ADVANCES IN DEVELOPMENTAL B I OC HEMISTRY
Volume 5
1999
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ADVANCES IN DEVELOPMENTAL B I OC HEMISTRY
Volume 5
1999
This Page Intentionally Left Blank
ADVANCES IN DEVELOPMENTAL BIOCHEMISTRY
Editor:
PAUL WASSARMAN Department of Cell Biology and Anatomy Mount Sinai School of Medicine New York, New York
VOLUME 5
1999
@ JAI PRESS INC. Stamford, Connecticut
Copyright 0 1999 /A/ PRESS INC. 100 Prospect Street Stamford, Connecticut 06907
All rights resewed. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any way, or by any means, electronic, mechanical, photocopying, recording, filming or otherwise without prior permission in writing from the publisher.
ISBN: 0-7623-0202-X Manufactured in the United States of America
CONTENTS
vi i
LIST OF CONTRIBUTORS PREFACE Paul M . Wassarman
IX
GENETIC CONTROL OF MESODERM PATTERNING AN D DIF F E RE NTIAT1ON DURING DROSOPHllA EMB RYOGENES IS Manfred Frasch and Hanh T. Nguyen
1
ACROSOMAL PROTEINS OF ABALONE SPERMATOZOA Victor D . Vacquier, Willie 1. Swanson, Edward C. Metz, and C. David Stout
49
CAPACITATION OF THE MAMMALIAN SPERMATOZOON Gregory S. Kopf, Pablo E. Visconti, and Hannah Galantino-Homer
83
OVARIAN NITRIC OXIDE: A LOCAL REGULATOR OF OVULATION, OOCYTE MATURATION, AND LUTEAL FUNCTION Lisa M . Olson
109
THE REGULATION AND REPROGRAMING OF GENE EXPRESSION IN THE PREIMPLANTATION EMBRYO Richard M . Schultz
129
ROLES OF METAL LOPROT EASE-DIS I NTEGRINS I N CELL-CELL INTERACTIONS AND IN THE CLEAVAGE OF TNFa AND NOTCH Carl P. Blobel
165
V
vi
CONTENTS
A N I NTIMATE B IOC HEMISTRY: EGG-REGULATED ACROSOME REACTIONS OF MAMMALIAN SPERM Harvey M. Florrnan, Christophe Arnoult, lrnrana C. Kazam, Chungqing Li, and Christine M.B. O’Toole
199
INDEX
235
LIST OF CONTRIBUTORS
Chrisrophe Arnoult
Laboratoire de Biophysique Moleculaire et Cellulaire CNRS Grenoble, France
Carl P. Blobel
Cellular Biochemistry and Biophysics Program Memorial Sloan-Kettering Cancer Center New York, New York
Harvey M . Florrnan
Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts
Manfred Frasch
Brookdale Center for Developmental Molecular Biology Mount Sinai School of Medicine New York, New York
Hannah Callantino-Homer
Center for Research on Reproduction and Women’s Health University of Pennsylvania Medicz! Center Philadelphia, Pennsylvania
irnrana C. Kazarn
Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts
Gregory S. Kopt‘
Center for Research on Reproduction and Women’s Health University of Pennsylvania Medical Center Philadelphia, Pennsylvania vii
...
Vlll
LIST OF CONTRIBUTORS
Chunying L i
Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts
Edward C. Metz
Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego La Jolla, California
Hahn T. Nguyen
Division of Cardiology Albert Einstein College of Medicine Bronx, New York
Christine M.B. O’Toole
Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts
Lisa M . Olsen
Department of Obstetrics and Gynecology Washington University School of Medicine St. Louis, Missouri
Richard M. Schultz
Department of Biology University of Pennsylvania Philadelphia, Pennsylvania
C. David Stout
Department of Molecular Biology The Scripps Research Institute La J o b , California
Willie 1. Swanson
Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego La Jolla, California
Victor D. Vacquier
Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego La J o b , California
Pablo E. Visconti
Center for Research on Reproduction and Women’s Health University of Pennsylvania Medical Center Philadelphia, Pennsylvania
PREFACE Advances in Developmental Biochemistty was launched as a series by JAI Press in 1992 with the appearance of Volume 1. The series is inextricably linked to the companion series Advances in Developmental Biology, which was launched at the same time. As stated in the preface to Volume I-“Together the two series will provide annual reviews of research topics in developmental biology/biochemistry, written from the perspectives of leading investigators in these fields. It is intended that each review draw heavily from the author’s own research contributions and perspective. Thus, the presentations are not necessarily encyclopedic in coverage, nor do they necessarily reflect all opposing views of the subject.” Volume 5 of the series follows these same guidelines. Volume 5 of Advances in Developmental Biochemistry consist: of seven chapters that review specific aspects of development in several different organisms including mollusks, flies, and mice. Five of the seven chapters address aspects of fertilization, including capacitation of sperm (Chapter 3), the acrosome reaction (Chapter 7), gamete adhesion (Chapters 2 and 6), and oocyte maturation and ovulation (Chapter 4). In Chapter 1, Frasch and Nguyen discuss the genetic control of mesoderm patterning and differentiation during Drosophila embryogenesis. The authors present a comprehensive description of the origins, genetics, patterning, and specification of mesoderm, from gastrulation through organ development. Their insights into the genetic and molecular mechanisms that regulate interactions during mesoderm development provide a framework for understanding of mesoderm development not only in Drosophila, but also in other invertebrate and vertebrate species. IX
