Current Topics in Developmental Biology - Volume 1

CURRENT TOPICS IN

DEVELOPMENTAL BIOLOGY VOLUME 1

ADVISORY BOARD VINCENT G. ALLFREY

DAME HONOR B. FELL, F.R.S.

JEAN BRACHET

JOHN C. KENDREW, F.R.S.

SEYMOUR S. COHEN

S. SPIEGELMAN

BERNARD D. DAVIS

HEWSON W. SWIFT

JAMES D. EBERT

E. N. WILLMER, F.R.S.

MAC V. EDDS, JR.

ETIENNE WOLFF

CONTRIBUTORS HANS JOACHIM BECKER

MARION 0. MAPES

J. R. COLLIER

PAUL A. MARKS

P. F. GOETINCK

A. S. SPIRIN

EUGENE GOLDWASSER

F. C. STEWARD

ANN E. KENT

MAURICE SUSSMAN

JOHN S. KOVACH

H. TIEDEMANN

JOSHUA LEDERBERG

CURRENT T O P I C S I N

DEVELOPMENTAL BIOLOGY EDITED BY

A. A. MOSCONA DEPARTMENT OF ZOOLOGY THE UNIVERSITY OF CHICAGO CHICAGO, ILLINOIS

ALBERT0 MONROY ISTITUTO Dl ANATOMIA COMPARATA UNIVERSITA DI PALERMO PALERMO, ITALY

VOLUME 1

1966

ACADEMIC PRESS New York London

COPYRIGHT @ 1966,

BY

ACADEMICPRESSINC.

ALL RIGHTS RESERVED, NO PART O F THIS BOOK MAY B E REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEAXS, WITHOUT WRITTEN PERhfISSION FROM T H E PUBLISHERS.

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United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W.l

LIBRARY OF CONGRESS CATALOG CARD

NUMBER: 66-28604

PRINTED I N T H E UNITED STATES OF AMERICA

LIST

OF CONTRIBUTORS

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

HANSJOACHIM BECKER,Zoologisches Znstitut der Universitat Munchen, Munich, Germany ( 155) J. R. COLLIER,Rensselaer Polytechnic Institute, Troy, New York, and Marine Biological Laboratory, Woods Hole, Massachusetts ( 39) * P. F. GOETINCK, Department of Animal Genetics, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, Connecticut ( 253 1 EUGENEGOLDWASSER, Argonne Cancer Research Hospital and Department of Biochemistry, University of Chicago, Chicago, Illinois (173) ANN E. KENT, Laboratory for Cell Physiology, Growth and Development, Cornell University, Zthaca, New York (113) JOHN S. KOVACH,Department of Medicine, Columbia University College of Physicians and Surgeons, New York City (213) JOSHUA LEDERBERG, Department of Genetics, Stanford University School of Medicine, Palo Alto, California ( i x ) MARION0. MAPES,Laboratory for Cell Physiology, Growth and Development, Cornell University, Zthaca, New York (113) PAULA. MARKS,Department of Medicine, Columbia University College of Physicians and Surgeons, New York City (213) A. S. SPIRIN,A. N . Bakh Znstitute of Biochemistry, Academy of Sciences of the Union of Soviet Socialist Republics, MOSCOW,U.S.S.R. ( 1 ) F. C . STEWARD, Laboratory for Cell Physiology, Growth and Development, Cornell University, Zthaca, New York (113) MAURICESUSSMAN,Department of Biology, Brandeis University, Waltham, Massachusetts ( 61 ) H. TIEDEMANN, Max-Planck-Znstitut f u r Meeresbiologie, Wilhelmshaven, Germany (85) * Present address: Biology Department, Brooklyn College, Brooklyn, New York. V

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PREFACE The organizers of this publication, as well as others interested in the mechanisms of differentiation and development, believe that there is an urgent need for an annual publication devoted specifically to topical discussions of current work, concepts, and prospects in developmental biology. The study of development and differentiation is, at present, in the midst of a searching reevaluation of its objectives and methodologies. Among embryologists there is a growing emphasis on cellular, genetic, and molecular approaches to the analysis of developmental phenomena; on the other hand, among non-embryologists, biochemists, virologists, molecular biologists, geneticists, etc., there is an increasingly active awareness of the fundamental biological significance of the processes of embryonic differentiation and related developmental problems. Nevertheless, an “interdisciplinary communication gap” exists caused largely, apparently, by the absence of suitable sources of concisely summarized, mutually understandable topical information concerned with problems of common interest. There is, at present, no publication platform designed specifically as a “meeting-ground for critical review and discussion of current work on developmental processes in the context of today’s interdisciplinary approaches and concepts. Current Topics in Developmental Biology is intended to meet, at least, part of this need. Its organizers hope that the volumes might be useful not only as a source of topical information and interdisciplinary exchange of views, but might also aid in evaluating concepts and in crystallizing guidelines in the field of developmental biology. The design of this work takes into account the increasing pace at which biological information accumulates and turns over. This serial publication will thus be devoted exclusively to brief topical reviews on sharply delimited subjects with emphasis on regulatory mechanisms at the molecular, biochemical, cellular, and histological levels. The articles will be solicited from active investigators and reviewed in accordance with the editorial policy. The articles should focus primarily on the writers‘ work and views and include only an essential minimum of historical background. The contributors should aim at balancing factual material with discussion and provocative projection of their topic, Publication will be prompt in yearly volumes. vii

viii

PREFACE

Each volume will usually cover several topics, but sometimes one topic may be dealt with in more than one article. In addition, we hope to include in each volume Remarks or a Conspectus by a scientist whose research experience and scholarship confer special interest and significance upon his views on the problems of development and differentiation. The authors contributing to the first volume have had a particularly difficult ,task because of lack of precedent and because of the interdisciplinary aims of this publication. The editors wish to thank them for their patient cooperation in the efforts to increase the usefulness of this publication. We wish to record our gratitude to the staff of our publishers, Academic Press, for friendly support and professional assistance. Finally, we wish to thank our colleagues, too numerous to list, who have encouraged us in undertaking this task.

September, 1966 A. MONROY A. A. MOSCONA

Joshua Lederberg DEPARTMENT O F GENETICS STANFORD UNIVERSITY SCHOOL OF MEDICINE P A L 0 ALTO, CALIFORNIA

Right now is a particularly awkward time to frame any useful commentary on developmental biology. The field has had enough fancy; more recently its methodology has been under enormous pressure to accommodate the inspirations of molecular biology and the models of development that can be read into microbial genetic systems. But now, as this volume amply shows, it is responding. Why did the editors invite me into this “tender trap” to begin with? The main excuse may have been an unguarded remark I once made that “embryology should be studied with embryos.” Since, at the time, most of my colleagues, and I myself, were professing to be studying embryology better than the embryologists could, by applying ourselves to regulation and quasi-heredity in microbes, e.g., antigenic variation in Salmonella, this profession may have endeared me to the guild. A less endearing remark I made a few years later that “embryology was about to begin” may have been the final goad to the editors to make sure that I would read this book, and see that it indeed had begun. For that at least, I am duly grateful. I hope that my colleagues in molecular biology will read this volume, and the ones to follow, especially as more and more of ,them become impatient to furnish the one missing concept or technique that will illuminate the whole problem of development, once and for all. If I have any criticism to offer of the organization of the pioneering volume of this serial publication, it would be just against the spirit of my earlier remark about embryos-namely, that the “developmental” analysis of bacteriophage is so cogent that its omission is inexcusableeven if it were beyond the persuasive capabilities of the editors to collect a pledge. (Dr. Sussman quotes some of the texts in his article. ) Despite the mechanistic flavor of the now classic work on tissue induction, embryology has historically had more than its share of mystiix

X

REMARKS

cism, with some mysterious property of “organization” always in the background to inhibit bold experiments. There is relatively little of that now, but the working hypothesis should be brought out into the open. The point of faith is: make the polypeptide sequences at the right time and in the right amoun’ts, and the organization will take care of itself. This is not far from suggesting that a cell will crystallize itself out of the soup when the right components are present. And it might be worth thinking of experiments capable of such a result at that! This faith has no foundation except a modicum of empirical success in accounting for a problem that most of us would have thought to be the ultimate bastion of jealous Nature’s secrecy, the biochemistry of gene replication. True, “organizational” factors doubtless play a large role in the integrity of the hereditary process. But now is the time to study them, when they are a challenge to explicit experimentation, rather than a lid over a porthole. For the most part, organization seems to be turning out to be quite comprehensible, even to the unaided human mind, as one more level of macromolecular chemistry. Should we be hopeful that developmental biology will be cleaned up in one more decade? It probably could be done, in an orderly way, but not before there is a concensus both about the nature of the problem (which can perhaps be found) and especially the choice of experimental material. The central problem is twofold: (1) How is the time-ordered sequential program of protein synthesis generated from the cell’s informaton, and (2) What is the character of epinucleic heredity, i.e., the restrictive information transmitted in tissue lines that cannot sensibly be attributed to DNA-sequence codes. In thinking about (1)one can hardly help but be profoundly influenced by the Jacob-Monod operonl models, which had their roots in part in studies, like those of Barbara McClintock, on inhibition of proximate genes in a chromosome field, But the authors of these models would be the first to decry a slavish adherence to their detailed manifestation as seen in bacteria. Even at a cytological level, as we now know, bacterial chromosomes are importantly simpler in structure than the metazoan. As soon as some concrete fads were brought out it was as foolish to persist in the analogy as it would have been to ignore it in the early attack on bacterial genetics. The logical design of metazoan chromosomes is quite different too. Coordinate regulation of genes in a biosynthetic sequence in bacteria is almost always correlated by -

1 Beautifully reviewed in Jacob‘s Nobel Prize lecture (Science 152:1470-1478, 1966).

REMARKS

xi

close linkage of these genes, i.e., to form an operon. As against dozens of examples in bacteria, it is not clear whether there are any in metazoa. Note, for example, the nm-linkage of the hemoglobin-a and -/3 factors; and certainly as no mere coincidence or vestige of duplication, fl and y are linked, and these are competitive, not coordinated, in synthesis. Even more spectacular, each of the genes for a recognizable step in the biosynthesis of the ABO blood group system, I , Le, Se, H, is dispersed in the chromosome set. If the corresponding enzymes are coordinated, it must be by some quite different mechanism, not coordination within an operon. The Rh complex might be cited as a counter-example, if C, D,E are regarded as linked genes. However, the position effect leading to qualitatively distinguishable products from this system hints that these components concern different segments of a single molecule, i.e., that the Rh complex is a single cistron, not an operon containing a series of cistrons. Many other blood group factors, of uncertain but possible affinity to the ABO mucopolysaccharide, are also dispersed, We should then be searching for some other correspondence principle, evolved as an alternative to the operon, whereby genes on different chromosomes can still be coordinated. The work to prove may be harder than the wit needed to think of a number of possibilities. The variability in total DNA content of the nucleus among plant or animal species of similar complexity points to the triviality of function of large parts of it. Organisms that have a generation time larger than thirty minutes can afford to be extravagant with DNA synthesis. G. L. Stebbins2 has suggested that the excess DNA is analogous to the interrecord gaps on a computer tape-which can sometimes be used to regulate the pace of a tape-controlled process. To turn a computer into a clock may be an extravagance, but it is sometimes cheaper than designing a new piece of hardware. At any rate, this is one way of rescuing human self-esteem from derogation by a salamander’s thirtyfold excess of DNA. For point (2), epinucleic heredity, it might still have been argued about whether there is any problem, Are there many examples, relevant to normal differentiation, where single cells transmit epigenetic information to a clone? (The skeptic had the advantage that the only somatic clone that occurs naturally is the zygote itself.) The ideal model for this process now is the heterochromatism of X-chromosomes in mammals (for which studies on human material have, once again, been consequential). 2

Science 152:1463-1469,1966.

xii

REMARKS

As this phenomenon can be studied in cell culture, some crucial answers should soon be available on how the choice of heterochromatized chromosomes is initiated and perpetuated. There is much more contention about tactics, and this may be the most glaring weakness. Epochs of revolutionary advance in biology have usually been connected with the convergence of many workers on some common, or reasonably comparable, experimental material: witness the role of Drosophila and maize for the growth of cytogenetics, and of E . coli B and its phages for the early delineation of the new virology. This convergence can, of course, be carried to a fault: Whatever else may have been brought up instead, we might have missed quite a bit but for the idiosyncrasy of E. L. Tatum and his students in working with another strain K-12, which was exhibitionistic enough to display sexuality, lysogenicity, and specialized transduction, all missing in the E . coli B/Tphage system. If any single experimental system in developmental biology had a fraction of the convergent attention that was given the T phages, we might be more optimistic about the pace of further work, but embryology suffers from being a traditional field, and seems to need the impulse of more novelty than frog gastrulae would now offer. Nor can we perceive who would or could play the disciplinary or rather disseminatory role that Max Delbruck did for phage. I have little doubt in my own mind that the mouse should be that central material, but this is a prejudice possibly based on expectations of utility from and for genetics, biochemistry, cytology, immunology, psychology, oncology, and medicine rather than on any significant personal experience. At the other pole, some very simple system like a rotifer or a nematode needs to be conventionalized-as much for the same array of ancillary fields as for embryology. Such conventions can hardly be imposed by any authority, but it might not be completely amiss for some group of investigators to attempt to find common ground by voluntary agreement, and to advertise the wisdom of their choice by their own good example. My final remark as an outsider is that embryology is the branch of biology closest to human affairs, if only in the sense that man’s intellect is the enduring morphogenesis of his brain. More broadly, the semilethal mutants that we count up only by the numbers in fruitflies are the congenital anomalies, mental retardation and recurrent stillbirth in man. A chemical factor that induces only a barely significant change in brain development in an experimental animal could have revolutionary consequences in a human context. The human life span is an almost

REMARKS

xiii

incidental side issue of his embryology. I would even put embryology ahead of genetics in the practical sphere, knowing that we can hardly be more than a generation away from the techniques for calculated manipulation of development that would take a millenium to match by any realizable program of artificial or natural selection. Finally, the genetic mechanism itself, like the determination of sex or the very need for a sexual process in reproduction, controls but is also an outcome of development.

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v

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vii

Contributors Preface

Remarks JOSHUA

CHAPTER

1.

...........................................

ix

LEDERBERG

On “Masked” Forms of Messenger RNA in Early Embryogenesis and in Other Differentiating Systems

A . S . SPIEUN I . Introduction .................................................. I1. On “Masked” mRNA in Unfertilized Eggs ....................... I11. On “Masked” mRNA in Early Developing Embryos and in a Number of Other Differentiating Systems ............................. IV . Experimental Data on the Existence of mRNA-Carrying Postribosomal Particles in Animal Cells ..................................... V. Experimental Data on the Existence of Masked Polyribosomes . . . . . . VI . General Conclusion and Hypothetical Scheme .................... VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ...................................................

CHAPTER

2 3

7 11 28 33 36 36

2. The Transcription of Genetic Information in the Spiralian Embryo

J. R . COLLIER I. I1. I11. IV. V. VI . VII . VIII .

Introduction .................................................. Nucleic Acid and Protein Synthesis during Embryogenesis ......... Cleavage and Early Development ............................... Oogenesis and Gene Transcription .............................. Morphogenesis and Gene Transcription .......................... Precocious Segregation and Gene Transcription . . . . . . . . . . . . . . . . . . . Discussion ................................................... Summary .................................................... References ...................................................

xv

39 40 42 44 48

53 55 57 58

xvi

CONTENTS

CHAPTER

3. Some Genetic and Biochemical Aspects of the Regulatory Program for Slime Mold Development

MAURICESUSSMAN I . Introduction ................................................. I1. The Succession of Developmental Events and the Regulated Appear-

ance of Specific Biochemical Products ......................... I11. Alteration of the Overall Developmental Program in Mutant Strains . . IV FR-17, a Temporally Deranged Mutant .......................... V . The Regulatory Program for UDP-Gal Polysaccharide Transferase . . . VI . Specific Requirements for RNA and Protein Synthesis in the Transferase Program ............................................. VII . The Role of Protein Synthesis during Genetic Transcription . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

CHAPTER

61 62 65 66

67 70 76 82

4. The Molecular Basis of Differentiation in Early Development of Amphibian Embryos

H . TIEDEMANN I . Introduction .................................................. I1. Test Methods for Inducing Factors ............................. 111. Isolation and Properties of the Mesoderm-Inducing Factor . . . . . . . . . . 117 . Characteristics of the Mesoderm-Inducing Factor . . . . . . . . . . . . . . . . . V . Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . RNA Metabolism ............................................. VII . Intracellular Distribution of the Inducing Factors . . . . . . . . . . . . . . . . . VIII Changes in Cell Affinities ..................................... IX . Formation of Complex Organ Structures ........................ X . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ...................................................

.

CHAPTER

5.

85 87 88 96 98 101 105 106 109 110 110

The Culture of Free Plant Cells and Its Significance for Embryology and Morphogenesis

F. C. STEWARD.ANN E . KENT. AND MARION0. MAPES I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . The Historical Setting: The Concepts of Haberlandt . . . . . . . . . . . . . . 111. Chemical Stimuli to Growth and Morphogenesis . . . . . . . . . . . . . . . . . . IV . From Plant Tissue and Organ Cultures to Free Cell Cultures . . . . . . . . V . Free Plant Cells in Culture: Their Morphology and Division . . . . . . . .

113 115 116 117 123

xvii

CONTENTS

VI . VII . VIII . IX .

From Free Cells to Flowering Plants: The Case of the Carrot Plant . . Free Cells to Plants: Other Examples ........................... Other Relevant Studies on Embryogenesis and Morphogenesis . . . . . . Concluding Remarks .......................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CHAPTER

129 136 146 149 151

6. Genetic and Variegation Mosaics in the Eye of Drosophila

HANSJOACHIM BECKER I. I1. I11 IV .

.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Mosaics and the Development of the Eye . . . . . . . . . . . . . . . . . Variegation ................................................... Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CHAPTER

155 156 161 169 171

7. Biochemical Control of Erythroid Cell Development

EUGENE GOLDWASSER I. 11. I11 . IV . V. VI . VII .

173 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . System under Study .......................................... 174 The Role of Other Hormones in Control of Erythropoiesis . . . . . . . . . . 182 The Role of Nonhormonal Substances in Erythropoiesis . . . . . . . . . . . . 184 Erythropoietin as Inducer of Red Cell Differentiation . . . . . . . . . . . . . . 185 Models of Erythroid Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 206 References ................................ . . . . . . . . . . . . . . . . . . .

CHAPTER

8. Development of Mammalian Erythroid Cells

PAULA . MARKSAND I. I1. 111. IV . V. VI . VII .

JOHN

S. KOVACH

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 215 Sites of Erythroitl Cell Development ............................ Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 Structural and Biochemical Aspects of Erythroid Cell Differentiation . . 220 233 Messenger HNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Comment ...................... . . . . . . . . . . . . . . . . . . . 245 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

xviii

CONTENTS

CHAPTER

9. Genetic Aspects of Skin and limb Development

P. F. GOETINCK

. . .

Introduction .................................................. Differentiation of the Embryonic Chick Skin ..................... Differentiation of the Embryonic Limb .......................... Summary and Concluding Remarks ............................. References ...................................................

253 254 263 277 281

Author Index

285

Subject

....................................................... Index ......................................................

296

I I1 I11. IV

CHAPTER 1

ON “MASKED”

FORMS OF

MESSENGER RNA IN EARLY EMBRYOGENESIS AND IN OTHER DIFFERENTIATING SYSTEMS A. S. Spirin A. N. B A K H INSTITUTE O F

BIOCHEMISTRY,

ACADEMY OF SCIENCES O F THE UNION OF SOVIET SOCIALIST

REPUBLICS,

MOSCOW, U.S.S.R.

.

..

I. Introduction . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . 11. On “Masked mRNA in Unfertilized Eggs . . . . . . . . . . A. Evidence for the Existence of Stored “Masked” mRNA in Eggs .............................. B. On the Possible Nature of Masked Forms of mRNA 111. On Masked mRNA in Early Developing Embryos and in a Number of Other Differentiating Systems . . . . . . . . . . IV. Experimental Data on the Existence of mRNA-Carrying Postribosomal Particles in Animal Cells . . . . . . . . . . . . . . A. Early Observations . . , . . . . . . . . . .. . ... . . . . .. . . B. “Idormosomes” of Early Embryos . . . . . . . . . . . . . C. mRNA-Containing Postribosomal Particles in the Cytoplasm of Virus-Infected Cells . . . . . . . . . . . . . D. mRNA-Carrying 4 5 s Particles in the Cytoplasm of . .. . Nondifferentiating Animal Cells . . . . . . , E. mRNA-Containing Nuclear Particles . . . . . . . . . . . . F. Conclusion and Some Speculations . . . . . . . . . . . . . V. Experimental Data on the Existence of Masked Polyribosomes .......................................... A. Inactive Stored Oligoribosomes of Early Embryos B. “Repression” of Polyribosomes . . . . . . . . . . . . . . VI. General Conclusion and Hypothetical Scheme . . . . . . VIII. Summary ....................................... References ......................................

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1

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2 3 3

5 7 11 11 13 20

23 26 27 28 28 31 33 36 36

2

A. S. Sl’IHIN

1. introduction

The discovery of messenger RNA (mRNA) and the formulation of certain principles of regulation of gene activity in bacterial and viral systems opened up extensive opportunities for speculations concerning the mechanisms of cellular differentiation and development in the Metazoa (Monod and Jacob, 1961; Jacob and Monod, 1963). Because of the accomplishments of the above approach, attention was devoted almost exclusively to the gene level of regulation of protein synthesis as the only way of explaining biological differences. It was implied, by analogy with bacterial systems, that the mRNA synthesized on active (derepressed) genes and passing into the cytoplasm is always immediately and automatically expressed in protein synthesis. However, it soon began to appear that in a number of cases a rather long period may elapse between the synthesis of mRNA and its expression in the cell. This raised the question about a “masked” or “blocked” form of mRNA. This question was particularly clearly formulated for the first time with regard to the unfertilized egg’ (see Section 11, A ) . Indications of another kind also appeared, namely, that the actively functioning template may at a particular moment be “repressed” by a protein inhibitor (see Section V, B ) . Thus it turned out that the question of regulation of protein synthesis in differentiating systems cannot be considered only in terms of regulation at the gene level. In this article an attempt is made, on the basis of an analysis of a number of facts (regrettably, not very numerous), to discuss the possible forms in which mRNA may exist in an inactive state, and their relation to processes of early embryogenesis, differentiation, and development. The term “masked mRNA” is here used to designate a form of mRNA in which it does not manifest its protein-synthesizing activity, regardless of whether this inactive state precedes the active state or is a result of “repression” of a formerly active template. It should be noted that not everything stated here is well substantiated. Moreover, many purely hypothetical considerations are advanced. The author understands that many biochemists working on embryological problems and many embryologists working on the biochemical or “molecular-biological” aspects will disagree with the concepts stated here and * In the most general form the idea of “unmasking” of an active principle in embryonal development was expressed as far back as 1936 by Waddington et al. The question of “masked” mRNA, as one of the possibilities, was repeatedly raised by Brachet ( 1962).

1.

“MASKED”

FORMS OF

mRNA

3

might perhaps attempt to disprove them by experiments; in this case the objectives of this article will have been fuElled, since its aim, aside from setting forth facts, is to stimulate new experiments along lines which may not have been pursued before. The author hopes that future investigators will generously overlook the errors and fallacies of his temporary hypotheses. II. On “Masked“ mRNA in Unfertilized Eggs

A. EVIDENCE FOR

EXISTENCE OF STORED “MASKED”MRNA IN EGGS The presence of stored mRNA in unfertilized eggs is indicated by the aggregate of the following groups of facts: 1. Non-nucleated fragments of the sea urchin eggs are capable, after their parthenogenetic activation, of cleaving and forming blastulas ( Harvey, 1936, 1940). Since at least the processes of cell division require protein synthesis (Hultin, 1961a) and the nuclear source of templates is absent, it is reasonable to suppose that the development of parthenogenetic merogons is determined by preformed maternal mRNA stored in the egg cytoplasm. Active protein synthesis in non-nucleated parthenogenetic merogons was actually demonstrated by direct experiments with incorporation of labeled amino acids; parthenogenetic activation is marked by intensive protein synthesis in non-nucleated egg fragments just as it is in nucleated fragments (Tyler, 1963; Denny, 1963; Denny and Tyler, 1964; Brachet et al., 196313). 2. In the presence of actinomycin D, when mRNA synthesis is excluded, the eggs are capable of cleaving after fertilization and of reaching the blastula stage; incorporation of labeled amino acids shows that fertilization in the presence of actinomycin initiates protein synthesis in the eggs, this synthesis being on the whole no less intensive than in the absence of actinomycin (Gross and Cousineau, 1963, 1964). This enabled Gross and his associates most clearly to express and substantiate the idea that the egg contains preformed stored mRNA which conditions the initiation of protein synthesis during fertilization, as well as its course during cleavage and early blastula formation (Gross and Cousineau, 1963, 1964; Gross et al., 1964; Gross, 1964). 3. RNA preparations isolated from unfertilized sea urchin eggs are capable of stimuIating the incorporation of labeled amino acids in a cellfree system (Maggio et al., 1964); this may be regarded as an indication of the presence of a fraction of messenger RNA in the preparations. THE

4

A. S . SPIRIN

4. On studying RNA synthesis by means of labeled nucleic acid precursors in maturing eggs of the frog (Brown and Littna, 1964) and of the sea urchin (Gross et a?.,1965) it is possible directly or indirectly to reveal fractions of labeled nonribosomal and non-4 S ( “soluble”) type RNA which are preserved in the mature egg and even survive fertilization. It may be thought that these labeled fractions reflect the synthesis and accumulation of messenger RNA during oogenesis to provide for the mature egg. Despite the storage of mRNA in the mature unfertilized egg, protein synthesis in it is, as has been repeatedly shown, completely absent (see, for example, Nakano and Monroy, 1958; Monroy, 1960; Gross and Cousineau, 1964; Gross, 1964). Protein synthesis is also practically absent in the homogenates of unfertilized sea urchin eggs (Hultin and Bergstrand, 1960; Hultin, 1961b; Wilt and Hultin, 1962; Tyler, 1963). In a number of special investigations it was shown that the reason for the inactivity of the protein-synthesizing apparatus of the mature egg cannot be either the absence or inactivity of the enzymes serving the protein synthesis, or the lack or defectiveness of “soluble” RNA; all these components of the protein-synthesizing apparatus are found abundantly in the mature egg and are quite active (Hultin, 1961b; Nemer and Bard, 1963; Maggio and Catalano, 1963). Stimulation of incorporation of labeled amino acids into nonfractionated homogenates from unfertilized eggs upon addition of exogenous template, polyU (Wilt and Hultin, 1962; Tyler, 1963), also attests to the adequacy of these components to serve in protein synthesis. On the basis of the same experiments the assumption that the cellular sap of the unfertilized egg contains special inhibitors of protein synthesis may be rejected. In direct experiments it was shown that the cellular sap of unfertilized eggs may serve as a normal medium for the incorporation of labeled amino acids by active preparations of ribosomes from developing embryos (Hultin, 1961b). At the same time it was found that ribosome preparations from an unfertilized egg remain inactive both in liver cell sap, where ribosomes of developing embryos function normally ( Hultin and Bergstrand, 1960; Maggio et al., 1964), and in extract from developing embryos (Hultin, 1961b). On the basis of such data Hultin (1961b) suggested that the reason for the inactivity of the protein-synthesizing apparatus of the unfertilized egg must lie in the very ribosomal particles. However, it was soon shown that the ribosomes of an unfertilized egg are capable of normally incorporating labeled amino acid upon addition of an exogenous

1.

“MASKED” FORMS OF

mRNA

5

template, polyU, polyUC, or polyUG (Nemer, 1962; Wilt and Hultin, 1962; Nemer and Bard, 1963; Tyler, 1963; Brachet et al., 1963a; Maggio et al., 1964). Consequently, the assumptions that the inactivity of the protein-synthesizing apparatus is due to “modifications” of ribosomes, or their defectiveness, or their “blocked state” in the unfertilized egg, have not been confirmed. In the aforementioned experiments with exogenous artificial templates the ribosomes of unfertilized eggs appeared quite normal. All of the aforesaid leads to the conclusion that the reason for the inactivity of the protein-synthesizing apparatus of the unfertilized egg is the masked state of its stored mRNA. As a result, the ribosomes of the unfertilized egg are not programmed by functionally active mRNA; fertilization or the parthenogenetic agents induce “unmasking” of the stored mRNA, the result of which is initiation of protein synthesis. However, it is necessary to make two reservations. Firstly, the proposition that the ribosomes of the unfertilized egg are not programmed by mRNA need not imply that mRNA in masked form cannot be attached to the ribosomes; it means only that in this case mRNA and ribosomes do not form active protein-synthesizing complexes. Secondly, the possibility must not be excluded that a certain portion of ribosomal particles in the unfertilized egg may be in a “‘blocked or “inhibited” state. The recent experiments by Monroy et al. (1965) show that treatment of ribosome preparations (100,000 g pellet) from unfertilized sea urchin eggs with trypsin leads not only to unmasking of mRNA and appearance in the preparations of their own endogenous protein-synthesizing activity, but also to a rise in the level of amino acid incorporation in response to stimulation with exogenous templates.

NATURE OF MASKEDFORMS OF MRNA B. ON THE POSSIBLE It follows from the foregoing that the unfertilized egg contains a store of mRNA. The state of this mRNA must satisfy a number of obvious requirements enumerated below, which make it possible to arrive at certain conclusions concerning the possible nature of stored mRNA. 1. Until fertilization mRNA stored in the egg must be reliably protected against possible involvement in protein synthesis as a template. It has been stated above that in the mature egg the absence of protein synthesis is apparently due not so much to inactivity of the ribosome apparatus itself as to the inaccessibility of the mRNA present there; since ( a ) upon addition of an exogenous template to the homogenate of un-

6

A. S . SPIRIN

fertilized eggs the ribosomes exhibit activity; ( b ) no protein synthesis is observed in the homogenate of unfertilized eggs without templates added from outside; ( c ) deproteinization of the homogenate leads to isolation of RNA which possesses stimulatory ( template) activity. Moreover, it is important to remember that in the process of oogenesis there are active ribosomes, while the accumulating mRNA is, at least in part, not involved in protein synthesis. This inactivity or inaccessibility of the stored mRNA of eggs may very likely be due, predominantly, to the fact that it forms complexes with some intracellular structures or macromolecules. 2. The stored mRNA of an unfertilized egg must be efectively protected against any degradation by intracellular enzymes. Effective protection of mRNA against degradative influences, during the period from the maturation of the egg until its fertilization, presupposes that RNA forms complexes with intracellular structures or macromolecules that completely prevent it from interacting with other macromolecular substances, enzymes in particular. One possibility is that in the stored form mRNA is surrounded by some macromolecular protective coat. 3. This protection must be remoued us a result of fertilization and during the subsequent development, under the action of definite physiologic factors; i e . , it must in a certain sense be regulated. The protection may be removed by either an enzymatic mechanism (appearance of an enzyme which destroys the coat) or a dissociation mechanism (dissociation of the macromolecules forming the coat under the action of some low-molecular-weight or high-molecular-weight substances of the type of allosteric effectors). The latter case suggests a protein coat of close-packed protein molecules. In the former case this is not so clear but is also probable; the experiments by Monroy et al. (1965) in which the proteolytic enzyme activated the endogenous templates in ribosome preparations from unfertilized eggs provide experimental evidence that the protective envelope of mRNA is a protein. A protected form of mHNA where the mechanism of this protection is well established is known in nature in the case of the small RNA-containing viruses. They fully satisfy the requirements formulated for the stored form of mRNA of the egg: (1) The RNA of the virus is inaccessible to the protein-synthesizing apparatus; ( 2 ) it is protected against degradative influences; ( 3 ) the protection is removed by the action of certain factors of the infected cell (“uncoating” of the virus). In this case

1.

