurogenesis, 559 sensow differentiation, 553 Delta/Notch pathway, 347 Depression, neurogenesis-associated, 247 Dermomyotome axes, 139-140 Desert hedgehog characterization, 384 crainofacial development, 470 Development. see also specific o r g a n s asymmetry early; 23-27 overview, 21-23 functional analysis mutant genes, 13 -14 in vitro approaches, 14 in vivo approaches, 14-15 MRF gene expression during, 258 preimplantation, 10-11 prepattern, 21-23 Dhh. see Desert hedgehog Diabetes mellitus Diencephalon, 84-85 Differentiation blastomeres, 151 cardiac GATA factors, .9":4/" . . . .- -.3 4 . 2 MEF2, 341-342 modulation in cardiac chamber, 343 SRF, 342

chrondrocyte, 28 t-284 diencephalon, 95 digestive tract.~ 307-308 embryonic, I55-156 endoderm, 301-302 follicle, 578 forebrain olfactory bulb, 95 pallium, 91-95 subpatlial, 89-9 t gonadotrope, 509-501 mechanisms, 152 otfacto~" bulb, 95 ovary. 384 proximal-distal, 313-314 sensory, inner ear hair ceil, 555-557 lateral inhibition, model, 553,555 organ of corti, 558 pattern regulation, 552-553 supporting cell. 557-558 Sertoli ceil, 380-382 skeletal muscle ceil-cell interactions in, 263-264 embryonic origins, 254-256 inductive mechanisms, 262-263 migratory progenitor cells, 265-266 MRFs in characterization, 256-258 functional groups, 258. 260-261 gene expression, 258 gene regulation, 262 negativetpostive factors, 264 study, histoLv, 253 terminology, 254 transcription factors regulating, 266-269 somite, 128-129 stem cells alternative models, 240-24 t BMP-induced, 238-239 testis, 380-382 urogenital, 374- 375 visceral endoderm, 164-166 yolk sacs, t 61 Diphtheria toxin, 510-51 I DKKt, 44 DLX transcription factors crainofacia! development, 471-472 DlxI/~

forebrain differentiation. 89-92 neuronal subtype specification, 89 skull formation, 450 tooth development, 437-438 Dlx3, dermal development, 580

Subject Index

696 DLX transcription factors (continued) DIx5

bone formation, 290 suballial neuron differentiation, 8 9 - 9 t Dlx6. bone formation. 290 Dmrtl

conservation. 387 testis differentiation. 382 DNA complementary, libraries, 11- 13 Dorsal-ventral axis. 108-109 Dorsoventral axis. 139-140 D r e h e r mutants, 122 Duodenum. see Intestines DV axis. see Dorsal-ventral axis Dynorphin B peptide, 337-338 Dysplasia. see Campomelic dysplasia

E Ear inner anatomy, 540-54 t cell proliferation. 547 compartment boundaries, 552 complexity, 539 development, 541-452 morphogenesis description, 547 expression domains. 547. 549-550 semicircular canal. 547 neurogenesis. 558-559 otic placodes gene expression. 546 patterning, 543-546 specification. 542-543 regionalized expression, 550-552 sensory differentiation hair celt, 555-557 lateral inhibition, model. 553. 555 organ of corti. 558 pattern regulation. 552-553 supporting cell. 557-558 sensory," organ specifications, 552 stria vascularis, 559-560 Early gastrula organizer definition. 40 patterning signals, 41 Ebfl, 91 ECM. see Extraceltular matrix Ectoderm CNC patterning, 432-"4.~,. .~.5 dental, 440

d e c a l , 570-57 t myotome formation, _62-26~ oral, 440 E d g l , 227 EGF. see Epidermal growth factors EGO. see Early gastrula organizer EGR transcription factors, 5 t 0 Embryo AP axis blastocyst. 29 conserved properties, 40 duplication. 22 EGO response, 4 t - 4 2 node development, 3 8 - 4 0 organizer function, 42-.44 polarity specification. 29-32 relationship, 25-26 chondrocranium characterization, 457 morphology dermatocranium. 464-465 nasoethmoidal, 457-460 occipital region, 463 orbitotemporal, 460, 462 otic region, 462-463 splanchnocranium, 463-464 gene expression, t 1-13 hematopoiesis, 202-205 lineages, differentiation, 1.55-156 nonneural ectoderm. 570-571 patterning, 2 - 3 periderm. 583-584 stem cells, in organizer function. 4 2 - 4 3 E m x 2 on, 374-375 En2, 115

Endoderm CNC patterning. 433 definition. 301 development, 156, t 6 4 - t 66 differentiation, 301-302 formation digestive tract. 307-3t0 formation analysis, 303-304 AP patterning, 304 during gastrulation, 303-304 gastrulation to midgestation, 304-305 liver, 316 - 318 lung basis, 311-312 differentiation. 313-314 models. 314 primary buds, 312 respiratow system, 312-313

Subject Index stem cells. 315 trachea, 312 vascular, 314 -mesoderm, spatial relation to, 304-305 pancreas cell lineages, 320-322 dorsal/ventral buds. 319-320 origins, 318-319 stem cells, 322 thyroid gland. 310-31i visceral, characterization, 161 yolk sac. 302-303 rote of cardiac induction, ~ .". . .-.,.~. o visceral anterior region activity source, 2 anterior patterning, 4 5 - 4 8 cardiac induction, .~" . . . -ooy ... CNC patterning, 427 differentiation, 3 0 - 3 t early patterning, 304 function, model, 47-48 Liml expression, 43 nascent, AP axis formation, 29-30 nascent, net movement of cells, 32 Endothelial cells arterial, 218 cadherin-positive, 200 cell-celt junctions, 226 generation cell number regulation, 2 t 5, 217-2 t 8 lineage specification, 213-215 origin, 212-213 hemogenic, 219-220 lymphatic, 218-219 organ specific, 219 type specification, 220 venous, 218 Endothelin- I CNC patterning, 432 crainofacial development, 465 skull formation, 449-450 Engraitedl/2, 112-117 Eomes, 167 Ephrins B 1, retina patterning, 525 B2 endothelial ceil expression, 218 retina patterning, 525 vascular development, 225 B4 expression, 218 vascular development, 225 -eph, rhombomere border formation, 119