X
PREFACE
In Chapter 2, Vacquier and coauthors present a detailed account of their research on two acrosomal proteins of abalone sperm. This research has significantly advanced our understanding of how species-specificity evolves. Knowledge of the behavior and structure of these sperm proteins has suggested possible mechanisms of evolution of species-specific gamete interactions and has demonstrated that these proteins may play essential roles in establishing reproductive isolation between species. In Chapter 3, Kopf and coauthors summarize recent studies on capacitation of mammalian sperm. The chapter examines the role of media constituents, membrane events, and transmembrane and intracellular signaling during sperm capacitation. The authors provide detailed information about the biochemical and molecular events that form the basis of this critical event in mammalian fertilization. In Chapter 4, Olson discusses ovarian nitric oxide, a local regulator of ovulation, oocyte maturation, and luteal function in mammals. Although this is a relatively new area of investigation, there is compelling evidence that nitric oxide is a local regulator of all of these key ovarian processes in mammals. The author provides an experimental basis for this conclusion and for future studies. In Chapter 5 , Schultz reviews research on the regulation and reprogramming of gene expression in preimplantation embryos. The presentation focuses on the molecular basis of the maternal-to-zygotic transition (zygotic genome activation) in which maternal transcripts that direct early mammalian development are replaced by transcripts expressed from the embryonic genome. In a detailed analysis, the author summarizes the latest exciting findings in this area of research. In Chapter 6, Blobel discusses the roles of metalloprotease-disintegrins in cell-cell interactions and in the cleavage of T N F a and Notch. A brief summary of the known properties of snake venom disintegrins and metalloproteases is followed by a discussion of the role of membrane-anchored metalloprotease-disintegrins in cell-cell interactions and proteolysis of extracytoplasmic or extracellular protein domains. This timely and exciting area of research bears on aspects of mammalian fertilization and on cell fate decisions during development. In Chapter 7, Florman and coauthors review the mechanisms of egg-regulated acrosome reactions of mammalian sperm. Among the aspects discussed are the nature of primary signal transducers and targets stimulated by binding of acrosomeintact sperm to receptors in the egg zona pellucida. In a detailed presentation, the potential roles of several sperm components including G proteins, cation channels, tyrosine kinases, calcium, and cellular pH are considered. I am grateful to the authors for their excellent contributions, as well as for their cooperation and great patience during the preparation of this volume. I hope that the final product justifies the wait. Paul M. Wassarman Series Editor
GENETIC CONTROL OF MESODERM PATTERNING AND DIFFERENTIATION DURING DROSOPHlLA EMBRYOGENESIS
Manfred Frasch and Hanh T. Nguyen
I. Introduction
..........
A. Formation and Morphogenetic Movements of the Mesoderm . . . . . . . . . . . . . . 3 C. Genes Regulating Mesoderm Invagination: concertina, folded gastrulation, Rho, RhoGEF. . . . . . . . . . . . . . . . . . . . . . . . . . 4 D. heartless, A Gene Regulating the Dorsal Spreading of the Invaginated Mesoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 111. Embryonic Origin and Morphogenesis of Mesodermal Tissues. . . . . . . . . . . . . . . .5 A. Somatic Musculature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 B. Visceral Musculature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 C. Fat Body and Gonadal Mesoderm. . . . . . . . . . .9 D. Dorsal Vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 E. Hemocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 F. Dorsal Median (DM) Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Advances in Developmental Biochemistry Volume 5, pages 1-47. Copyright 0 1999 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0202-X
1
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MANFRED FRASCH and HANH T. NCUYEN