“MASKED” FORMS OF

mRNA

7

it is well known that the protection is effected by formation of a coat from numerous regularly packed protein molecules. The following question arises: Is it not possible for an analogous mechanism of effective protection of mRNA to exist in the normal cell when it is biologically necessary? The preceding discussion agrees with our hypothesis that the stored masked form of mRNA in the egg is a complex of mRNA with protein; the molecules of this protein form a protective coat around the molecule (or aggregate of the molecules) of mRNA; it cannot be excluded that the protein is packed around the RNA molecule regularly, in the manner of organization of the capsids of small viruses. This hypothesis leads to several consequences. Firstly, if such mRNA-protein structures analogous to small viruses exist, by their chemical composition they must exhibit a predominance of protein over mRNA; at any rate, they must contain more protein than the ribosomal particles of the cell. Secondly, since the cell apparently has a set of mRNA molecules of different molecular weights there must be mRNA-protein structures of different sizes; at the same time, if their organization resembles that of spherical viruses, the size distribution of these structures cannot be continuous but must show a number of discrete groups. Some special questions are whether the proposed inactive mRNA-protein complexes of the egg exist as separate free particles or as large aggregates, whether they are complexed with ribosomes or are closely connected with the membranes of the cell, etc. It is as yet difficult to answer these questions even hypothetically for lack of experimental data. That at least part of the protein-protected mRNA is in association with the ribosomes of the cell appears plausible to us and is also consistent with the results of the recent experiments by Monroy et al. (1965). 111. On Masked mRNA in Early Developing Embryos and in a Number of Other Differentiating Systems

It has recently been established that the synthesis of new mRNA and its appearance in the cytoplasm begins in the developing egg during the very first hours after fertilization (Wilt, 1963, 1964; Brown and Littna, 1964; Spirin and Nemer, 1965). This means that as early as the first half of the cleavage period the nuclei of the embryo begin to function actively in the sense of transferring the genetic information to the cytoplasm. However, the importance of this synthesis of cytoplasmic mRNA

8

A. S . SPIRIN

to the cleaving embryos is not immediately apparent; various groups of data indicate that this synthesis apparently plays no essential role in development at this stage. Thus, activated non-nucleated egg fragments (Harvey, 1936; Briggs et al., 1951), eggs with inactivated and destroyed nuclei (Dalcq and Simon, 1932; Neyfakh, 1959, 1961, 1964), or eggs treated with actinomycin D and not synthesizing new RNA (Gross and Cousineau, 1963, 1964; Brachet and Denis, 1963; Brachet et al., 1964) are capable of cleaving and even of forming blastulas. The inhibition or complete cessation of mRNA synthesis by actinomycin does not lead to diminution in protein synthesis throughout the stage of cleavage and early blastula formation in the sea urchin (Gross and Cousineau, 1964; Gross et al., 1964); the inhibition of mRNA synthesis begins to show only during the late blastula-gastrula stages, when this inhibition blocks the further increase in protein synthesis and then leads to its diminution (Gross et at?., 1964). Recently it was reported that during cleavage and blastula stages actinomycin-treated embryos synthesize exactly the same set of soluble proteins as normal (control) embryos, as judged from gel electrophoresis fractionation ( Spiegel et al., 1965). Thus examination of the factual material as a whole reveals a certain discrepancy between the latest biochemical data which show that the nucleus is active in mRNA synthesis from the very Erst hours of development, and all the evidence showing that the nucleus and its products apparently play no essential role during the first stages of embryogenesis (cleavage and early blastula). This discrepancy may be eIiminated if we assume that the mRNA synthesized during the first stages of embryonic development is temporarily inactive, i.e., is in a masked form. Apparently this mRNA is necessary not for immediate realization but for ensurance of the subsequent stages of development (transition to gastrulation, gastrula). Certain data concerning plant seeds may suggest that they contain a stored inactive form of mRNA which becomes active during imbibition and germination. In this respect imbibition and germination of seeds are apparently very similar to fertilization or parthenogenetic activation of the egg. For example, the ribosomes from nongerminated cells are incapable of synthesizing protein in vitro and begin to incorporate amino acids intensively only after addition of the exogenous template (polyU ) ( Marcus, 1964; Marcus and Feeley, 1964, 1965; Allende et al., 1966). At the same time the ribosomes from germinating seeds are active. Actinomycin D, while inhibiting RNA synthesis in imbibing seeds, exerts no influence

1.

“MASKED” FORMS OF

mRNA

9

on the rate of protein synthesis in them (Dure and Waters, 1965). It may consequently be assumed that the protein synthesis during the imbibition and germination of seeds is in the main determined not by mRNA synthesized at the moment of germination but by preexisting templates stored in the seeds in a masked form. The masked state of mRNA is apparently responsible for the inactivity of the preparations of ribosomes isolated from resting seeds in a cell-free system devoid of exogenous template. The data furnished by studies on the dynamics of RNA and protein synthesis in some differentiating cells of metazoans may serve as another indication of the existence of a temporarily inactive stored form of mRNA. These are primarily the cells of the erythrocytic series (erythroblast + orthochromatic normoblast + reticulocyte + erythrocyte), It has turned out that RNA synthesis ceases soon after transition to the orthochromatic normoblast stage. However, the preponderant part of hemoglobin is synthesized in the orthochromatic normoblast after cessation of RNA synthesis. It follows that mRNA for the synthesis of hemoglobin is synthesized in advance (De Bellis et al., 1964). The synthesis of silk fibroin may serve as an analogous example; in the cells of the silk gland this synthesis is preceded by intensive RNA synthesis, apparently including mRNA synthesis. The maximum synthesis of fibroin takes place in the almost complete absence of RNA synthesis (Smirnov et al., 1964). Thus the two processes-synthesis of template RNA for specific protein and synthesis of the protein itself-are separated in time. First there is an accumulation of mRNA, apparently in an inactive form, and then there is the realization of these templates, i.e., their transformation to an active state. mRNA in an inactive form is apparently also stored in the cell during the synthesis of embryonic hemoglobin. The synthesis of hemoglobin in the chick embryo begins at a definite stage. Actinomycin D may prevent the formation of hemoglobin, but only if the treatment with the antibiotic begins at earlier stages of development when no hemoglobin synthesis is as yet taking place. Administration of an antibiotic before the very beginning of the hemoglobin synthesis no longer affects the protein synthesis (Wilt, 1965). These data support the assumption that the mRNA’s which serve as templates for hemoglobin are produced in the cell in advance, before the beginning of the protein synthesis. Wessels and Wilt (1965) also used another model of differentiating cells-embryonic epithelium of the mouse pancreas-to study the action

10

A. S. SPIRIN

of actinomycin on differentiation and RNA synthesis. Cells which had already begun to differentiate proved insensitive to the action of the antibiotic despite the 70-90% inhibition of RNA synthesis in them. Such cells continued to develop, synthesizing specific proteins. However, if the cells were given the antibiotic before they had begun differentiating, they lost their capacity for further development. The authors drew the conclusion that the synthesis of mRNA for the specific proteins characterizing the given type of cells is completed before the synthesis of these proteins; therefore only at earlier stages of development, when the templates are still being synthesized, does the inhibition of RNA synthesis by the antibiotic prevent the subsequent synthesis of the corresponding specific proteins and thereby stop further differentiation of the cells. Interesting indications of the presence of masked mRNA may be obtained even by studying unicellular organisms which exhibit differentiation-type phenomena. Thus in the development of the Acetabularia the formation of the cap is accompanied by a considerable increase in the activity of one of the cellular phosphatases. The cap may be regenerated in nucleate acetabulariae, as well as after their enucleation. The increase in the activity of the phosphatase during regeneration of the cap is entirely equal in both cases (Spencer and Harris, 1964). If it were proved that the increase in the activity of the phosphatase is due to the synthesis of this enzyme, it would mean that the mRNA for the synthesis of this protein had been present in the cytoplasm beforehand in an inactive masked state. Unmasking of mRNA should thus occur at a defink moment before formation of the cap. The transition of a bacterial cell to spore formation may also be regarded as a model of differentiation. This process was studied in particular in the Bacillus cereus with the aid of inhibitors of RNA and protein synthesis ( Actinomycin D, base analogs, and chloramphenicol ) ( Rosas del Valle and Aronson, 1962). It was found that spec& mRNA needed for spore formation (apparently for the synthesis of spore proteins) is synthesized in a relatively short time, in the main between 8.5 and 9.5 hours after development of the culture. The bulk of the spore proteins is synthesized after 12 hours of development of the culture, i.e., several hours later. It follows that in this case as well mRNA first accumulates without participating in the protein synthesis and only begins to function as an active template some time later. Thus on the basis of a number of experimental data the possibility that mRNA’s exist in a masked form in the cells of differentiating systems

1.

“MASKED” FORMS OF

mRNA

11

may be considered highly probable. In this form they are not directly active in the protein synthesis. These mRNAs apparently accumulate in the cells to provide for the subsequent stages of differentiation. In this stored form they are stable. The masked (inactive) state of the mRNA’s is temporary; at a certain moment they become activated with a resulting synthesis of corresponding new proteins. The formation of masked mRNA apparentIy occurs only in those cases where the cells have to switch to the synthesis of a new set of proteins in connection with the transition to the next phase of differentiation. IV. Experimental Data on the Existence of mRNA-Carrying Postribosomal Particles in Animal Cells

A. EARLYOBSERVATIONS One of the ways of experimentally revealing and identifying masked forms of mRNA in cells is to look for intracellular mRNA-containing particles other than the usual active ribosomes and polyribosomes; this search may in the first place be directed toward the postmicrosomal ( postribosomal) fraction. The first substantiated indication of the existence of postrnicrosomal particles carrying bound mRNA in the cytoplasm of animal cells was the work by Hoagland and Askonas ( 1963). As a result of long-continued centrifugation of postmicrosomal supernatant of rat liver the authors obtained a pellet containing proteins and a certain amount of RNA (X-fraction). This pellet was resuspended and the free RNA was removed by passage through a DEAE-Sephadex column a t an ionic strength of about 0.05 (incidentally, under these conditions not only any free RNA, but also ribosomes and their subunits, are adsorbed). As it turned out, the eluate contained not only proteins but also RNA. It follows that the X-fraction contained some bound form of RNA. On the basis of the stimulatory activity of the X-fraction in the cell-free system it was concluded that it was mRNA. The conclusion was confirmed by observations of more rapid incorporation of a2P into this RNA than into ribosomal and soluble RNA. On the basis of the data furnished by Hoagland and Askonas the following properties of the supposed mRNA-containing complex may be noted: ( 1 ) Like the greater number of free proteins, it is not adsorbed by DEAE-Sephadex at an ionic strength of about 0.05; ( 2 ) it is not sensitive to ribonuclease; (3) trypsin does not affect it in low concentrations but destroys it in high concentrations; (4)its addition to a cell-free

12

A. S. SPIRIN

system stimulates amino acid incorporation into ribosomes. Whether this last fact is connected with the manifestation of template activity of the complex itself or with the destruction of the complex during incubation and liberation of free active mRNA is unknown. The attempt by Hoagland and Askonas to study the sedimentation properties of the complex and its RNA by sucrose gradient centrifugation can probably be considered unsuccessful. While studying the X-fraction isolated and passed through DEAE they discovered the complex to be a heterogeneous material sedimenting at below 20 S; the RNA from it had a sedimentation coefficient of 4 S. At the same time the distribution of stimulatory activity over the gradient was shifted to the heavier side. This apparently reflects degradation of the greater part of the component and its RNA in the sucrose gradient centrifugation experiments. The properties of the RNA-containing complex described by Hoagland and Askonas warrant the assumption that this is protein-coated RNA. At any rate, its insensitivity to RNase and failure to be adsorbed on DEAE appear to indicate that this RNA is not exposed to the external environment to any considerable extent; the same properties do not agree with the idea that the carriers of mRNA in this case are ribosomal subunits. The next communication concerned with postribosomal particles carrying newly synthesized nonribosomal RNA was based on sucrose gradient centrifugation of cytoplasmic extract of early embryos of the loach ( M i s gurnus fossilis L . ) Belitsina et al. (1964). It was shown that components rapidly labeled in both RNA and protein are found on the sedimentation profile in a region lighter than 75-80 S ribosomes, Their sedimentation coefficients lie in the region from 20-30 S to 60-70 S. Their distribution is not continuous but discrete. Since no synthesis of ribosomes and ribosomal 28 S and 18 S RNA had as yet been observed at the investigated stage of embryogenesis, it was conjectured that the detected labeled components are complexes of newly synthesized RNA with some proteincontaining structures. These complexes were further identified in a subsequent work (Spirin et al., 1964). Some time later several reports were published on mRNA-containing postribosomal particles found through the gradient centrifugation of cytoplasmic extracts of other animal cells (Shatkin et al., 1965; Joklik and Becker, 1965b; Henshaw et at., 1965; McConkey and Hopkins, 1965) and also in extracts of nuclei of animal cells (Samarina et al., 1965, 1966; Henshaw et a,?., 1965).

1.

“MASKED” FORMS OF

mRNA

13

B. “INFORMOSOMES” OF EARLY EMBRYOS As was already noted above, when early embryos of the loach (Misgurnus fossilis L. ) were incubated with radioactive precursors of nucleic

C ) subsequent fractionation of acids ( adenine-I4C and ~ r i d i n e - ~ ~the the cytoplasmic extract by sucrose gradient centrifugation revealed several

600

I

I

I I

I

600

I I I

I I

I

I

tI

400

I

I

I

200

~

10

20

30

Fraction No

FIG.1. Sucrose gradient sedimentation of labeled cytoplasmic ( nuclei-free ) extract of the loach embryos ( N . V. Belitsina and L. P. Ovchinnikov, this laboratory). Solid line, UV absorption; dotted line, radioactivity.

labeled components moving slower than 75-80 S ribosomes, but much faster than the bulk of free proteins and RNA (Belitsina et al., 1964; Spirin et al., 1964). When the fractionation was carried out with cytoplasmic extracts of embryos incubated with labeled amino acids (leucineI4C and lysine-14C) the same fractions between ribosomes and soluble proteins turned out to contain labeled protein (Belitsina et al., 1964;

14

A. S. SPIRIN

Spirin et al., 1964). The same thing was also observed upon fractionation of labeled cytoplasmic extracts of early sea urchin embryos (cleavage, blastula, gastrula) (Spirin and Nemer, 1965). Figure 1 shows a profile of distribution of the components after sucrose gradient centrifugation. During the analysis of the profiles of label distribution in the postribosomal zone it was possible to distinguish up to six or seven discrete components. The approximate sedimentation coefficients of the components containing labeled RNA and labeled protein were calculated with the 75 S ribosome as a reference; the results are shown in Table I. TABLE I VALUES OF SEDIMENTATION COEFFICENTS FOR LABELED POSTRIBOSOMAL COMPONENTS OF THE CYTOPLASM OF THE LOACH AND SEA URCHIN EMBRYOS Loachb Sea urchina Component No.

0

b 0

d

e

Uridine-3H incorporationc

Leucine-14C incorporntiond

66 f 2

65 f 1

57 'r 2 49 f 2 40 2 2 31 f 2 20 f 2

57 f 1 5Of1 39 f 1 31 f 1 22 f 1

Leucine-l4C, Lysine-ldC, or KH,32P04 incorporation,c 68 f 3 63 2 3 56 f 2 49 f 2

41 f 2 31 2 2 23 f 2

From Spirin and Nemer, 1965. From L. P. Ovchinnikov and M. A. Ajtkhozhin, this laboratory. Data from 12 experiments. Data from 5 experiments. Data from 7 experiments.

The particles pelleted from the postribosomal zone of the sucrose gradient (from 65 to 40 S were used) possessed, as was shown, stimulatory activity upon introduction into a cell-free system (Spirin et al., 1964). This apparently indicated that they contained mRNA. However, as in the experiments by Hoagland and Askonas (1963), it was not clear whether the stimulation occurred through direct interaction of the described particles with ribosomes or whether the stimulatory action was possessed only by the mRNA liberated by the destruction of the complexes during incubation. Isolation of RNA from the zone of 2&65 S particles by direct phenol

1.

“MASKED” FORMS OF

15

mRNA

deproteinization of the corresponding sucrose gradient fractions has shown that by its sedimentation characteristics newly synthesized (labeled) RNA of these particles is not ribosomal 28 S and 18 S RNA, but at the same time coincides by its profile with rapidly labeled RNA extracted from the polyribosomal zone (i.e., apparently with mRNA of polyribosomes ) ( Nemer and Infante, personal communication; M. A. Ajtkhozhin and L. P. Ovchinnikov, this laboratory). This is illustrated in Figure 2 by comparative data on sedimentation distribution of labeled RNA from the polyribosomal zone (Fig. 2a) and the postribosomal zone (Fig. 2b).

w

OOE

40

004

20

220

Fraction No

E

g

“

n

k

Fraction No

FIG. 2 . Sucrose gradient sedimentation of labeled RNA isolated by phenol deproteinization from informosomes ( a ) and polyribosomes ( b) of the loach embryos ( M. A. Ajtkhozhin and L. P. Ovchinnikov, this laboratory). Ribosomes were added in deproteinization mixture to have ribosomal RNA as a reference. Solid Iine, UV absorption; dotted line, radioactivity.

Moreover, Nemer and Infante in their subsequent experiments with sea urchin embryos isolated RNA directly from each single component of the postribosomal zone and found the 20 S component to contain only 10 S labeled RNA. The 31 S component contained only 13 S RNA, the 40 S component contained 18 S RNA, and the 49 S component contained 22 S RNA (Nemer and Infante, personal communication). Finally, one of the most important proofs of the informational nature of the labeled RNA of the postribosomal particles was furnished by the hybridization experiments by Spirin and Nemer ( 1965). The experiments demonstrated that labeled RNA from the 20-65 S zone of cytopIasmic extract of sea urchin embryos is highIy capable of forming hybrids with sea urchin DNA; under these conditions ribosomal RNA yielded a

16

A. S . SPIRIN

very low level of hybrid formation with DNA. In subsequent experiments Nemer and Infante showed hybrid formation of labeled RNA separately from each postribosomal component ( Nemer and Infante, personal communication). Thus on the basis of all accessible criteria-( 1 ) the ability to be relatively rapidly labeled in uiuo, ( 2 ) the ability to stimulate incorporation of amino acids in a cell-free system, ( 3 ) the sedimentation distribution which does not coincide with that of ribosomal RNA and at the same time is analogous to the distribution of rapidly synthesized RNA of polyribosomes, and (4) the high ability to hybridize with homologous DNA-it may be assumed that the labeled RNA of postribosomal structures detected in cytoplasmic extracts of the loach and then of the sea urchin is mRNA. The newly synthesized mRNA in these structures is bound, and it is liberated as a result of deproteinization. These structures also contain newly synthesized protein. To identify the postribosomal structures, their density distribution was studied by means of CsCl gradient centrifugation (Spirin et aE., 1964). For this purpose loach embryos were preincubated with either labeled nucleic acid precursors or labeled amino acids, and cytoplasmic extracts of cells were fractionated by sucrose gradient centrifugation. Labeled components were collected from the zone between 75 S and 30 S and pelleted in a preparative rotor. Simultaneously the zone of 75 S ribosomes was taken from the sucrose gradient and the particles were sedimented. The preparations were resuspended in a small volume of phosphate buffer with MgCl, and were fixed with formaldehyde to prevent subsequent secondary changes in their composition under the action of CsC1, as was described by Spirin et al. (1966a). Only after that was the suspension mixed with a CsCl solution. As a result of centrifugation the investigated components were distributed in the density gradient as shown in Figs. 3 and 4.’

* In order to reveal simultaneously all the structures that may be distributed in the wide range of buoyant density from p = 1.3gm/cm3 to p = 1.8 gm/cm3 a special technique of precreated steep CsCI density gradient stabilized by a sucrose gradient was elaborated. The gradient was made by a gradual mixing of “heavy” and “light” solutions, using an ordinary mixer for linear gradients. The “heavy” CsCl solution was made in 10% sucrose and 0.01 M phosphate buffer, pH 7.9, with 0.01 M MgC1,; the summary density was about 1.8 gm/cm3. A mixture consisting of 1 part saturated CsCl prepared in 5% sucrose with the same phosphate buffer and MgCI, and 2 parts formaldehyde-fixed suspension of the sample (Spirin et al., 1966a) in the same buffer was used as the “light” solution; the final density of this “light” solution containing the preparation was about 1.3 gm/cm3. As a result, the gradient of the density before centrifugation was from 1.8 to 1.3 gm/cm3, the gradient of sucrose concentra-

1.

‘‘MASKED” FORMS OF

mRNA

17

The distribution of ultraviolet-absorbing material in the density gradient, in experiments conducted with components from the 75 S zone (Fig. 3 and 4, upper), shows that 75 S ribosomal particles of the loach occupy the band of buoyant density of e = 1.55 gm/cm3. In a series of experiments conducted in this laboratory it was shown that the buoyant density of ribonudeoprotein particles treated with formaIdehyde as determined in CsCl under standard conditions of concentration Mg” depends practically linearly only on the RNA-protein ratio; a different amount of charging with protein, or a different particle conformation, does not appreciably affect this dependence (see articles by Lerman et al., 1966; Gavrilova et al., 1966). In such a case, using the aforesaid dependence and ignoring the differences in the densities of RNA from different sources, it may be considered that 75 S ribosomes of the loach contain about 53% RNA and 47% protein. The presence of labeled RNA and labeled protein in the band corresponding to ribosomes apparently attests that some of them retain sRNA, perhaps fragments of mRNA, and nascent peptide. Besides the aforementioned main band with e = 1.55 gm/cm3, all the experiments with 75 S material reveal a small additional less dense component which is not dearly revealed in uItravioIet absorption but which possesses high specific activity both in nucleic acid and protein label (Fig. 3 and 4, upper). As the examination of the label distribution in the case of postribosomal material shows, this less dense component is most likely due to the presence in the 75 S zone of a certain number of the largest labeled postribosomal particles. No labeled components denser than ribosomes are found in the 75 S material. In the study of postribosomal material labeled in RNA in CsCl density gradient (Fig. 3, lower) the most characteristic thing was that the main or at least a considerable part of labeled RNA was distributed in bands less dense than the band of ribosomes. As Figure 3 shows, this material is concentrated for the most part in the region of buoyant density e from 1.45 gm/cm3 to 1.49-1.51 gm/cm3. The study of the material in gradual equilibrium CsCl gradient revealed up to five discrete density bands, at tion was from 10% to 5% and the gradient of the ribonucleoprotein particles in question was from zero at the bottom to maximum at the top. The centrifugation was carried out at 39,000 rpm for 9-10 hours; under these conditions the particles being studied concentrated in equilibrium bands. After the end of the centrifugation run the density was checked refractometrically with due correction for the contribution of the sucrose to the refraction index and the density; practically the linear gradient was observed to be from 1.75 gm/cm3 at the bottom to 1.33 gm/cm3 at the top.

18

A. S. SPIRIN

1.45, 1.47, 1.49, 1.51, and 1.53gm/cm3 (L. P. Ovchinnikov and N. V. Belitsina, this laboratory). If we keep to the aforementioned linear dependence of the buoyant density on the RNA-protein ratio and assume that the particles contain no other components except RNA and protein, the material distributed in bands less dense than ribosomes will contain

3,O.I

I55

150

30

20 !OO

10

00

I 0 N W

k

n 0

I00

50

Froction

No

Froction No

FIG. 3. CsCl density gradient centrifugation (see footnote, p. 16) of ribosomes (upper) and infonnosomes (Zuruer) of the loacli embryos labeled in RNA ( a and h are independent duplicate experiments; N. V. Belitsina, this laboratory). Solid line, UV absorption; dotted line, radioactivity.

correspondingly from 25 to 43% RNA (the considerable predominance of protein over RNA and the very values of RNA content strongly resemble the situation in the case of small “spherical” viruses). Hence the bulk of postribosomal material containing newly formed RNA (apparently mRNA) is represented by particles less dense than ribosomes and apparently unrelated to ribosome subunits or ribosome precursors. They may be regarded as unique mRNA-protein complexes with predominance of the protein moiety.

1.

“MASKED” FORMS OF

mRNA

19

Density distribution analysis of postribosomal particles labeled in protein (Fig. 4,lower) reveals their localization in the same zone that was noted for labeled RNA; band Q = 1.55gm/cm3 in all probability corresponds to the admixture of ribosomal particles with nascent peptide; the bands with buoyant density from 1.45 gm/cm3 to 1.51 gm/cm3 apparently

’

1.54

I

750

20 200

500

100

250

10

z 0

N (D

k

ri

6

$

75

50

190

25

50

..

Froction No

10

20

FIG. 4. CsCl density gradient centrifugation (see footnote, p. 16) of ribosomes (upper) and informosomes (lower) of the loach embryos labeled in protein ( a and b are independent duplicate experiments; N. V. Belitsina, this laboratory). Solid line, UV absorption; dotted line, radioactivity.

reflect the same basic components of postribosomal material which are revealed in the nucleic acid label. It follows that these bands really contain precisely the postribosomal material which on sucrose gradient centrifugation is revealed as several discrete components simultaneously labeled in nucleic acid and protein. These components revealed as newly synthesized mRNA-protein complexes, less dense than ribosomes and apparently representing a new type of ribonucleoprotein particles, were named “informosomes” ( Spirin et al.. 1964).

20

A. S . SPIRIN

In addition to informosomes the density distribution analysis of the postribosomal fraction reveals other components which also contain labeled RNA but which are denser than ribosomes; their buoyant density lies in the region of 1.60-1.65 gm/cm3 (Fig. 3, lower). However, these dense postribosomal components containing labeled RNA do not contain labeled protein, as can be seen in Fig. 4 (lower).It follows that in addition to informosomes other structures, possibly also carrying mRNA, may be discovered in the postribosomal fraction; these structures differ from informosomes in having a much higher buoyant density and in absence of newly synthesized protein molecules. It is not excluded that they may be ribosomal subunits, particularly 40 S subunits, with newly synthesized mRNA attached (see below). As a matter of fact, attachment of an mRNA molecule to a 40 S particle must lead to a substantial increase in buoyant density, compared with the density of usual ribosomes. Since no synthesis of ribosomes is detected during the stages of embryogenesis being studied, these may be only complexes of preexisting 40 S particles with newly synthesized mRNA; this idea finds corroboration in the absence of amino acid label in this dense material. C. MRNA-CONTAINING POSTRIBOSOMAL PARTICLES IN THE CYTOPLASM OF VIRUS-INFECTED CELLS Reports published in 1965 showed that RNA-containing particles sedimenting in the region between 30 S and 74 S of sucrose gradient could be revealed in the cytoplasm of HeLa cells infected with vaccinia virus (Shatkin et al., 1965; Joklik and Becker, 1965b). Shatkin et al. showed that 5-6 hours after infection newly synthesized (labeled) RNA having virus DNA-like base composition may be revealed, on the one hand, in polyribosomes and, on the other hand, as heterogeneous material whose bulk sediments as a broad peak in the region between 30 S and 70 S with a maximum at about 50 S. In noninfected cells no such material could be detected and only labeled 45 S particles with newly formed RNA with a base composition similar to that of ribosomal 16 S RNA were revealed. In virus-infected cells the DNA-like RNA associated with polyribosomes was found to be rather unstable, as could be seen by the disappearance of the label after actinomycin block: about half of it degraded to acidsoluble fragments within half an hour. At the same time the DNA-like RNA associated with postribosomal structures was found to be relatively stable and could not be seen to decay in the presence of actinomycin during half an hour. The authors concluded that the stability of the newly

1.

“MASKED” FORMS OF

mRNA

21

synthesized DNA-like RNA of the postribosomal zone “may be due to association with 45 S ribosomal subunits or with protein.” In the work by Joklik and Becker (1965b) the localization of newly synthesized ( pulse-labeled ) virus-specific mRNA in the zone between 20 S and 60-70 S was also shown by sucrose gradient centrifugation of a cytoplasmic extract of infected cells. A distribution of mRNA in the postribosomal zone of the vaccinia virus-infected HeLa cells, very similar to that of the Joklik-Becker experiments, can be seen in Figure 5, lower. Unlike Shatkin et al., Joklik and Becker, using an actinomycin block, succeeded in showing that at least part of the pulse-labeled mRNA of the postribosomal zone rapidly passes into polyribosomes. The authors assumed without any experimental proof that the mRNA-containing postribosomal particles observed by them represent complexes of mRNA with 40 S ribosomal subunits. Apart from absence of acceptable evidence, the assumption of Shatkin et al. and the assertion of Joklik and Becker that the label in the postribosoma1 zone is due to mRNA associated with 40 S ribosomal subunits does not quite agree with the cited profiles of label distribution: (1) The profile of label distribution does not coincide with the peak of 40 S particles observed as ultraviolet-absorbing material but is always broader; ( 2 ) the presence of the label ahead of 40 S particles could be easily explained, since attachment of mRNA must make 40 S particles heavier, but it is difficult to explain from this standpoint the presence of a considerable part ( u p to 50%) of the label in the zone directly behind the 40 S particles*; ( 3 ) the label distribution in the postribosomal zone, especially in Joklik and Becker’s experiments, is not even but displays a number of more or less discrete reproducible peaks (half of which sediment slower than 40 S particles). (For illustration, see also Fig. 5, lower.) Our caIculations of the sedimentation velocities of the peaks on the basis of 10 curves of label distribution in Figs. 1 4 of Joklik and Becker’s article (196513) yielded the results shown in Table 11. A comparison of the values obtained with those in the cases of labeled postribosomal components of the loach and sea urchin embryos (Table I ) cannot but reveal their striking similarity. All of the aforesaid warrants the conclusion that at

* As should have been expected, in experiments with in oitro complex formation of the sinall ribosomal subunit with template polynucleotides a shift in the sedimentation coefficients of the particles is always observed in the direction of an increase and never in a direction of a decrease (Takanami and Okamoto, 1963; Henshaw d al., 1965).

TABLE I1 VALUESOF SEDIMENTATION COEFFICENTSFOR LABELEDPOSTFUBOSOMAL COMPONENTS OF THE VACCINIA VIRUS-INFECTED HELA CELLSa

THE

CYTOPLASM OF

From Fig. 3c

From Fie. "

From Fig. 2C

l b

+

~~

1 min

2 min

3 min

-

-

65

60 48 42 37

29 20

61

49 43 37 31 00

55 49 43 38 25

-

5 min

58 ( ? )

5 min DNase 63 ( ? )

-

5 min 62 48 42 34

22

5 min then puromycin added

5 min after removing puromycin

68 60 54 ( ? ) 48 42 35 29 23 ( ? )

66

52

From Fig. 40

10 min

66 60 ( ? ) 56 46

44

35 26 20

43 38 33 27 ( ? ) 22

4 min, and 6 min in the presence of puromycin 66 ( ? ) 61 55 45

39

28 22 ( ? )

Average values of s

66&2 61 & 1 55&1 48 & 1 43 & 1 38 & 1 3421 28 & 2 21 & 1

Calculated from data of Joklik and Becker (1965b), Figs. 1, 2, 3, and 4, using the 60 S ribosomal subunit as a reference. Assuming the 60 S point as a reference the sedimentation velocity of the 40 S ribosomal particle is 43 & 2s in this calculation. b Uridine-3H incorporation. c Uridine-lW incorporation. a

9 * VJ

5

1.