697 Epidermal growth factors crainofacial development, 465 labyrinth branching morphogenesis, 171-t73 skull formation, 452 Epithelium lens maintenance, 532 to mesenchyme conversion aggregation, 408 - 4 i 0 ceil adhesion, 408-410 celt matrix interactions, 410 epithelial basement membranes, 4 t 0 - 4 I2 stroma, 407-408 pigmented, eye, 523, 525 thickening stage, dental, 454-455 ERRfl, I67 Er~4, 424 Escargot, 169-170 Estrogen dermal development, 58 I pituitary vascularization, 504~ Esxt, t72 Ets2, 168 Ewing's sarcomas, 247 Excretory system, see Kidney Extracellular matrix cartilage-specific, 280-28 I CNC, 424 dermal development, 572-574 epithelial to mesenchyme conversion, 4 ! 0 germ cell association with, t 87 Extraceitular proteinases, 223 Extraemb~/onic lineages, see also specific lineages analysis, 156-158 blastocyst, t 59-161 chorioattantoic placenta development, t 61-162 membranes, development, 162-164 overview, 155 - 156 primitive endoderm, t 56 spongiotrophoblast, 168-169 trophoblast development, 156 giant cell development, 169-170 signaling pathways, 166-168 yolk sacs, t 6 I Extraemb~,onic tissues, 2 Eya. see Eyes Absent genes Eye basic structure, 519 development conservation, 533 Pax6 effects, 527-528 ectopic, 533-534 endothelial cells, 219 lens development

Subject Index

698 Eye ( c o n t i n u e d ) epithelial celt maintenance, 532 fiber. 532 gene regulation, 53I induction. ~.z,,~ ~.8-530 morphological/biochemical events. _ _8 multiple pathways, 534 placode to vesicle, 530-53I optic stalk, development. 523. "" ~, pigmented epithelium, 523,525 retina development cell type specifications, 526-527 dorsoventral/nasotemporaI patterning. 5 25 ,.-~enetic determination. 521 -~_,.-'~'~,5 25 RA role. 525-526 Eyes Absent genes, E y a l conservation/divergence, 533 inner ear development, 549-550 lens development, 530-531 myotome formation. 268 renal mesoderm, 398-399

F Fate mapping emb~'o, advantage. 191 gastrulation studies, 39 hematopoietic system, 2 0 1 - 2 0 2 Mes/Met domains. 11"_~-113 testing, stem cell potential. 246 Feedback loops. 62-64 Fertilization. 5.8 Fetus. see Embryo FGE see Fibroblast growth factors Fibroblast growth factors chondrocytes. 286-287 crainofacial development. 465-466 dorsal CNS patterning. 121 - 122 endoderm formation. 303 FGF-1 angiogenic function. 2 2 3 - 2 2 4 lens development, ~.~,.-533 ""'~ FGF-2 angiogenic function, 223-224 endothelial differentiation, 215 hematopoietic cell differentiation. 215 lens development, 532-533 metanephros development, 408 neural stem cell renewal. 242 trophoblast development, 167 FGF-3 inner ear development, 543.545-546 sensory differentiation. 558 FGF-4, 166 - 167

FGF-8 anteroposterior tetencephaton patterning, 84 AP axis polarity, 3t cardiac induction, 337 "9 CNC patterning, 4~z forebrain morphogenesis, 86 mes/met development, 115 - 1 t 6 mes/met pattern development, 112-113 pallial telencephalon patterning, 82-83 pituitary development, 506 skull development, 449 tooth development, 438..440 genes, effects on lung development, 314 heart. 340 hematopoietic specification, 203 hormonal expression, 511 labyrinth branching morphogenesis, 171-173 mesoderm formation. 203 pituitary' development, 505 pituitars, vascularization, 504 renal tubule induction, 407 skull formation. 452 Fibronection description. 222 gene. effects on heart, 340 Flat-top mutation, 243 Flk- 1 glomerular development, 4 1 2 - 4 t 4 hematopoietic specification, 203 mesoderm formation, 203 F molecule model. 55 Fn 1. see Fibronection FOG-2 cardiac myogenesis, 343 vascular development, 219 Follicle cycling, 578-579 differentiation. 578 formation. 574-575,577 morphogenesis. 578-579 spacing, 577-578 Follicle-stimulating hormone functions, 500 transcription factors, 510 Fotlistatin. 468 Forebrain anterior neural plate, 79 cell-type specifications, 87-88 diencephalon cell specification, 95 differentiation, 95 induction/patterning, 8 4 - 8 5 differentiation neuronal, 91-95

699

Subject Index pallium, 91-95 subpallial neurons. 89-9 t morphogenetic mechanisms, 85-87 neurogenesis, control of. 87-88 neuronal subtype specification, 88-89 organization, 75-77 prosencephalon early morphogenesis. 79 neural plate, patterning subdivisions, 78-79 organization, 75, 77 telencephalon anteroposterior, patterning, 83-84 organization, 77 pallial, patterning, 82-83 subpalliaI, patterning, 79-82 Forkhead/winged helix genes, 404 Foxc. see Fork_head/winged helix genes FRIZZLED, 82-83 FSH. see Follicle-stimulating hormone Furin. 67-68 Fzd5, 172

G Gap junctional communication, 6 0 - 6 t Gastrointestinal tract, see specific regions Gastrulation conserved properties, 40 description, 38 node development, 38-40 vertebrate organizers embry'ological studies, 41-42 function, 42-44 genetic studies, 41-42 inhibitory.' signals, 44 specification, 44-45 GATA transcription factors A m h regulation by, 381-382 -FOG2. vascular development, 2 I9 GataI, hematopoietic specification/differentiation, 204 Gata2

hematopoietic specification/differentiation, 204 liver hematopoiesis, 203-204 pituitao~ development, 381-382 Gata4

endoderm development, 165 -FOG, cardiac myogenesis, ~4_-o43 Gata6, endoderm development, 165 Gbx2