IV. Mesodermally-Expressed Genes Determining Broad Areas and Specific Tissues in the Mesoderm , . . , . . . . . . . . . . . . . 11 A. tinman, a Homeobox Gene Required for the Formation of Visceral Musculature, Dorsal Somatic Muscles, and the Heart . . B. hagpipe, a Homeobox Gene Required for Visceral Muscle Forma C. serpent, a GATA Gene Regulating Fat Body and Hemocyte Development . . . . . . . . . . . . . . . . . . . . . . . . .15 D. The Roles of clift, zJh-1, tin, and Abd-A in Gonadal Mesoderm Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 E. huttonless, A Homeobox Gene Required for DM Cell Formation . . . . . . . . . . 17 V. Early Patterning Mechanisms of the Mesoderm. . . . . . . . . . . . . . . . . . . . . . . . . . . 17 A. Dorsoventral Patterning. . . . . . . . . . . . . . . B. Anteroposterior Patterning. . . . . . . . . . . . . C. A Combinatorial Model of Mesoderm Patt D. Molecular Aspects of Early Mesoderm Patterning . . . . . . . . . . . . . . . . . . . . . . 26 VI. Patterning and Specification within the Developing 27 Heart and Visceral Mesoderm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... A. The Diversification of Heart Cells. . . . B. Anteroposterior Patterning of the Midgut Visceral Mesoderm. . . . . . . . . . . . . 28 VII. Patterning and Specification of Body Wall Muscles . A. A Role of twist in Muscle Development . . . . . . . . . . . . . . . . . . . . B. The Founder Cell Concept of Somatic Muscle De C. Genetic Mechanisms in Formation and Specification of Founder Cells. . , . , . 3 0 VIII. Genetic Control of Mesodermal Tissue Differentiation . . . . A. The Role ofmej2 in the Differentiation of Somatic Muscles,Visceral Mesoderm, and the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 B. Distinct Enhancer Modules of mej2 Linking Patterning and Differentiation Events . . . . . . . . . . . . . . . . . . . . . . . . . 36 XI. Concluding Remarks . . . . . . . . . . . . . . . . .40 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof .................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
1.
INTRODUCTION
The mesodermal germ layer forms during gastrulation and gives rise to a variety of internal tissues and organs in all triploblastic animals. The composition, structure, and function of many of these tissues have clear similarities in different species, indicating that at least some of the mechanisms that regulate their development have been evolutionarily conserved. In recent years, the fruit fly Drnsophila has become one of the premier model systems to study the processes of gastrulation, patterning processes that subdivide the mesoderm, and regulatory events that control the differentiation of individual mesodermal tissues. Much has been learned about the genetic hierarchies that are responsible for the invagination and spreading of the mesoderm
Genetic Control of Mesoderm Patterning and Differentiation
3
into the interior of the embryo, progressive subdivisions generating the anlagen ofthe mesodermal derivatives, and differentiation of these anlagen into the mature tissues. It has become evident that many of these steps involve an interplay between mesoderm-intrinsic regulators and spatially-restricted inductive signals that are released from ectodermal cells. We summarize the insights into the genetic and molecular mechanisms of these regulatory interactions that have been obtained in recent years and that have provided a framework for the understanding of mesoderm development not only in Drosophilu, but also in other invertebrate and vertebrate species.
II. A.
MESODERM FORMATION, GASTRULATION, AND MESODERM MIGRATION Formation and Morphogenetic Movements of the Mesoderm
The Drosophila mesoderm is specified during the blastoderm stage in the ventral quadrant ofthe embryo, corresponding to about a20-cell-wide area that extends between about 5-95% egg length (0%=posterior pole) along the anterior/posterior axis. Gastrulation initiates at cellular blastoderm with the apical flattening and constriction of mesodermal cells, which subsequently elongate and invaginate to form a ventral furrow. Upon completion of the invagination, the hollow tube of mesodermal cells collapses and contacts the inside of the ectoderm. Thereafter, the mesodermal layer spreads dorsally and finally forms a monolayer of cells that extends from the ventral midline to the border between the dorsal ectoderm and amnioserosa (Poulson, 1950; Sonnenblick, 1950; Leptin and Grunewald, 1990; Sweeton et al., 1991). Until this stage (stage 10; 4.5 hrs. after egg laying), no clear morphological differences among individual mesodermal cells can be discerned and mesodermal cells are not yet committed to particular developmental fates (Beer et al., 1987). Genetic and molecular studies have defined a number of genes that have essential roles in the specification and morphogenetic movements of the early mesoderm. as summarized below. B.