“MASKED”

FORMS OF

mRNA

23

least some of the mRNA-containing postribosomal particles of HeLa cells infected with vaccinia virus are not complexes of mRNA with 40-45 S ribosomal subunits. The data of Shatkin et al. on the stability and preservation of mRNA in the postribosomal zone in case of actinomycin block also hardly agree with the idea of transit mRNA-40 S complexes. True, Joklik and Becker’s work asserts the opposite, i.e., that in case of actinomycin block the mRNA of this zone rapidly passes to the polyribosomes, but the factual material of ,this work shows that only a portion of labeled RNA of the postmicrosomal zone behaves in this manner, while the other portion, larger in label balance, looks like stable and nontransit labeled RNA. It is interesting that, as can be inferred from the aforementioned work, it is precisely the mRNA, which accounts for the marked predominance of the label in the peaks in the 40-45 S region, that rapidly disappears in case of actinomycin block, whereas the stable mRNA of the postribosoma1 zone forms a more even label distribution in the observed peaks throughout the region between 65 S and 20 S. An analysis of these data warrants still stronger adherence to the opinion that in the postribosomal zone of HeLa cells infected with vaccinia virus there are at least two types of mRNA-containing structures differing in behavior: ( 1) particles containing stable (stored) mRNA and forming a number of discrete components with various sedimentation velocities, from 66 S to 20 S, and ( 2 ) particles carrying transit mRNA which rapidly passes to polyribosomes; these particles sediment for the most part in the 40-45 S region and may coincide in position with the 40 S ribosomal subunits. It is not excluded that the former may be particles analogous to informosomes discovered in embryos. The latter may be the supposed transit complexes of mRNA with 40 S ribosomal subunits, although the works cited furnish no experimental data in favor of this supposition. IN D. MRNA-CARRYING 45 S PARTICLES DIFFERENTIATING ANIMALCELLS

THE

CYTOPLASM OF NON-

The two recent communications dealing with nondifferentiating animal cells, namely, rat liver cells ( Henshaw et al., 1965) and uninfected HeLa cells (McConkey and Hopkins, 1965), yielded data to the effect that in the cytoplasm newly synthesized DNA-like RNA possessing stimulatory activity may also be found in the postribosomal zone, but in this case as a single labeled discrete component which in the sucrose gradient profile

24

A. S. SPIRIN

exactly coincides with the position of the 45 S ribosomal subunit (see also Fig. 5, upper). The authors drew the conclusion that they were dealing with mRNAcarrying 45 S ribosomal subunits and speculated that this may be a form of mRNA transfer from nucleus to cytoplasm and then directly to polyribosomes. I

75s

i'

I

60s

.c

0

t

455

t

I

;! Ip,

0

I I

I

I

3000

I

n

I

75s

60s

455

'!

t

t

t

I

5

I 0 '

I I

%

I

0

1500

500

0

0

r

'i I

I

1500

i

I I

I

0 1000 C

500 C

10

20

30

Fraction No

FIG. 5. Sucrose gradient sedimentation of uridine-l*C labeled cytoplasmic extract of HeLa cells, normal (upper) and infected with vaccinia virus (lower) (Spirin et al., 196Bb). In both cases uridine-14C was given during 30 minutes, in parallel. Solid line, UV absorption; dotted line, radioactivity.

1.

“MASKED” FORMS OF

mRNA

25

This hypothesis about the mRNA complex with the smaller ribosomal subunit as a transit form of mRNA in the cell has become particularly popular in the past year, apparently under the influence of two groups of data (although it was also independently expressed somewhat earlier; see Lerman et al., 1965). On the one hand, the works of Girard et al. (1965) and Joklik and Becker (1965a) have shown that newly synthesized 60 S and 45 S ribosomal subunits pass from the nucleus into the cytoplasm independently of each other and are then found at once as complete 75 S ribosomal particles in the polyribosomal fraction, avoiding the stage of free 75 S monoribosomes. This means that the association of 60 S and 45 S subunits occurs in the presence of mRNA. On the other hand, data on the possibility of specific attachment of a template polynucleotide to the small ribosomal subunit have already been pubIished ( Takanami and Okamoto, 1963). Hence the tempting hypothesis that the newly synthesized small ribosomal subunit combines with mRNA in the nucleus and transports the mRNA to the cytoplasm where it combines with a 60 S subunit and forms a directly active protein-synthesizing complex. The actual discovery of mRNA-containing particles sedimenting at the same velocity as the small ribosomal subunits is regarded as experimental confirmation of this hypothesis. However, two important circumstances must be noted here: 1. The coincidence of the 45 S ribosomal subunit with the peak of postribosomal mRNA is, of course, not incontestable proof that both peaks are represented by particles which are the same in their nature. A mere coincidence of the sedimentation velocities of two different types of particles -free 45 S ribosomal subunits and some other mRNA-carrying particles -cannot be excluded. In any case, not one of the working groups cited above offers any proof that mRNA is associated precisely with the small ribosomal subunit and not with some other structure with the same sedimentation velocity. 2. The exact coincidence of the peak of 45 S ribosomal subunits with the peak of labeled mRNA is evidence against rather than for the complex of the 45 S subunit with mRNA. It is most unlikely that the sedimentation velocity of the 45 S particle would not change at all as the result of attachment of 18 S (Henshaw et al., 1965) or 1 5 1 6 S (McConkey and Hopkins, 1965) mRNA to it. Since in this case both the density of the particles (the RNA-protein ratio will increase from 1:1 to 2: 1) and their molecular weight will increase (the latter almost 50%), such complexes should sediment rather noticeably faster than free subunits (unless we make two hardly likely assumptions at the same

26

A. S. SPIRIN

time: The attachment of mRNA to the subunit leads to conformational changes in the particle consisting of unfolding or loosening, and these changes by chance coincidence compensate precisely for the increase in density and molecular weight). Thus, although on the basis of a number of observations the existence of natural complexes between the small ribosomal subunit and mRNA in the cytoplasm of animal cells appears quite feasible and might explain a number of features of the passage of mRNA into the cytoplasm and its entrance into polyribosomes, there is as yet no incontestable experimental proof in favor of it. Moreover, some observations (sedimentation velocities) are hard to reconcile with the existence of such complexes in the form in which this is usually implied, At the same time it is more or less evident that some bound transit form of mRNA does exist in animal cells. In this respect the following alternatives may be considered: (1) These may be complexes of mRNA with the small 45 S ribosomal subunits; ( 2 ) the transit form may be a complex of mRNA not with a complete ribosomal subunit but with its slower sedimenting precursor, which has not yet combined with protein; ( 3 ) the discovered mRNA-containing 45 S complexes may be informosome-type particles; and (4)lastly, there may exist transit mRNA-carrying particles which have nothing to do with either ribosomes or informosomes. All these alternatives need accurate experimental analysis, in which respect studies of the density behavior of the particles after their preliminary stabilization with formaldehyde (Spirin et al., 1966a) may prove very helpful.

E. MRNA-CONTAINING NUCLEARPARTICLES The first indication of the possible existence of special mRNA-containing particles in the nucleus, as a transport form of mRNA, was furnished by Beermann’s electron microscopic observations ( Beermann, 1964). In the nuclei of the salivary glands of the Chironomus he noticed that some RNA-synthesizing puffs produced characteristic large round particles, larger than ribosomes. The particles were sensitive to ribonuclease and trypsin, hence the assumption that they were mRNA-containing ribonucleoproteins. The particles passed into the nuclear sap, approached pores of the nuclear envelope, and entered them. As they penetrated the pores their shape changed into rods or threads. On the cytoplasmic side of the nuclear envelope they seemed to disappear. Beermann held that, while mRNA is in the nucleus, it is bound with some transport protein with which it forms characteristic large particles. As soon as a particle

1.

“MASKED” FORMS OF

mRNA

27

passes a nuclear pore, mRNA leaves its vehicle behind and is picked up by ribosomal structures of the cytoplasm. In connection with these data it is not clear whether mRNA-containing postribosomal particles of the type found in the cytoplasm must form and exist in the nucleus. It is possible that the transport of an mRNA molecule from the site of its synthesis on the chromosomal DNA to the cytoplasmic ribosomes is a multistage process presuming the existence of a specific intranuclear mRNA-carrier of the type of particles described by Beermann, then transmission of the mRNA molecule to the cytoplasmic carrier (maybe a 45 S particle) and then formation of polyribosomes. Another possibility is that the large particles observed by Beermann are specific aggregates of mRNA-containing postribosomal particles; on passing into the cytoplasm these aggregates dissociate into individual particles. In any case, Beermann’s data do not settle the question of the presence or absence of small ( postribosomal ) mRNA-containing particles in the nucleus. In the work of Georgiev’s group the existence of postribosomal, apparently mRNA-containing particles in nuclear extracts of animal ceIls (rat liver and Ehrlich ascites tumor cells) were shown by sucrose gradient centrifugation analysis (Samarina et al., 1965, 1966). The particles were revealed as a rather homogenoiis peak with a sedimentation velocity of about 30 S. The particles contained newly synthesized DNA-like RNA after deproteinization sedimenting as a two-component material in the region of 15-25 S. The authors supposed that they were dealing with particles which are a transport form of mRNA. They did not exclude the possibility that this may be a complex of mRNA with the small ribosomal subunit. The nuclear extract of liver cells was also analyzed by Henshaw et al. ( 1965) , the authors discovering postribosomal particles containing labeled RNA in the form of a rather broad peak with the maximum in the region of 45-55 S. Neither the nature of the discovered mRNA-containing nuclear complexes nor their relation to the postribosomal particles of the cytoplasm is as yet clear, and no experimental data with regard to this are available.

F. CONCLUSION AND SOMESPECULATIONS 1. It may be considered almost established that mRNA in a bound (associated) form is likely to be found in postribosomal particles of the cytoplasm and apparently also of the nucleus.

28

A. S . SPIRIN

2. The aggregate of available data may serve as an indication of the possible existence of at least two forms of bound mRNA in the postribosoma1 cytoplasmic fraction: a stable form apparently capable of accumulating and persisting for a certain time as postribosomal particles without destruction; and a transit form which rapidly passes into polyribosomes. 3. The stable form of bound mRNA apparently forms a series of discrete components with sedimentation coefficients from 20 S to 70 S and constitutes mRNA-protein complexes with considerable predominance of the protein component (“informosomes”) , 4. The transit form of bound mRNA in the cytoplasm constitutes uniform particles with a sedimentation coefficient of 45 S which coincides with that of the small ribosomal subunit (mRNA-containing 45 S particles ) . 5. As a hypothesis it may be assumed that the stable (informosomal) form of postribosomal mRNA has to do with regulation of protein synthesis in the processes of development and differentiation; in such masked form only that mRNA may accumulate whose realization must occur not directly, but at a definite subsequent period of development. The involvement of this mRNA in the protein synthesis must be preceded by uncoating, i.e., removal of the protein envelope (unmasking under the action of some physiological “inducers”). It is possible that informosomes are the form of existence of the masked mRNA of the egg. 6. The function of mRNA-containing 45 S particles is, in all probability, a rapid and immediate incorporation of the mRNA associated with them into active protein-synthesizing polyribosomes. 7. In accordance with these assumptions nondifferentiating cells or cells which have finished their differentiation must not contain informosomes, and the only type of mRNA-containing postribosomal particles in them must be transit 45 S complexes. Formation of informosomes must have something to do with preparation for transition to another stage which is characterized by switching on the synthesis of a new set of proteins, i.e., it must be typical only of differentiating cells. It follows that differentiating cells exhibit both types of mRNA-containing postribosomal particles. V. Experimental Data on the Existence of Masked Polyribosomes

A. INACTIVE STOREDOLIGORIBOSOMES OF EARLY EMBRYOS It is known that early embryos, sea urchin embryos in particular, exhibit during the very first hours after fertilization intensive protein syn-

1.

“MASKED” FORMS OF

mRNA

29

thesis in the polyribosome fraction, as can be seen after pulse incubation of the embryos with labeled amino acid and subsequent fractionation of cytoplasmic extracts by sucrose gradient centrifugation ( Monroy and Tyler, 1963; Stafford et nl., 1964; Spirin and Nemer, 1965). Protein-synthesizing activity is observed mainly in the zone of very heavy polyribosomal complexes ( >200 S). At the same time a parallel study of the distribution of newly synthesized mRNA in the polyribosomes (after 2hour incorporation of labeled uridine) shows that during the initial stages of development ( cIeavage stage) these heavy, active polyribosomes are programmed mainly with old, preexisting ( unlabeled ) mRNA; newly synthesized mRNA associated with ribosomes is localized in the region of barely active or inactive “light” polyribosomes ( oligoribosomes) from 90 to 200 S (Fig. 6; Spirin and Nemer, 1965). This agrees with the idea that during the cleavage stage newly synthesized mRNA is not directly utilized and the protein synthesis is realized with the participation of only the old, maternal mRNA. It follows that at this stage the “light” polyribosomes containing newly synthesized mRNA may be regarded as temporarily inactive oligoribosomal complexes. It was conjectured that they may constitute complexes of one or several ribosomes with an informosome (Spirin and Nemer, 1965). The recent experiments by Monroy et al. (1965) may be interpreted as an indication that in unfertilized eggs a considerable portion of inactive stored mRNA is already attached to ribosomes. As a result of treatment of a ribosomal pellet with trypsin, formerly inactive preparations are transformed into active preparations even without addition of exogenous mRNA. It may be assumed that, if informosomes are a stored form of mRNA in unfertilized eggs, they may form inactive oligoribosomal complexes with ribosomes. Removal of informosomal protein will mean activation (unmasking) of such structures with formation of working polyribosomes. Thus it may be supposed that in the cytoplasm of early embryos informosomal particles are present not only in a free form in the postribosomal fraction, but also in association with ribosomes, forming oligoribosomes inactive in the protein synthesis. It is possible that in the cytoplasm there is an equilibrium: inactive oligoribosomes % free ribosomes ( complex of ribosomes with an informosome)

+ free informosomes

Activation of the complexes occurs when the informosomal protein is

30

A. S. SPIRIN

cast off (unmasking), which is induced by some physiological factors during transition to a new phase of development. It is not implausible that this also constitutes the activation of the protein-synthesizing apparatus of the egg during fertilization. I

I

3000

I

m

0)

E

V .-

5

1500

i 8

I

10

20

Fraction No

-.-.-,

FIG. 6. Sucrose gradient sedimentatLon of labeled cytoplasmic extract of the sea urchin embryos: incorporation of uridine-3H and leucine-14C into polyribosomes at the cleavage stage (Spirin and Nemer, 1965). Solid line, UV absorption; dotted line, uridine-sH; leucine-14C. radioactivity: -o+,

In addition to the foregoing data on early embryos we may refer to the work in which inactive oligoribosomes (tetrads of ribosomes) were directly observed by sedimentation analysis and electron microscopy in a developing down feather of the chick embryo (Humphreys et al.,

1.

“MASKED” FORMS OF

mRNA

31

1964; Bell et al., 1965). These structures, consisting of four ribosomes (158 S ) , did not synthesize any protein until the fourteenth day of the chick‘s embryonic development and were ribonuclease-resistant; in vivo actinomycin treatment of the cells did not affect their amount. After the fourteenth day of development they began to synthesize protein, became sensitive to ribonuclease and actinomycin, and changed to the usual form of polyribosomes. It is not excluded that this case is an example of existence of masked oligoribosomal complexes analogous to the inactive oligoribosomes of early sea urchin embryos. As in early embryogenesis these complexes accumulate in the cell for a definite stage of development; unmasking occurs at a very definite moment under the action of some specific physiological factors.

B. “REPRESSION” OF POLYRIBOSOMES Of late it has become clear that the phenomena of regulation of mRNA activity are not limited only to cases of unmasking of earlier inactive templates. Several observations show that the reverse situation, where actively functioning templates are at a certain moment “repressed and become inactive, may be the normal mode of regulation of protein synthesis in differentiating systems. The first work which clearly demonstrated repression at the level of mRNA was McAuslan’s experiments ( McAuslan, 1963) on synthesis of thymidine kinase in pox virus-infected HeLa cells. The following data were obtained: 1. Synthesis of viral thymidine kinase is induced immediately after infection. 2. Synthesis of mRNA for thymidine kinase is completed within 2 hours of infection: If Actinomycin D is given to infected cells before the 2 hours are up, the earlier it is given, the lower the subsequent rate of the enzyme synthesis in the cells; if the antibiotic is given 2 hours after the infection or later, the maximum rate of synthesis is observed. 3. mRNA for thymidine kinase is stable: In the presence of actinomycin given 2-3 hours after infection the enzyme synthesis continues at least 15 more hours. 4. Six hours after infection the synthesis of thymidine kinase ceases (although general protein synthesis, according to the data on labeled amino acid incorporation, continues); it follows that within 6 hours the stable template for thymidine kinase ceases functioning (repression of mRNA). 5. Formation of the repressor depends on some mRNA whose synthesis ceases within 4 hours of the infection: No repression occurs if Actinomycin D is given within the first 4 hours after infection (for example, between the second and fourth

32

A. S . SPIRIN

hours). 6. The repressor agent is a protein (apparently synthesized on the aforementioned mRNA ) : Puromycin inhibits formation and accumulation of the repressor. 7. The establishment of repression requires a certain critical amount of protein synthesized: Repression is established only after a certain period of active protein synthesis regardless of whether or not ,the protein synthesis was interrupted as a result of puromycin treatment. 8. In McAuslan’s opinion the latter denotes that the repressor protein might act stoichiometrically rather than catalytically. Analogous data on repression of stable functioning mRNA were obtained by Garren et al. (1964) on another model while studying the stimulation of the synthesis of some liver enzymes (tryptophan pyrrolase and tyrosine transaminase) with hydrocortisone. Intraperitoneal injection of hydrocortisone to adrenalectomized rats led to induced synthesis of tryptophan pyrrolase; the synthesis lasted 5 hours, then slowed sharply and, 7 hours after the injection, ceased altogether. If the RNA synthesis was inhibited with Actinomycin D at the moment of hydrocortisone injection, no induction of the enzyme synthesis was observed. If Actinomycin D was given 4 hours after the injection, the synthesis of the enzyme continued during the subsequent hours at the same rate without slowing down or ceasing. Even 7 hours after the injection, when the synthesis of the enzyme had ceased altogether, it could be restored by inhibitors of RNA synthesis. The latter circumstance indicates that mRNA for tryptophan pyrrolase is preserved, in a nonfunctional state, even after 7 hours. It follows that immediately after the injection of hydrocortisone the stable mRNA for tryptophan pyrrolase is synthesized and 4-5 hours later the synthesis of mRNA for the repressor which acts on the tryptophan pyrrolase mRNA begins; the repressor (apparently protein) is unstable and, to maintain the repression of the tryptophan pyrrolase mRNA, continuous synthesis of the repressor mRNA and the repressor itself is required. Since in the foregoing cases it is a matter of repression of already actively functioning templates, it may be said that precisely polyribosomes are the object of the repressor protein action. Unfortunately there are no direct data on what happens to the polyribosome. Two possibilities appear the most plausible to us: (1) Numerous molecules of repressor protein form a protective coat around the mRNA molecule, excluding it from protein synthesis. In this case either the ribosomes may completely dissociate from mRNA or some of them may remain in association with the mRNA-repressor complex. This will mean that the process of repression is a transformation of mRNA into the masked form of the type of

1.

“MASKED” FORMS OF

mRNA

33

informosomes or their complexes with ribosomes (inactive oligoribosomes). An indirect indication of the plausibility of such a mechanism, when repression requires cooperative assembly of a coat from a large number of protein molecules, is furnished by McAusIan’s data (1963) on the critical amount of the repressor for the establishment of repression. ( 2 ) One or several molecules of the repressor specifically attach themselves to the start or the end of the mRNA chain and thereby prevent the ribosomes from moving along the chain. Attachment of the repressor to the start of the chain will make it impossible for ribosomes to associate with mRNA and the latter will quickly discharge the ribosomes; mRNA turns out to be in a free repressed form and, thus, may rapidly become degraded. Attachment of the repressor to the end of the mRNA chain will simply stop the movement of ribosomes and the mRNA will retain all the ribosomes; in this form the mRNA will be protected by the ribosomes themselves and will apparently turn out to be stable. (Incidentally, the regulation of the rate of attachment of ribosomes to the start of the mRNA chain and their detachment from the end of the chain may explain the differential stability of the RNA‘s with different functions in the cells,) VI. General Conclusion and Hypothetical Scheme

All of the aforementioned data and considerations warrant the idea that there are many different forms of existence of mRNA in differentiating cells. The following forms of mRNA in the cytoplasm of differentiating animal cells may be discussed: (1) Actiue, in functioning polyribosomes; ( 2 ) Repressed, in polyribosomes with inhibited activity or inactive; ( 3 ) Degrading, in a free form or a form charged with but few ribosomes; ( 4 ) Stored, in inactive oligoribosomes (of the type of ribosome-infornosome complexes) ; ( 5 ) Stored, in postribosomal informosome-type particles; ( 6 ) Transit, in postribosomal 45 S particles. The variety of forms of mRNA existence must apparently be the inevitable result of the presence of regulatory mechanisms which govern the activity of mRNA, i.e., reguIation at the level of translation. Such regulation may be regarded as a necessary aspect of the complex intracellular regulatory system which ensures development and differentiation. A simultaneous abrupt switching-on of a sufficiently intensive synthesis of a new set of proteins at a very definite moment of development requires that a large amount of new mRNA should be immediately and

34

A. S. SPIRIN

simultaneously put into operation, for which purpose mRNA must be available in a stored masked form at the moment it has to be switched on. Contrarywise, a rapid and efficient switching-off of the synthesis of old proteins during transition to a new stage of development presupposes primarily inactivation of a portion of the functioning mRNA, i.e., its tranSYNTHESIS OF MESSENGER KNA I N /

/

\

/

\

/

\

J

I N A CT I V E OLICOSOMES

c4]

NUCLEUS

\

f -REGULAT1ON

ACTIVE POLYSOMES

REPRESSED I'OLYSOMES

['I

[*I

t DEGRADATION OF mRNA [33

FIG. 7. General hypothetical scheme presenting interrelations and transitions of different forms of cytoplasmic messenger RNA.

sition to a masked form (repression), possibly with subsequent degradation. The scheme shown in Fig. 7 may be suggested as an attempt to determine the interrelations of the different forms of mRNA existence and thereby to point out possible spheres of application of regulators. Within the framework of this scheme one may try to conceive certain events of early embryogenesis. In the unfertilized egg all of the stored (maternal) mRNA exists in

1.

“MASKED” FORMS OF

mRNA

35

the form of informosomes (5) and inactive oligoribosomes ( 4 ) . Fertilization or activation of the egg induces uncoating (removal of the protein protection) and transformation of inactive oligoribosomes ( 4 ) into active polyribosomes ( 1): Protein synthesis is switched on. The pool of inactive oligoribosomes which change to active polyribosomes is continuously replenished by the large accumulated surplus of stored informosomes and stored ribosomes of the egg. This store ensures the development of the embryo all through the stage of cleavage and early blastula, and apparently also in large measure accounts for the development during late blastula and possibly gastrula, RegardIess of the intensive realization of the stored forms of mRNA and ribosomes, synthesis of new mRNA also begins during the very first hours after fertilization; however, new mRNA also appears in the form of informosomes, i.e., in an inactive form, and wilI be realized (i.e., incorporated into active polyribosomes ) only during later stages of development. It is possible that during passage from one stage of development to another, before gastrulation in particular, not only a programming of a new portion of stored ribosomes, but also a reprogramming of already working ribosomes, takes place. This must be due to somehow induced repression of a certain part of mRNA in the polyribosomes. A portion of active polyribosomes (1) change to a repressed form ( 2 ) and the synthesis of some proteins specific only of early stages correspondingly ceases. The mRNA of the repressed polyribosomes may further degrade, releasing the ribosomes. If the synthesis of certain specific proteins is switched off but temporarily, the mRNA may be preserved in the form of repressed polyribosomes with their subsequent derepression and reversion to an active state ( 1) . Apparently by the late blastula and gastrula stage a portion of newly synthesized mRNA is used for immediate realization. This means that it appears in the cytoplasm not in the form of informosomes but as transit 45 S particles ( 6 ) and is immediately incorporated in the newly organized active polyribosomes (1).This direct flow of mRNA into polyribosomes must be particularly characteristic of the synthesis of less finely regulated and less stage-specific protein systems. In nondifferentiating cells, which are no longer due for abrupt switching from one set of proteins to another under the action of internal physiologic factors of selfdevelopment, this direct flow ( 6 ) -+ (1) should predominate or be even the only one.

36

A. S. SPIRIN

From the standpoint of the foregoing hypothetical scheme it is also possible to examine phenomena of regulation at the translation level in other cases of cellular differentiation, in the process of virus infections, in hormonal action, etc. In conclusion it should be noted again that the author in no way intends to explain with this scheme the phenomena under consideration. His only aim is to suggest in this schematic form certain concrete experimental objectives. This article was written only to stimulate appropriate investigations and lays no claim to the correctness of the scheme. VII. Summary

There is indirect evidence of the presence in unfertilized eggs of a “masked” mRNA that is stored in a temporarily inactive, protected form; expression ( “unmasking”) of this mRNA takes place after fertilization. The presence of “masked mRNA in other embryonic systems may be inferred from various experimental evidence. It is suggested that the “unmasking” of stored mRNA plays a role not only in early embryogenesis but also in later development, where it may precede the appearance of new proteins and the transition to a new phase of differentiation. Furthermore, mRNA synthesized in such a form is thought to be stored in order to support successive stages of development. Recently, mRNA-containing postribosomal ribonucleoprotein particles in animal cells have been discovered. In a number of cases, the relation of such particles to “masked forms of mRNA may be discussed. In particular, mRNA-containing postribosomal particles of early embryos (fish and sea urchin), referred to as “informosomes,” may be tentatively considered as a distinct “masked nucleoprotein form of mRNA. Certain data on inactive polyribosomes or on repression at the level of polyribosomes may also be considered as having a bearing on the question of “masked forms of mRNA. ACKNOWLEDGMENT The author is grateful to Dr. N. V. Belitsina for her extensive help in writing this article and in preparing the manuscript, which was translated from the Russian by David A. Myshne. REFERENCES Allende, J., Bravo, M., and Basilio, C. (1966). In press. Beermann, W. (1964). J. E i p t l . Zool. 157, 49. Belitsina, N. V., Ajtkhozhin, M. A., Gavrilova, L. P., and Spirin, A. S. (1964). Biokhimiya 29, 363.

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mRNA

37

Bell, E., Huniphreys, T., Slayter, H. S., and Hall, C. E. (1965). Science 148, 1739. Brachet, J. (1962). J. Cellular Comp. Physiol. 60, Suppl. 1, 1. Brachet, J., and Denis, H. (1963). Nature 198, 205. Brachet, J., Decroly, M., Ficq, A., and Quertier, J. (1963a). Biochim. Biophys. Actn 72, 660. Brachet, J., Ficq, A., and Tencer, R. (1963b). Exptl. Cell Res. 32. 168. Brachet, J., Denis, H., and de Vitry, F. (1964). Deuelop. Biol. 9, 398. Briggs, R., Green, E., and King, T. (1951). J. Exptl. Zool. 116, 455. Brown, D. D., and Littna, E. (1964). J. Mol. Biol. 8, 669. Dalcq, A., and Simon, S. (1932). Protoplasma 14, 497. De Bellis, R. H., Cluck, N., and Marks, P. A. (1964). J. Clin. Inoest. 43, 1329. Denny, P. C. (1963). Am. Zoologist 3, 505. Denny, P. C., and Tyler, A. (1964). Biochem. Biophys. Res. Commun. 14, 245. Dure, L., and Waters, L. (1965). Science 147, 410. Garren, L. D., Howell, R. R., Tomkins, G. M., and Crocco, R. M. ( 1964). Proc. Natl. Acad. Sci. US.52, 1121. Gavrilova, L. P., Ivanov, D. A., and Spirin, A. S. ( 1966). 1. Mol. Biol. 16, 473. Girard, M., Latham, H., Penman, S., and Darnell, J. E. (1965). J. Mol. Biol. 11, 187. Gross, P. R. (1964). J. Exptl. ZooZ. 157, 21. Gross, P. R., and Cousineau, G. H. (1963). Biochem. Biophys. Res. Commun. 10, 321. Gross, P. R., and Cousineau, G. H. (1964). Exptl. Cell Res. 33, 368. Gross, P. R., Malkin, L. I., and Moyer, W. A. (1964). Proc. Natl. Acad. Sci. U.S. 51, 407. Gross, P. R., Malkin, L. I., and Hubbard, M. (1965). J. Mol. Biol. 13, 463. Harvey, E. B. (1936). Biol. Bull. 71, 101. Harvey, E. B. (1940). B i d . Bull. 79, 166. Henshaw, E. C., Revel, M., and Hiatt, H. H. (1965). J. Mol. Biol. 14, 241. Hoagland, M. B., and Askonas, B. A. (1963). Proc. Natl. Acad. Sci. US.49. 130. Hultin, T. (1961a). Experientia 17, 410. Hultin, T. (1961b). Exptl. Cell Res. 25, 405. Hultin, T., and Bergstrand, A. (1960). Deuelop. Biol. 2, 61. Humphreys, T., Penman, S., and Bell, E. (1964). Biochem. Biophys. Res. Commun. 17. 618. Jacob, F., and Monod, J. ( 1963). I n “Cytodifferentiation and Macromolecular Synthesis” (M. h c k e , ed.), p. 30. Academic Press, New York. Joklik, W. K., and Becker, Y. (1965a). J. Mol. Biol. 13, 496. Joklik, W. K., and Becker, Y. (1965b). 1. Mol. Biol. 13, 511. Lerman, M. I., Vladimirzeva, E. A., Terskikh, V. V., and Georgiev, G. P. (1965). Biokhimiya 30, 375. Lerman, M. I., Spirin, A. S., Gavrilova, L. P., and Golov, V. F. (1966). J. Mol. Biol. 15, 268. McAuslan, B. R. ( 1963). Virology 21, 383. McConkey, E. H., and Hopkins, J. W. (1965). J. Mol. Biol. 14, 257. Maggio, R., and Catalano, C . (1963).Arch. Biochem. Biophys. 103, 164.

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Maggio, R., Vittorelli, M. L., Rinaldi, A. M., and Monroy, A. (1964). Biochem. Biophys. Res. Cornmiin. 15, 436. Marcus, A. ( 1964). Federation Proc. 23, 268. Marcus. A., and Feeley. 1. (1964). Proc. Nail. Accrd. Sci. t7.S. 51, 1075. Marcus, A,. and Feeley, J. (1965). J. Biol. Chem. 240. 1675. Monod, J., and Jacob, F. (1961). Cold Spring Harbor Symp. Qiianf. Biol. 26, 389. Monroy, A. (1980). Experientia 16, 114. Monroy, A., and Tyler, A. (1963). Arch. Biochem. Biophys. 103, 431. Monroy, A., Maggio, R., and Rinaldi, A. M. (1965). Proc. Natl. Acad. Sci. U.S. 54, 107. Nakano, E., and Monroy, A. ( 1958). Expil. Cell Res. 14, 236. Nenier, M. (1932). Biochem. Biophys. Res. Commun. 8, 511. Nemer. M., and Bard, S. G. (1963). Science 140, 664. Neyfakh. A. A. (1959). Zh. Obshch. Biol. 20, 202. Neyfakh, A. A. (1961). Zh. Obshch. Biol. 22, 42. Neyfakh, A. A. (1964). Nature 201, 880. Rosas del Valle, M., and Aronson, A. I. (1962). Biochem. Biophys. Res. Commun. 9, 421. Samarina, 0. P., Assrian, I. S., and Georgiev, G. P. (1965). Dokl. Akad. Nauk S S S R 163, 1510. Eamarina, 0. P., Krichevskaya, A. A., and Georgiev, G. P. (1966). Nature 210. 1319. Shatkin, A. J., Sebring, E. D., and Salzman, N. P. (1965). Science 148, 87. Smirnov, V. N., Spirin, A. S., Kullyev, P., and Zbarsky, I. B. (1964). Dokl. Akad. Nauk SSSR 155, 957. Spencer, T., and Harris, H. (1964). Biochem. I. 91, 282. Spiegel, M., Ozaki, H., and Tyler, A. (1965). Biochem. Biophys. Res. Commun. 21, 135. Spirin, A. S., and Nemer, M. (1965). Science 150, 214. Spirin, A. S., Belitsina, N. V., and Ajtkhozhin, M. A. (1964). Zh. Obshch. Biol. 25, 321; see Federation Proc. 24, T907-TG15 ( 1965) (translation). Spirin, A. S., Belitsina, N. V., and Lerman, M. I. (1966a). J. Mol. Biol. 14, 611. Spirin, A. S., Belitsina, N. V., Ovchinnikov, L. P., Gendon, Yu. Z., and Tchemos, V. I. (1966b). In preparation. Stafford, D. W., Sofer, W. H., and Iverson, R. U. (1934). Proc. Natl. Acad. Sci. US. 52, 313. Takanami, M., and Okanioto, T. (1963). J. Mol. Biol. 7, 323. Tyler, A. (1963). Am. Zoologist 3. 109. Waddington, C. H., Needham, J., and Brachet, J. (1936). Proc. Roy. SOC. B120, 173. Wessels, N., and Wilt, F. H. (1965). J. Mol. Biol. 13, 767. Wilt, F. H. (1963). Biochem. Biophys. Res. Commun. 11, 447. Wilt, F. H. (1964). Deuelop. Biol. 9, 299. Wilt, F. H. (1965). J . Mol. Biol. 12, 331. Wilt, F. H., and Hultin, T. (1962). Biochem. Biophys. Res. Cornmun. 9, 313.