CNS AP patterning, 114- l 15 mes/met domain expression, 114 pattern development, I t 2-1 ! 3 patterning, model, I t 6-117

GDF type t node flow, 60 patIiat teIencephalon patterning, 82-83 type 7, dorsal CNS patterning, I21.-122 type 8 (see Myostatin) GDNE see Gliat-derived neurotrophic factor Germ cells characterization, 181 early appearance, t 82- t 83 *'~ "~ 4 in gonad development., 38.-.-o8migration adverse behavior during, 187- t 88 guidance oK 186-187 process, t 85-t 86 proliferationl~urvival during, 188-189 specification, 183-185 Germinal vesicle, see Oocytes GFP. see Green fluorescent protein GH. see Growth hormones GHRH. see Growth hormone-releasing hormone GJC. see Gap junctional communication Glial cells derived neurotrophic factor, 398, 40t-403 missing- I, 170 neuron generation, 87- 88 Gliat growth factor, see Neuregulin Gti zinc finger transcription factors GIil

dermal tumorigenesis, 579 diencephalon, 84-85 Gli2

CNS patterning, I2 t diencephalon, 84-85 lung development, 3 i 2 tooth development, 439 Gli3

CNS patternin, 12 t lens fiber development, 532 lung development, 3 t2 optic stalk development, 522 palIiat telencephalon patterning, 83 tooth development, 439 Gtomerular, 412-414 Gonad development genes involved in, 374-376 germ cells, 383-384 Leydig cells, 384 origins, 372-373 studies, 382-383 differentiation, 375-376 major cell lines, 373 mesonephros, 37o - ~, 4

Subject Index

700 Gonadotrope. 509-510 Gonadotropin-releasing hormone. 510 Goosecoid. 477 Gorlin syndrome, 579 Grb2, 171-172 Green fluorescent protein expression, 182 guidance of. 186-187 motility, 185 Growth hormone-releasine hormone in diencephalon. 95 expression, 51 1-512 Growth hormones expression. 511-512 functions. 500 transcription factors. 510 Gut tube regionalization. 305

N Hairy genes, 138 - 139 Hand genes cardiac looping, 355 crainofacial development, 355 spongiotrophoblast development. 168-169 ventricular specification, 348-349 HB-EGF. 156 Head CNC fate mapping, 424-426 migration. 423-424 pattern formation ectoderm. 432-433 endoderm. 433 experimental evidence, 426-427 information source. 427-428 mesoderm. 428. 432 primordial cells, 423 primordial cells. 422 skull development regulation calvarium formation. 451-452 intra-BA identity, 449-451 molecular evidence, 442-443 neurocranium formation, 450 skeletogenesis, 442 studies, 441-442 skull organization characterization, 456-457 chondrocranium, embryonic. 457 morphology dermatocranium. 464-465

nasoethmoidaI. 457-460 occipital region, 463 orbitotemporal, 460. 462 otic region, 462-463 splanchnocranium, 463-464 units/divisions, 457 Heart apoptosis, 34 t cellular proliferation, 341 chamber formation. 347-348, 351 development, 332-334 disease, congenital, 356-357 endothelial cells. 219 induction activin. 337 BMR 336, 337 cerberus, 338 CFC. 338 conserved pathway, 334 endoderm. 334-336 FGE 337 timing/stability, 340 visceral endoderm, 338-339 Wnts/anti-Wnts, 336-337 inflow/outflow patterning Delta/Notch pathway. 347 early model. 345 RA signaling, 346-347 left-right axis, 351,354-356 morphogenesis conserved pathway, 334 field, 339 looping, 351,354-356 segmental model, 344 myogenesis cardiac chamber, 343 GATA factors. 342-343 MEF2.341-342 SRF. 342 precursors, migration of, 340-341 regionality development, 343-344 plasticity of, 344 structure, 332-334 ventricular specification hand genes. 349-350 Irx4. 350-351 MEF2 proteins, 350 Nkx2-5 homolog, 348-349 versican, 350 Helix transcription factors HNF3, endoderm development, 165-166 HNF4, endoderm development, 164-i 66

Subject Index H N F r , pancreatic expression, 321 HNF313

anterior patterning, 47 endoderm formation, 303 liver development, 316-3 t 7 organizer function, 42-44' Hemangioblasts, 214 Hematopoiesis colonization theory of. 201 definitive, 203-204 genes affecting cell migration, 204 fetal liver, 203-204 lineage specification/differentiation, 203-204 proliferative potential, 205 regulation, 202 Hematopoietic system characterization, 191 colonization, 205 compartments, origins amphibians, 196, 197 colonization theory, 201 fate mapping, 201-202 mammals. 198-199 mesodermal precursors, 200-201 multiple, 199-200 evolution, 205 genetic prograrning, 205-206 growth factors, response to, t93-194 hierarchy, 192 progenitors identification, 192-194 location, 195 - 196 specification, genes involved, 203 stem cells fate of, redirecting, 206 identification, 192-194 location, 195 - t 96, 199-20 t proliferative potential, 205 Heparan sulfate proteoglycan, 187 Hepatocyte ga'owth factor function, 317 -SE role in muscle development, 266 ureteric bud ~owth, 404 Hepatocyte ~owth factors, 17 t-173 Hereditary hemorrhagic telangietasia, 227 Herpes simplex virus 1, 510 Hes 1

pancreatic expression, 321 paraxial mesoderm expression, 138-139 sensory differentiation, 555 Hesxl

anterior neural development, 47

701 expression in AVE, 45 pituitary development, 507-509 Heteroxtaxia, 354 Hex

characterization+ 21T expression, 30-3 t HGE see Hepatocyte growth factor; Hepatocyte growth factors HGM proteins, 9, 377 Hhex