Genes Determining the Mesodermal Anlage: Dorsal, twist, snail
The formation of the Drosophilu mesoderm is largely determined by autonomous regulatory mechanisms that act within the nuclei or cells that acquire mesodermal fates. Mesoderm formation is initiated by a nuclear gradient of the NKKB-related morphogen Dorsal, which is synthesized from maternally provided, ubiquitously distributed mRNA (reviewed in Rusch and Levine, 1996). Peak levels of nuclear Dorsal protein are present along the ventral midline of blastoderm embryos and are required to activate two zygotic genes, mist and snail, which are essential for mesoderm formation. Whereas the expression of twist extends to both poles of the embryo and is tapered toward the lateral sides of its expression domain, snailexpression is limited to a
4
MANFRED FRASCH and HANH T. NGUYEN
ventral area between about 5-95% egg length with sharp lateral borders. It is precisely the area of cells coexpressing twist and snail that gives rise to the mesoderm. twist encodes a basic helix-loop-helix (bHLH) protein and snail a zinc-fingercontaining protein, indicating that the products of both genes act as transcription factors (Boulay et al., 1987; Thlsse et al., 1988). Mutations of either of these two genes cause a complete absence of invagination and differentiation of the mesoderm (Grau et al., 1984; Simpson, 1983). Genetic evidence also suggests that the two genes play different roles in mesoderm development. snail is thought to function largely, although not exclusively, to repress nonmesodermal genes in the prospective mesoderm and thus has a predominantly permissive role in mesoderm formation. By contrast, twist appears to have a key role in activating downstream genes that are required for the processes of invagination, patterning, and differentiation of the mesoderm (Kosman et al., 1991; Leptin, 1991). Recent studies have identified some twist target genes and have functionally defined the interaction of Twist protein with sequences In their enhancer elements (see below).
C. Genes Regulating Mesoderm Invagination: concertina, folded gastrulation, Rho, RhoCEF
The complete lack of mesoderm invagination in twist mutant embryos indicates that twist activates one or several target genes that mediate the cell shape changes required for gastrulation. Observations with particular alleles of snail that fail to repress neuroectodermal genes ventrally but still allow mesoderm invagination suggest that snail may also activate some genes that mediate gastrulation movements (Ip et al., 1994; Hemavathy et al., 1997). folded gastrulation (fog) is presently the only known gene that is expressed in a twist-dependent manner i n ventral areas of the blastoderm and that is required for regulating mesoderm invagination. In fog mutants, the concerted apical constriction of mesodermal cells does not occur, leading to their failure to invaginate, Sincefog encodes a secreted protein, it is thought to act as a signal that coordinates apical constrictions of mesodermal cells (Costa et al., 1994). While the receptor of Fog has not yet been identified, concertina (cta), the a-subunit of a trimeric G-protein, is likely to be involved in the intracellular transduction of theFog signal. This is indicated by the gastrulation phenotype of embryos lacking maternally derived Cta activity, which is very similar to the defects observed infog mutant embryos (Parks and Wieschaus, 1991). Other effectors of cell shape changes during gastrulation include the Rho GTPase and its exchange factor, RhoGEF.fog requires RhoGEF to elicit invagination events, and in embryos that are mutant for RhoGEF or express a dominant negative form of Rho, mesoderm invagination does not occur (Barrett et al., 1997; Hacker and Perrimon, 1998). Taken together, these observations suggest a signaling pathway in which twist and snail transcriptionally activate f o g and probably additional genes encoding signaling molecules that bind to transmembrane receptors on ventral cells of the blastoderm embryo (Barrett et al.,
Genetic Control of Mesoderm Patterning and Differentiation
5
1997; Morize et al., 1998). Activated receptor molecules may transmit these signals through a trimeric G-protein that includes the G a subunit Cta, which may directly or indirectly activate RhoGEF and, in turn, Rho. Rho has been shown to induce cell shape changes in cultured cells, at least partly by triggering a reorganization of the actin cytoskeleton (Ridley and Hall, 1992). However, the molecular targets of Rho that induce changes in mesodermal cell morphologies during gastrulation in Drosophila remain to be identified. D. heartlesr, A Gene Regulating the Dorsal Spreading of the lnvaginated Mesoderm
The collapse of the hollow mesodermal tube after invagination occurs during a wave of mitosis in the mesoderm and may therefore be a passive event. By contrast, the subsequent process of dorsal spreading of the mesoderm appears to be regulated by specific signaling mechanisms. It has been shown that the heartless gene is a critical component in regulating dorsal migration of the mesoderm (Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido et al., 1997). In heartless mutant embryos, the mesoderm invaginates normally; however, it subsequently fails to spread towards the dorsal ectoderm and remains as a multilayered mass near the site of invagination. Interestingly, heartless encodes an FGF-receptor molecule that is specifically expressed in the mesoderm in a mist-dependent manner, indicating that the dorsal migration of the invaginated mesoderm is regulated by FGF-signaling. Because the ligand of Heartless has not yet been identified, it is unknown which cells produce the FGF signal molecules that are expected to trigger mesoderm migration. However, stainings of embryos with antibodies that recognize epitopes that are phosphorylated by activated receptor tyrosine kinase (RTK) molecules have revealed specific staining of cells in the lateral edges of the mesoderm, presumably corresponding to activated Heartless receptors (Gabay et al., 1997). This indicates that the FGF signals are released from lateral or dorsal cells of the ectoderm andor amnioserosa and trigger thc directed movement and spreading of the responding mesodermal cells toward the source of the signals. Because the ectoderm later releases important differentiation signals to the underlying mesodermal cells (see below), the internal spreading of the mesoderm below the ectoderm is a prerequisite for normal differentiation of the mesoderm at later stages of development.
ill.