CHAPTER 2

THE TRANSCRIPTION OF GENETIC INFORMATION IN THE SPlRALlAN EMBRYO*

I. R. Collier HENSSELAER POLYTECHNIC INSTITUTE, TROY, N E W YORK, AND MARINE BIOLOGICAL LABORATORY, WOODS HOLE, MASSACHUSETTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Nucleic Acid and Protein Synthesis during Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Cleavage and Early Development . . . . . . . . . . . . . . . . . . IV. Oogenesis and Gene Transcription . . . . . . . . . . . . . . . . . . V. Morphogenesis and Gene Transcription . . . . . . . . . . . . . . VI. Precocious Segregation and Gene Transcription . . . . . . . VII. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Summary ....................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

39

40 42 44 48 53 55 57 58

Introduction

From a common genome embryogenesis produces a multicellular organism of a variety of cell types; the cytoplasm that contains the genome is either heterogeneously organized with respect to its influence on differentiation from the beginning or it becomes so during the course of embryogeny. It is the interplay of those molecules responsible for the initiation of cytoplasmic diversity and for its final result of cellular differentiation that is of paramount interest. The spiralian egg is unique because of its determinant cleavage, i.e., the blastomeres resulting from the early divisions of the egg are not equal

* This paper is dedicated to my friend and fellow scientist, Dr. Albert Tyler, on the occasion of his sixtieth birthday.

39

40

J. R. COLLIER

in their ability to differentiate. The blastomeres can be separated from each other, and when they are reared in isolation they differentiate just as they would have in the intact embryo. This means that the individual blastomere exerts a particular influence upon its genome, an influence that is intrinsic and stable to the extent that cellular interactions and the external milieu are relatively unimportant. Because of these two features, the determinant cleavage and the separability of the blastomeres, the spiralian egg is promising material for studies of the operation of the genome in cells that give rise to different phenotypes. The cytoplasmic localization (or segregation) responsible for determinant cleavage is not restricted to the spiralian egg-it is a universal phenomenon. But in the spiralian egg this localization occurs early in the history of the egg, i.e., it is precocious in relation to the origin of the ultimate functional and structural characters of a cell. From this precocious segregation* of ooplasmic substances, which may alter the expression of the genome, arises another advantage of the spiralian egg for the study of the operation of the gene: The determination of the ultimate character of a cell is temporally separated, often widely spaced, from the actual differentiation of that cell. In other words, the fate of an early blastomere is clearly decided long before it actually develops the properties of a muscle, nerve, or liver cell. Thus these eggs afford the possibility of studying those processes responsible for the determination of a cell separated in time from the synthetic processes associated with differentiation. This report presents some recent experiments of the author and a brief review of other work with spiralian embryos. The use of spiralian embryos in recent embryological research has declined, and it is hoped that the work presented below will illustrate some aspects of these forms that are favorable for investigations of genetic transcription in embryogenesis. II. Nucleic Acid and Protein Synthesis during Embryogenesis

The relation of nucleic acids and protein syntheses to the phases of embryogenesis is valuable because these syntheses are the results and the operational criteria of gene transcription.

' The term precocious segregation, introduced by Lankester (1877), so aptly describes this important feature of the spiralian egg that I believe it should be retained.

2.

THE TRANSCRIPTION O F GENETIC INFORMATION

41

In the ZZyanassa embryo (Collier, 1961a) there is no net synthesis of ribonucleic acid (RNA) until about 1 day after gastrulation. At that time cytochemical observations ( Collier, 1965b) show pronounced nuclear RNA synthesis with a subsequent increase of RNA in the cytoplasm. In all succeeding stages there is a continuous synthesis of RNA. This onset of intense RNA synthesis precedes by 1 to 1% days the beginning of morphogenesis. Protein synthesis, as judged by the incorporation of leucine-l*C into the total protein, roughly parallels the pattern of RNA synthesis, except for a period of increased protein synthesis which begins one day earlier than the onset of detectable RNA synthesis, and which reaches a maximum by the fourth day and later declines. Just after gastrulation an increase in the incorporation of 32P and of other precursors into RNA begins, but net RNA synthesis does not occur until later. These bulk syntheses in the Zlyanassa embryo suggest that extensive gene transcription in initiated at gastrulation. There is indirect evidence, which will be presented later, for the possibility that transfer and/or ribosomal RNA synthesis is initiated at this time and that specific mRNA formation related to the differentiation of specific organ primordia begins somewhat later in development. Protein and RNA syntheses during cleavage will be considered in a later section. Morrill (1961) and Morrill and Norris (1966) have separated electrophoretically and identified by enzymatic activity 23 proteins during the course of Ilyanassa development. Some of these proteins were detected at all stages, others disappeared when organ primordia began to form, and still others appeared only after the onset of the formation of organ primordia. Morrill et al. (1964) have made a similar electrophoretic analysis of hydrolytic enzymes in Limnaea palustris, and Morrill (1965a) has also studied changes in electrophoretically mobile bands of phosphatase during the development of Physa acuta. To the extent that these “enzymic bands” represent distinct proteins they are a reflection of genomic transcription and indicate a repeated transcription for some proteins or the operation of stable mRNA and a repression and derepression of part of the genome for the corresponding shut off and initiation of synthesis of other proteins. It is important that some of these “enzymic bands” be established as discrete polypeptides and used as markers in experiments designed to study the mechanism of repression and derepression of the genome during development. Goldberg and Cather (1963) have found five isomers of lactic acid dehydrogenase in the embryo of the prosobranch gastropod Argobuc-

42

J. R. COLLIER

c h u m oregonese. In the egg and early cleavage stages there are present five isomers; two are lost by the blastula stage but reappear during the differentiation of the veliger. These isomers are formed by a tetrameric combination of two distinct polypeptides. The variation in number of isomers suggests that the relative abundance of the polypeptides is not always the same in different stages of development, although it is possible that conditions for recombination and availability of polypeptides can account for variations in isomeric content. If the former should be the case the synthesis of lactic acid dehydrogenase may be a suitable pathway for studying the regulation of differential rates of gene transcription during embryogenesis. 111. Cleavage and Early Development

That protein synthesis is necessary for cleavage in the llyanassa embryo is indicated by the reversible block to cleavage caused by puromycin and by the observation that puromycin represses the incorporation of amino acids into proteins by 80% (Collier, 1965b). Similarly, Morrill ( 1965b) has observed that treatment of uncleaved Limnaea eggs with puromycin, 400pg/ml, reversibly blocks cleavage by the 4-cell stage. Morrill has found also that nuclear division occurred in the treated eggs while cytokinesis was suppressed. These observations indicate that protein synthesis is required for cleavage in these two molluscan eggs; similar findings for sea urchin eggs and other cells suggest that protein formation is a general requirement for cell division. That deoxyribonucleic acid (DNA)-dependent RNA synthesis is not required for cleavage and gastrulation is indicated by the author’s finding that cleavage and gastrulation of Zlyanassa is not blocked by a 6-hour treatment from first cleavage with Actinomycin D at concentrations of 10, 25, 50, and 100pg/ml. At the highest concentration cleavage is retarded, but upon removal of the Actinomycin D cleavage continues and gastrulation occurs. It is quite significant that eggs treated in this manner fail to develop beyond the gastrula stage, and it is particularly pertinent to the questions of protein synthesis and cleavage that they do cleave, gastrulate, and become ciliated. The effect of Actinomycin D on RNA, DNA, and protein synthesis in later stages of embryogenesis will be considered in a later section; but it is pertinent to this discussion that in the 5-day embryo Actinomycin

2.

THE TRANSCRIPTION OF GENETIC INFORMATION

43

D at 10 pg/ml represses about 65% of the RNA synthesis with no effect on DNA and protein synthesis; at 50 \ig/ml these syntheses are repressed by 950/0,43%, and 23?>. Feigenbaum and Goldberg (1965) have reported results similar to those of the author for the effect of Actinomycin D, 10-50 p.g/ml, on the early development of Ilyanassa; in addition they observed that nuclei of treated embryos were abnormal in the staining of nucleoli and the distribution of chromatin. Morrill (1965b) has observed that the egg of Lirnnaea pal’ustris when treated from the two-cell stage for 48 hours with Actinomycin D, 100 pg/ml, develops to the equivalent of a 1%- to 2-day normal embryo. At 60pg/ml the head differentiates and other organs are inhibited, and at 40 p g h l only the shell is malformed. The results cited above show that DNA-dependent RNA synthesis is not essential for cleavage, cell movement and adhesion, and ciliation in the embryos of llyanussa and Limnuea. In view of the data supporting the requirement of protein synthesis for cleavage it seems likely that gene transcription is not concurrent with cleavage and probably not with cell movements and adhesion nor ciliation. This leads to the conclusions that transcription necessary for protein synthesis occurs at an earlier time in the life cycle and that either stable messengers are involved or that unstable but shielded messengers are rendered active as required during cleavage. While there is no direct evidence that there exists a shielded messenger RNA that is thrust into active form upon demand, it is a possible alternative to the existence of stable messengers. The events of protein synthesis following fertilization in the sea urchin egg and the likelihood of the operation of a “masked-unmasked’ messenger complex in this case (Monroy, 1965; Tyler, 1965) sustain this possibility. That there may exist geneticaIly functional cytoplasmic DNA is not germane unless it can be shown that this DNA is not affected by Actinomycin D or that it is initially protected from actinomycin and made available as a primer subsequent to treatment. Therefore prior to gastrulation gene transcription may not be concurrent with the visible events of early embryogenesis. This suggests that transcription for these events, at least some, probably all, of which require protein synthesis, occurs prior to cleavage. In some forms it has been demonstrated that there occurs during oogenesis, particularly in early stages, an intense period of protein and RNA synthesis. This leads to

44

J. R. COLLIER

the expectation that precocious transcription of the genome required to support these early steps in development occurs during this period. IV. Oogenesis and Gene Transcription

Transcription that may occur during oogenesis would be expected to result in the synthesis of one of the RNA's, ribosomal, transfer (tRNA), or messenger, or of all three. The formation of several specific messengers may be anticipated, but until more sophisticated methods can be used to study embryos it will not be possible to consider the diversity of these RNA's. This discussion is restricted therefore to mRNA as a category of RNA rather than to specific kinds of messengers. Figure 1 (Davidson, 1965) is a photograph of a section of an ZZyanassa

FIG. 1. Section of an oocyte of Ilyanussa obsoleta showing the presence of lampbrush chromosomes. ( This unpublished illustration was kindly contributed by Dr. Eric Davidson. )

2.

THE TRANSCRIPTION OF GENETIC INFORMATION

45

oocyte and shows the presence of the lampbrush chromosome in this oocyte. From our general knowledge of the role of the lampbrush chromosome and of RNA synthesis contributed by Gall (1963) and Edstrom ( 1964), the presence of these chromosomes during oogenesis may be interpreted to mean that the DNA of the chromosome is functioning as a primer for RNA synthesis. That this transcription is restrictive, i.e., does not involve the indiscriminate transcription of the whole chromosome, is indicated by the observation of Gall (1963) that there is variation among the loops of the chromosome in the intensity of their incorporation of uridine into RNA. Edstrom (1964) has found that the nucleolar organizer regions are producing RNA with a ribosomal RNAlike base composition; that other regions of the chromosomes yield RNA with a DNA-like base composition has been demonstrated by Gall (1963). That these chromosomes are producing different RNA’s at different sites has been suggested also by the study of the variation of base composition of the RNA’s produced in different regions of a given chromosome in Chironomus ( Edstrom and Beermann, 1962 ) . From the presence of lampbrush chromosomes in the Zlyanassa oocyte it can be inferred that gene transcription occurs at this stage and that this transcription involves the formation of ribosomal RNA and a variety of mRNA’s. Similarly, the presence of a large nucleolus in the germinal vesicle of the Zlyanassu egg suggests that ribosomal RNA has been synthesized during oogenesis. Unfortunately, the small size of the Zlyanassu chromosome may prevent a detailed study of the type that has been so successful with chromosomes of the amphibians and the chironomids. Other approaches will probabIy be required for the study of RNA production during oogenesis of Ilyanassa and similar forms. Allen (personal communication) has observed by radioautography the incorporation of ~ i d i n e - ~into H oocytes of Autolytus edwawi. Figure 2, A-D, shows radioautographs of oocytes exposed to ~ r i d i n e - ~for H 10 minutes, 30 minutes, 2 hours, and 20 hours. Parts A and B have the same exposure time; the number of grains are a relative measure of RNA synthesis. Parts C and D have different exposure times that prevent comparison with the number of grains in other figures, but they are informative in estimating the approximate time required for transfer of nuclear RNA to the cytoplasm. Allen’s results (personal communication) show that the most rapid synthesis of RNA occurs in the nucleolus. In Part 2, B the grains over nonnucleolar regions of the nucleus may indicate synthesis of RNA in

FIG. 2. Radioautographs of Autolytus edujarsi oocytes incubated with uridine-3H for ( A ) 10 minutes, ( B ) 30 minutes, ( C ) 2 hours, and ( D )20 hours (Courtesy of Dr. M. J. Allen).

2.

THE TRANSCFUPTION OF GENETIC INFORMATION

47

these regions or merely the shift of nucleolar RNA into the nuclear sap at this time. From Parts C and D it is clear that no major transfer of nuclear RNA synthesis has occurred within 2 hours and that only a moderate transfer has been achieved after 20 hours, Thus, it appears clear that the nucleolus is extremely active in RNA synthesis during oogenesis and that transfer of the RNA to the cytoplasm is a relatively slow process at this stage of development. Tweedell (1964) has found that the incorporation of th~midine-~H into the cytoplasm of the egg of Pectinaria gouldii occurs principally during early oogenesis. Thymidine was incorporated either not at all or only very slightly in the growing and mature primary oocytes. Similarly, the mature oocyte failed to incorporate thymidine either before or after germinal vesicle breakdown. Of particular interest in his study of uridine-3H incorporation into this egg are Tweedell's (1964, 1965) observations that some very young oocytes were labeled only over the cytoplasm and that in other stages of oogenesis the uridine incorporated into the nucleus was not removed by ribonuclease or deoxyribonuclease. The first of these findings suggests that cytoplasmic RNA synthesis is primed by an RNA or by cytoplasmic DNA, which is present at this stage. An alternative, of course, is that the RNA was transferred to the oocyte cytoplasm from other cells. The failure to remove the label from the nucleus by either nuclease raises the possibility that a natural DNA-RNA hybrid exists at this stage. These are interesting findings and certainly deserve further attention. It would be of particular interest to find out whether the cytoplasmic DNA is functional in RNA synthesis at this stage; the observation that thymidine is incorporated into the cytoplasm of very young oocytes may be pertinent to this question. Das et al. (1965) have prepared radioautographs of the unfertilized and fertilized eggs of Urechis caupo following a short pulse of ~ r i d i n e - ~ H and have found that the nucleoIus and the chromosomes synthesize RNA in the unfertilized egg. RNA synthesis was not observed in the fertilized egg nor in the 2- to 4-cell stage. Shortly after fertilization the nucleolus diminished in size but did not completely disappear. About half of the label remained in the nucleolar remnant, which represented about onetenth of the original nucleolus. The authors suggest that the bulk of the unlabeled nuclear RNA was probably synthesized at an early stage of oogenesis; this is in agreement with the results of Allen and with observations generally made on nonspiralian eggs. The chromosomal RNA

48

J. R. COLLIER

synthesis observed just before fertilization by Das et al. suggests that some transcription occurs in the mature oocyte at that time. The cytochemical studies of Davenport and Davenport (1965) on three molluscan eggs, the slug Deroceras gracile, the clam Lima scabra, and the chiton Zschnoradsia australis, have yielded the interesting observation that the cytoplasmic basic proteins present in young oocytes are very likely bound to cytoplasmic RNA. These basic proteins are not demonstrable cytochemically after early oogenesis, although in Lima they appear to persist for a longer time. Is the cytoplasmic RNA, the bulk of which is ribosomal RNA, masked by basic proteins? It is clearly desirable to learn more about synthesis of proteins, and especially of basic proteins, during oogenesis. Indeed, an attractive possibility is that during oogenesis protein synthesis is controlled by the binding of cytoplasmic RNA's to basic proteins. V. Morphogeneris and Gene Transcription

The embryologist has long recognized that the crucial events of embryogenesis are operated by differential gene activity; yet only recently has the hope of a direct attack on this problem been possible and even now the path of meaningful experimentation is less than clear. In all too many embryos there is a disappointing absence of genetic markers; chromosomes are small, numerous, and indistinguishable from each other; and in general the genetic mechanism of the embryo does not lend itself to analysis. While it may be assumed that transcription is temporally linked to morphogenesis, more precise information is required before useful questions can be asked about the regulatory mechanism that controls the expression of the genome. The author (Collier, 1965b) has attempted to determine the time relation of gene transcription to the differentiation of particular structures. This work, which will be reported in detail elsewhere, was done with the embryo of the marine mud snail Zlyanassa obsoleta; principaIly it is a study of the effect of Actinomycin D on the synthesis of RNA, DNA, and proteins on differentiation. The effects of different concentrations of Actinomycin D on macromolecular syntheses was studied first so that a concentration that would give a maximum repression of RNA synthesis and a minimum interference with DNA and protein synthesis could be found. Table I shows the effect of Actinomycin D on the incorporation of

2.

49

THE TRANSCRUPTION OF GENETIC INFORMATION

precursors and presumably on the synthesis of RNA, DNA, and proteins. A concentration of 10pg/ml did not affect DNA and protein synthesis nor did it completely suppress RNA synthesis. A concentration of 25 pg/ml did not completely suppress RNA synthesis, but it did repress DNA synthesis by 44.2% and protein synthesis by 24.0%. Thus, a concentration of Actinomycin D, 10 pg/ml, that represses RNA synthesis to about 65% but does not repress DNA or protein synthesis has been determined. TABLE I EFFECTOF ACTINOMYCIN D ON RNA, DNA, AND PROTEINSYNTHESIS IN THE Ilvanassu EMBRYO^ Inhibition of incorporation intoc