AP axis polaris; 3 I expression in cloche, 2t4-215 HHT1. see Hereditary hemorrhagic telangietasia HIF- lc~, 225 Hindbrain, 117-t t 9 Histories, 9 HL-rb9, 319- 320 Hmx3, 549 HNE see HeIix transcription factors H o x a t , 544-545 Hox genes CNC patterning mesoderrn, 428, 432 sources, 427-428 crainofacial development, 474 ectodermal development, 433 gut specification, 305 hindbrain patterning, 117-118 neural stem cell renewal, 242 rhombomere formation, 1 t 9 skull formation, 443,449 small intestine, 309 th~oid growth, 3 t 1 HSPG. see Heparan sulfate proteoglycan HSV 1. see Herpes simplex virus 1 Huntington's disease, 247 Hyaluronic acid, 350 Hyd, 68 Hypothalamic-reieasing hormones, 502 Hypothalamus, 504 Hypoxia, t 73

ICM. see Inner celt mass IKK, 580 Ileum. see Intestines I'mfa], 169 Indian hedgehog bone formation, 287-288 characterization, 285 crainofacial development, 470 stomach development, 308, 310

Subject Index

702 Inner cell mass derivatives. 157 description, 156 differentiation, 23, 2 5 - 2 6 isolation, stages, t58 Oct4, t 60 surface. 28 trophobtast development. 166 Insl3. 386 Integrins dermal development. 573-574 description. 222-223 germ cell migration. 188 hematopoietic cell migration, 204 Integumentary structures, see specific organs Intestines development, 309-310 differentiation. 307-308 patterning. 307-308 Intrauterine growth restriction. 174 Inversin gene. 354 lrx. 350-351 lsI1

pancreatic bud formation. 319-320 pituitar?' development. 505 Isomerism. 354 Itga-4. 170 IUGR. see Intrauterine growth restriction iv mutation. 58-59

J Jagged gene. 553.555 Jak2. 204 Jejunum. see Intestines JNK. see c-Jun N-terminal kinase c-Jun N-terminal kinase. 405

K Keratin. 579-580 Kidney development classical models, 404-405 glomerular. 412-414 intermediate mesoderm, 397-399 mesenchymal induction. 405.407 PKD-association. 414-415 vascularization. 412-414 Wilms' tumor-association. 415-416 Wnt signaling, 405.407 epithelial to mesenchyme conversion aggregation. 408-410 cell adhesion. 408-410 cell matrix interactions, 4 t 0

epithelial basement membranes, 4 t 0 - 4 1 2 stroma, 407-408 metanephric mesenchyme, 399-400 nephric duct initiation. 400-401 origins, 372. 395-396 tubules~ induction, 407 ureteric bud outgrowth inhibitors/enhancers. 4 0 3 - 4 0 4 RET/GDNF pathway. 401-403 Klf4. 580-581 Kreisle~ 544

KROX20. 117-118

L Labyrinth branching morphogenesis. 171-173 description, 161 development. 161 - 162. 170 LacZ, 267 Laminar patterning, 91-95 Laminins. 411 Lateral ganglionic eminence description. 77 development. 81 - 82 forebrain differentiation, 89-90 olfacto~' bulb differentiation, 95 specification, 82 Lateral plate mesoderm G D F 1 expression, 60 left-side expression in, 62 node and. line between. 64 node to, signal transfer, 64 LCR. see Locus control region LDL gene family, 94 Lefl

crainofacial development. 471 labyrinth branching morphogenesis, 172 tooth development, 439-440 LEF/TCE 86-87

t~h~.J

characterization, 47 left-right asymmet~', 59-61 midline, 6 4 - 6 5 retinoic acid effects, 67 Lefo,2 characterization. 47 expression, retinoic acid effects. 67 expression in left-right asymmetry, 5 9 - 6 I -nodal. regulatory relationship, 6 2 - 6 4 Legless mutation. 59 Leukemia inhibitory factor expression, 512 implantation process, 156

703

Subject Index Leukocyte inhibito~ ! factor, 407 Leydig cells, 373,384

LNg inner ear neurogenesis, 559 paraxial mesoderm, 138-139 sensor: differentiation, 555,558 LGE. s e e Lateral ganglionic eminence LH. s e e Luteinizing hormone Lh_r ,~ ,-) CNC patterning, 4_~_ pituitary development, 507-508 retina development, 521-522 LIF. s e e Leukemia inhibitor5' factor; Leukocyte inhibitory factor LIM homeodomain, L i m t anterior neural development, 4 6 - 4 7 gonad development, 376 organizer function, 43-44 renal mesoderm, 397 urogenital differentiation. 374-375 Lineage specification, t 5 I Liver fetal, hematopoiesis, 203-204 formation cell interactions. 3 t7-318 endodermal competence, 31.6-317 mesodermal signals, 3 t 7 nonendodermal tissues, 3 t 8 regenerative capacity, 318 LMR s e e Lateral plate mesoderm Locus control region, 202 Lung development basis, 311-312 buds, 3 t 2 mesoderm differentiation, 314 models, 3 t4 proximal-distal differentiation, 313-314 respirator' system, 3 t 2-313 stem cells, 3 t5 trachea, 312 vascular, 3 t 4 Luteinizing hormone expression, 502 functions, 500 transcription factors, 510 Luteinizing hormone-releasing hormone, 435

M MAR 53 t MAR s e e Mitogen-activated protein MAP kinases, s e e Mitogen-activated protein kinases M a s h 1 , 88-89

t 68- i 69 Math l, 555, 558 Math5, 526 Matrix metaJtoproteinases, 223 Matrix remodeling, 223 Maturation promoting factor description, 7 inactivation, 8 Medial ganglionic eminence description, 77 development, 8 t-82 forebrain differentiation.. 89-90 specification, 82 Medial-lateral axis, 78 MEF2 proteins cardiac myogeneis, 341-342 ventricular specification, 350 Meiosis, 7-8 MelanocytestimuIating hormone, 499 Mental illness, 247 Mes. s e e Mesencephalon Mesencephalon AP patterning, 1 t 0 - I 12 domains, gene expression patterns, 112- t 13 fate mapping, 1 t 3 - t 14 Mesenchymal cells renal development, 405, 407 renal function, 386 Mesenchyme epidermal appendage morphogenesis, 574 to epithelial conversion aggregation, 408-4 t 0 cell adhesion, 408-410 cell matrix interactions, 410 epithelial basement membranes, 410-412 stroma, 407-408 pituitary development, 504-506 Mesoderm cardiac, 335 ,,) -,, cells, ,.6~ CNC patterning, 428,432 differentiation, 314 -endoderm, spatial relation to, 304-305 endothelial celt origin and, 2t2-213 formation, 203 hepatic signals, 317 paraxial molecular clock, 138-t39 progenitor cells, 132 presomitic, cell transition, t 32-133 progenitor cells, 254 renal, 397-399 Mesp t, 340 Met. s e e Metencephalon