EMBRYONIC ORIGIN AND MORPHOGENESIS OF MESODERMAL TISSUES
The determination of the anlagen of different mesodermal derivatives and their morphogenesis is initiated shortly after the dorsal migration of the invaginated mesoderm has been completed (see IV.). As a result of morphological studies and the availability of early molecular markers for these derivatives, we now have a
MANFRED FRASCH and HANH T. NCUYEN
6
relatively clear picture of the developmental origins and subsequent morphogenesis of the major mesoderm tissues. A.
Somatic Musculature
The mature larval somatic musculature (body wall musculature) is composed of syncytial striated muscle fibers. Individual fibers consist of single syncytia which include between approximately 5 and 15 nuclei, depending on the particular muscle type. Each larval segment includes a defined set of muscle fibers, and the abdominal segments A2 to A7 (and A1 with minor modifications) have an identical pattern of 30 different muscles on either side. Each of these muscles can be identified based on its specific shape, orientation, size, position, and epidermal attachment sites (Figure 1B). It is thought that most, if not all of the somatic muscles are derived from segmentally repeated areas of the early mesoderm that extend from the ventral midline to the dorsal border. These areas correspond to the stripes of high-twist expression that are observed at stage 10 ( 5 hours of development) in the mesoderm (see 1V.B. and V1.A.). The formation of muscles is preceded by the appearance of a special type of myoblasts called muscle founders (Bate, 1990). Muscle founders can be identified based upon their specific expression of molecular markers, including homeobox genes that mark subsets of founders and an enhancer trap insertion, rP298-lacZ, which appears to mark most, if not all, of these cells (Figure 1A) (Dohrmann et al., 1990; Nose et al., 1998). rP298-lacZ-expressing muscle founders arise from four distinct domains along the dorsoventral mesoderm axis that give rise to the dorsal, dorsolateral, lateral, ventrolateral, and ventral groups of muscle fibers, respectively. Most of the muscle founders develop into muscles near the positions where they are formed, but several of them first undergo cell migration to specific locations before developing into muscles. Beginning at stage 12, the muscle founders fuse with surrounding myoblasts, which are thought to be undetermined and are called fusion-competent cells. The resulting muscle precursors continue to recruit additional myoblasts until the syncytia reach their final size. At the same time, they form filopodial extensions, which form specific contacts with muscle attachment cells of the epidermis. The expression of myofiber-specific genes is also initiated during this period and the final differentiation and innervation of the muscle is completed by stage 17 (16 hours of development).
B.
Visceral Musculature
The visceral musculature, which surrounds the endoderm of the foregut, midgut and hindgut, is formed by mononucleate, highly elongated muscle cells. The midgut musculature consists of two layers, an inner layer of circular muscles and an outer layer of longitudinal muscles. By contrast, fore- and hindgut have only a single layer of circular muscles. As described below, each of these types of gut muscles
Genetic Control of Mesoderm Patterning and Differentiation
7
has a diffcrent origin and undertakes a specific developmental pathway during its m orphogenesis. Circular Muscles of the Midgut
The circular muscles ofthe midgut are derived from eleven rectangular, segmentally distributed fields of mesodermal cells, which are located in the dorsal mesoderm in the region between parasegments 2 and 12 of the early embryo (stage 10;Figure 1C)(Azpiazu and Frasch, 1993;Dunin-Borkowski et al., 1995). At late stage 10,these cells ofthe inidgut visceral mesoderm primordia detach from the ectoderm and, at the same time, the surrounding cells of the somatic and fat body mesoderm spread into their former positions. These processes lead to the segregation of the midgut visceral mesoderm toward the interior, where it forms a second mesodermal layer dorsally while the outer mesodermal layer consists of somatic mesoderm. During this segregation process, the visceral mesoderm primordia elongate along the anterior-posterior axis withn each segment and ultimately merge with one another to form a continuous band of cells along the middle body region. The cells of this band elongate during late stage 12 in the dorsoventral direction and subsequent migration and further elongation around the midgut endoderm leads to the formation of the circular midgut musculature (Figure 1D) (Goldstein and Burdette, 1971; Sandborn et al., 1967). Longitudinal Muscles of the Midgut
The precursors of the longitudinal midgut muscles originate near the caudal end of the mesoderm (Campos-Ortega and Hartenstein, 1997). The earliest available marker, DHLH53E identifies the anlage of the longitudinal visceral mesoderm in the caudal mesoderm, which is already present during the late blastoderm stage, thus indicating that it is determined prior to gastrulation (Georgias et al., 1997). During stage 1 1, the cells of the caudal visceral mesoderm divide into two bilateral clusters from which they start migrating toward the anterior on either side ofthe embryo (Figure 1E). This migration occurs in close association with the midgut visceral mesoderm that will form the circular muscles and results in the scattering of the caudal visceral mcsoderm cells along the future midgut. During midgut formation, these cells elongate and align with one another to form longitudinal rows of fibers that are evenly distributed around the midgut. These migration events have been examined by PGal expression from certain tirrrnudhcZ and nief2/fucZ reporter constructs which is specifically detected in caudal visceral mesoderm and longitudinal gut muscles from stage 1 1 until late stages of embryogenesis (Figure 1F) (M.F., unpublished data; Nguyen and Xu, 1998). Foregut and Hindgut Muscles
The precursors of the foregut and hindgut muscles (Figure 1C) appear to be derived from the anterior- and posterior-most regions of the mesoderni, respectively.