Concentration of Actinomycin Db

RNA

DNA

10 25 50 100 200

64.5 72.7 95.4 94.3 96.7

0.0 44.2 72.5 82.3 92.0

~~~~

Protein

0.0

24.0" 21.4

-

46.5

~~

D for 6 hours, then labeled precursor ( 14C or 3H labeled uridine, thymidine, leucine, or valine) was added, and incubation continued for an additional 4 hours. Each value is a mean of at least 3 measurements. b Measured in pg/ml. c Measured in %'s. d Valine used instead of leucine. a Five-day embryos were incubatedn Actinomycin

When Actinomycin D is used at a concentration of 50 pg/ml or greater, RNA synthesis is almost completely repressed and DNA synthesis is also repressed more severely than at lower concentrations. It is significant that at 200yg/ml there is a greater inhibition of protein synthesis although the block to RNA synthesis is not greater than at 50pg/ml. This result suggests that Actinomycin D has a nonspecific side effect on protein synthesis and dictates caution in the interpretation of results obtained at higher concentrations. Even though eggs treated with Actinomycin D at first cleavage cleave and gastrulate, as described above, one must recall that treatment at this stage, or at any interval prior to and including the third day of development, blocks the later differentiation of the embryo. This finding indicates that, while cleavage and gastrulation can proceed independently of DNAprimed RNA synthesis, some of the events occurring during the first 3 days are essential for later development and require DNA-dependent

50

J. R. COLLIER

RNA synthesis. When embryos older than 3 days, i.e., just after the stomodeal invagination has appeared and before the invagination of the cells of the shell gland, are treated with Actinomycin D at concentrations of 25 pg/ml the subsequent inhibition of differentiation is entirely dependent upon the stage at which it was treated. In this study embryos were treated for 6 hours with actinomycin (25 pg/ml), removed to sea water, and allowed to continue development for at least 5 days beyond the time when the controls were fully differentiated. Then the treated embryos were examined to determine what structures had developed. Several hundred embryos were observed at all stages of treatment, 25 to 30 from each experimental group were carefully examined, and the differentiation of several structures was judged relative to the control embryos. Preliminary experiments indicate that actinomycin at 10 pg/ml yields comparable results. At a lower concentration (6.5 pg/ml) there is a differential effect on differentiation, i.e., some structures are blocked but others that are partially or completely inhibited at 25 pg/ml differentiate. Thus, there is a spectrum of effects obtained when actinomycin is used at various concentrations, but at any given concentration the effects are reproducible. It is relevant to point out that the results described below were obtained when reasonably low concentrations of Actinomycin D were used. When 4-day embryos were placed in Actinomycin D for 5 hours and then allowed to develop in the absence of actinomycin, they formed a nearly normal foot, an operculum, and a much reduced velum (see Table 11); all other structures were virtually suppressed. However, in embryos exposed for 6 hours to actinomycin 24 hours later, differentiation was more complete; in addition to the foot, operculum, and velum, there was also partial differentiation of otocysts, shell, esophagus, and intestine and complete differentiation of the eyes. It is significant that, except the foot, operculum, and velum, none of these organs developed in embryos exposed to actinomycin 24 hours earlier, and that the other major organs, i.e., the stomach, digestive gland, and heart, failed to appear following these treatments. The partial differentiation of the foot and operculum occurring when the embryos were treated with actinomycin at least 24 hours before their appearance shows that the transcription for these structures occurred at least 24 hours before their formation. The failure of these structures to differentiate completely indicates that the transcription of that part of

TABLE I1

THEREPRESSION OF MORPHWENESIS IN Ilyunassu BY ACTINOMYCIN Da Time of treatment

Foot

Otocyst

Operculum

Shell

Velum

Eyes

Esophagus

Stomach

Digestive gland

Intestine

Heart

1.8 0.2 1.o 0.0 0.1 0.2 0.0 0.0 1.8 0.3 Day 4 1.1 2.3 2.8 1.4 2.2 0.0 0.0 3.0 1.3 2.3 Day 5 a Embryos were treated at either 4 or 5 days for 6 hours with Actinomycin D, 25 pg/ml, and allowed to complete differentiation in plain sea water. A nonnal organ was scored as 3.0. All values are the mean from the scores of at 25 embryos.

0.0

0.0 their least

81: 8 c, M

3ci 8

3

0 Y

F=!

E

52

J. R. COLLIER

the genome responsible for their development was incomplete. This result suggests that the transcription process is not a short-term pulse-like process but that it begins prior to differentiation and continues over a period of several hours. That the actinomycin does not block proliferation and growth is shown by the fact that embryos treated on the fifth day, when the foot and operculum anlagen have just appeared, develop a nearly normal foot and an operculum. This point is more dramatically shown by a similar consideration of the differentiation of the otocysts and eyes as described in Table 11. If a structure differentiates after treatment with actinomycin the interpretation is that the transcription of the part of the genome responsible for its formation occurred before the time of exposure to actinomycin. For example, transcription required for the formation of the eye has not occurred by the fourth day, as shown by the failure of the eye to develop in embryos treated with actinomycin then; but a 5-day embryo treated with actinomycin forms a pair of normal eyes (which appear after 6% to 7 days of development), meaning that transcription must have taken place between the fourth and fifth days. Also, the stomach and the digestive gland fail to differentiate in embryos treated on either the fourth or fifth day, indicating that transcription of these structures occurs after the fifth day of development. It is clear from Table I1 that a time schedule for gene transcription can be made from studies of this type. Such a schedule for the Zlyanassa embryo is given in Table 111. TABLE I11 GENESCHEDULE FOR llyanassa EMBRYOGENESIS Transcription for Stages of Development Oogenesis

Day 1 Cleavage Epiboly

Day 4 Foot

Operculum

Day 5

Day 6

Eyes Shell Esophagus Otocysts Intestine

Digestive gland Heart Stomach

A general correlation was expected between the appearance of an organ primordium and gene transcription. This correlation was found, but it was not predictable that transcription would occur from 1to 2 days prior to morphogenesis and that the time interval between transcription and morphogenesis would not be the same for all structures. Further, this study has indicated that transcription is continuous over several hours.

2.

THE TRANSCRIPTION OF GENETIC INFORMATION

53

It is not possible to decide from the available data whether the transcription suppressed by actinomycin treatment of older embryos involves only or principally messenger RNA synthesis. However, it appears clear that the synthesis of new RNA is required for differentiation, that all of this RNA is not made at one time, and that its synthesis occurs before the morphogenesis of each structure. Thus, the requirement for new RNA synthesis is periodic during the morphogenetic phase of embryogenesis. Evidence for the synthesis of mRNA during the time of organ primordium formation has been presented previously (Collier, 1965a). The most compelling reasons for thinking that the new RNA synthesis suppressed by actinomycin treatment in the above experiments is principally of mRNA are that fractionation of RNA synthesized in the presence of Actinomycin D (25 yg/ml) has shown that neither tRNA nor ribosomal RNA synthesis is completely repressed and that a reduction in incorporation of precursors occurs in the fraction of RNA containing mRNA ( Collier, 1965b ) .

VI.

Precocious Segregation and Gene Transcription

Among the spiralian embryos there are many examples of precocious segregation, i.e., the cytoplasmic localization of essential, formative materials during early cleavage. The crucial role of this early localization in later differentiation suggests that the materials allocated to particular blastomeres may affect the reading of the genome. This effect could be immediate or delayed or both. Davidson et al. (1965) have reported that removal of the polar lobe* from the Zlyanassa egg results in a repression of incorporation of uridine-3H into RNA. During the first 9-10 hours of development the incorporation of uridine by lobeless embryos and normal ones is nearly the same, but from this stage onward to 72 hours the incorporation by the lobeless embryo is repressed. Cell counts were made and the difference shown to be on a per cell basis. The uptake of uridine into the acidsoluble pool of both normal and lobeless embryos was found to be comparable. Davidson et al. (1965) conclude: “WhiIe it is difficult to exclude

* Polar lobes of the Ilyunassu egg are enucleate cytoplasmic protrusions that form one at a time from the first maturation division to the 8-cell stage. The third polar lobe, the one referred to here, occurs during the first cleavage and forms with the two hlastomeres a trefoil figure.

54

J. R. COLLIER

with absolute certainty all other alternatives, the evidence we have presented points most directly to a delayed effect of polar lobe cytoplasm on gene action in the postgastrular Zlyanussa embryo.” Earlier studies by the author (Collier, 1961b) have shown that removal of the polar lobe from the Zlyanassa egg represses the incorporation of leucine-14Cinto proteins of both early and late stages of embryogenesis. More recently, the author (Collier, 1965b) has measured RNA and DNA contents of lobeless and normal embryos during later stages of development. These measurements were begun after 4 days of development (at the time of shell gland formation) and show that the absence of the polar lobe cytoplasm does not repress bulk RNA synthesis in those cells that are formed in the lobeless embryo but that the formation of certain groups of cells is prevented by the absence of the polar lobe. When these results were used to compare the protein synthesizing capacity of lobeless embryos and normal, it was clear that per microgram of RNA the lobeless embryo incorporated considerably less leucine into the total protein than did the normal embryo. Similarly, the incorporation of leucine into protein per microgram of DNA was less for the lobeless embryo. Thus, whenever protein synthesis is used as a criterion, it is seen that the information of the genome is less effectively executed in a cellular milieu lacking the polar lobe cytoplasm. This situation could arise from a repression of gene transcription or from a partial failure of the protein synthesizing mechanism at the translation level. The former possibility must still be considered, even though the synthesis of total RNA was the same for both normal and lobeless embryos, because these measurements were made spectrophotometrically with a coefficient of variation of about 8%, a sensitivity not sufficient to detect the fluctuation expected from a variation in mRNA synthesis. While the author’s experiments, because of analytical crudities, do not necessarily support the conclusions of Davidson et al. (1965), they are not contrary to their interpretation. In the work of Davidson et al. it is possible that the incorporation of ~ r i d i n e - ~into H RNA was a measure of terminal addition to transfer RNA rather than a measure of nascent RNA synthesis. Should this be the case, the interpretation of precursor incorporation to reflect gene activity is not justified. A recent study of the fine structure of the Zlyanassa polar lobe by Crowell (1964) has shown that this region of the egg contains a vesicular endoplasmic reticulum, what appear to be free ribosomes, and numerous

2.

THE TRANSCRIPTION OF GENETIC INFORMATION

55

double membrane vesicles. These observations suggest, as do the earlier findings of RNA in the polar lobe (Collier, 1960), that the polar lobe cytoplasm is itself equipped for in situ protein synthesis. Further studies of this kind are needed to determine the extent of localization of these structural components in the polar lobe. VII. Discussion

That cleavage and gastrulation can proceed without parallel DNAdependent RNA synthesis in some spiralian embryos suggests that the spiralian egg has been supplied with suitable messengers prior to the onset of cleavage and that these mRNAs are not replenished during cleavage. The mRNA’s may be assumed to be stable, since cleavage, an operation demonstrated to require protein synthesis, proceeds in the presence of or following treatment with Actinomycin D. The author’s finding that Zlyanassu embryos fail to differentiate, but progress to the gastrula stage, when treated with Actinomycin D at any time during the first 3 days of embryogenesis shows that transcription essential for later differentiation occurs during that time. If transfer and ribosomal RNAs as well as messenger RNA’s are products of the genome (see Spiegelman and Hayashi, 1963) it can be concluded that the genome is not entirely at rest during those early stages dominated by cleavage and gastrulation. At this point it is interesting to compare what is known about transcription in the spiralian embryo to similar findings for the sea urchin embryo (see Gross, 1964; Monroy, 1965; Tyler, 1965). Cleavage in both the spiralian and the echinoderm embryo can proceed without the concurrent synthesis of RNA, as judged from actinomycin experiments, and is therefore presumably not under the immediate control of the genome. Actinomycin treatment blocks gastrulation in the sea urchin embryo (Gross, 1964), but not in the ZZyanassu embryo. Gastrulation in this spiralian embryo occurs by epiboly rather than by invagination, as in the echinoderm embryo. However, it is doubtful that the mechanism of gastrulation is of particular significance in respect to the problem of genomic control. It appears more likely that this failure of the actinomycin-treated echinoderm embryo to gastrulate may be related to the fact that cell differentiation occurs at or soon after gastruIation in the echinoderm and that at this time further programming by the genome is necessary for continued development. In the IZyanassu embryo the period

56

J. R. COLLIER

of cellular differentiation and of intense protein and RNA synthesis is several hours postgastrula, at which time this embryo also stops development when transcription is repressed. In this embryo gastrulation is not closely linked with cellular differentiation and therefore occurs without the immediate control of the genome. Apparently pregastrula mRNA synthesis is essential for later differentiation in both the echinoderm and the spiralian embryo, since treatment with Actinomycin D prior to gastrulation suppresses subsequent development. Thus, similarities in the general features of genomic control are apparent in these two embryos; differences in the details of their development should be carefully noted and used in selecting the one suitable for the analysis of particular aspects of development. The author's experiments with Actinomycin D during postgastrular stages of Zlyanassa have permitted a more detailed analysis of gene transcription and morphogenesis than has so far been possible with the sea urchin embryo. This work indicates that transcription occurs several to many hours before morphogenesis and that relatively stable messengers are involved. The provisional gene schedule deduced from this work suggests an approach for an inquiry into the regulatory mechanism of gene activity. At present a major difficulty is the lack of a suitably refined analytical procedure for detecting the molecular events that result from the transcription of a limited portion of the genome. Finally, a caution on the use of Actinomycin D. Although the action of this antibiotic is reasonably well established (see Reich and Goldberg, 1964), Wiesner et al. (1965) have reported that when mouse fibroblasts are treated with Actinomycin D nuclear and cytoplasmic RNA's are degraded at the rate of 1 to 1.5%per hour. Honig and Rabinowitz (1965) report that Actinomycin D has an effect on protein synthesis in tumor cells that is unrelated to the breakdown or cessation of template RNA production. This effect was alleviated by incubation with glucose. A similar effect of Adinomycin D on protein synthesis was indirectly suggested by the author's results reported above (see Table I ) . It is unlikely that the use of Actinomycin D in this study of the Zlyanassa embryo resulted in extensive degradation of RNA, because the differentiation of some organs was virtually complete after treatment with Actinomycin D at certain stages. However, it is important to investigate whether RNA becomes degraded in embryos treated with actinomycin. Also needed is an investigation of the effect of actinomycin with glucose in order to determine whether the failure of an organ to develop after

2.

THE TRANSCRIPTION OF GENETIC INFORMATION

57

treatment results from the prevention of the formation of a protein required for the activation of the genome. This experiment should be done even though the latter possibility seems remote-a low concentration of actinomycin ( 10 pg/ml), at which no inhibition of protein synthesis was detectable, resulted in a block to differentiation comparable to that obtained at higher concentrations. The experiments of the author and of Davidson et al. (1965) have not conclusively demonstrated that the precocious segregation of cytoplasmic materials in the spiralian egg influences the transcription of the genome, but the experiments of Davidson et al. are certainly very suggestive of this possibility. This experimental approach may well lead to one of the more significant contributions that is to be made from the study of the spiralian embryo. Should these cytoplasmic materials of the spiralian egg indeed effect the reading of the genome, a resolution of the nature of the effector molecules would be a contribution to the general problem of gene regulation. Returning to the problem of embryonic determination, it is possible to envision that the cytoplasmic components of a given cell may have a delayed effect on the genome and in this way entirely account for determination. An interesting alternative is that these precociously localized cytoplasmic materials function only as initiators for the transcription of a limited number of genes (regulator genes?) whose product, either a protein or an RNA molecule, would later activate specific parts of the genome required for the differentiation of a particular cell. That these or similar mechanisms may be responsible for embryonic determination is not unlikely, and the spiralian embryo is certainly very suitable material for an investigation of this problem. VIII. Summary

The synthesis of nucleic acids and proteins in the spiralian embryo is briefly reviewed. A study of the effect of Actinomycin D on the synthesis of RNA, DNA, and protein and on differentiation in the Ilyanassa embryo is described. From these results it is concluded that in this spiralian embryo: 1. Cleavage and gastrulation do not require concurrent transcription of the genome. 2. The genome is not completely repressed during early development. 3. Transcription of the genome precedes visible morphogenesis.

58

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4. The messenger RNAs that program the protein synthesis required for differentiation are probably stable. From the relation of the time of treatment with Actinomycin D and subsequent differentiation a tentative schedule of gene transcription is presented for this embryo. Some results on the role of the polar lobe in development are reviewed, and the relation of precocious segregation to the reading of the genome is discussed. ACKNOWLEDGMENTS The author thanks Marjorie M. Collier for her assistance in the work reported in this article and in the preparation of the manuscript. Unpublished work of the author presented in this article and the preparation of the manuscript were supported by grant AM 08316-02 of the Division of Arthritis and Metabolic Diseases of the National Institutes of Health. REFERENCES Collier, J. R. (1960). Erptl. Cell Res. 21, 126. Collier, J. R. (1961a). Exptl. Cell Res. 24, 320. Collier, J. R. (1961b). Acta Embryol. Morphol. Exptl. 4, 70. Collier, J. R. (1965a). Science 147, 150. Crowell, J. (1964). Acta Ernbryol. Morphol. Exptl. 7, 225. Das, N. K., Luykx, P., and Alfert, M. (1965). Deuelop. Biol. 12, 72. Davenport, R., and Davenport, J. C. (1965). Exptl. Cell Res. 39,74. Davidson, E. H., Haslett, G. W., Finney, R. J., Allfrey, V. G., and Mirsky, A. E. (1965). PTOC.Natl. Acnd. Sci. US.54, 696. Edstrom, J.-E. (1964). In “The Role of Chromosomes in Development” ( M . Locke, ed.), p. 137. Academic Press, New York. Edstrom, J.-E., and Beermann, W. (1962). J. Cell BioE. 14, 371. Feigenbaum, L., and Goldberg, E. (1965). Am. Zoologist 5,198. Gall, J. G. (1963). In “Cytodifferentiation and Macromolecular Syntliesis” ( M . Locke, ed.), p. 119. Academic Press, New York. Goldberg, E., and Cather, J. N. (1963).J. CelluZur Comp. Physiol. 61, 31. Gross, P. R. (1964). In “Differentiation and Development, Proceedings of a Symposium Sponsored by the New York Heart Association,” p. 21. Little, Brown, Boston, Massachusetts. Honig, G. R., and Rabinovitz, M. (1965). Science 149,1504. Lankester, E. R. (1877). Quart. J. Microscop. Sci. 17, 2. Monroy, A. (1965). In “The Biochemistry of Animal Development” (R. Weber, ed.), Vol. 1, p. 73. Academic Press, New York. Morrill, J. B. (1951 ). Am. Zoologist 1, 464. Morrill, J. B. (1965a). Am. Zoolopist 5, 198. h4orrill. J. B., and Norris, E. ( 1966). Acta Embqol. Morphol. Exptl. (in press). Morrill, J. B., Norris, E., and Smith, S. D. ( 1954). Acta Embryol. Morphol. Exptl. 7, 155.

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Reich, E., and Goldberg, I. H. ( 1964). In “Progress in Nucleic Acid Research and Molecular Biology” ( J . N. Davidson and W. E. Cohn, eds.), Val. 3, p. 183. Academic Press, New York. Spiegelman, S., and Hayashi, M. (1963). cold Spring Harbor Symp. Quant. Biol. 28, 161. Tweedell, K. (1964). Biol. Bull. 127, 394. Tyler, A. (1965). Am. Naturaht 99, 309. Weisner, R., Acs, G., Reich, E., and Shafiz, A. (1965). J. Cell Biol. 27, 47.

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CHAPTER 3

SOME GENETIC AND BIOCHEMICAL ASPECTS OF THE REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT* Maurice Sussmn DEPARTMENT OF BIOLOGY, BRANDEIS UNIVERSITY, WALTHAM, MASSACHUSETTS

I. Introduction

.....................................

61

11. The Succession of Developmental Events and the Regu-

lated Appearance of Specific Biochemical Products .... 111. Alteration of the Overall Developmental Program in Mutant Strains ...................................... IV. FR-17, a Temporally Deranged Mutant .............. V. The Regulatory Program for UDP-Gal Polysaccharide Transferase ...................................... VI. Specific Requirements for RNA and Protein Synthesis in the Transferase Program ........................... VII. The Role of Protein Synthesis during Genetic Transcription ............................................ References ......................................

62 65 66

67 70 76 82

1. Introduction

The developmental cycle of a cell or multicellular organism comprises a complex progression of phenomic alterations which themselves are expressions of underlying changes in macromolecular constitution. This progression is under rather precise temporal, spacial, and quantitative The work reported here was performed with the aid of grants from the National Science Foundation (GB-1310) and National Institutes of Health (C-4057). 61

62

MAURICE SUSSMAN

control, i.e., developmental events occur in fixed chronological order, at specific intracellular sites and/or within specific cells of a multicellular assembly, and to fixed amounts or extents. Such precision dictates the operation of an overall regulatory program whose molecular basis represents one of the main current mysteries of developmental biology. Previously, it was difficult even to ask meaningful questions about the genetic and biochemical properties of such regulatory programs let alone to answer them. But recent conceptual and technological advances in molecular genetics and RNA and protein biosynthesis, as well as insights into specific metabolic controls which operate during bacterial growth, now make both the asking and the answering feasible. The intent of this essay is to describe the beginnings of that kind of experimental approach to slime mold development. II. The Succession of Developmental Events and the Regulated Appearance of Specific Biochemical Products

Figure 1 is a schematic summary of the developmental cycle carried out by Dictyostelium discoideum. Thick-walled dormant spores germinate into amoebae which feed on live or dead bacteria and increase exponentially in number. Upon entering the stationary growth phase, the amoebae prepare to form multicellular aggregates and then do so. Each aggregate becomes further organized into a conical, finger-like aerial projection (pseudoplasmodium or slug) which lies on its side and migrates over the substratum. This is followed by a complex series of morphogenetic movements which result in the construction of a fruiting body (or fruit) consisting of a lemon-shaped mass of spores at the top surmounting a cellulose-ensheathed stalk, made up of tightly packed, vacuolated cells, which rests upon a basal disk. The developmental fate of a given cell appears to be determined by its position within the aggregate and pseudoplasmodium and thus may be the result of a matrix of cell interactions (Raper, 1940; Bonner, 1944, 1950). In general, two experimental systems are available for biochemical and serological analyses. Vegetative amoebae are harvested, washed, and dispensed on a solid substratum. The latter can be either non-nutrient agar, or a 2-inch Millipore filter resting on an absorbent pad saturated with inorganic salts-streptomycin solution inside a 60-mm petri dish ( M . Sussman and Lovgren, 1965). Morphogenetic synchrony is good on agar, even better on millipore filters. Alternatively, single aggregates,

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

63

slugs, and fruits may be employed for histochemical study (Gregg and Bronsweig, 1956). A variety of specific products, detectable by serological and biochemical means, appear and in some cases disappear at particular stages of this sequence. The patterns of their appearance are generally deranged

0

f

-0 \ Spor.e

8,

Amoeba

Growth,and

rnultiplicotion

I

19

‘Qz

A

-

Aggregation

Pseudo plasmodium

FIG.1. The developmental cycle of Dictyostelium discoideum.

in mutants with altered morphogenetic capacities. Thus, a considerable number of new antigenic determinants are acquired. One of these appears and accumulates just prior to and during aggregation (Sonnebom et at., 1964). Species which can coaggregate with cells of D. discoideum also accumulate a serologically detectable material which cross-reacts with this antigen; but a member of another genus, which cannot coaggregate, does not. In vivo, the antiserum (prepared against homogenates of aggregating cells and absorbed out with homogenates of vegetative amoebae) specifically inhibits D. discoideum aggregation

64

MAURICE SWSSMAN

without affecting cell viability. Preliminary fractionation suggests that this antigen is a lipoprotein associated with the cell membrane. The act of slime mold cell aggregation may be closely allied to analogous phenomena in animal cells (Moscona and Moscona, 1952; Moscona, 1957; Trinkaus and Groves, 1955), i.e., it may be mediated by the formation of divalent cationic bridges (DeHaan, 1959) and directed by specific macromolecular components of the cell surface (Moscona, 1963; Humphreys, 1963) of which the “aggregation” antigen (Sonnebom et al., 1964) may be one. At high cell density, as on growth plates or in shaken liquid suspension ( Gerisch, 1960), random collisions are sufficiently frequent to ensure rapid aggregation. At lower initial cell densities, however, the appearance of locally high cell concentrations is assured by a chemotactic mechanism ( Runyon, 1942; Bonner, 1947; Shaffer, 1953). Although investigation of this process is impeded by the lack of a quantitative assay system, it is already clear that chemotaxis is promulgated by the controlled release of a specific chemotactic factor which is unstable as the result of extracellular enzymic destruction (M. Sussman et al., 1956; Shaffer, 1956). Several steroids have some chemotactic activity, among them A-22-stigmastene, which is present in the amoebae in considerable amounts ( Heftmann et al., 1959). Production of the chemotactic agent apparently continues in migrating slugs and may play a role in later morphogenetic movements ( Bonner, 1949). Other antigenic determinants successively appear at later developmental stages. Some are concentrated in the spore contingent of the mature fruit, others in the stalk (Gregg, 1961; Takeuchi, 1963; Sonneborn, 1962). In addition, at least one antigen carried by vegetative cells is preferentially lost by the spores but not by stalk cells (Sonneborn et al., 1965). The synthesis of at least three polysaccharides and one disaccharide accompanies the developmental sequence. One of the former is a starchlike polymer which accumulates to a level of approximately 2% of the dry weight during cell aggregation and slug migration and disappears rapidly from the trichloroacetic acid ( TCA )-soluble fraction during fruit construction (White and Sussman, 1963a,b). An analogous glucose polysaccharide is subsequently found in the fruiting stalk (C. Ward and Wright, 1966), but the relationship between the two is not yet clear. A second polysaccharide fraction, a glucose polymer, almost certainly cellulose, insoluble in TCA and alkali, appears during fruit construction and ultimately makes up about 4% of the dry

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

65

weight of the fruiting body (White and Sussman, 1963a; C. Ward and Wright, 1966). Histological studies ( Raper and Fennell, 1952; Muhlethaler, 1956; Gezelius and Ranby, 1958) have shown the fruiting body stalk to be sheathed in a cellulose wall. The third fraction is an acid mucopolysaccharide (White and Sussman, 1963b), composed of galactose (55% ), galactosamine (25% ), and galacturonic acid ( 15% ) . It reacts serologically with sera against slime mold spores and with pneumococcic capsular type VII antiserum. It first appears in early pseudoplasmodia, accumulates to about 2% of the dry weight, and is found only in the spore mass of the mature fruit. The disaccharide is trehalose (glucose l,l-aD-glucoside), present in low concentration in vegetative amoebae but comprising 5 7 % of the spore dry weight. It is lost during germination, presumably serving as a carbon and energy source (Clegg and Filosa, 1961) . A variety of enzyme activities have been followed during development. These include several DPN- and TPN-linked dehydrogenases (Wright and Anderson, 1959; Wright, 1960), succinic dehydrogenase and cytochrome oxidase ( Takeuchi, 1960), alkaline phosphatases ( Gezelius and Wright, 1965) and esterases (Solomon et al., 1964), UDPG synthetase (Wright, 1960), UDP-galactose polysaccharide transferase ( M. Sussman and Osborn, 1964), UDPG cellulose transferase (C. Ward and Wright, 1966), UDPG trehalose transferase (Roth and Sussman, 1966), and trehalose (Ceccarini, 1966). The specific activities of at least some of these enzymes change drastically during morphogenesis. The detailed program for one of them ( UDP-Gal polysaccharide transferase) is described in Section V. Finally, the appearance of the mature fruiting body is followed closely by the fabrication of a yellow pigment concentrated in the spore mass. The chemical composition is unknown, but the pigment may be a carotenoid possibly related to phytol ( Ennis, personal communication). 111. Alteration of the Overall Developmental Program in Mutant Strains

Mutants can be isolated which, taken as a group, run a very wide gamut of morphogenetic aberrations (R. R. Sussman and Sussman, 1953; Rafael, 1962; Kahn, 1964; Sonneborn et al., 1963). Some strains construct morphologically altered fruiting bodies ( e.g., “fruity, bushy, forked, curly, glassy”), Others lack pigment or produce altered pigments. Still

66

MAURICE SUSSMAN

others are morphogenetically deficient, i.e., their development stops at a stage prior to the construction of a mature fruiting body and prior to the appearance of stalk cells and spores. These include stocks which either completely fail to aggregate or form loose, amorphous clumps (“aggregateless”). Still others are morphogenetically deficient. Of the latter, some mutants cannot aggregate at all while others can begin the morphogenetic sequence but stop at a stage short of the appearance of mature fruiting bodies with spores and stalk cells. Such mutants are incapable of accumulating the specific molecular products described above, since these normally arise at developmental stages subsequent to the morphogenetic block (White and Sussman, 1981, 1963a,b; Sonneborn et a,?., 1963; M. Sussman and Osborn, 1964). Thus, the effects of these mutations to morphogenetic deficiency are not confined each to a single metabolic activity but ramify into a wide variety of biosynthetic processes. It is particularly interesting to note that many of the deficient stocks, when mixed with one another or with the wild-type, can develop synergistically to the terminal morphogenetic stage ( M. Sussman, 1954; M. Sussman and Lee, 1955). IV. FR-17, a Temporally Deranged Mutant

Strain FR-17 (Sonneborn et al., 1963) grows normally but constructs amorphous flattened, papillated aggregates which develop no further in gross aspect but which are composed of terminally differentiated spores and stalk cells intermixed chaotically. In addition, all products characteristic of mature, wild-type fruits appear to be present in FR-17. Those whose presence has already been demonstrated include the aggregation antigen, a stalk-specific antigen, a spore-specific antigen, the starch-like polysaccharide, ,the mucopolysaccharide, and the corresponding UDPGal polysaccharide transferase, cellulose, pigment. The morphogenetic anomalies encountered in FR-17 are accompanied (and perhaps caused) by a drastic temporal derangement of the developmental program. In the mutant, most developmental events, including the accumulation of products listed above, start sooner and are accomplished faster than in the wild-type under comparable conditions, so that the mutant attains its terminal morphogenetic state (the papillated, flattened mass of spores and stalk cells) in about half the time required by the wild-type to construct a mature fruit. However the morphogenetic aberrations ultimately expressed by the mutant make it

3.

REGULATORY PROGRAM

FOR SLIME MOLD DEVELOPMENT

67

likely that at least some parts of the program are not correspondingly accelerated. The growth rate of the mutant vegetative amoebae is approximately equal to that of the wild-type, and this, too, argues against a general acceleration of all metabolic activity. Thus, in contrast to the variants discussed above, FR-17 provides an example of a mutation whose regulatory consequences accelerate the flow of developmental events without significantly changing the kinds of products fabricated or the terminal states of cytodifferentiation. The topographical relationships among the developing cells, however, both along the way and terminally, are affected drastically indeed. V. The Regulatory Program for UDP-Gal Polysaccharide Transferase

Galactose is incorporated into the mucopolysaccharide by the following transfer reaction: UDP-Gal

+ acceptor

-

galactosyl-acceptor

+ UDP

The reaction can be followed in crude pressates or sonicates by measur~ C UDP-Gal into the ethanol-insoluble ing the transfer of g a l a c t o ~ e - ~from fraction (M. Sussman and Osborn, 1964). The completed mucopolysaccharide or a smalIer-molecular-weight precursor can serve as the acceptor, and enzyme activity is assayed in the presence of a standard concentration of the latter, Under assay conditions, the incorporation is linear with time, directly proportional to enzyme concentration and the concentration of acceptor. Incorporated counts are recovered quantitatively as galactose after acid hydrolysis. Figure 2 shows the developmental kinetics of the transferase in D. rliscoideum wild-type. The activity first appears in early pseudoplasmodia, about 1 hour before the mucopolysacchande itself can be detected. The enzyme accumulates rapidly to a peak of specific activity which is attained shortly before the cessation of mucopolysaccharide synthesis and then it rapidly disappears. Mixed extracts from active and inactive stages show no evidence of inhibitors or destroyers of enzyme activity that might mask the presence of transferase in the latter stages. During the period of its accumulation all of the activity remains associated with the cells when they are concentrated by centrifugation at low speed. However, within a 2 hour period after the peak of activity is reached the bulk of the enzyme is released by the cells and up to 80% is found in the super-

68

MAURICE SUSSMAN

natant of centrifuged cells (Table I ) , This release is preferential, since the specific activity of the released enzyme is about thirtyfold greater than that of the small residuum still associated with the cells. As Fig. 3 indicates, most of the mucopolysaccharide is synthesized before the re-

FIG.2. The developmental kinetics of UDP-Gal polysaccharide transferase activity in D. discoideum. Dotted line: the accumulation of the mucopolysaccharide as measured by its bound, nondialyzable galactose content (White and Sussman, 1963b ) . TABLE I TIMECOURSEOF ENZYMERELEASE Enzyme BSSOC. with cells

Enzyme released in supernatant

Time, hrs.

% of Total act.

Spec. act.

% of Total act.

10.5, 14.5 17 19 21.5 22.5

100 98 98 90 42 24

121 407 1390 1385 530 304

0 2 2 10 58 76

Spec. act.

1750 9200 11,200

lease of enzyme, and the former remains bound to the cells long after the extrusion of the latter (M. Sussman and Lovgren, 1965). That the activity of the enzyme is under control of the over-all developmental program is indicated by the study of five morphogenetically

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

69

FIG. 3. Relation between the release of enzyme and the synthesis of polysaccharide. The data for the former are taken from Table I. The same samples were assayed for mucopolysaccharide content by quantitative complement fixation ( White and Sussman, 1963b).

deficient mutants. Two of these, Agg-204 and FR-2, do not reach the morphogenetic stage at which the mucopolysaccharide normally appears. They fail to synthesize it and, as Fig. 4 shows, do not accumulate any

t

FIRST SPORES

I

4 FIRST MATURE FRUITS

IOOO.

C

:-', /

I I

500I

>

I

I I

\

\

\ I

--WILD-TYPE o FR-17 6 FR-2 A Agg-204

t \ t 1

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FIG.4. The developmental kinetics of transferase activity in three mutant strains of D. discoideum.

70

MAURICE SUSSMAN

transferase activity. Two other mutants, KY-3 and KY-12 (Yanagisawa and Sussman, 1966), yield normal migrating slugs which fail to develop into mature fruits. The transferase does appear to accumulate in normal fashion but is not released or destroyed, events which in the wild-type accompany the last stages of fruit construction. The peak of specific enzyme activity attained by KY-3 is somewhat less than that of the wildtype and KY-12 accumulates about half as much. In FR-17, the synthesis of mucopolysaccharide is accelerated in approximate accord with the overall temporal derangement. As seen in Fig. 4, the developmental kinetics of transferase activity in the mutant are like that of the wild-type except that it appears sooner and accumulates and disappears faster. In summary then, the activity and location of UDP-Gal polysaccharide transferase seems to be entrained by the overall developmental program, and the control pattern for this enzyme includes the following steps: ( a ) initial appearance shortly after cell aggregates are transformed into slugs; ( b ) rapid linear accumulation to a peak of specific activity which is attained at a late stage of fruiting body construction; ( c ) preferential release by the cells of the bulk of the enzyme followed by its rapid destruction; ( d ) coincident disappearance of the small residuum still associated with the cells (possibly also preceded by release). VI. Specific Requirements for RNA and Protein Synthesis in the Transferase Program