Mash2,

Subject Index

704 Metanephric mesenchyme, 399-400 Metanephros. s e e Kidney Metencephalon AP patterning, 110-112 domains, gene expression patterns. 112-113 fate mapping. 113-114 M g a t , 67 MGE. s e e Medial ganglionic eminence Microinjection of proteins, t 4 Migration germ cells adverse behavior during, 187-188 guidance oL 186-187 process. 185-186 proliferation/survival during, 188-189 hematopoietic cell, 204 Mitogen-activated protein. 287 Mitogen-activated protein kinases, 8, 167 Mixer. 166 Morphogenesis branching chorioallantoic, 171-173 organ formation. 298-299 budding, 298-299 cardiac. 334. 344 endoderm. 304-305 epidermal appendage, 574 follicle. 578-579 forebrain, 85-87 inner ear, 547, 549-550 node, 38- 40 olfactow bulb. 84 prosencephalon, 78-79 Mouth development, 433-434 M o x 2 , 267 MPCs. s e e Myogenic precusor cells MPF. s e e Maturation promoting factor MRFs. s e e Myogenic regulatory factors MSH. s e e Melanocytestimulating hormone Msx transcription factors crainofacial development, 474 Msxl

formation, 290 myotome formation. 267 skull formation, 450 tooth development bud-to-cap, 440--441 initiation, 439 patterning, 437-438 Msx2

formation, 290 skull formation. 452 tooth patterning, 437-438

mTEAD-2 n, 9-10 Mueller cells, 526 Mi.ilterian duct characterization, 373-374 differentiation. 385-386 Muscle. s e e Skeletal muscle MyoD. s e e Myogenic regulatory factors Myogenesis. s e e u n d e r Differentiation Myogenic precusor cells. 269 Myogenic regulatory" factors cell-cell interactions, regulation of. 263-264 characterization, 151-t52, 253 conservation. 256-258 ectoderm. 263 expression, 254 functional groups, 258.260-261 gene expression, 258 gene regulation, 262 muscle specification, 268-269 myotome formation. 265, 266-269 satellite cells, 269, 271 Myosin. 556 Myostatin, 264 Myotome formation ectoderm role, 262-263 genetic analysis, 264-265 positive/negative signals, 264 transcription factors regulating, 266-269 waves of, 131

N NCSC. s e e Neural crest stem cells Nephric duct. 400-401 Nephron, 396 Nerve gowth factors expression, 511 neural stem cell survival, 239 Netrin-1 inner ear development, 547 pituitary development, 506 Neural axis, 2-3 Neural crest stem cells adult/fetal, comparison, 245 cell cycle status. 245 characterization. 235 childhood cancer and, 247 differentiation alternative models, 240-24 t BMP role, 238-239 " hematopoietic stem cells, comparison, 9_4_~-244 lineage, 237-238 neuregulin, 239-240

Subject Index neuronal potential, 241-242 nonneural derivative potential. 245-247 occurrence, 236-237 other nerve cells, association, 237 outstanding issues, 248 response to nervous system inju~', 247 self-renewal, regulation, 242-243 survival, role of neutotrophins, 239 in vivo function, 244-245 Neural plate anterior, 78-79 prosencephalic, 78-79 Neuregulin, 239-240 NEUROD 1,509 Neurodl, 559 Neuron restrictive silencing factor, 243 Neurons development, 247 differentiation in forebrain, 89-91 laminar patterning, 94 subtype, forebrain, 88-89 Neuropilin- 1,223 Neurosphere cell, 246 Neurotrophin 3 inner ear neurogenesis, 559 neural stem cell survival, 239 Neurulation, 78-79 Neutotrophins, 239. see also specific Neutotrophins NGE see Nerve growth factors Ngn2, 526 -527 Nkx genes Nkx2.1, 474 Nkx2-3

L-R asymmetry, 68 nodal regulation, 66-67 small intestine, 310 Nkx2-5

cardiac looping, 356 cardiac morphogenesis, 339 CHD, 357 heart formation, 348-349 Nkx5.1, 474 Nkx6.1, 474 Nodal gene, cardiac looping, 355 left-fight asymmetry, 59-61 Lef-o~'2regulation, 62-64 Nkx2-3 regulation, 66-67 Node development, 38-40 flow, 59-6I, 64 genes regulating, 66-67 and LPM, line between, 64

705 Noggin crainofacial development, 468 myotome formation, 265 Nose. see Olfactory Notch signaling cardiac patterning, 347 myogenic differentiation, 263 sensorv~ differentiation. ~.~-~' son-Ate borders and AP polarity, t36-138 vascular development, 226 NRSE see Neuron restrictive silencing factor NT-3. see Neurotrophin 3

O Oct4

germ cell expression, 183 ICM role, 160 Olfacto~' bulb development, 435 differentiation, 95 Oligodendrocyte precusor cells, 246 Oocytes asymmetry, 7-8 development initial stages, 6 - 7 transcription during, 8-10 fertilized asymmetry in, 1 morphological study, t maturation, 10-t I OPCs. see Oligodendrocyte precusor cells Organogenesis. see also specific organs functional compartments, 299 molecular control of, 299-300 morphogenesis, 298-299 process, 297-298 progenitor tissue remodeling, 298 Orthc,denticle transcription factor Otxt, inner ear development, 549 Otx2

anterior neural development, 4 6 - 4 7 anterior patterning, 47 CNS AP patterning, 114-115 expression in Mes/Met domains, 113-114 function, 44 inner ear development, 549 rues/met domain patterning, model, 116-117 rues/met pattern development, 112-1 t3 retina developmenL 521 Ossification description, 279, 281 types, 280