heinocytes
DM cells
dorsal vessel fat bodyfgonadalins.
longit. gut musc.
circ. midgut musc.
somatic muscles
Genetic Control of Mesoderm Patterning and Differentiation
9
Figure 7. Developmental origin and morphogenesis of individual mesodermal tissues in the Drosophila embryo. The left column shows the anlagen of different tissues and the right column the corresponding tissues after differentiation. Anterior is to the left and dorsal is up, unless noted otherwise. (A) Stage 11. Segmental repeats of founder cells of somatic muscles (arrows)asvisualized by pCal stainingwith enhancer trap line rP298. (B) Mature somatic muscle pattern as visualized by staining for myosin heavy chain protein (filet preparation courtesy of S. Knirr). (C)Stage 11. Clusters of the circular midgut muscle primordia between PS2 and PS 1 2 are stained for bap mRNA, as is the hindgut visceral mesoderm at the posterior of the embryo. (D)The differentiated circular midgut musculature is visualized with a bap//acZ reporter gene. (E) The caudal visceral mesoderm prior to its migration (stage 1 I), as visualized with a mef2//acZ reporter gene. (F) Differentiating longitudinal midgut muscles (arrows), stained as in E. (G)Segmental anlagen of the fat body (arrows)and gonadal mesoderm (in posterior segments) in a stage 11 embryo stained forsrp mRNA. ( H l ) Cross sectioned embryo (stage 13)stained forsrp mRNA and fasciclin Ill protein to visualize the differentiatingfat body and midgut visceral mesoderm, respectively. (H2) Stage 14 embryo (cross section) stained for Tinman protein to visualize the differentiating gonadal mesoderm and dorsal vessel. (I) Stage 11 embryo stained for Even-skipped protein to visualize the segmental heart anlagen (note that only pericardial progenitors are positive). 0) Dorsal view of dorsal vessel formation short before completion of dorsal closure. The internal rows of nuclei belong to cardioblasts and the external ones to pericardial cells (nuclear pGal staining of an enhancer trap line). (K) Stage 11 embryo stained for D M cell precursors in a bt///acZ enhancer trap insertion (Chiang et al., 1994). (L) Ventral view of late-stage embryo showing differentiated D M cells (stained as in K). (M)Blastoderm embryo (ventrolateral view) stained for srp mRNA expressionto show the mesoderm anlagen of the hemocytes (arrow). (N)Late stage embryo stained with antibodies against peroxidasin to visualize the differentiated migratory hemocytes. Abbreviations: cvm: caudal visceral mesoderm; en: endoderm; fb: fat body; gm: gonadal mesoderm; hg vm: hindgut visceral mesoderm; hp: heart progenitors; mg vm: midgut visceral mesoderm.
While the exact location of the foregut muscle anlagen has not yet been defined, it appears that the anlagen of the hindgut muscles are formed just posteriorly to those of the longitudinal midgut muscles (M.F., unpublished observations). During foregut and hindgut formation, these cells spread along the ectdermally-derived portions of the gut and form circular gut muscle fibers.
The mature fat body is composed of a larger lateral lobe and a smaller dorsal lobe on either side of the embryo (Figure 1H1). The precursors of the dorsal lobe are largely derived from the dorsal mesoderm in parasegment 13 (which does not give rise to midgut visceral mesoderm; see II.B.l) (Riechmann et al., 1998). By contrast, the precursors of the lateral lobe arise from segmentally repeated clusters of cells that are located in parasegments 4 to 13 (Hoshizaki et al., 1994; Azpiazu et al., 1996; Riechmann et al., 1997). One field of fat body precursors is
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MANFRED FRASCH and HANH T. NLIJYEN
located just ventrally to the inidgut visceral inesoderm primordia in each of these parasegments (Figure IC). However. in parasegments 10 to 12. these fields take up only half their normal size and the missing (anterior) portions are occupied by the precursors ofthe gonadal mesoderm (Cumberledge et al., 1992; Brookman et al.. 1992; Riechmann et al.. 1998). A second. smallcr field offat body precursors is located more ventrally and posteriorly in each ofthe parasegments 4 to I ? . During fat body morphogenesis? these clusters of cells merge with one another and move between the somatic and visceral mesoderm to form the continous lateral lobes 0 1 the fat body. The three clusters of gonadal mesoderm primordia in parasegments 10 to 12 also coalesce and, together with the germ cells, form the gonads (Figure lH2). D.