The following experiments (M. Sussman, 1965; M. Sussman and R. R. Sussman, 1965) demonstrate that both the accumulation and subsequent disappearance of the transferase can be halted by coincident inhibition of protein synthesis (by cycloheximide) and the prior inhibition of RNA synthesis ( by actinomycin). Figure 5 shows the cycloheximide reversibly inhibited amino acid incorporation into TCA-insoluble material in strain FR-17. Under the same conditions, uridine incorporation was not altered. Figure 6 summarizes the effect of cycloheximide on transferase accumulation and disappearance. The cells were harvested, washed, dispensed on Millipore filters, preincubated for 16 hours at 15"C, and then switched to the standard temperature of 22°C. The preincubation at 15°C for 16 hours is equivalent to 2 hours of development at 22°C. Thus all of the morphogenetic and biochemical events ( including appearance and disappearance of transferase) occur 2 hours sooner in cells preincubated in this way, and

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

71

n W

G 2000c 0

a K 0

u

z 2"

m

g - 1000-

0

a 0

z E

a E n u

I

v

3

4

5

. HOURS

6

7

I

8

FIG.5. 14C-Amino acid incorporation in the presence and absence of cycloheximide. I, control; 11, cycloheximide added at 4 hours; 111, cycloheximide added at 4 hours, removed at 6 hours.

4

HOURS

FIG.6. Effect of cycloheximide on the accumulation and disappearance of the transferase. I, control (open and closed circles are from different experiments); 11, cycloheximide added at 8 hours; 111, added at 6 hours; IV, added at 5 hours and removed at 7 hours.

72

MAURICE SUSSMAN

the remainder of the developmental sequence is subsequently completed in 12 hours instead of the 14 hours for cells not preincubated. Since this writer preferred a 12-hour to a 14-hour working day, all subsequent experiments involving strain FR-17 include the period of preincubation. As Fig. 6 indicates, the transferase activity appeared in the control cells 6.5 hours after the temperature switch, reached a peak at 10.5 hours, 2000

-

a a 0

xa

HOURS

FIG.7. Uridine-SH incorporation in the presence and absence of actinomycin.

and thereafter disappeared. Addition of cycloheximide at 6 hours prevented the elaboration of any transferase activity. When the agent was added at 8 hours (by which time the cells had accumulated about 30% of the peak activity), further increase in transferase activity stopped immediately and also its subsequent disappearance was prevented. When cycloheximide was added at 5 hours and removed at 7 hours, the rise in enzyme activity commenced after a lag period and proceeded at a rate usually equal to that of the control but sometimes slower. Both the

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

73

duration of the lag and the subsequent rate of accumulation depended upon the time at which cycloheximide was added and removed. Actinomycin D was found ( M . Sussman and R. R. Sussman, 1965) to reduce uridine incorporation to about 10-15% of the control (Fig. 7 ) . Sucrose density gradient centrifugation revealed that this residue was confined to the 4 S region and presumably reflected terminal labeling of transfer RNA by derived cytosine (Franklin, 1963). In contrast, amino acid incorporation was slightly accelerated. The effect of actinomycin on accumulation and disappearance of transferase is illustrated in Fig. 8.

FIG.8. Effect of actinomycin on transferase accumulation and disappearance in FR-17. The time values next to the curves represent the times at which actinomycin was added.

When the agent was added 2 hours after the temperature switch, transferase accumulation was completely inhibited. When actinomycin was added at 3, 4, and 5 hours, the activity appeared at the usual time but accumulated to levels that were 20, 55, and 80% of the control peak value; furthermore, in the first two activity did not disappear subsequently, and in the third it disappeared at a slower rate. When actinomycin was added at 6 hours or later, transferase activity at the normal time accumulated to 100-1200/0 of the control peak and thereafter declined, although at a rate usualIy slower than in the control. Figure 9 summarizes the relation between the time of actinomycin addition and the amount of enzyme activity ultimately detected. Figures 10 and 11 illustrate similar experiments using the wild-type.

74

MAURICE SUSSMAN

50-

00 -

-

v

/

4

I

8

12

TIME OF ACTINOMYCIN ADDITION (HOURS)

FIG. 9. Relation between the time of actinomycin addition and the amount of enzyme activity that subsequently accumulated in FR-17. Dotted line: the time course of actual enzyme accumulation taken from the control curve of Fig. 8.

TIME (hours) DEVELOPMENTAL STAGES IN CONTROL:

*9 f f

FIG. 10. Effect of actinomycin on accumulation and disappearance of transferase in wild-type D. discoideum.

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

75

Here the transferase activity appeared 12.5 hours after deposition of cells on the Millipore filters, accumulated to a peak at 21-22 hours, and thereafter declined. Exposure to actinomycin prior to 8 hours prevented the rise in transferase activity; exposure after 15 hours did not prevent the full rise. When added between these periods, the drug did not affect the time of the enzymic increase but did restrict the amount of activity accumulated, in a manner similar to that observed with FR-17. In the wildtype, however, actinomycin did not at any time dissociate enzyme accumulation from disappearance as it did in the mutant.

/D / / /

I

5 10 15 20 TIME OF ACTINOMYCIN ADDITION (hours)

FIG. 11. Relation between the time of actinomycin addition and the amount of enzyme activity that subsequently accumulated in the wild-type ( C ) . Curve D, the time course of actual enzyme accumulation in the wild type taken from the control curve of Fig. 10. Curves A and B, the corresponding curves for FR-17 taken from

Fig. 9.

In summary, then (Fig. 12), treatment of FR-17 with actinomycin permits the delineation of a sensitive period between 2 and 6 hours after the temperature switch (i.e., between 4 and 8 hours after the start of the morphogenetic period ) during which specific RNA is synthesized, the presence of which is required for the subsequent accumulation of the UDP-Gal transferase. Within this period, a simple linear relation exists between the time of actinomycin addition and the amount of enzymic activity that can be elaborated. A corresponding period of actinomycin sensitivity is detected in the wild-type except that it extends between 7.5 and 15.5 hours after the start of morphogenesis. Thus, in the latter, the period begins later ( 7.5 versus 4 hours) and lasts longer ( 8 versus 4 hours) than in the former but there is approximate correspondence with the general acceleration of development observed in the mutant ( including

76

MAURICE SUSSMAN

the time of appearance of the enzyme itself and the rate at which its activity increased). It would appear, therefore, that the synthesis of at least some developmentally significant RNA is entrained by the overall regulatory program. Furthermore, the finding that transcription of one particular segment of the genome does not continue throughout the developmental sequence but in fact occupies less than a third of the total time is of great significance. It should also be noted that in both strains 7-

WILD TYPE

PERIOD OF ACTINOMYCIN SENSITIVITY FOR ACCUMULATION-'

ENZYME ACCUMULATES ENZYME D i s m w m

-.I

I -n

DISAPPEARANCE

I

10

-

ACCUMULATION

, 20

25

DISAPPEARS 30

35

TIME

FIG. 12. Schematic outline of the developmental program for UDP-Gal polysaccharide transferase in D. discoideum wild-type and strain FR-17.

there is a lag of 4-5 hours between the beginning of the actinomycinsensitive period and the appearance of transferase activity. This raises the possibility that temporal controls may operate at the level of translation of mRNA into protein. VII. The Role of Protein Synthesis during Genetic Transcription

Assuming that the actinomycin-sensitive period represents the time of transcription of the structural cistron( s ) for the UDP-Gal polysaccharide transferase, the lag of 4-5 hours mentioned above implies that athe mRNA has a correspondingly long functional life span. The question arises

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

77

whether this functional capacity is affected by the inhibition of protein synthesis coincident with or subsequent to transcription. The data presented below (M. Sussman, 1966) indicate that the nascent message is indeed rendered unusable by such interference but that after transcription it is no longer sensitive. It will be recalled that, in FR-17, the actinomycin-sensitive period extends between 2 and 6 hours after the temperature switch. When cycloheximide was added at 3 hours and removed at 5 hours (Fig. lk),

6oool

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6-8 hourr

4b-6h hourr

CYCLOHEXIMIDE AT; 3 - 5 hOUrl

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0 k

u a

in

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8 1 HOURS CONTROL

0

-

A

0

2

o

0

-

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ACTINOM

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a

0

CYU.

o

0

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0

L ACTINOM.

CYCL.

FIG. 13. Effect of cycloheximide treatment during and after the period of transcription. Ordinate: cpm galactose incorporated per hour per milligram protein ( 918 cpm = 1 mpmole). Abscissa: time of incubation of the cells after the temperature switch from 12" to 22°C.

transferase activity did not appear at the usual time (6.5 hours) but instead required 3 more hours ( a total of 4.5 hours after removal of cycloheximide) and then accumulated to a level 8590% of that reached by the control. If, after cycloheximide was removed, actinomycin was added to prevent subsequent RNA synthesis, no enzyme appeared.* In agreement with previous results, addition of actinomycin at 5 hours to previousIy untreated celIs permitted the enzyme to appear at the usual time and to acoumulate to 75% of the peak level in the controls. Figure 13b illustrates the result of adding cycloheximide at 4.5 hours and removing

* In repeat experiments, cells treated in this fashion accumulated up to 10% of activity attained by the controls.

78

MAURICE SUSSMAN

it at 6.5 hours. Transferase activity appeared at 9.5 hours ( 3 hours after the controls and 3 hours after removal of cycloheximide) and accumulated to the same level as in the controls. However, when actinomycin was added immediately after removal of the cycloheximide, the transferase accumulated at a lower rate and to a level only 40% of the control, this despite the fact that actinomycin added at 6.5 hours to untreated cells permitted full accumulation. Figure 13c illustrates the result of addb

W

I

z

w

40001

I

2000

6

4

0 0 0

€

HOURS

0

-

A

0

A

0

CYCL.

-

ACTINOM

FIG. 14. Dependence of enzyme accumulation on RNA synthesis after the removal of cycloheximide. Ordinate and abscissa as in Fig. 13.

ing cycloheximide at 6 hours (i.e., at the end of the transcription period) and removing it at 8 hours. When actinomycin was added immediately after removal of cycloheximide, the cells could still accumulate enzyme to a level approximately the same as in the controls. Nevertheless, the appearance of activity was delayed 1.5 hours after removal despite the fact that overall protein synthesis as reflected by 14C-amino acid incorporation resumed immediately. When actinomycin was not added immediately after removal of cycloheximide at 8 hours, the cells failed to accumulate the full complement of enzyme. This paradox stems from the fact that the 6-8 hour exposure to cycloheximide delayed the appearance

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

79

of spores by only 1.5 hours (whereas the 2 4 or 4%-6$ hour exposures delayed it by 3 hours). Thus, in the former, the accumulation of enzyme was stopped by the premature entrance into dormancy. Figure 14 shows the results of an experiment in which actinomycin was added at different times after the cells had been exposed to cycloheximide between 3 and 5 hours. When added immediately after removal of cycloheximide, actinomycin permitted the enzyme to accumulate only to a level of about 5% of the control peak; when added 2 hours after removal k LA

I-0

2E 100-

0

of cycloheximide, it permitted accumulation of about 50% of the control peak; when added after a 4-hour lapse, it permitted normal accumulation. Figure 15 is a summary of two experiments of this kind and it shows in more detail the relation between the amount of enzyme activity accumulated and the time at which actinomycin was added during this second period of transcription. The combined results of these experiments are schematically summarized in Fig. 16. They suggest that, when the cells were exposed to cycloheximide during the normal period of transcription, the RNA synthesized during the 2-hour exposure plus that fraction made during the previous 30-60 minutes did not subsequently give rise to active UDP-Gal transferase. Instead, a second round of RNA synthesis followed after removal

80

MAURICE SUSSMAN

of cycloheximide and this second round accounted for part or all of the delayed accumulation of transferase activity depending on whether part or all of the first round had been made nontranslatable by treatment with the drug. Thus in FR-17, the transcription of a specific region of the genome is not automatically restricted to a particular period of time but may be considerably extended. It is noteworthy that the net amount of enzyme activity elaborated as a result of RNA synthesis before and after cycloheximide exposure together approximated the total which accumulated in the undisturbed system. This may reflect the operation of a feedback system which may conceivably regulate the amount of transcription

=

CYCLO. C 0 NT ROL

0

4 8 1 2 0 ' ' " '

HOURS I ' PERIOD OF RNA SYNTHESIS FOR ENZYME ACCUMULATION \ YO OF TOTAL ENZYME ACTUALLY ---a ACCUMULATED 0

n

4 "

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-

-

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CYCLO.

=

8

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FIG. 16. 13-15.

A schematic summary and interpretation of the data presented in Figs.

or translation or both. The data also indicate that the translation, normally lagging 4-5 hours behind transcription, can be made to lag at least 7-8 hours without affecting the amount of enzyme which accumulates. The mechanism by which cycloheximide renders the RNA nontranslatable is not yet clear. It certainly does not interfere with net incorporaH Sussman, 1965). But the RNA made in its presence tion of ~ r i d i n e - ~(M. is considerably more sensitive to RNase in neutralized TCA precipitates than in control preparations, a result to be expected if the former were not bound to protein. Furthermore RNA, pulse labeled with 32P in the presence of cycloheximide is distributed differently after sucrose gradient. centrifugation (Fig. 17), being skewed toward lower molecular weights

3.

REGULATORY PROGRAM FOR SLIME MOLD DEVELOPMENT

81

than is R N A synthesized by control cells ( M . Sussman, 1966). Corresponding studies in yeast have revealed essentially the same result (Fukuhara, 1965). However, the data cannot distinguish between the possibility that cycloheximide has a differential effect on the kind of R N A synthesized or that it renders the nascent R N A less stable. The main import of these experiments bears upon the control of the transcription process during development. The most naive picture would be one in which, given an initial triggering, the R N A polymerase would 1-19.700

H3.1001 cpm

Icpm

3000

k 2000

I000

10

20

30

20

30

4000

3000

k 2000

I000

10

TUBE

NO.

FIG. 17. Sucrose density centrifugation of RNA synthesized during a 2-hour period in the presence (upper graph) ancl the absence (lower graph) of cycloheximide. The cells were harvested and then centrifuged at 5000 g, and the pellets were frozen and then thawed in the presence of 1% sodium dodecyl sulfate (SDS). The clear extracts were then layered over a 1540% sucrose-SDS gradient and spun 17 hours at 18°C. in an SW-25 Spincorotor. The tubes were emptied from below, passed through a Gilford recording spectrophotomer to measure OD,,,, and collected in 1-ml aliquots. The TCA-precipitable radioactivity of each fraction was then measured.

82

MAURICE SUSSMAN

contribute a sequential reading of the genome in an automatic fashion uninfluenced by further signals from the overall program. The appearance of messages specific for early phage proteins in T-2 infected E. coli seem to occur in this manner (Cohen et al., 1963). The results described in this section fail to support that view and suggest that transcription at least of the transferase-specific RNA is continually open to external control and may be extended if need be to guarantee the subsequent appearance of enzyme. REFERENCES Bonner, J. T. (1944). Am. J. Botany 31, 175. Bonner, J. T. (1947). J. Exptl. Zool. 106, 1. Bonner, J. T. (1949). J. Exptl. Zool. 110, 259. Bonner, J. T. (1950). Biol. Bull. 99, 143. Ceccarini, A. (1966) Science 151, 454. Clegg, V. S . , and Filosa, M. (1961). Nolure 192, 1077. Cohen, S. S., Sekiguchi, M., Stern, J. L., and Barner, H. D. (1963). Proc. Null. Acad. Sci. U S . 49, 699. DeHaan, R. L. (1959). J . Embryol. Exptl. Morphol. 7, 335. Franklin, R. M. (1963). Biochim. Biophys. Acta 72, 555. Fukuhara, H. (1965). Biochenz. Biophys. Res. Commun. 18, 297. Gerisch, G. (1960). Arch. Entroicklungsmeclz. Organ. 152, 632. Gezelius, K., and Ranby, B. G. ( 1958). Exptl. Cell Res. 12, 265. Gezelius, K., and Wright, B. E. (1965). J. Gen. Micr(iliol. 38, 309. Gregg, J. H. (1961). Develop. Biol. 3, 757. Gregg, J. H., and Bronsweig, R. (1936). J. Cellular Comp. Physiol. 47, 483. Heftmann, E., Wright, B. E.. and Liddel, C . U. (1959). J . Am. Chem. SOC. 81, 6525. Humphreys, T. (1963). Deoelop. Riol. 8, 27. Kahn, A. J. (1964). Develop. Biol. 9, 1. Moscona, A. (1957). PTOC.Nntl. Acad. Sci. US.43, 184. Moscona, A. (1963). Proc. Natl. Acad. Sci. U . S . 49, 742. Moscona, A., and Moscona, H. (1952). J. Anat. 86, 287. Muhlethaler, K. (1956). Am. J. Botany 43, 673. Rafael, D. E. (1962). Bull. Torrey Botan. Club 89, 312. Raper, K. B. (1940). J . Elisha Mitchell Sci. SOC. 56, 241. Raper, K. B., and Fennell, D. ( 1952). Bull. Torrey Botan. Club 79, 25. Roth, R., and Sussman, M. (1966). To be published. Runyon, E. H. (1942). Collecting Net 17, 88. Shaffer, B. M. (1953). Nature 171, 957. Shaffer, B. M. (1956). Science 123, 1171. Solomon, E., Johnson, E. M., and Gregg, J. H. (1964). Deoelop. Biol. 9, 314. Sonnebom, D. R. (1962). Ph.D Thesis, Brandeis University. Sonnebom, D. R., White, G. J., and Sussman, M. (1963). Deoelop. Biol. 7, 79. Sonnebom, D. R., Sussman, M., and Levine, L. (1964). J. Bacteriol. 87, 1321.

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Sonnebom, D. R., Levine, L., and Sussman, M. (1965). J. Bacteriol. 89, 1092. Sussman, M. (1954). J. Gen. Microbiol. 10, 110. Sussman, M. (1965). Biochem. Biophys. Res. Cornrnun. 18, 763. Sussman, M. (1966). In manuscript. Sussman, M., and Lee, F. (1955). Proc. Natl. Acad. Sci. U S . 41, 70. Sussman, M., and Lovgren, N. (1965). Ex$. Cell Res. 38, 97. Sussman, M., and Osborn, M. J. (1964). Proc. Natl. Acad. Sci. U S . 52, 81. Sussman, M., and Sussman, R. R. (1965). Biochim. Biophys. Actu 108, 463. Sussman, M., Lee, F., and Kerr, N. S. (1956). Science 123, 1171. Sussman, R. R., and Sussman, M. (1953). Ann. N . Y. Acad. Sci. 86, 949. Takeuchi, I. (1960). Deuelop. Biol. 2, 343. Takeuchi, I. (1963). Develop. B i d . 8, 1. Trinkhaus, J. P., and Groves, B. (1955). Proc. Natl. Acad. Sci. U.S. 41, 78. Ward, C., and Wright, B. E. ( 1966). Biochemistry (in press). Ward, J. M. (1959). Proc. 4th Intern. Congr. Biochem., Vienna, 1958 Vol. 6 , p. 1. Pergamon Press, Oxford. White, G. J., and Sussman, M. (1961). Biochim. Biophys. Acta 53, 285. White, G. J., and Sussman, M. (1963s). Biochim. Biophys. Actu 74, 173. White, G. J., and Sussman, M. (1963b). Biochim. Biophys. Acta 74, 179. Wright, B. E. (1960). Proc. Natl. Acad. Sci. U S . 46, 798. Wright, B. E., and Anderson, M. L. (1959). Biochim. Biophys. Actu 31, 310. Yanagisawa, K., and Sussman, M. (1966). I n manuscript.

This Page Intentionally Left Blank

CHAPTER 4

THE MOLECULAR BASIS OF DIFFERENTIATION IN EARLY DEVELOPMENT OF AMPHIBIAN EMBRYOS H . Tiedemann MAX-PLANCK-INSTITUT

FUR

MEERESBIOLOGIE, WILHELMSHAVEN, GERMANY

I. Introduction ..................................... 11. Test Methods for Inducing Factors . . . . . . . . . . . . . . . . . . 111. Isolation and Properties of the Mesoderm-Inducing Factor .......................................... IV. Characteristics of the Mesoderm-Inducing Factor ...... V. Mechanism of Action ............................. VI . RNA Metabolism ................................. VII. Intracellular Distribution of the Inducing Factors . . . . . . VIII. Changes in Cell Affinities .......................... IX. Formation of Complex Organ Structures .............. X. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ......................................

85 87 88 96 98 101 105 106 109 110 110

1. Introduction

In most embryonic cells during cleavage the daughter nucA, and hence the genomic material, are uniformly distributed to the blastomeres; at the same time the cleavage brings about the formation of more or less sharply demarcated regions of the egg cytoplasm and of the cell surface into different groups of cells. These groups become then progressively “determined as specialized tissues and organs. The time of onset of this determination in different areas of the amphibian egg and embryo was investigated by Hans Spemann and his school (see Spemann, 1938, for a review) by transplantation and isolation experiments. If a certain region of an embryo after being isoIated forms the same tissue as in situ, this 85

86

H. TIEDEMANN

region is said to be “determined.” In such a region all components which are needed for its differentiation can be intrinsic from the beginning of development. On the other hand the differentiation of a certain region can be channeled toward a state of determination by interactions with other parts of the embryo. One of the best-known interactions of this kind is the initiation of lens development in the head ectoderm by the eye cup. This kind of interaction between two groups of embryonic cells has been called embryonic induction (Spemann, 1901; see also Mangold, 1931, for a review). It was not until the last decade that the process of embryonic induction and differentiation in early embryonic development was investigated extensively by biochemical methods. These studies suggested that these processes may be controlled by specific chemical factors. The isolation and identification of these factors required, of course, the availability of an appropriate test system. The ectoderm of early amphibian embryos in the gastrula stage provides such a system. During gastrulation the ectoderm on the back of the embryo comes into contact with the layer of prospective mesoderm and is induced to form the nervous system. This is referred to as neural induction or neuralization. When ectoderm isolated from early gastrula stage, i.e., prior to the time of neural induction, is cultured in a physiological salt solution, it fails to differentiate into nervous tissues and instead forms strands of cuboidal nondescript cells. However, as will be explained later on, addition of appropriate extracts to this salt solution results in the differentiation of the ectodermal explant into epidermal and neural tissues, or into muscle, notochord, renal tubules, and gut epithelium. This clearly indicates that gastrula ectoderm has the competence to differentiate even into tissues which it does not form in normal development and therefore can be used to test “factors” which might induce the formation of different tissue types. Experimentally such factors can be tested only by their “inducing” action on gastrula ectoderm. In normal development they or their counterparts might be transferred from the inducing tissues into the tissue to be induced. Thus, it is generally thought that a neural-inducing factor is transferred during gastrulation from the prechordal plate and the mesoderm (the so-called archenteron roof ) into the dorsal ectoderm, where it initiates the development of the neural system. However, other such factors may be intrinsic to the region in which they become functional. Thus the region which early in development gives rise to mesodermal tissues, muscle, and notochord is thought to contain a factor that initiates

4.

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

87

digerentiation in this direction, the so-called mesoderm-inducing factor (see Section 111). II. Test Methods for Inducing Factors

Depending on the question being asked, three methods have been used to test for inducing factors. The tissue culture method (Becker et al., 1959; Becker and Tiedemann, 1961; Yamada and Takata, 1961) is schematically shown in Fig. 1. A sheet-like fragment of ectoderm excised Ind.

\

Y

FIG. 1. Test methods for inducing factors. In the “tissue culture m e t h o d (below) ectoderm is cultured in a salt solution to which inducing factors are added. In the “sandwich method” (above) a pellet which contains the inducing factors is enveloped by isolated ectoderm. Ec, Ectoderm; Ind, Inducer; Sol., salt solution with inducing extract.

from the early gastrula embryo is cultured on a cover slip in a depression slide in a physiological salt solution to which different concentrations of the inducing factor are added. To prevent curling of the cell sheet, which would make it impermeable to the inducing factors, the explanted ectoderm is held flat with silk. As will be shown, the developmental response of the ectoderm depends on the inducing factors added. Advanced differentiation occurs only if, following exposure to the inducing factors, the ectoderm is cultured under conditions favorable for progressive histological organization. Inducing factors can also be tested in solid form. The extracts are precipitated with ethanol and then dried in vacuum to form a pellet. In the so-called sandwich method (Holtfreter, 1933) (Fig. 1) pellets of

aa

H. TIEDEMANN

inducing agents are put inside a flap of ectoderm. The third, or "implantation method ( Mangold, 1923), is especially convenient for large-scale testing. The pellet is implanted into the blastocoel of an early gastrula so that during gastrulation it comes into contact with the ventral ectodenn on which it may exert an inducing effect (Fig. 2). When implants of crude preparations of inducers are tested in this manner, the result may depend on the region of the host which reacts with the inducer (Vahs, 1956). When more highly purified preparations are used such regional differences are far less.

A

B

FIG.2. Implantation method for testing inducing factors. A, insertion of a graft into the blastocoel. B, position of the graft after gastrulation.

The implantation method can be used to test inducing factors in different dilutions by mixing them in different proportions with inert proteins which have no inducing ability, such as y-globulin, egg albumin, or serum albumin. Besides the percentage and size of the induced structures, their tissue content and specificity also serve as an important criterion in the purification procedure of the factors. 111. Isolation and Properties of the Mesoderm-Inducing Factor

By using the testing procedures described it was found that the ability to cause embryonic inductions in gastrula ectoderm is present not only in embryonic but also in various adult organs and tissues, especially of vertebrates. Some of the adult organs tested preferentially induce tissues of a certain type. Brain (without meninges) induces forebrain, eyes, and nose (Becker and Tiedemann, cit. Tiedemann, 1963a). This tissue contains the so-called neural-inducing (or neuralizing) factor. On the other hand, guinea pig bone marrow evokes inductions in which muscle, notochord, and renal tubules prevail (Toivonen, 1940). These tissues are

4.

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

89

normally derived from the mesodcrm, and such inductions are therefore called mesodermal inductions and the factor producing them the mesoderm-inducing factor. Initial attempts in the 1930’s to isolate inducing factors ran into difficulties which today are being accounted for. The inducing factors are bound to cell structures. After differential centrifugation of tissue homogenates activity is found largely in the so-called microsomal fraction (Kawakami et al., 1961; Tiedemann et al., 1962) and much less in the fraction containing cell nuclei ( Tiedemann et al., 1962; Suzuki and Kawakami, 1963). The first step in any purification procedure must therefore involve the release of the inducing factors from the structures to which they are bound. Thus far only the mesoderm-inducing factor could be isolated in a highly purified form. A good source is 9- to ll-day chick embryos (Tiedemann and Tiedemann, 1956a, 1959; Tiedemann, 1963a,b; Tiedemann et al., 1964). Several methods have been employed for the extraction of the factor. In one, an acetone powder of chick embryos is extracted with a mixture of 30% acetic acid and 10% acetone, or with 1 M formic acid, followed by fractionation of the extract with acetone and ether. In another method, chick embryos are extracted with phosphate buffer (containing deoxycholate ) or with pyrophosphate. The inducing factors are then purified by precipitation with ammonium sulfate. Such preparations, however, contain, besides the mesoderm-inducing factor, a neuralizing factor. Two methods have been found effective for the separation of the two. Treatment of the extract with protamine sulfate results in the precipitation of the neuralizing factor; the mesodermal factor is recovered from the supernatant, from which it can be further purified either by ion exchange chromatography on CM-cellulose or by zone electrophoresis. The other method is based on the fact that the mesodermal factor is soluble in phenol. The phenol method (Tiedemann and Tiedemann, 1956b) had been originally developed at the time when the inducing factors were supposed to be RNA in nature. (Similar methods for the isolation of RNA were developed independently at the same time in two other laboratories [Kirby, 1956; Schuster et al., 19561.) However, after separation of the aqueous and phenol phases, the former (which contains RNA and polysaccharides) was found to have only a very small inducing capacity. On the other hand, the material precipitated at the interface caused neuralization of the ectoderm and induced forebrain and hindbrain, while the precipitate obtained from the phenol phase by addition of methanol induced tail and trunk structures, i.e., mesodermal

90

H. TIEDEMANN

structures. The mesoderm inducer thus recovered is insoluble in 574 trichloroacetic acid (TCA) but is soluble in 0.5% TCA in 85% ethanol. The factor can be partially separated from inactive proteins by chromatography on CM-cellulose ( Fig. 3 ) . ( Chromatography on DEAE-cellulose as used by Kawakami and Iyeiri [1964] was less satisfactory in our hands.' ) E 0.7 0.6

0.5 0.1

0.3 0.2

0.1

800

FIG. 3. procedure pH 4.2 is All buffers

1200

1600

2000 ml

Chromatography of a mesoderm-inducing protein purified by the phenol on CM-cellulose. 390 mg protein dissolved in 175 ml of 0.05M acetate adsorbed on 21 gm CM-cellulose (0.56meq/gm capacity; 23 x 2.1 cm). contained 6 M urea.

The mesoderm-inducing factor as eluted from CM-cellulose can be further purified by electrophoresis on Sephadex G 100. The elution diagram from the gel after electrophoresis is shown in Fig. 4. The main frac-

* A sharp separation could be obtained only if all buffers were made up with 6 M urea. The effect of nrea is not yet fully understood. Besides cleaving hydrogen bonds it may also interfere with the interaction of unpolar regions in proteins.

4.

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DIFFERENTIATION IN AMPHIBIAN EMBRYOS

tion has no inducing capacity, whereas the component eluted thereafter, which is only 5-107. of the main fraction, is highly active. This active fraction can be further purified either by submitting it to a second electrophoresis or by zone centrifugation. Of the three fractions which r E 1.4 1.2 1.0 0.8 0.6

0.4 0.2

.- _

- -1

100

I no inductions

1

I1

300 ml

200 111

100% large

inductions

1

IV 100% large-

medium ind.

I

V no inductions

FIG. 4. Zone electrophoresis of fraction I11 (62 mg protein) from CM-cellulose chromatography on a Sephadex G 100 column ( 7 5 x 2.3 cm; 0.05 M phosphate pH 7.5 in 6 M urea; 24 hours; 500 volts, 75 mA). The protein was washed into the column about 20cm before electrophoresis. Cathode on the right, anode on the left side.

partially separate after the second electrophoresis, only the slowest is active as a mesoderm inducer (Fig. 5). Centrifugation in a sucrose density gradient (for 72 hours) partially separated two fractions (Fig. 6 ) ; the active fraction is the one sedimenting faster. Whether this is the pure mesoderm-inducing factor needs further investigation. The slower-sedimenting fraction also has some activity (probably as a result of partial overlapping). The amino acid composition of the active fraction as well as of the slower-sedimenting fraction is shown in Table I. The molecular weight of the active fraction

92

H. TIEDEMANN

E

E280

-

10

20

30

t t Fraction

16 no

23 20%

ind. small ind.

LO

50

60

f

.T

37

60

90%

47%

large inductions

small inductions

FIG.5. Reelectrophoresis of electrophoresis fraction I11 on Sephadex G 100. Cathode on the right, anode on the left side. E 0.16 0.1 I 0.12

0.10 0.08 0.06

0.04 0.02 Fraction 5 Fraction

15

10 6

10

20 16

21

25 26

FIG. 6. Zone centrifugation of the mesoderm-inducing protein purified by chromatography and electrophoresis ( 7 2 hours, Spinco rotor SW 39, 38,000 rpm, 0.05 M tris, pH 7.5 in 6 M urea in 4-12% sucrose, 3°C.).

4.

93

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

TABLE I AMINO ACID COMPOSITION OF THE FAST-MOVING(ACTIVE)FRACTION AND THE SLOWER-MOVING FRACTION (FIG.6)a, Amino acid

Fast-moving protein

Slow-moving protein

Aspnrtic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Proline Phenylalanine Tyrosine Histidine Lysine Arginine

7.69 15.75 15.38 7.32 5.49 5.86 2.93 13.60 3.66 4.39 2.93 1.09 2.93 5.12 5.86

10.75 14.86 7.12 9.27 7.25 10.21 4.56 3.89 4.03 6.85 3.22 0.26 2.28 8.73 6.72

a The protein was hydrolyzed for 24 hours at 110°C with 1 N HCI under N,. The amino acids were separated on a chromobead Kation-exchanger and analyzed with an Auto-Analyzer. No correction for the loss of amino acids is made. Tryptophan and the cystein/cystine ratio were not determined. All values are mole % (mole fraction x 100). Mole fraction = pmole amino acid/Z pmole amino acids.

-I

I 0

(38,000)

(24,000)(12,700) (12,600)

FIG. 7. Relative positions of different proteins after sedimentation for 72 hours in the Spinco SW 39 rotor at 38,000 rpm.

94

H. TIEDEMANN

can be approximately estimated by comparison with the sedimentation velocity of proteins of known molecular weight under identical experimental conditions (Fig. 7). From the relative position of the mesodermal factor a molecular weight in the range of about 25,000 can be calculated. This relatively low molecular weight is in accordance with the observa-

FIG. 8. Section through a Triturus larva with large mesodermal induction after implantation of a purified inducing protein.

tion that the factor is not inactivated by phenol. It is well known that other small proteins e.g., ribonuclease, insulin, and trypsin, do not lose their biological activity after treatment with phenol. These proteins are easily renatured in aqueous solution and thereby refold into the thermodynamically favored, active form. From 1 to 2 mg of the highly purified mesoderm-inducing fraction is

4.

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

95

FIG. 9. Induction of a tail after implantation of a crude phenol-extracted inducing fraction into the blastocoel of an early Triturus gastrula (implantation method, Fig. 2 ) . M.i., induced muscle; N.i., induced notochord; Pi., induced pronephric tubules.

96

H. TIEDEMANN

obtained from 1.0 gm of chick embryo trunks ( -10% of dry weight). After 500-1000 dilution this fraction, when tested by the implantation method, induces only muscle, notochord, and renal tubules, i.e., mesodermal tissues (Fig. 8) in 80-100% of all experimental cases. After 5000 to 10,000 dilution this fraction still induces in 3 0 4 0 % of all experimental cases smaller mesodermal structures. The quantity of the factor ( -10,000 diluted) implanted into the blastocoel of a single gastrula amounts to approximately 3 x pg. (The effective quantity may indeed be smaller, since the pellet dissolves only partially during the time when the ectoderm is responsive to induction.) When a less p u r s e d fraction is implanted, induction of tails with neural tubes results (Fig. 9 ) , ie., a neuralizing as well as a mesodermalizing effect is obtained. When the mesoderm-inducing factor is tested by the sandwhich method it is found to induce also gut epithelium (besides muscle, notochord, and renal tubules; Kocher-Becker, unpublished). Takata and Yamada ( 1960) had observed that guinea pig bone marrow, a mesoderm inducer, is able to induce also structures of endodermal origin. Since the highly purified fraction used in our experiments also induces both mesodermal and endodermal tissues, it might be speculated that the primary result of the induction is a common precursor which later segregates into mesodermal and endodermal tissues.* On the other hand it cannot be ruled out that our inducing fraction still consists of two or more factors that are closely related chemically. The reason why much less gut epithelium is induced when tested by the implantation method is obscure. A mesoderm-inducing protein fraction has been prepared from mammalian liver by Wang et al. (1963), who also used the phenol method of preparation. This fraction also induced endodermal and small neural structures (Chuang, 1963; Wang et al., 1963). Yamada (1962) succeeded in the purification of a mesodermal factor from bone marrow. The comparison between the mesoderm-inducing factors from the three sources (bone marrow, liver, and chick embryo) may prove very interesting. IV. Characteristics of the Mesoderm-Inducing Factor

The protein nature of the inducing factors is also suggested by their sensitivity to proteolytic enzymes; indeed, both the mesodermal and the neural factors are inactivated by treatment with trypsin and pepsin

* If this should be true the factor might better be called “vegetalizing” factor instead of “mesodermal” factor.

TABLE I1 DECREASE OF INDUCTIVE ABILITY AFTER INCUBATION WITH TRYPSIN" Induced regionb

9-day-old chick embryo extract Trypsin-incubated series Control series Triturus gastrula and neurula extract Trypsin-incubated series Control series

No. of cases

Positiveb

102 138

28 79

2 28

34

21 17

52 72

4 33

0 3

0 8

4

22

Size Of Large Medium

5

Small

Arch- Deuterenceph- enceph- Spinealon alon caudal

Unspecific

1 2

3 29

7 43

9 19

2 11

0 3

0 0

2 19

a Incubation for 90-120 minutes with 0.5-1.0 mg trypsin/ml at 25"C, then stopped with soy bean trypsin inhibitor (Tiedemann et d., 1960, 1961). 6 Measured in %.

CL)

-a

98

H. TIEDEMANN

(Hayashi, 1958; Tiedemann and Tiedemann, 1956a; Tiedemann et d., 1960). The results of experiments with trypsin are shown in Table 11; a marked drop of the inducing activity as a result of this treatment is evident. The polypeptides formed during the incubation with trypsin were also tested and were found to be devoid of inducing capacity. Similar results were obtained with extracts from Triturus gastrulae and neurulae ( Tiedemann, 196313). Previous experiments had indicated that the inducing ability of tissues is not destroyed by treatment with RNase (Brachet et al., 1952; Kuusi, 1953; Englaender et aZ., 1953; Hayashi, 1955, 1959). This would seem to indicate that RNA is not directly involved in the induction process or, at least, that it does not represent the actual activity of the inducing factors. Some more detailed experiments further support this. It has already been mentioned that RNA prepared from chick embryos, from Tritumcs gastrula and neurula, or from liver has only a weak inducing capacity ( Tiedemann and Tiedemann, 195613) . When tested on explants of isolated ectoderm this RNA induces in a small percentage of cases mesenchyme and melanophores, and in some cases neural tissue (KocherBecker and Tiedemann, unpublished; Yamada and Osawa, quoted by Yamada, 1961). The inducing ability of isolated RNA was not enhanced when the ectoderm was pretreated by a hypertonic shock which, presumably, increases the chances of the RNA to enter the cell (Tiedemann, unpublished). Other investigators (Niu, 1958) have stated that RNA is the inducer. However, we have not been able to duplicate these observations. Because a mRNA belongs to each polypeptide chain an inducing ability of RNA would not have been contradictory to the inducing capacity of proteins. In experiments of this kind it has to be kept in mind that the lack of effect of the RNA may result not solely from an inability to penetrate into the egg but also from its rapid breakdown by the intracellular nucleases. V. Mechanism of Action