Subject Index

706 OT. s e e Oxytocin Ovary, differentiation, 384 Oxytocin diencephalon, 95 expression, 502 functions. 500

P PA. see Posterior anterior interfaces

Paired-box genes crainofacial development, 475-476 Pax2

inner ear development. 549 mes/met domain patterning, model, I16-117 mes/met pattern development, 113 metanephros development. 408 optic stalk development. 522 renal mesoderm. 397-399 reproductive tract. 385 Pax3

muscle development, 266 myotome formation. 267-268 Po~v5

mes/met development, 115 mes/met domain patterning, model. 116-117 Pax6

CNC migration. 423-424 conservation/divergence, eye. 533-534 dosage effects, eye. 527-528 ectopic eye development, 533-534 laminar patterning, 94 tens development. 530-531 optic stalk development, 522 pituitary development, 509 retina development, 521-522. 526 tooth development, 440 PcLv7

muscle development, 266 muscle satellite cells. 271-272 myotome formation. 267-268 P~v& thyroid growth, 311 Pax9

gut tube regionalization, 305 hematopoietic specification/differentiation. 204 tooth development, 44I Paired-related genes. 476-477 Pallium, 9 t-95 Pancreas formation ceil lineages, 320-322 dorsal/ventral buds. 319-320

origins, 318-319 stem cells, 322 function, 319 Paneth cells. 310 Para-aortic splanchnopleura angiogenesis, 225 CFU-S in. 196 hematopoietic progenitor source, t 99 hematopoietic stem cell source, 199 mesodermal precursors, 200-20I ParaHox genes Cdxt/2

characterization, t 67 gut tube regionalization. 305 role in gut tube regionalization. 305 Pdrl

gut tube regionaiization. 305 skull formation. 443.449 small intestine formation, 309 Parathyroid hormone. 284-286 Parathyroid hormone related peptides. 284-286 ParcLvis, 267 Parkinson's disease. 247 PAS. see Para-aortic splanchnopleura Patch. 466-467 Patterning AP .'aVE role. 4 4 - 4 8 in endoderm. 304 mes/met, regulation. 110-1 I2 CNS. 107-109 diencephalon, 84-85 digestive tract. 307-308 dorsal ventral in CNS, 120-122 DV, in forebrain, 88 establishment, 1-3 inflow/outflow, cardiac, 346-347 laminar, 91-95 rues/met domain, models, 116-117 prosencephalic neural plate, 78-79 retina. 525 telencephalon anteroposterior, 83-84 pallial, 82-83 subpallial, 79-82 Pb. see Polar body PBX genes, 478-479 Pdyn. see Preprodynorphin gene Pericytes. 226-227 Periderm. 583-584 Peripheral neuroectodermal tumors, 247 PFR see Prospective floor plate Pie- 1,243

Subject Index Pitl, 512

Pituitary gland anatomy, 499-500 development cell proliferation, 502, 504 committed cell type expansion, 510-5 t 2 genetic roles, 506-509 induction, 504-506 morphology during ontogeny, 502 multihormonat cells, 512 transcription factors cell specification synergy. 509-5t0 signaling molecule interactions, 506 vascularization, 502 hormones appearance, 502 functions, 499-500 PITX gene family Pitxt, crainofaciat development, 478 Pitx2

crainofacial development, 478 expression on left side. 65-66 heart looping, 354-355 mouth development, 433-434 pituitar? development, 506-507 Pitx3, lens development, 532 Pitx4, pituita_~' development, 506-507 PKA. see Protein kinase A PKD. see Polycystic kidney disease Placenta chorioallantoic development, 161 function, i 61-162 development, pregnancy complications and, 173-174 evolution, t 63 species differences, 162-i63 Placodes, neurogenic, 434-435 Ptatelet-derived growth factors crainofacial development, 466-467 glomerular development, 413-414 lung development, 313 pericyte recruitment, 226 smooth muscle ceil recruitment, 226 PNET. see Peripheral neuroectodermal tumors Podl, 413 Podocyte cells, 4I 1 Polar body, 26-27 Polarity AP axis somitogenesis, 134, 136-138 specification, 29-3 t vascular, 227 Polyadenytation, 10

707 Polycystic kidney disease, 414-415 PoIycystin- 1,415 POMC. see Pro-opiomelanocortin Posterior anterior interfaces, 138 POU inner ear development, 549 neuronal subtype specification, 89 pituitary development, 509 Pou5fl (see Oct4)

senso~J differentiation, 556, 558 Pregnancy complications, t73-174 Preprodynorphin gene, 338 PRL. see Prolactin Progenitor cells allocation to pa_raxiat mesoderm, t32 migratory muscle, 265-266 Progenitor tissues, 298 Prolactin characterization, 500 expression, 5 t t Pro-opiomelanocortin characterization, 499 expression, 502 HSVt, 510 Propl, 509 Prosencephaton description, 75, 77 early morphogenesis, 79 Prospective floor plate, 64-65 Protein kinase A, 285-286 Protein kinase C, 580 ProxI

inner ear development, 549 tens development, 532 Prox2, 549 Proximal-distal differentiation, 313-314 PTFt, 321

R RA. see Retinoic acid RAR, 403 Rathke's cysts, 5 t 2 Rax. see Rx Receptor type tyrosine kinase, 401-403 Rectum development, 310 Remodeling, bone, 298 Reproductive tract development, 385-386 Respiratocv system, see under Lung RET. see Receptor type tyrosine kSnase Retinaldehyde dehydrogenase 2, 545 Retinoic acid crainofaciaI development, 468-469

708

Subject Index

Retinoic acid (continued) Hox expression, 67. 1 19 inner ear development, 545 L-R asymmetry. 67 lung development, 312 retina development, 525-526 sensory differentiation. 557 signaling in cardiac patterning, 346-347 trophoblast, 167 Rhombomeres border formation. 119 junctions, 118- I 19 segmentation, 119 transcription factors. 117-118 Rieger syndrome, 507 RNA double-stranded. 14 messenger inhibiting translation. 14 - 15 oocyte maturation. 10-11 preimplantation development, 10-111 Rpx, 45 Rx. 521-522