Dorsal Vessel
The mature dorsal vessel, or heart, consists of a tubelike structure extending along the dorsal midline. Its lumen is formed by two longitudinal rows of cardioinyocytcs called cardioblasts or cardial cells (Figure 1J) (Rugendorff et al., 1994). There are about six cardioblasts on either side in each middle body scgment, which are surrounded by pericardial cells that are not myogenic (Mills and King, 1965). Like the inidgut visceral mesoderm and fat body, the anlagen of the heart are also formed as segmentally repeated clusters during stage 10 (Dunin-Borkowski et al., 1995; Azpiazu et al., 1996). The clusters of the heart anlagen are located at the dorsal margins of the mesoderm between the clusters of midgut visceral mesoderm precursors in each ofthe parasegments 2 to 12 (Figure 11). During the morphogenetic movements that lead to the multiple layers in the mesoderm, the heart progcnitors remain in contact with the ectoderm. As a result of cell rearrangements during stage 1 I , heart progenitors from adjacent parasegmental clusters come into contact to form a continuous longitudinal band of cells along the dorsal crest of the mesoderm on either side of the embryo. Following stage 12, the cardioblasts form a row at the dorsalmost positions of the mesoderm, and upon dorsal closure of the germ band. the cardioblast rows from either side ofthe embryo join at the dorsal midline t o form the heart tube.
E.
Hemocytes
Hemocytes function mainly as macrophages that remove debris from apoptotic cells in the developing embryo (Tcpass et al.. 1994). In addition, they appear to deposit extracellular matrix components of the basement membrane (KuscheGullberg et al.. 1992; Hijrtsch et al., 1998). Similar to the anlage of the caudal visceral mesoderm (H.B.),the blood cell anlage can already be detected at blastoderm stage, i.e.. prior to gastrulation. At this stage, the blood cell anlage occupies a small area of the head mesoderm that is locatcdjust anteriorly to the position of thc future ccplialic furrow (Figure 1 M ) (Rehorn et al., 1996).After gastrulation, the progeni-
11
C;enetic: Control of Me5oderr-n Patterning and Differentlatirjn
tors of hcmocytcs delaminate into the interior of the embryonic head and subsequently scatter throughout the embryonic body cavity (Figure 1 N). F.
Dorsal Median (DMj Cells
Pairs of dorsal median (DM) cells are located medially on the dorsal surface of' the ventral nerve cord and close to the segmental boundaries in each ofthe three thoracic and in the first seven abdominal segments (Figure 1 L). DM cells have glia-like properties since they develop laterally extended processes that pioneer and ensheath axons of motoneurons exiting from the dorsal midline of the nerve cord (Gorczyka et al.. 1994). The DM cell precursors form in small segmental clusters along the ventral midline ofthe mesoderm in stage 1 1 embryos in close contact with the mesectoderm (Figure 1K) (Chiang et al., 1994; Dunin Borkowski et al., 1995). During ncuroblast segregation and ganglion mother cell proliferation, the DM cell precursors. together with the rest of the ventral mesodermal cells, separate from the epidermis and assume their final locations on the dorsal surface of the CNS.