In general two different modes of action of the inducing factors can be visualized. They might ( a ) originate a new chain of reactions or ( b ) act by removing a repressor. Experiments carried out in the last few years on the inducing ability of gastrula ectoderm and of extracts from gastrulae support the hypothesis that embryonic induction has to be considered an antagonist to repression. Isolated gastrula ectoderm has no inducing ability. However, after

4.

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

99

treatment with ethanol the ectoderm acquires the ability to induce forebrain and, in a few cases, hindbrain structures (Holtfreter, 1933; Rollhauser, 1953). Following a treatment with phenol (Tiedemann et al., 1961), besides these structures, it induces in some cases head muscle and small pieces of notochord. A similar activation is observed if extracts from gastrulae and neurulae ( cleared by ultracentrifugation for 2 hours at 105,OOOg) are treated with phenol (Table 111). In these experiments neural structures were preferentially induced, besides some mesenchymal tails. These results thus suggest that the inducing factors are present in the ectoderm and possibly in other tissues of the embryo, although in an inactive form. Some experiments with inducers from other sources point to the same conclusion ( Becker et al., 1961; Kocher-Becker and Tiedemann, unpublished). Extracts of muscle of chick embryo have a neuralizing effect on amphibian ectoderm and induce forebrain and hindbrain in 42% of all experimental cases. After treatment with phenol at 60°C, the same extract acquires a strong mesoderm-inducing capacity and gives rise to trunks and tails (Table 111).The suggested interpretation is that in the extracts the mesodermal factor is associated with an inhibitor or a repressor which is either inactivated or released by the phenol treatment. A conversion of the neural factor into the mesodermal factor could be excluded by subjecting purified neuralizing factors to the same treatment. The autoinduction effects should also be mentioned in this context (Barth, 1941; Holtfreter, 1944, 1947). Following a short treatment with weak acid or weak alkali (and indeed with a number of different chemically unrelated substances) isolated ectoderm differentiates into neural tissue in the absence of any additional exogenous inducing factors. The ectoderms of different amphibian species differ in degree of susceptibility to this treatment. Ambystoma ectoderm can easily be “autoneuralized” whereas Triturus alpestris ectoderm is much more resistant. Muscle, notochord, and renal tubules as well as gut epithelium are formed after the addition of lithium chloride or lithium carbonate to the salt solution in which the isolated ectoderm is reared (Masui, 1960, 1961). The autoinduction phenomena may be considered as bringing further support to the contention that the inducing factors are present within the ectodermal ceIls in an inactive or masked condition. However, the very minute quantities of material available in such types of experiments make any biochemical approach very difficult. The chemicals mentioned certainly modify the interaction between inducers, inhibitors, and recep-

TABLE III INDUCING ABILITYOF 1 0 ~ , 0 0 0X g SUPERNATANT FROM Triturus GASTRULAE A,No. of cases

Position

(%)

Size of induction

Neural

20 26

5 54

Small Small-medium

0 27b

59 63

42 83

Medium Large

-

CHICK EMBRYOMUSCLE

Archenteron

Deuteron

Spinocaudal

Nonspeciik

-

0 1%

5 15

12 0

4 73

20 10

31

Triturus gastrulae

Control Phenol-treated Chick embryo muscle Control Phenol-treated

-

-

2

ffM 5:

7 0

a The control fraction was precipitated with 2 volumes of 96% ethanol. The phenol-treated fraction was extracted with phenol at 60°C for 8 hours, then precipitated and carefully washed with ethanol. b Balancers, induced. c Mesenchymatic tails, induced.

!2:2

4.

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

101

tors and thereby release the inhibitions and initiate differentiation. The action of Li+ can be explained in the following way. Li+ is a more highly hydrated ion than Na+ or K + and by partially substituting for K+ and Na+ it will change the conformation of proteins. As a consequence, the interaction between different proteins and between proteins and nucleic acids will probably be modified. Thus, the autoinduction effects are not contradictory to the existence of special inducing factors and inhibitors. As was already mentioned the purified factors from chick embryos and other sources are chemically related to the factors in amphibian embryos (p. 98). VI. RNA Metabolism

The suggestion of a close relationship between RNA metabolism and differentiation was presented many years ago by Brachet (1957) and Caspersson ( 1950). New technical and conceptual tools have recently been made available to tackle this problem and important results have been obtained (see Brachet, 1965, for a review). Particularly rewarding has been the use of substances which interfere specifically with certain steps of either genetic transcription or translation processes. One of the most interesting of such substances is Actinomycin D, which is known to interfere with genetic transcription and has been used extensively in studies on the mechanism of differentiation. Actinomycin penetrates very slowly into intact amphibian embryos and, in fact, the differentiation of intact embryos raised in the presence of this drug is not inhibited. If, however, actinomycin is injected into the eggs it stops development at the onset of gastrulation (Brachet and Denis, 1963) . Also, differentiation of the dorsal blastoporal lip * explanted together with adjacent ectoderm and endoderm into 2.5 pg/ml of Actinomycin D for 2 to 12 hours is completely inhibited (H. Tiedemann, unpublished). If treated with a lower dose of actinomycin (0.5 pg/ml) for 2 hours, the explants survive and the cells divide. Notochord and myoblasts differentiate more or Jess completely, whereas newal tissue does not differentiate at all, or very poorly, in contrast to the control explants which contain much neural tissue. This suggests that the delay of some preparatory processes for the differentiation of neural tissue, which de-

* A group of cells determined already in the early gastrula to form muscle and notochord; they give rise to the dorsal roof of the archenteron and induce neural tissue in the dorsal ectoderm.

102

H. TIEDEMANN

pends on the synthesis of RNA, hinders the tissue from neural differentiation. On the other hand, the preparatory processes for differentiation to muscle and notochord seem to have taken place already. If the isolated axial system of the embryo (neural tube, notochord, and myomeres) at the tail bud stage is treated with Actinomycin D (0.5 yglml; 2 1 1 hours) in some cases the neural tissue develops further but at a much slower rate than in normal development. Isolated ectoderm induced to form muscle and notochord by guinea pig bone marrow, then disaggregated, treated with Actinomycin D (2.5 yg/ml), and reaggregated, completely fails to differentiate (Toivonen et al., 1964). This suggests that the ability to respond to the inductive stimulus is suppressed by actinomycin. On the other hand, the neural-

FIG. 10. Operation scheme for the isolation of gastrula ectoderm. The shaded area was isolated and used for all experiments on the RNA metaboIism of ectoderm.

and mesoderm-inducing ability of the blastoporal lip is not reduced as a result of a treatment with high concentrations of actinomycin." This suggests that either a relatively stable messenger for the mesoderm- and neural-inducing proteins or the factors themselves are already present in the blastoporal lip of the early gastrula. Although other interpretations are conceivable, at present a protein fraction with neural- and mesoderminducing capacity has actually been extracted from gastrula tissue (see p. 98). The differentiation into specialized tissues does not depend on the overall rate of RNA synthesis. Brown and Gurdon (1964) have observed that an anucleolate Xenopus mutant which does not form ribosomal RNA differentiates normally until the swimming tadpole stage and dies

* In order to preserve the neural-inducing activity Actinomycin D has to be removed completely by treatment with ethanol before the blastoporal lip is implanted into the blastocoel of an early gastrula.

4.

103

DIFFERENTIATION I N AMPHIBIAN EMBRYOS

18000

14000

10000

E

8

6000

2000 Fractions

FIG. 11. Neurulae were incubated for 24 hours with 14C02. The RNA was isolated and centrifuged on a sucrose density gradient (5-2070 sucrose).

18000

14000

E 2.00

10000

g

1.50 6000

1.00 0.50

2000 Fractions

FIG.12. Isolated gastrula ectoderm was reared for 2 days in salt solution and incubated with 14CO, for 24 hours. The RNA was isolated and centrifuged on a sucrose density gradient ( 5 2 0 % sucrose).

104

H. TEDEMANN

thereafter. Therefore the differentiation to specialized tissues up to that stage can proceed in the absence of new synthesis of ribosomal RNA. Tiedemann et al. (1965) compared the synthesis of ribosomal RNA and transfer RNA in neurulae with that in isolated fragments of gastrula ectoderm (Fig. 10) that were maintained for 2 days in salt solution (and therefore attained the same chronological age as the neurulae). It turned out that the ribosomal RNA's ( -18 S and -28 S ) and the transfer RNA (-4 S ) were synthesized not only in the intact neurulae

LOO

E 300 2.00

;

200

1.50 1.00

100 0.50

Fractions

FIG.13. Neurulae, respectively isolated ectoderm were incubated with 14CO, for 1.5 hours. The isolated RNA was centrifuged on a sucrose density gradient. Solid line, neurula RNA; broken line, ectoderm RNA.

but also in the cells of the isolated ectoderm which divided but did not undergo differentiation into specialized tissues ( Fig. 11, 12). As is shown in Fig. 13, the radioactivity of pulse-labeled neurula RNA (after a 1%-hour incubation with 14C02) is higher than that of pulselabeled ectodermal RNA. Both are heterogeneous and contain highmolecular-weight RNA species ( Brown and Littna, 1964; Tiedemann et al., 1965) which could be polycistronic messengers or precursors of ribosomal KNA. Also the rate of protein synthesis is only slightly lower in the ectoderm as compared to whole embryos. The methods available at present do not distinguish more than quantitative differences between the neuralized and the nondifferentiating ectoderm. In fact, what one

4.

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

105

would like to recognize are specific messengers for specialized tissue proteins. One would also like to know whether the inducing factors affect the processes of transcription (DNA + RNA) or translation (RNA + protein) .* VII. lntracellular Distribution of the Inducing Factors

The earliest embryonic stage from which inducing factors could be extracted is the gastrula of the amphibian embryo. In the gastrula as well as in older embryos the mesodermal factor is found predominantly in the microsomal fraction and to a lesser extent in the cell nuclei (page 89). It is conceivable that the inducing factors (masked or not) (Meyer, 1939) or their specific KNA’s are already present in the unfertilized egg and that their final distribution within the egg results from the movements of the ooplasm and the egg cortex which follow fertilization. Once these movements have been completed, the concentration of the inducing factors will be highest in the vegetal region and lowest in the animal region. A prelocalization of inducing factors in the cytoplasm or the egg cortex would be compatible with the differentiation of distinct egg regions into different Organanlagen being controlled by the cytoplasm rather than by the cell nuclei (Spemann, 1914, 1938; Boveri, 1899; Wilson, 1904). Fitting here is the observation ( Gurdon and Brown, 1965) that the synthesis of different RNA components in nuclei transplanted form older embryos into enucleated eggs as well as the morphological appearance of the transplanted nucIei is affected by the cytoplasm. The mesoderm-inducing capacity of cytoplasmic fractions derived from older embryos is much higher than the inducing capacity of similar preparations from early developmental stages, Therefore, it can be supposed that the mesodermal factor is continuously synthesized in the cytoplasm of tissue cells which differentiate into “mesodermal” organs; perhaps it plays a role in “determining” or stabilizing them in their mesodermal state, possibly by some sort of a feedback mechanism. Whether this would be accomplished by the transfer of inducing factors from the cytoplasm to the nuclei or by other means is unknown. Specu* The inhibition of differentiation by Actinomycin D proves (means) that all the mRNA is not already present in the cytoplasm, but that it must be synthesized de nouo in the nucleus. However, the experiments with Actinomycin D cannot tell us whether the regulation of RNA synthesis is exerted primarily in the nucleus or in the cytoplasm.

106

H. TIEDEMANN

lation along these lines is encouraged by suggestive evidence of temporal differences in gene activity and their susceptibility to regulation by extrinsic factors. This follows from different experimental approaches. Hybridization experiments between DNA and complementary RNA have suggested differences in mRNA in diferent organs of an organism (McCarthy and Hayer, 1964). Cytochemical observations on polytene-chromosomes (Beermann, 1963), as well as the different activity of pulse-labeled RNA in ectoderm and in whole embryos, also favor such an assumption. It is, however, not possible to choose, at present, between the many conceivable regulatory mechanisms without a better knowledge of the operational organization of the genome in higher organisms. VIII. Changes in Cell Affinities

One of the early detectable changes in differentiating cells is a change in their contact properties, i.e., in their mutual adhesiveness or affinity. These changes represent an expression of differentiation and play an important role in organizing the cells into distinct groupings and tissues. Holtfreter (1939) found that in the amphibian embryo differential cell affinities first arise in the gastrula and neurula stages. The ectoderm as it differentiates into neural tissue loses its affinity for endoderm; when ectodermal and endodermal explants are placed in contact they tend to separate from each other. On the other hand, both ectoderm and endoderm develop a strong affinity for mesoderm; when explants from these three layers are placed in contact, the mesoderm is found interposed between the ectoderm and the endoderm. These normally occurring changes in cell affinities suggested the following experimental question: would ectodermal cells acquire an affinity for endoderm after exposure to the mesodermal inductor? Such a change of affinities has actually been observed. When the highly active mesodermal factor was implanted into the blastocoel of a Triturus gastrula, gastrulation continued. However, after 14 to 18 hours the blastopore reopened and the endoderm reappeared in the blastopore and in part spread over the ectoderm as a single layer ( Kocher-Becker et al., 1965). A neural plate did not develop. Thus, under the influence of the mesodermalizing factor, the induced ectoderm developed an affinity for endodermal cells stronger than that of endodermal cells for each other. When fractions with lower mesodermalizing activity were tested, the formation of the neural plate was not wholly prevented (Fig. 14) but

4.

DIFFERENTIATION IN AMPHIBIAN EMBRYOS

107

the final outcome was the same: the endodermal cells moved out and after 10-14 days such embryos consisted of an external coat of endoderm surrounding muscle, notochord, and large masses of renal tubules (Fig. 15) in the caudal part of the embryos. Occasionally, areas of irregularly shaped neural tissue were also present. Thus, ectodermal cells induced by the mesodermalizing factor to differentiate into mesoderm acquire an affinity, or a preferential adhesive-

FIG. 14. Triturus neurula partially overspread by endoderm after implantation of a highly purified mesodermal factor into the gastrula blastocoel (implantation method, Fig. 2 ) . A small neural plate has formed. Np = Neural plate. (From Kocher-Becker et ul., 1965.)

ness, for endoderm. These differentiation-dependent changes in cell affinities are compatible with the assumption that the properties of the chemical groupings in the cell surface that mediate ceIl contact may vary with changes in the developmental characteristics of the cells. The mesodermalizing factor “converts” ectodermal into mesodexma1 cells with the consequent modification of their adhesive and associative properties and a resulting affinity for endodermal celIs. A slight effect could also be obtained by treatment of amphibian gastrulae with LiCl ( Kocher-Becker, unpublished) and also by treat-

108

H. TIEDEMANN

ment with lysine-rich histones (Brachet, 1964) from calf thymus.” The histones might have reacted with cell surface constituents and modified their original properties, or they might have entered the cell nucleus and inhibited the DNA-dependent synthesis of RNA which, in turn, may be involved in the synthesis of cell surface constituents. This may

FIG. 15. Section through the caudal part of a 14-day-old Triturus larva, treated with highly purified mesodermal factor. An external coat of endoderm ( E n . ) surrounds large masses of renal tubules ( R.T. ), Magnification -50 x (Photograph Dr. Kocher-Becker ).

suggest that modification or loss of certain chemical groupings in the cell surface enables ectodermal cells to take up contact with endodermal cells. Such a loss of certain groupings could also happen as one of the first steps if the mesodermalizing factor “transforms” ectodermal into mesodermal cells. As M. H. Moscona and Moscona (1963) have shown, the adhesive and associative properties of embryonic cells are under * This observation was confirmed in our laboratory. Histones extracted from chick embryos or calf thymus, however, do not show an inducing capacity.

4.

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109

metabolic control. The ability of trypsin-dissociated embryonic chick cells to reaggregate normally is suppressed rapidly by puromycin ( a n inhibitor of protein synthesis) and more slowly by Actinomycin D (which blocks DNA-dependent RNA synthesis ) . This suggests that DNA-dependent RNA’s may function in regulating the synthesis of enzymic or structural cell surface constituents that are involved in the mutual selective association of these embryonic cells. Whether these propositions apply also to gastrula and neurula cells of amphibian embryos has yet to be tested. However, one related point can be made: the surface constituents responsible for mutual reaggregation of dissociated cells include glycoproteins (A. A. Moscona, 1963; Humphreys, 1963). It was found that the reaggregation of dissociated amphibian neurula cells, as of certain other cells (A. A. Moscona, 1963), is retarded by lop4 M Na-periodate ( Tiedemann, unpublished ) , which oxidizes neighboring OH-groups in carbohydrates. This may mean that carbohydrates are a significant constituent in the surface of embryonic amphibian cells as well. As pointed out by Holtfreter (1939, 1943; Townes and Holtfreter, 1955), the succession of changes in cell affinities that take place during early development is essential for the morphogenetic movements of gastrulation and neurulation. The intercellular transmission of inducing factors or inhibitors might be enhanced, retarded, or otherwise controlled by changes in differential cell adhesiveness. Thus, changes in cell affinities not only represent an outcome of differentiation but, in turn, may themselves be an important factor in regulating differentiation. IX. Formation of Complex Organ Structures

The two inducing factors mentioned here as being able to modify early development of amphibian embryonic cells have been referred to as the mesoderm- and the neural-inducing factors. This is, of course, merely a descriptive simplification, since embryonic mesoderm develops into a variety of tissues, such as muscle, notochord, renal tubules, and blood elements, and embryonic neural tissue gives rise to the different components of the central and peripheral nervous systems. When gastrula ectoderm is induced its differentiation is switched into one of two alternative directions, depending on the inducing stimulus. After “neural” induction the ectoderm acquires the competence to form brain and certain elements of the peripheral nervous system, but no other tissues. Precisely which parts of brain or nervous tissues develop and how

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far they differentiate depends possibly on the time, duration, site of the inductive effect and on secondary progressive sequences of interactions between different cells and regions which are, as yet, poorly understood. The same applies to the consequences of the “mesodermal” induction, which, presumably, triggers a progression of interdependent processes that lead to the development of the various mesodermal tissues and structures. Furthermore, various “ectodermal” and “mesodermal” tissue derivatives affect each other, and development is both dependent on and channeled forth by these interactions. Thus, the neural factor alone induces in the ectoderm forebrain with eyes and nose; when the mesodermal inductor is also present muscle and endomesenchyme too are formed. In turn, the neural tissue responds to their presence by giving rise to hindbrain and ear vesicles. (For further information on these, see Toivonen and SaxBn, 1955a,b; Saxen and Toivonen, 1961; Tiedemann and Tiedemann, 1964.) The chemical basis of these later interactions remains to be examined. X. Summary

At least two chemical factors take part in the determination of the axial system in amphibians, the neural factor and the mesodermal factor. The latter could be highly purified and characterized as a protein of relatively low molecular weight. Besides the inducing factors special inhibitors seem to take part in the control of differentiation. The process of induction and differentiation therefore has to be considered as an antagonist to repression. One of the first changes called forth by the mesodermal factor is a change of cell affinities in the reacting cells. Ribosomal RNA and transfer RNA are synthesized in isolated ectoderm, which does not form specialized tissues, as in whole differentiating embryos of the same age. Pulse-labeled RNA, which may include informational RNA, has, however, a lower activity in isolated ectoderm. ACKNOWLEDGMENT Our investigations referred to in this article were supported by research grants from the “Deutsche Forschungsgemeinschaft.”

REFERENCES Barth, L. G. (1941). 1. Exptl. Zool. 87, 371. Becker, U., and Tiedemann, H. ( 1961). Verhandl. Deut. Zool. Ces. p. 259. Becker, U., Tiedemann, H., and Tiedemann, H. (1959). 2. Naturforsch. 14b, 608. Becker, U., Tiedemann, H., and Tiedemann, H. (1961). Embryologin 6, 185.

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Beermann, W. (1963). In “Induktion und Morphogenese,” p. 64. Springer, Berlin. Boveri, T. (1899). In “Festschrift f. C. von Kupffer,” p. 383. Fischer, Jena. Brachet, J. ( 1957). “Biochemical Cytology.” Academic Press, New York. Brachet, J. ( 1964 ). Nature 204, 1218. Brachet, J. (1965). Progr. Biophys. Mol. Biol. 15, 97. Brachet, J., and Denis, H. ( 1963). Nature 198, 205. Brachet, J., Kuusi, T., and Gothie, S. (1952). Arch. Bwl. (Liege) 63, 429. Brown, D. D., and Gurdon, J. A. (1964). Proc. Natl. Acad. Sci. U.S. 51, 139. Brown, D. D., and Littna, E. ( 1964). J. Mol. Biol. 8, 669. Caspersson, T. (1950). “Cell Growth and Cell Function.” Norton, New York. Chaung, H. (1963). Acta Biol. Exptl. Sinica 8, 370. Englaender, H., Johnen, A. G., and Vahs, W. (1953). Experientia 9, 100. Gurdon, J. B., and Brown, D. D. (1965). J. Mol. Biol. 12, 27. Hayashi, Y. (1955). Embyologia 2 , 145. Hayashi, Y. (1958). Embryologia 4, 33. Hayashi, Y. (1959). Develop. Biol. 1, 247. Holtfreter, J. ( 1933).Arch. Entruicklungsmech. Organ. 128, 584. Holtfreter, J. ( 1939). Arch. Entwicklungsmech. Organ. 139, 227. Holtfreter, J. (1943).1. Exptl. Zool. 94, 261. Holtfreter, J. (1944). J. Exptl. Zool. 95, 307. Holtfreter, J. (1947). J. Exptl. Zool. 106, 197. Humphreys, T. (1963). Deoelop. B i d . 8, 27. Kawakami, I., and Iyeiri, J. ( 1964). Exptl. Cell Res. 33, 516. Kawakami, I., Iyeiri, J., and Matsumoto, A. ( 1961). Embryologia 6, 1. Kirby, K. S. (1956). Biochem. I. 64, 405. Kocher-Becker, U., Tiedemann, H., and Tiedemann, H. (1965). Science 147, 167. Kuusi, T. (1953). Arch. B i d . (Liege) 64, 189. McCarthy, B. S., and Hayer, B. H. (1964). Proc. Natl. Acud. Sci. U.S. 52, 915. Mangold, 0.( 1923). Arch. Mikroskop. Anut. Entwicklungsmech. 100, 198. Mangold, 0. (1931). Ergeb. Biol. 7, 196. Masui, Y. (1960). Mem.Konan Unio., Sci. Ser. 4, Art. 17, 79. Masui, Y. (1961). Experientia 17, 458. Meyer, B. ( 1939). Naturwissenschaften 27, 277. Moscona, A. A. (1963). Proc. Natl. Acad. Sci. US.49, 742. Moscona, M. H., and Moscona, A. A. (1963). Science 142, 1070. Niu, M. C. ( 1958). Proc. Natl. Acad. Sci. U.S.44, 1264. Rollhauser, J. ( 1953). Arch. Entruicklungsmech. Organ. 146, 183. Sax&, L., and Toivonen, S. (1961). J. Embryol. Exptl. Morphol. 9, 514. Schuster, H., Schramm, G., and Zillig, W. (1956). Z. Naturforsch. l l b , 339. Spemann, H. (1901). Verhand2. Anat. Ges. Jena 15, 61. Spemann, H. (1914). Verhandl. Deut. Zool. Ges. p. 216. Spemann, H. ( 1938). “Embryonic Development and Induction.” Yale Univ. Press, New Haven, Connecticut. Suzuki, A,, and Kawakami, I. (1963). Embryologiu 8, 75. Takata, C., and Yamada, T. (1960). Embryologia 5, 8. Tiedemann, H. ( 1963a). In “Biological Organization at the Cellular and Subcellular Level” (R. J. C. Harris, ed.), p. 183. Academic Press, New York.

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Tiedemann, H. ( 1963b). In “Induktion und Morphogenese,” Springer, Berlin. Tiedemann, H., and Tiedemann, H. (1956d) Z. Physiol. Chem. 306, 7. Tiedemann, H., and Tiedemann, H. ( 1956b). Z. Physiol. Chem. 306, 132. Tiedemann, H., and Tiedemann, H. (1959). 2. Physiol. Chem. 314, 156. Tiedemann, H., and Tiedemann, H. (1964). Reu. Suisse Zool. 71, 117. Tiedemann, H., Tiedemann, H., and Kesselring, K. (1960). Z. Naturforsch. 15b, 312. Tiedemann, H., Becker, U., and Tiedemann, H. (1961). Embryologiu 6. 204. Tiedemann, H., Kesselrmg, K., Becker, IT., and Tiedemann, H. (1962). Develop. Biol. 4, 214. Tiedemann, H., Born, J., Kocher-Becker, U., and Tiedemann, H. (1964). Z. Nuturforsch. 20b, 608. Tiedemann, H., Born, J., Kocher-Becker, U., and Tiedemann, H. ( 1965). Z. Nutztrforsch. 20b, 997. Toivonen, S. (1940). Ann. Acud. Sci. Fennicae: Ser. A V I 55, 7 . Toivonen, S., and Saxkn, L. (1955a). Ann. Acud. Sci. Fennicae: Ser. A IV, 30, 1. Toivonen, S., and S a x h , L. (1955b). Exptl. Cell Res. Suppl. 3, 346. Toivonen, S., Vainio, T., and Saxkn, L. (1964). Reu. Suisse 2001.71, 139 (hommage a F. Baltzer). Tomes, P. I., and Holtfreter, J. (1955). J. Ex$. Zool. 128, 53. Vdhs, W. (1956). B i d . Zentr. 75, 360. Wang, Y., Mo, H., and Shen, S. (1963). Acta BioE. Exptl. Sinica 8, 356. Wilson, E. B. (1904). J. Exptl. Zool. 1, 1. Yamada, T. (1961). Advun. Morphog. 1, 1. Yamada, T. ( 1962). J. Cehlar Comp. Physiol. GO, Snppl. 1, 49. Ydmada, T., and Takata, K. (1961 ). Develop. Biol. 3, 411.

CHAPTER 5

THE CULTURE OF FREE PLANT CELLS AND ITS SIGNIFICANCE

FOR EMBRYOLOGY AND MORPHOGENESIS F . C. Steward," Ann E . Kent, and Marion 0 .Mapes LABORATORY FOH CELL

CORNELL

UNIVERSITY,

PHYSIOLOGY,

GHOWTH A N D DEVELOPMENT,

ITHACA, NEW YOHK

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Historical Setting: The Concepts of Haberlandt . . . 111. Chemical Stimuli to Growth and Morphogenesis . . . . . . IV. From Plant Tissue and Organ Cultures to Free Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Free Plant Cells in Culture: Their Morphology and Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. From Free Cells to Flowering Plants: The Case oE the Carrot Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Free Cells to Plants: Other Exaniples . . . . . . . . . . . . . . . . VIII. Other Relevant Studies on Embryogenesis and Morphogenesis . . . . . . . . . . . . . ........................ IX. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . ... .......

1.

113 115 116 117 123 129 136 146 149 151

Introduction

An ultimate aim of culture of free plant cells is to achieve a complete understanding of all the factors that control the development of sexually reproduced organisms from a single cell, the zygote. This objective lies a t

* This chapter records and interprets observations made in the course of a longterm investigation under the direction of one of us (F.C.S.); this has been supported 113

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the very core of experimental morphogenesis or embryology. Whereas in many lower organisms the zygote becomes a free living spore, the development of a zygote into an embryo in higher plants requires progressively greater degrees of protection and more specialized nutritional dependence upon the parental generation. Oogonia and archegonia in nonflowering plants and ovules in flowering plants meet these requirements in varying degrees and in marked contrast to the means by which similar ends are achieved in animals. Plants, however, present the additional feature of separate, asexually produced, cells, called spores. Spores are capable of development which is often independent of the plant, or may be enclosed within it. In a normal life cycle, spores may initiate events that lead to the formation of a sexual plant, or gametophyte, upon which the sex organs are formed and nourished. Conversely, the development of the nonsexual or sporophytic phase begins with the growth of a zygote. In both these cases, therefore, normal development stems from single free cells, i.e., spores and zygotes. In flowering plants, however, the predominant phase of development is the sporophyte, for the true sexual phase is much reduced and, in the case of the female gametophyte, is represented by the embryo sac which grows within and at the expense of the nucellus.* Flowering plants also present a unique feature, for a second nuclear fusion produces a nutritive tissue, the endosperm, which contributes to the early nourishment of the zygote and to its development into an embryo. In many plants ( e.g., those with exalbuminous seeds) endosperm has a transient role; in others (e.g., those with albuminous seeds ) endosperm develops apace, its cell divisions even outstripping those of the zygote. When embryo development is delayed, endosperm may accumulate in the form of a large liquid mass, as in the horse chestnut (Aesculus) or in the coconut ( Cocos); thus a naturally occurring balanced nutritive material provides both nutrients and stimuli for the later development of the young embryo. The content of endosperm is absorbed by, and no doubt transformed in, the cotyledons, throughout by a grant from the National Institutes of Health, Department of Health, Education and Welfare, Bethesda, Maryland. The participation of one of us (M.O.M.) as Research Associate, together with essential technical assistance, was made possible by the grant. Miss Kent was able to contribute to the work and to this account of it in her capacity as Research Assistant. * Nucellus: this comprises the bulk of the young ovule. Within the nucellus spores (megaspores) are formed by meiosis. From a megaspore the embryo sac, containing the egg, develops. The nucellus functions as a nutritive organ for the developing embryo sac and in the early development of a seed.

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which are the absorbing, or storage, organs of the embryo in the developing seed. In the culture of free cells which have been removed from mature higher plants, a special use was found for the materials which normally nourish young embryos; these materials, in the form of liquid endosperms, furnished the clue both to the vigorous growth of free cells and to their totipotent development. This is possible because, in the organization of flowering plants, the formation of tissues and organs occurs in such a way that all the living cells may retain the genetic message of the zygote and, if this information can be released and expressed in the free growth of cells, normal development may again ensue. Thus the culture of plants from cells opens vistas not yet achieved with animal cells. During the development of animal organs their cells seem subject to more rigorous restrictions and possibly irreversible controls which have precluded, at least to the present time, the totipotent development of animal cells in free cell culture (cf. Harris, 1964). Nevertheless, if one knew how to remove the restrictions which arise during development, the totipotent development of animal cells might also occur. Considerations of this sort give to the free culture of plant cells the great biological interest which it currently enjoys, for in this way light may be shed on morphogenesis and embryology. The present account focuses attention upon the newer knowledge of the culture of free plant cells and the factors that evoke organization, and special reference will be made to examples in which the growth of cultured cells recapitulates normal embryogeny to a surprising degree. This review, however, is concerned solely with cells of angiosperms. While cells and explants derived from lower organisms may be more responsive in culture, the knowledge that is gained from the behavior of cells and tissues of higher plants has greater significance because it bears upon the organization of the most advanced plants, in which the problems of growth and development are most acute. II. The Historical Setting: The Concepts of Haberlandt

The concept of the culture of free cells of angiosperms is usually traced to a somewhat obscure but remarkable article by Haberlandt published in 1902. By modern standards there was no really significant experimental evidence to show that cells isolated from the plant body could grow, but the noteworthy features are that Haberlandt foresaw the advantages of

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growing plants from free cells and that he anticipated that such development might recapitulate embryogeny. The passage in question is quoted here in translation: “Without permitting myself to pose more questions, or to prophesy too boldly, I believe, in conclusion, that it will be possible to grow, in this manner, artificial embryos from vegetative cells.* In any case the method of growing isolated plant cells in nutrient solutions could be a new experimental approach to various important problems.” Although Haberlandt’s 1902 prophecy was not substantially based on his own observations on free cells, his later morphological studies supported his ideas in so far as they related to cells within the plant body. The extensive pioneer investigations into embryo development, the formation of adventive embryos, polyembryony, and parthenogenesis ( Haberlandt, 1921a, 1922) emphasize his continuing preoccupation with ideas of totipotency of plant cells which are implicit in his earlier paper. In addition to this concept, Haberlandt expressed the view that cell division factors, or wound hormones, exert a morphogenetic influence; this was utilized in Haberlandt’s reinterpretation of parthenocarpy ( 1921a ) and the formation of adventive embryos (1921b). When he pierced the ovary of Oenothera a callus was induced which, when it penetrated the embryo sac and came into contact with its contents, formed an adventive embryo ( Haberlandt, 1922). Significantly, Haberlandt stressed that the wound hormone stimulated the cells to divide and that the special formative effect of the contents of the embryo sac induced embryogenesis. In the light of later work on the role of liquid endosperms in the culture of free cells, this was an acute observation. 111. Chemical Stimuli to Growth and Morphogenesis

It is an obvious physiological and biochemical problem to determine the stimuli which prompt individual cells, already located within the plant body, to grow, develop, and form adventive embryos in the manner indicated above. An earlier review (Steward and Mohan Ram, 1961) attempted to see how far the current knowledge of the factors that determine the growth of cells by division and by enlargement could contribute to this end. Reference may be made to this review not only for these ideas, but also for some cited examples of the phenomena in question. In summary, the following major points were made.

* “. , . aus vegetative Zellen kiinstliche

Embryonen zu ziichten.”

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In higher plants, cells of the plant body develop in organic contact with each other; though separated by cellulose walls, their protoplasts are united by the connections known as plasmodesmata. In this respect the organic union between adjacent plant cells is much closer than between animal cells, which are often free to move with respect to one another. Spores and zygotes are unique among the cells of the higher plant body in the sense that they are separate cells, they develop independently, and they are not connected to other cells by plasmodesmata. Paradoxically, therefore, higher plant cells have shown their greatest ability to express their innate totipotency by morphogenetic development when they grow as free cells unrestricted by their proximity to, or organic connection with, neighboring cells. Thus many early attempts to recapitulate normal development by starting with preformed masses of cells in large tissue explants, or by actual organ transplants, were limited at the outset by the fact that the tissue explant, or the isolated organ, was already predestined to grow as such. However, in order to grow, free cells must receive all their requirements exogenously. It was in this respect that the free cells used by Haberlandt were severely limited by the knowledge of his day. The best current means of satisfying the growth requirements of free cells is to provide them with ( a ) the full range of exogenous nutrients, both organic and inorganic; ( b ) the vitamins for which they may not be autotrophic; ( C ) the kind of stimuli to which the fertilized egg is normally subjected in the environment of the ovule and the embryo sac. All these requirements are most conveniently met by the use of liquid endosperms such as those obtainable in the central cavity of the coconut or from the vesicular embryo sac of a dicotyledonous tree such as Aesculus or Juglans to supplement a basal medium such as that of White (1963). It is, therefore, desirable to summarize briefly how this knowIedge emerged from the long history of plant cell and tissue culture. IV. From Plant Tissue and Organ Cultures to Free Cell Cultures

Despite the fact that the aseptic culture of tissue explants froin angiosperms and of their preformed organs, such as root tips, has been practiced for many years-certainly since 1920-the effective cultivation of free cells and their use in morphogenetic studies is a relatively recent development. Knudson was a pioneer in the use of aseptic culture methods in ways

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that contributed to the later development of tissue and organ cultures. He used these methods during studies of the organic nutrition of roots (1916) and also noted the longevity of isolated root cap cells in nutrient solutions ( 1919). From this beginning followed the early work of Robbins (1922) and that of White (1934) on the culture of excised roots. While the propagation of an organized system such as a dicotyledonous root by means of its severed tip stressed the maintenance of an existing organization, it paid little regard to the potentialities of the individual cells. Once the successful passage of root tips through several transfers and successive subcultures was achieved and their potentiality for indefinite culture established, this technique was slow to yield further useful morphogenetic knowledge. However, the recognition of thiamine as the component of yeast extract found to be necessary for the growth of tomato roots alerted investigators to the need to supply vitamins to tissues and organs grown under culture conditions if they were incapable of synthesizing them autotrophically; and, as the requirement of plants for minor (trace) elements became known these became accepted also for the growth of excised roots or of tissue explants (cf. Street, 1957). The use of surviving slices of storage organs was common in plant physiology, even in the nineteenth century, but it was Nobecourt (1938) and Gautheret (1938) who, by their work on carrot, really established the culture of explanted angiosperm tissue as a staple technique. Again, these pioneer studies became heavily preoccupied with the demonstration that the continued-even indefinite-aseptic growth of tissue explants, furnished with appropriate nutrients and vitamins, was feasible. This emphasis on long-continued subculture of tissues that had become adapted to culture conditions-the habituated carrot culture of Gautheret -certainly did not channel work in directions which were conducive to the study of morphogenesis. Perhaps the mistaken idea, assiduously fostered by some of the pioneers in this field, that the initial aseptic isolation of explants was a major problem helped to develop this trend. Furthermore, the prevailing preference for the slow, long-continuing growth obtainable on agar (when cultures become necrotic at their center and at their base) and for large inocula (which disguise the fact that their relative growth rate may in fact be small) tended to direct work of this kind into a rather rigid pattern, which was remote from the conditions of culture which are necessary to produce the fastest growth of tissue explants or of isolated cells and to foster their totipotent behavior. Therefore, the period 1920 to 1950 was one in which much general