S Satellite cells in adults, 269 MRFs. 269. 271 proliferation. 271-272 Sca-1. 192-193 Sclerotome axis, generation. 139-140 cells, function. 128-129 delineation. 140-141 formation. 131 subdivision. 129. 131 Sct/tal-1

characterization. 216 expression in cloche, 214-215 hematopoietic specification. 203 mesoderm formation, 230 Secreted factors of the Frizzled-related protein, 83 Segmentation description, 127 rhombomere regulation, 119 somite, rate oL 130-t31 Sensor.. neurons, 434-435 Sensu stricto, 77 Serrate- I, 557 Serrate-2, 557 Sertoli cells differentiation, 380-382 genotypes, .~82-.~8_~

Serum response factor, 342 Sex cord, 188 Sex determination, see also Gonad; Ovary evolution and, 386-387 genes involved in D ~ I , 378-380 SRY, 376-378 Tda, 378 Sexual development, 384-386 S F I see Steroidogenic factor 1 SFRP. see Secreted factors of the Frizzled-related protein Shaker2, 556-557 SHH. see Sonic hedgehog Signal transducer and activator of transcription, 2 8 6 287 Six-homeodomain transcription factors Sixl, myotome formation. 268 Six3

lens development, 530-53 I retina development, 521-522 Skeletal muscle differentiation cell-cell interactions in, 263-264 migratory, progenitor cells, 265-266 MRFs in characterization, 256-258 functional groups, 258, 260-261 gene expression. 258 gene regulation, 262 negative/postive factors. 264 study, histou, 253 transcription factors regulating, 266-269 embry, onic origins, 254-256 mesodermal progenitors, 253-254 myogenesis, inductive mechanisms, 262-263 regeneration, 269, 271.-272 specification. 268-269 transcriptional regulation, specificity, 262 Skeleton. see Bone Skin development factors. 572-574 tumorigenesis and, 579 embr3'onic ectoderm, nonneural, 570-571 mature cells, 570 characterization, 570 function. 570 stratification. 571-572 terminal differentiation early. 579-580 late. 581-583 late stage, transit to, 580-581 tumorigenesis, 579

Subject Index SMAD2. s e e a l s o Transforming growth factor-/3 anterior patterning, 47 endoderm formation, 303 mutants, 43 Small proline-rich region proteins, 581 SMG see Submandibular gland Smooth muscle cell recruitment. 226-227 Sna, 65-66, 169-170 S n a i l gene, 354 Snell's waltzer, 556 SOM. see Somatostatin Somatic cells, 373 Somatostatin, 95 Somites AP compartments, 143 AP polarity, role of Notch signaling, 134, 136-138 borders, role of Notch signaling, t34, 136-t38 compartments, lineage dorsal. 141 generation, 139-140 lateral. 140-141 ventral. 140-141 development, staging, 130 formation kinetics. 138-139 process, 127-132 number, variation, 130 prepatterning, 133-134 segmentation, rate of, 130-131 skeletal, muscle origins, 254 Sonic hedgehog crainofacial development, 470 dermal tumorigenesis, 579 diencephalon, 84-85 follicle development, 578 forebrain morphogenesis, 86 L-R asymmetry, 68 lung development, 312 mutants, in organizer function, 43 myotome formation, 264, 266 pancreatic buds, 319-320 prosencephalic neural plate patterning, 78 scletotome delineation, 140-141 stomach development, 308 tooth development bud-to-cap transition, 440-44 t ectoderm regionalization, 439-440 SOX transcription factors chrondrocyte differentiation, 28 t-284 ectopic eye development, 533-534 forebrain differentiation, 91 lens development fiber, 532 induction. 529

709 pIacode to vesicle, 530-531 regulation, 53 t sex determination, 91 testis differentiation, 380-381 Species differences, 162-164 Spongiotrophoblast, t 68-169 SPRRs. s e e Small proline-rich region proteins SRF. s e e Serum response factor SRY gene characterization, 376-378 conservation, 386-387 Stem cells CNS adult/fetal, comparison, 245 cell cycle status, 245 characterization, 235 differentiation BMP rote, 238-239 process, 241 function in adults, 237 hematopoietic stem cells, comparison, 243-244 lineage, 237-238 neuronal potential, 241-242 nonneural derivative potential, 245-247 occurrence, 235-236 other nerve cells, association, 237 outstanding issues, 248 response to nervous system injury, 247-248 survival, role of neutotrophins, 239 in vivo function, 244-245 emb~'onic characterization, 152-153 differentiation, 160 for hemangioblast study, 200 mouse, 158 trophoblast development, 166 epidermal, 570 hematopoietic description, 191 identification, t 92-194 location, 195-t96, 199-201 proliferative potential, 205 rate of, redirecting, 206 hepatic, 318 ligands, hematopoietic specification, 203 mesoderm formation, 203 neural adult/fetal, comparison, 245 cell cycle status, 245 characterization, 235 childhood cancer, 247 differentiation alternative models, 239 BMP role, 238-239

Subject Index

710 Stem cells (continued.) hematopoietic stem cells, comparison, 243-244 lineage, 237- 23 8 neuregulin effects. 239-240 neuronal potential. 24 t-~.4_ nonneural derivative potential, 245-247 occurrence, ..~)-236 other ner~'e cells, association. 237 outstanding issues, 248 response to nervous system injury', 247-248 self-renewal, regulation. 242-243 survival, neutotrophin role. 239 in vivo function. 244-245 pluripotent muscle-derived. 271-272 pulmonary. 315 source, pancreatic ducts as. 322 trophoblast isolating, 166-167 proliferation, 160 Stereocilia production. 557 Steroido_~enic factor 1 gonad differentiation. 375-376 hormonal development role. 509-510 testosterone production, 384 Stomach development, 308-309 Submandibular gland. 436 Subventricular zone description. 87 properties, 88-89 Superovulation. 21 SVZ. see Subventricular zone