IV. MESODERMALLY-EXPRESSEDGENES DETERMINING BROAD AREAS AND SPECIFIC TISSUES IN THE MESODERM A.
tinman, a Homeobox G e n e Required for the Formation
of Visceral
Musculature, Dorsal Somatic Muscles, and the Heart Structure and Expression of tinman
tirirmm (tin)encodes a homeodomain protein ofthe NK family (Kim and Nirenberg, 1989). Its closest vertebrate homologs, Nkx2-3, Nkx-2-5,Nkx-2-6. Nkx2-7. and Nkx2-8, share between 63% and 68%)amino acid identity within the homeodomain (reviewed in Harvey, 1996). Binding site selection experiments have defined an optimal binding site with a consensus sequence 5'-TNNAGTG-3' for Nkx2-5. The most commonly found Tinman binding sites share this motif ;r,d contain the sequence YTCAAGTG-3' (Chcn and Schwartz, 1995; Gajewski et al., 1997; Xu et al., 1998). I n additicn t o the homeodomain. Tinman contains ahighly conserved dei'ppiiclc ~ t - y u c r i ~LCc :it11 unl\riovvii funLiiui1 iicili it> N-tzimiinua ~ d l l e dilie TN dumain. Initial ririrticiri expression occurs at cellular, blastoderm in the prospective mesoderm and is confined to the trunk mesoderm where it is initally modulated in a periodic pair-rule pattern (Bodmer et al.. 1990; A q i a z u and Frasch. 1993). During imxgination and dorsal migration of the mesoderm, the entire trunk mesoderm expresses tiiiriiciri mRNA i n a uniform pattern. However. shortly before the mesoderm reaches the dorsal border of the ectoderm. firirtictri expression decreases in the ventral poi-tions and its levels increase i n the dorsalmost cells 01' thc mcsodcrm. Be-
12
MANFRED FRASCH and 1 iANH I. NCIJYCK
tween stages 10 and I 1. tirirrrciri mRNA and protein expression is entirely restricted to the dorsal portions ofthe mesoderm. These domains occupy about one third of the distance between the dorsal margin and the ventral midline on either side. At early stage 12. the tirrrriar7 domains are further refined and become restricted to cells ;it the dorsal margins of the mesoderm that correspond to heart progenitors. riririirrrr remains expressed i n heart cells until late embryogenesis. This expression includes I'our out 01' six cardioblaxts and four pericardial cells per hemisegment, While rirr1?1(iti rnKNA is exclusively observed in the heart of late-stage embryos. tiririiuri protein perclures in segmentally repeated cells of the visceral mesoderm and in the gclnadal mcsotlerm. in addition to its continued expression in cardioblasts and pericat-dial cells (Figure 1 H2;Manfred Frasch, unpublished data).
Cenetic and Developmental Function of tinman The genetic function of tirirriari has been investipted with ethylmethanesulfonate~(EMS-) induced alleles, including one that carrics a small delet i o n in the coding region and another with a stop codon in the homeodomain. which arc presuniably null-alleles (AzpiaLu and Frasch, 1993; Bodmer. 1993).The ina.jor defects in IiomoLygous mutant embryos are observed in tissues derived from the clot-sal mesoderm. Notably, both the dorsal vessel and midgut visceral mesoderm ;ire entircly missing in the absence oftirirrim function (Figure 2A-D). The absence ol'an enhancer trap marker that is specific for all dorsal somatic muscles in tirlrriur? mutant embryos strongly indicates that tirrriiurz is also rcquired tor the formation 01' dot-sal body wall muscles (Figure 2E.F) (Yin and Frasch, 1998). This is consistent with the severe disruption ofthe muscle pattern in dorsal areas and the concomitant clongation of dot-solateral muscles into the areas normally occupied by dorsal types ol'niuscIes. Earl!. markers for the precursor cells ofeach of these tissues are also not expressed in firir)/(/r? mutants. Specifically. the homcobox gene l x q p i p o . which niai-ks the anlagcn of t h e midgut visceral mesoderm (see 1II.B.). is not activated. rr-.~kipp(~d and l~irh~hir-d (see V.A.) I'ail to be expressed and the liomeobox genes in early heart precursors (Azpiazu and Frasch. 1993; Bodmet-. 1993; Jagla CI d . . 1997).The expression oftlie homeobox gene r r i s h . which is normally cxprcss~ilin muscle founder cells. is also missing in this area in f i r r r i i m r mutimts d o t - a a l l ~locntccl . (Yin and Fi-asch. I998). Thus. i t appcars that rirrriiciri is required tor the specification 01a i i dorsai iiicsocicrmai tissues 'oeiween siages i O ;ind i i u i ' c l i i h i - ~ ~ i ~ i i i Thi:; z~is. function is consistent with its specific expression in dorsal portions of the mesode rni d 11r i n p this per i od t Iiu s s ug ge s t i ng t li at tin r r i m ful fi I1 s its major de \T Iop men t a I l'un c t i c) 11s during t h i s second . dorsal Iy rest I-ict ed phase of me s ode rni al expression . Apai-t I'rorii these m;i,jor defects i n dorsal mesodermal derivatives. r i r r r r r m m w tants also exhibit inore subtle defects in other areas ofthe mesodcrni. Fot-example.a specific subset ol' \,entral and lateral body w;ill m u s c l e s and thcir founder cells ;ire not forincd (1:igui-c 2G.H) ( A q i a l u and F3xcIi, 1993).hlorc o~ e t-. r i r l r r i r l r i rnutant embryos lack the DM c e l l s i i n d thcit-pi-ecursors (Figure 21.5) ( G o r c ~ y k ; t c;d.. c 1994:
bnman
wild type
The tunction of tinman i n the tormation o t mesodermal tissues. The left column shows wild-type embryos and the right column tinman mutant embryos of the iaine stage and stained with the same markers. (A, Bi Keither cardioblastj nor pericardial cells are formed in tin mutants (embryos stained as i n Figure 11). ( C , Di 'The midgut inusculatul-e (stained tor PCaI expression from ,In enhancer trap insertion) i s completely !??d +? lt 'l r 7 l t.ypr?w.i(:n +