T Tabby gene, 44 1 Tal-l, 216 TATA box binding proteins. 9 T-box transcription factors TBrl. in laminar patterning, 92 Tbxl CHD role, 357 retina patterning, 525 T cells CD31. HSC expression, 192-193 CD34, HSC expression, 192-193 hematopoietic cell migration, 204 hematopoietic specification/differentiation, 204 Tcfl, 172 Tcfl 5. see Paraxis Tda gene, 378 rdgfl, 31 Telencephalon organization. 77 palliat, 82-83 subpat!ial, patterning, 79-82

Testis descent, 386 differentiation, 380-382 Testosterone, 58 I Tfm, 386 Thyroid gland development, 310- 3 t 1 Thyroid hormones, 510 Thyroid-specific enhancer binding protein lung development, 312 pituitaor development, 504, 507-508 thyroid development, 311 Thyroid stimulating hormone expression, 502 functions. 500 receptor, 311 Thyrotrope, 512 Thyrotropin-releasing hormone diencephaton, 95 expression, 512 Tie 1 receptors. 224 vascular polarity, 227 venous malformation disease and, 227 Tinman, 334 Tissue nonspecific alkaline phosphatase, 183 Titf]. see Thyroid-specific enhancer binding protein TNAR see Tissue nonspecific alkaline phosphatase Tooth characterization, 436 development bud stage, 455 bud-to-cap transition, 440-441 cap stage, 455-456 cells, origins, 436-437 dentition patterning, 437-438 ectoderm regionalization, 440 enamel knot. 44 1 epithelial thickening stage, 454-455 initiation, 438-440 molar, 456 morphogenesis, 574 Trachea. see under Lung Transcription factors, see also specific f a c t o r s laminar patterning, 92, 94 during oogenesis, 8-10 Transforming growth factors crainofacial development, 465 hematopoietic specification, 203 mesoderm formation, 203 secondary palate development, 434 Transforming growth factor-fl anterior neural development, 4 6 - 4 7 crainofacial development, 467 dorsal CNS patterning, 121-122 germ cells, 187

711

Subject Index myotome formation, 264 node flow, 60 organizer formation. 44-45 paliial telencephalon patterning, 82-83 "9 t pancreatic expression, ~_ pericyte recruitment. 226 signaling, prosencephalic neural plate, 78-79 signaling, vasculogenesis, 222 smooth muscle cell recruitment. 226 vascular development, 225-226 TRH. s e e Thyrotropin-releasing hormone Trophoblasts branching, 16 i development, 156, 166-268 giant cell development, 169-170 proliferation, 160 stem cells proliferation, 160 TSH. s e e Thyroid stimulating hormone TSHR. s e e Thyroid stimulating hormone receptor Tumor angiogenesis, 227 Tumorigenesis. dermal. 579 Tumor necrosis factor. 441 Twist

characterization, 452 crainofacial development, 473-474 Tyrosine ldnase receptors, 91

U UDP-Nacetylglucosamine:a-3-sDS-mannosideB- 1,2-N' acetylglucosaminyl transferase I, 67

V "Vascular channel regression, 227 Vascular endothelial growth factor angiogenic function. 223 bone formation, 291 description, 216 endothelial number regulation, 215, 217-218 expression in hemangioblasts, 214 function, 200 glomerular development, 412-413 pituitary vascularization, 504 receptors expression, 200 VEGF-R t, 223 VEGF-R2, 215, 223 VEGF-R3, 218-220 VEGF-C, 218-220 Vascular permeability factor, 216 Vascular system, s e e a l s o s p e c i f i c e l e m e n t s channel formation, 222-225

development associated endothelial cells cell number regulation, 215,217-218 lineage specification, 213-215 origin, 212-213 blood islands, 2 t 2 definition, 2 i 1 process. 220. v?') pulmonary, 314 morphogenesis ( s e e Angiogenesis) Vasopressin, 500 Vaxl, 522. 525 Veal2, 525 Vcam-1, 170 VCFS/DGS. s e e VelocardiofaciaI/DiGeorge syndrome VEGE s e e Vascular endothelial growth factor "vein segregation, 225-226 Velocardiofacial/DiGeorge syndrome, 357 Venous malformation disease, 227 Ventral ectodermal ridge, 132 Ventricular zone cardiac specification, 349-35 I CNS, 109 forebrain, 87 VER. s e e Ventral ectodermal ridge Versican, 350 Vertebrate hematopoietic system characterization, 191 hierarchy, 192 organizers gastralation f;anction, 42-44 inhibitory signals, specification, 44-45 in gastrulation embryological studies, 41-42 genetic studies, 4 t - 4 2 VLDLR in laminar patterning, 94 von Willebrand factor. 219 VPF. s e e Vascular permeability factor VZ. s e e Ventricula,r zone

W Waardenburg syndrome, 268 WAGR, 375 Witms' rumor renal development association, 415-416 suppressor gene A m h regulation by, 381-382 as metanephric mesenchyrne marker, 399-400 sex determination, 379 urogenital differentiation and, 374-375

712

Subject Index

Winged helix lforkhead genes, 479 Wingless factors cardiac induction, 336-337 CNC patterning. 432 crainofacial development, 471 dorsal CNS patterning, I " 1-i ")'~ follicle formation, 555,557 forebrain morphogenesis. 86 labyrinth branching morphogenesis. 172 mes/met development. 115 Miillerian duct differentiation, 385-386 myotome formation. 264-265 organizer formation, -~' - 4 5 pallial telencephalon patterning. 82-83 pituitary development, 505-506 renal development. 405,407 somite patterning. 141 WNT. see Wingless factors Wolffian ducts characterization. 373 differentiation. 386 WT1. see under Wilms" tumor

X X chromosome characterization. 371 ~'~ evolution. 386-387

Xgsc, 4 I Xotx2, 41 Js _ 0.~-304

Y Y chromosome characterization. 371-372 evolution. 386-387 role in maleness. 377 Yolk sacs differentiation, 161 HSC in, 195-196 visceral endoderm, 302-303

Z Zic3.67 Zona pellucida 3 gene, 14-15 Zygote, 26-27

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