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experience was gained on the feasibility of culturing tissues and organs from selected dicotyledonous plants ( although monocots were strangely recalcitrant), but from which little was learned by these means about morphogenesis. This trend was changed when physiologists turned to the culture procedure as an experimental technique to help them solve other problems, for they modified many of the time-honored culture practices. First there was a return to culture in liquid as the best means to secure both rapidity and uniformity in the growth of replicated tissue explants and to achieve greater control over the many factors that affect that growth. To obtain large number of accurately comparable explants in numbers which permit statistically valid physiological experiments, fresh explants withdrawn from the intact organ were used; thus the technique no longer depended upon the habituated cultures used previously. Also it was realized that the then conventional nutrient media with their content of salts (supplying both major and minor nutrient elements), of sugars, and of vitamins fell far short of allowing the malcirnum growth of which the tissues were capable. The limiting factors in these media were those that permit the cells to divide; this led to the recognition that there are both naturally occurring and synthetic substances which, when added to the otherwise complete culture medium, support growth by cell division at rates far in excess of those that occur in the unsupplemented medium. While knowledge of auxins, which are concerned primarily with cell enlargement, had been familiar since 1928 and while the concept of cell division factors was known to Fitting (1909) and to Haberlandt ( 1921c), the evidence in tissue cultures for what are now termed kinins (Miller et al., 1955) or cytokinins (Skoog et al., 1965) came much later; the role of coconut milk ( C M F ) in stimulating the growth of carrot explants was a prominent early example (Caplin and Steward, 1948). The recognition of such exogenous growth factors became very important when the size of the tissue explants or of the initial inocula was much reduced, for the larger the explant, or the more inoculum transferred, the more these requirements were apt to be satisfied accidentally, or endogenously. I t was the fact that the zygote of dicotyledons develops in an environment of special nutrients and stimuli which enable it to grow that suggested the use in tissue culture systems of the fluids which surraund young embryos. The prototype of this effect was that obtained by using coconut milk (coconut water), the liquid endosperm of the coconut, as

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a supplement to a basal medium in which small carrot explants were grown (Caplin and Steward, 1948). By these means small (2.5 mg) t'issue explants, excised far enough from the cambium so that they would not otherwise grow, were now enabled to grow at relative rates not achieved previously; the basis of this growth induction was a greatly enhanced cell division. From this observation a standardized system evolved; this was based on 'the use of carrot phloem* explants and a basal medium supplemented with coconut milk. This system permitted the factors that promote maximum growth to be investigated and the effectiveness of different chemical substances on growth to be assayed (Caplin and Steward, 1952). However, the coconut milk supplement that promoted optimum growth of carrots did not remove the obstacles to rapid growth of all angiosperm tissue. For example, potato tuber tissue, long known to be capable of wound healing by cell division, failed to proliferate under conditions that sufficed for the carrot. Out of this dilemma developed the first use, by Steward and Caplin (1951), of synergistically active mixtures (such as coconut milk 2,4-D; coconut milk NAAt ) as combined additives to the basal medium; such combinations have allowed many otherwise recalcitrant tissues to grow; indeed, the first monocotyledonous tissue culture was obtained in this way by Morel and Wetmore (1951). It should, therefore, be recognized that the conditions necessary to evoke the full and innate capacity of a somatic cell to grow in culture are complicated. The major complication is that the accessory substances which must be added to a basal tissue culture medium to make it the equivalent of the environment of the embryo sac are many, and their interactions are complex. An earlier hope that the cell division factor present in coconut milk might be a single substance was too optimistic. In fact, the conclusions to be drawn from studies of coconut milk or of other liquid

+

+

* Phloem is the tissue essentially concerned with translocation in angiosperms. It may form directly from a shoot apex (primary) or during growth in girth from a cambium (secondary). In the carrot root most of the tissue is of secondary origin and the phloem, composed largely of unspecialized living cells (parenchyma) and of a few conducting elements (sieve tubes and associated companion cells), lies immediately outside the cambial ring, which is clearly visible in a transverse slice. 2,4-D: 2,4-dichlorophenoxyacetic acid, a synthetic substance which has many of the properties of an auxin, of which indoleacetic acid ( I A A ) is the best-known naturally occurring example. NAA: naphthaleneacetic acid, another synthetic auxin.

+

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endosperms, such as those of horse chestnut ( Aesculus) or of corn ( Z e a ) , are all similar. All these fluids greatly stimulate the growth of carrot cells and, in synergistic combination with such substances as 2,4-D or NAA, induce more recalcitrant cells to divide. However, active components which account for the cell division fostered by these fluids fall into two parts. The first of these, termed the “neutral” fraction because it adheres to neither anionic nor cationic resins, is required in relatively large quantity. Four hexitols ( sorbitol, mannitol, myoinositol, and scyllitol) have been isolated from this fraction and characterized (Pollard et al., 1961; Shantz and Steward, 1964). The neutral fraction alone will not foster rapid cell division, however; the simultaneous presence is required of the “active” fraction which is specifically related to cell division and which is effective at very great dilution (of the order of a few parts per million or less). The different constituents of the active fraction, which include kinin-like substances, seem to act independently of each other although only in conjunction with the neutral fraction. Progress in this whole area has been slow because the activities of the individual fractions and also their interactions must be understood ( cf. Steward et al., 1964‘1; Shantz and Steward, 1964). In addition to the interaction between the neutral and active fractions in the stimulation of growth and cell division it has been shown repeatedly that the system also responds to casein hydrolysate (as a source of reduced nitrogen) and individual constituents of the active fraction may also interact with auxin (Fig. 1). Although relatively large living cells were readily visible at the margins of cultured tissue or explants, and although such cells had been seen floating free in the liquid medium ( D e Ropp, 1955), a long interval elapsed before angiosperm cells were grown in suspension cultures in a way enabling them to be manipulated like microorganisms with the techniques familiar in microbiology. Early chemical devices to obtain separate and viable cells (for example by the use of pectinase) were, for the most part, unsuccessful. By far the best means of obtaining isolated cells was by growing many (50 to 100) tissue explants in one vessel; as a byproduct of this growth, cells were sloughed off into the medium from the margins of the explants a s they gently rubbed against one another in the slowly revolving ( 1 rpm) flasks. It was quickly found that any culture, rapidly growing under these conditions, would give rise to viable free cells. Cultures of carrot, potato, tobacco, Haplopappus, peanut, Jerusalem artichoke, and a variety of other plants (even of the

140

120Average fino1 100. fresh weight of 80 cultures

CH = Casein Hydrolysate a t 2 5 0 pprn IAA = Indoleacetic Acid a t Q5pprn INOS. Mp-inositol a t 25 pprn AFC = Aesculus Active Fraction Concentrate a t 50 mrn ..

-

60

~

40-

2GnTreotment

IAA

+ IN05 AFC

+

+

+

INOS AFC AFC

+

+

IAA

AFC *

Growth response w

r basal m u m controls.

+

INOS INOS +

+

IAA +

AFC AFC INOS + Portion of growth response which k grrotrr tho? a d d t i single r c y x x l s c s is ~ AFC due to synergistic interaction.

INOS IAA

FIG. 1. Growth-promoting effects and interactions of casei 1 hydrolysate. indoleacetic acid, myo-inositol and Aesculus active fraction concentrate on carrot phloem explants.

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parthenocarpic fruit of the banana) were obtained (Steward, 1961; Blakely and Steward, 1961) . This development marked the beginning, in this laboratory, of events that led to the simulated behavior of zygotes by somatic cells: It is when cells are free and removed from organic connection with the tissue explant from which they are derived that they organize as they grow. V. Free Plant Cells in Culture: Their Morphology and Division

Freely suspended cells can now be made to grow and divide in an environment which is very uniform with respect to light, gravity, aeration, and nutrition. By gentle agitation many of the “daughter” cells separate and then grow, but a free cell culture cannot be kept wholly composed of single cells, for during growth small clumps of cells are formed and their protoplasts are in organic contact via plasmodesmata. Cell suspensions composed entirely of single cells can, however, be obtained by filtration ( Blakely and Steward, 1964). Such single cell suspensions can be spread on agar and the genesis of colonies observed (Blakely, 1964). Single cell suspensions can also be inoculated into a liquid medium at so great a dilution that the only way to detect their presence is to allow the culture to grow, the cells to multiply, and colonies to form. By modern methods very dilute suspensions (of the order of five cells per milliliter or even fewer) will routinely develop into very dense suspensions of the order of 100,000 cells or more per milliliter in a few ( 2 to 3) weeks. However, there is nothing analogous to the aggregation of animal cells which, as in the case of sponges, reassociate to form a colony and to produce an organism. When free plant cells grow into plants, each plant represents the development from a small group of cells that has in turn grown from a single celI (Steward et al., 195Sb). Cells growing free do not resemble cells in the tissue from which they were derived. They are usually conspicuously free from storage products and their general chemistry may be very different from that of the tissue of their origin-so much so that they rarely contain the characteristic products which typify the parent tissue. Many examples have been examined to see whether, in culture, cells would produce such substances as special enzyme proteins (e.g., urease or papain), unusual nitrogen compounds, etc., for which the tissue of origin was notable. On the contrary, some free cells in a particular medium have produced substances in quantity which did not accumulate in the parent tissue. Such an ex-

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F. C. STEWARD, A. E. KENT, AND M. 0. MAPES

ample occurred in a strain of Haplopappus cells which responded in the presence of coconut milk to a particular concentration (0.5 ppm) of naphthaleneacetic acid by accumulating anthocyanins in great quantity whereas at another concentration (5.0 ppm) the cells vere virtually free from these pigments (Blakely, 1963). However, in most of the cases yet investigated in this laboratory (Krikorian, 1965) it seems as if the biochemical potentialities of the tissue to produce stored products which are expressed in the environment of the mature plant body are changed in the freely growing cells. In other words, the situation in the plant body which endows cells with a characteristic morphology also determines their metabolism. Thus the cells behave as they do by virtue of “where they are,” not only because of “what they are.” In short, cells in free culture respond more to their intrinsic capabilities than cells in the plant, where their behavior is more subject to extrinsic factors. The main characteristics of the cells as they grow free are ( a ) their prominent cytoplasmic strands, which are clearly visible under the phase microscope and which show very active streaming if the cells are able to grow; ( b ) their prominent nuclei (with nucleoli), which are often located and surrounded by granular cytoplasm at the intersection of strands which criss-cross the cell; ( c ) numerous and extensive aqueous vacuoles, even in cells which are able to divide; and ( d ) the presence in a given cell suspension of a variety of cell types, including some which grow in the form of small aggregates of dense, more minute cells, whereas others become large and internally septate or grow as filaments or even form new cells by “budding.” The fine structure of these cells as they grow in culture has recently been investigated with the electron microscope, especially for carrot cells (Israel and Steward, 1966). The growth factors that stimulate cell division also make their impact on virtually all the cytoplasmic organelles; Golgi bodies become more prominent with more highly developed lamellae and more abundant secretion of globules; this is especially evident near dividing nuclei, where the globules may contribute to the new cell wall. Mitochondria change from the more rounded condition, as in the resting cells, to elongated and somewhat branching amoeboidlike structures with abundant internal features typical of actively metabolising cells. But the effect of the growth substances is observed especially on plastids, which change from the large, rather structureless plastids of the resting cell and pass through all the developmental stages which produce, in the light, mature chloroplasts with grana and pigment.

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This is only another way of saying that when the quiescent cells undergo growth induction this affects every feature of their development as it occurs within the plant. Metabolically, however, the response to the growth factors is to be seen in a greatly stimulated rate of protein synthesis and an accentuated turnover (Steward et al., 1956; Bidwell et al., 1964) which is especially marked in the free cells (2.07. of protein carbon per hour). Just as the morphological effects of the cell division growth factors extend to all the cytoplasmic organelles, so also do they affect all aspects of metabolism. Freely cultured cells of different plants appear very much alike (Figs. 2-5); so much so that, unless they contain special pigments or have other recognizable features, the cells from diff erent plants are often difficult to distinguish when they are present in mixed liquid cultures. By their simplicity of form, eggs in embryo sacs are more similar than the plants to which they give rise; the same is also true of free cells, although, as soon as multicellular groups arise, cells in different parts of the tissue mass diverge in their appearance and behavior. The causal factors that determine this are still obscure, for often two attached “daughter” cells, the product of a single equational division, may differ in either their form or their composition. (This has been observed in both Haplopappus and carrot cells [Steward et al., 1958a; Blakely, 19631.) Under the circumstances of free cell culture one might have expected that the classical rules, or laws, of cell division (with Sachs’ and Errera’s laws as the guiding principles) would operate under ideal conditions and that cells would partition space like the similar liquid systems in equilibrium, to which they are often likened. In the case of carrot cells this is often not so, for, as single cells pass to the multicellular condition, they may diqplay a baffling array of patterns of growth (Steward et al., 1958a). Indeed one may question why there should be such a diversity of behavior in a population in which all the free cells originated from the same tissue explants. Equational division of isodiametric cells; the formation of long filaments by accurately transverse intercalary divisions and by curved walls in the cells at the tip; large cells which become multinucleate by internal septation and produce by their mutual compression a solid moruloid mass of small dividing cells; the formation of papillae by a method of budding to produce initials which give rise to filamentous outgrowths; and the occurrence of very dense small granular clusters, reminiscent of spores, have all been seen in the free cell cultures of carrot or of Haplopappus (Steward et al., 1958a; Blakely, 1963). In

126 F. C. STEWARD, A. E. KENT, AND M. 0. MAPES

FIG.2. A carrot cell (Daucus carota) as grown freely suspended in a liquid basal medium plus coconut milk. Photographed under phase contrast, showing the nucleus embedded in a central mass of cytoplasm suspended by cytoplasmic strands along which streaming occurs. FIG.3. A cell of PeZZioniu under similar conditions.

z

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2!

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127

128

F. C . STEWARD, A . E. KENT, AND M. 0. MAPES

fact, the conditions of culture favor, to some extent, one type of development or the other. By long-continued growth of carrot cells in completely submerged culture on a horizontal shaker, a clonal strain rich in long filamentous cells was produced (Blakely, 1961). Any or all of these forms may, by somewhat different routes, lead to organization within a cluster of cells with the eventual formation of both shoots and roots; the following facts, however, are clear. The probability of survival of a cell cluster to form a viable colony is greater if it originates from small isodiametric cells rather than from the longer filamentous ones ( Blakely, 1964). Also the organization and recapitulation of normal embryogeny is closer if the growth commences from the smaller and the more isodiametric cells than from the large and very highly vacuolated ones. Moreover, the route which cells take toward organized development may be, in part, a function of their prior history; notably, of the particular part of the plant body from which they may have been derived and even, in the case of cultivated plants, of the genetics of the strain in question. In particular the route toward organization may be a function of the prior cultivation of the cells and especially so if they have been in long-continued cultivation. Hence the extent to which cultured cells may recapitulate embryogeny is often a function of their origin and their prior history in cultivation. The conditions for the most rapid vegetative growth of cultured plant cells may not necessarily be the same as those which promote their organized development. Comparisons of the multiplication rate of cells in small tissue explants with that of the free cells derived from them suggest that the former undoubtedly grow faster. In other words there is something in the environment of the cell at the surface of an explant, or its presence in a small cluster, which is difficult to reproduce exactly in the ambient medium of free cells. Although single cells will give rise on agar plates to viable colonies, the chances that a particular cell will divide and survive are greater if it exists in a small colony (Blakely, 1964). In part this may be a matter of physical protection, for the cells en mmse are shielded from the casual episodes which often cause individual cells unpredictably to swell or burst or die. More probably it derives from the fact there is a certain division of labor in any colony of cells. In other words, the larger, less frequently dividing, more central cells of an explant or cluster may rework the stimuli to cell division (i.e., the content of the coconut milk) and secrete substances to neighboring, or attached, cells that may be even more effective than those originally in the medium.

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In this way some cells of the colony may simulate the role of cotyledons when they absorb the content of endosperm and convert it for the use of the embryo. This niay explain the effectiveness of the so-called nurse tissue technique by which it was demonstrated that single angiosperm cells, not in organic contact with others, will divide (Muir et al., 1958). In this technique the cell to be observed is placed upon a cultured explant, separated from it by a thin barrier (e.g., of filter paper) which prcvents organic contact but permits the flow of nutrients to enable it to grow. A similar effect was demonstrated in this laboratory in another way by pIacing pregrown carrot explants on the surface of an agar plate on which single cells were distributed at random. Cells close to the explant formed colonies more successfully than those which were unaffected by materials diffusing from the explant (Blakely, 1963). VI. From Free Cells to Flowering Plants: The Case of the Carrot Plant

The dramatic and, at this juncture, biologically most interesting feature of the free plant cells is their ability to grow in an organized way. This section therefore will summarize what we now know about the growth of plants from free cells. This will be done first with respect to the work of this laboratory to illustrate the points to be made from the examples that are best known to the authors. Subsequent reference will be made to other work and workers to show in perspective that these results are not isolated ones but are now typical of what has become a wellrecognized and increasingly successful technique and a productive field of work. The first culture of a complete plant, or plants, from a long-cultivated cell suspension occurred in cells which had originated from the secondary phloem (see footnote, p. 120) of the carrot root but which had been subcultured many times. Any peculiarities established in their initial formation in the dormant carrot storage organ had therefore had ample opportunity to be eliminated. Nevertheless, when cultivated on a basal medium supplemented with coconut milk, these cells gave rise to plantlets. In this respect these cells, though completely removed from the plant body, functioned like those described by Haberlandt which showed apomictic, parthenogenetic, or other kinds of reproduction by which embryos developed adventitiously. Whereas roots, continually growing as such, readily formed in the

130

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submerged rotated cultures (Steward and Shantz, 1956), the shoots developed best when the small clusters with initiated roots were sown on agar in stationary flasks. The first such plantlets very readily gave rise to storage roots while they were in the culture flask; their superficial cells, being in contact with the medium and exposed to a balance of growth factors heavily oriented toward cell division, again proliferated ( Steward et al., 1958b). As in the case of the normal biennial cultivated carrot, normal flowers were obtained only in the second cycle of growth, from the buds at the crown of the storage root (Steward et al., 1961). Thus the complete and normal life cycle was reproduced by cells cultivated from small secondary phloem explants. When such cells were examined in their freely growing state they showed patterns that were strongly reminiscent of embryonic growth (Steward et al., 1961). A prominent feature of the first successful culture of carrot plants was that within the cultured mass, embryo-like structures developed best from characteristic small nodules; these gave rise to “embryoids,” i.e., structures with roots and shoots with their two first cotyledonary leaves (Steward et al., 1958b; see Fig. 8). The conditions that prevailed when these cultures of free cells were sown on a stationary agar permitted these forms to develop abundantly. Using mature plants with storage roots that were routinely grown in this way from free cell cultures, the above process could be repeated: that is, explants were excised from the newly formed root, and free cell cultures were again established from these; a new crop of plantlets was obtained from the embryoids formed vegetatively in these cultures. Thus, the carrot life cycle was repeatedly recapitulated in culture, starting from single cells (Steward and Mapes, 1963); in each such cycle of growth and development the organism was reduced to a free cell stage, not by forming spores or gametes but by the establishment of freely suspended dividing somatic cells from the secondary phloem. These cells, therefore, behaved totipotently, and in this respect were like zygotes. The stimulus to grow which the otherwise mature cells of the storage root received was their contact with the basal tissue culture medium supplemented with the coconut milk (i.e., liquid endosperm of the coconut); but the organized growth developed best from freely suspended cells, in contrast to cells that were present in tissue explants. From an original strain (with the laboratory designation MIT-1) successive “life cycles” have thus been traversed; whenever explants were freshly obtained from a newly formed root, the free cells derived from

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them promptly organized and developed again into plants under the conditions prescribed (Steward and Mapes, 1963). On the other hand, the clonally propagated free cells, which were maintained for several years as a suspension culture in liquid medium, gradually lost their ability to give rise to organized structures although they continued to grow vigorously. First, shoot formation failed; then roots occurred with decreasing frequency. This suggested that the continuously cultivated free cells had lost an “epigenetic” factor which carrot cells normally acquire as they develop in situ in the plant body, and which is essential for the expression of their totipotency when freed. Until recently this failure of the continuously cultivated free cells had seemed to be irreversible. However, it is now known that by a sequential application of growth-regulating substances, i.e., by culturing the cells first on a coconaphthaleneacetic acid, then on coconut milk withnut milk medium out the naphthaleneacetic acid, cells which would hitherto only proliferate will again organize, producing roots, shoots, and entire plants. * But the most striking examples of totipotent nonzygotic embryonic development from free somatic cells of carrot occurred when the cell suspensions were established in the first instance not from mature tissue, as above, but from developing embryos. This observation was made initially in two ways. When embryos were isolated from immature seeds of plants which had been grown from cells, to demonstrate their viability, they were “germinated aseptically on the coconut milk-containing medium. However, in that medium, many free cells were sloughed off from the main plantlet; these cells grew on the coconut milk-containing medium and formed heart- and torpedo-shaped embryosf (Steward and Mapes, 1963). Indeed, these embryos in turn proliferated at their margins, and the process obviously could repeat itself. But even more striking were the events when a single embryo from an immature seed of the wild Daucus carota (Queen Anne’s lace) gave rise to a freely growing cell culture which, when spread on agar, formed a very large number

+

* These observations were reported to The American Society of Plant Physiologists in August, 1965 (Mapes et al., 1965) and will be published more completely in due course. f “Heart-shaped einliryo” and “torpedo-shaped embryo” are terms in descriptive angiosperm embryology. The zygote commonly gives rise to a globular embryo which develops two lobes (primordia of cotyledons) which, at this stage, make it heart-shaped; by later elongation of the axis and the cotyledons and the early development of a primordial root, or radicle, the stage described as “torpedo” is formed.

132 F. C. STEWARD, A. E. KENT, AND hl. 0. MAPES

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5

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5. THE CULTURE OF FREE PLANT CELLS

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133

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P. C. STEWARD, A. E. KENT, ANI) M. 0. MAPES

(order of 100,000) of organized plantlets on a single petri plate (Steward et al., 1963); these plantlets started from globular forms and showed all the familiar stages of embryonic development as it occurs from a zygote in an ovule (Steward et al., 1964b) (Figs. 6-12). Furthermore, these plantlets could be grown to complete plants and brought to normal flowering (without a special vernalizing treatment) in a period even shorter than that which is needed for these plants in nature. It is in fact more impressive, and statistically valid, to note the very large number of embryonic forms that develop in a free cell culture than to concentrate on growing one plant from one cell, interesting as this may be. For Daucus carota, therefore, the case is clear. Any normal diploid cell of the plant body is potentially able to produce a plant. It has full genetic and morphological totipotency. To express this it needs to be free from the restrictions which apply in situ in the plant body or in the previously developed organized mass of cells. To give effect to its totipotency, each cell must obviously grow. But growth alone is not enough, for, in cell culture as well as in normal development, even vigorous growth may occur in ways which preclude the normal morphogenetic development, as seen in zygotic embryos. While these restrictions may be imposed upon cells and persist in them by virtue of their culture or prior history, it is now equally clear that by appropriate exogenous applications of growth regulators (in a balanced or in a sequential manner) the cells may emerge in a form as totipotent as any zygote. A recent development promises to be the basis of an important concept. Differentiation clearly imposes certain restrictions on the behavior of the cells as they occur in situ in the plant body. Being an orderly and progressive process in time, differentiation may impose such restrictions sequentially. The release of cells from these restraints may not always be accomplished in a single step; a sequence of stimuli, applied in the correct order, may be needed (Kent and Steward, 1965) (Fig. 13). We now know that the cultivation of some cells, e.g., Nicotiana suaueolens, on a synergistic mixture of a basal medium containing coconut milk and napthaleneacetic acid is an essential prerequisite for their later organized totipotent growth. It was after their transfer to another medium (the basal medium plus coconut milk alone) that the cells grew in an organized way (Mapes et al., 1965). Other examples of this sequential effect in the application of growth-regulating substances will be described later. One should recognize, however, that all details of these complex responses cannot be fully specified. It is obvious that there are gradations

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to be observed from the free and fully zygotic behavior of cells grown from embryos, to the various and more devious devices by which previously cultured cells from more differentiated organs are induced to express their innate totipotency. These gradations, or variations, even involve the apparent loss of totipotency by long cultivation and its subsequent recovery. It should be recognized that there is no exclusive formula, or singIe magic nutrient, which will produce these results in

I. EXCISED EMBRYOS: in the Ioter staqer of embryaqeny a11 isolated embryos grow and form callus;

in lhe earlier stoqes this is more difficult. with appropriate treatment these regions have yielded free ~011sshown l o be capoble of !X. ROOT- secondary phloem. cambium ond secondary Xylem growth and morphoqenesis. I[. STEM

m.PETIOLE P.FREE

1

CELLS IN SUSPENSION- when derived from any young embryo they readily recapituiote embryoyany on Basal Medium +Coconut MilkICM); when derived from motura orqans they am more restricted in response,requirinq on intermediary or prior qrowth stimulus.

FIG. 13. The morphogenetic responses of free cells of Daucus carota as determined by their origin and culture.

identical manner for all strains under all conditions. The point of the carrot plant seems to be that for many of its living cells the controls that limit their behavior during development are relatively easily undone; inhibitors to their growth, whether by cell enlargement or cell division, seem relatively inconspicuous, and the appropriate balance of exogenous and endogenous factors (Lee, requirements for which the cells are heterotrophic or autotrophic ) can in given cases be achieved. Significantly this is accomplished most easily when the cells are brought into culture from young embryos. Nevertheless, with appropriately balanced or sequential treatments the same ends have been accomplished, using cells from mature parts of the plant body. Of all the living cells of the plant

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body those that are taken from fully developed green leaves seem the most reluctant to respond totipotently. It seems as if their metabolism, probably their protein metabolism, is so fully committed to photosynthesis and to chloroplasts that they lack, or have deemphasized, other metabolic features (probably mitochondrial) that are essential for their totipotent development. Having outlined the way this work developed with the carrot plant, one may now summarize, very briefly, its ways of extension and application to other angiosperms. This points to the possibility and even the probability that cells of any angiosperm would respond in a similar way. VII. Free Cells to Plants: Other Examples

In this laboratory success in culturing whole plants from cells other than those of the carrot plant was first achieved with other members of the Umbelliferae. This was in itself noteworthy because various unsuccessful attempts were made to bring the other free cell cultures then available to the point of nonzygotic embryonic development. For some reason the Umbelliferae are particularly prone to this behavior, even in species not otherwise noteworthy for apomictic, polyembryonic, or parthenogenetic development. Complete success in producing plants from cells was achieved with Coriundrum (where flowers were obtained) (Figs. 14-19) and with Sium, ,the water parsnip (Figs. 20-25). The procedures were essentially those adopted with carrot, and full use was made of the device of starting the free cells from an embryo and bringing them into the rapidly growing totipotent state by access to coconut milk. The precise conditions that applied to each of the examples in question are described in the legends to Figs. 14-25. It is interesting that Svobodova (1964, personal communication) has been able to establish a vigorous callus culture at the base of a small segment of a potato shoot. This culture, when removed from contact with the shoot, grows easily on a basal medium which, without further supplements (coconut milk, 2,4-D, etc.), would not support the growth of potato tuber cells. In fact, some plants have originated from the callus mass grown in this way. The suggestion may be made that the response of these potato cells was modified by the basipetal stimuli which they received while in contact with the shootstimuli which in the case of the tuber cells had to be supplied exogenously in the medium. It is curious, therefore, that three of the first successful demonstrations,

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in this laboratory, of the totipotency of somatic angiosperm cells involved members of the Umbelliferae. There seems no obvious reason, however, why cells of the Umbelliferae should be especially compatible with coconut milk. In search of an underlying morphological reason, the

FIG. 14. Cells of Coriundrztm sutiviim as grown freely suspended in a basal medium containing coconut milk showing both large cells and dense, globular, more embryonic, forms. FIG. 15. A heart-shaped stage in the development of a plantlet from free cells of Coriandrum. FIG.16-17. Later embryonic stages in the development from free cells of Coriandrum.

138

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FIG.20. Cells of Sium suaue as grown freely suspended in a basal liquid medium containing coconut milk showing both large cells and dense, globnlar. more embryonic, forms. FIG. 21. An early torpedo stage in the development of a plantlet from free cells of Sium. FIGS.22-23. Later embryonic stages in the development from free cells of Sium.

FIG. 18-19. Plantlets of Coriundrum grown aseptically from free cells showing a vegetative plant ( 1 8 ) and a plant with an inflorescence ( 1 9 ) .

FIGS.24-25. Plantlets grown aseptically froin free cells of Sium, showing plants with plumule and radicle (24) and a portion of a large population of plantlets in different stages of development as grown in one flask ( 2 5 ) . 140

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scant development of a suspensor* in the embryos of the Umbelliferae seemed suggestive. If the suspensor commonly makes some essential nutritive, or regulatory, contribution to proembryos ( other than its purely mechanical role of forcing them into the nutritive endosperm), it may be that this function is implicit in the proembryo itself in cases where suspensors are lacking. As a result, it may be more feasible to obtain a globular, proembryo-like mass in free cell culture that will grow into a viable embryo when the cells used are from plants in which a suspensor is not essential. Indeed, the argument may be extended. Semiparasitic or saprophytic flowering plants have extremely primitive embryos and when shed they lack cotyledons, but they are nevertheless adapted to develop independently without the aid of nutrients from the parent sporophyte. The outstanding case is that of orchids, where the rudimentary embryos readily become green and develop into globular masses ( protocorms) within which buds form (cf. the account below of free cell culture and development of orchid plants from cells derived from an orchid protocorm). A long-cultivated callusf culture of N . rustica of embryo origin was obtained from Dr. Mohan Ram in Delhi, India, and this readily yielded a vigorous cell culture. Nevertheless, it failed to organize. Even freshly established cultures of N . tabacum, which grew well, did not organize. However, it was drawn to the attention of F. C. Steward that in the work of Paulet and Nitsch (1963) it had been noted that N . suaveolens readily gave rise in conventional tissue culture to buds. Following this suggestion, free cell cultures were developed and grown from this material; the expectation was fulfilled, for they could both grow and organize. Under conditions described in the legend to Figs. 26-29, normal complete plants were grown from free cells and these plants were brought to flower. After this manuscript was prepared we noted the demonstration by V. Vasil and Hildebrandt (1965) that single cells derived from a tobacco hybrid N . glutenosa x N . tabacum could be grown into colonies and then into a flowering plant. The figures there shown (Fig. 1, a to 1 ) bear a striking resemblance to those previously published for carrot cells (cf. Figs, 1, 7, 11, and 12 in this paper, or Steward et al., 1958a), although

* The suspensor i\ L I S L I a~ ~chain ~ of cells attached to the lower extremity of an embryo which, in s a n e vascular plants, pushes the young embryo into the surronnding nutritive tissue. t The term callus applies to any loosely proliferated, unorganized mass of cells.

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FIG.26. A cluster of cells which developed in a basal liquid medium supplemented with coconut milk from a free cell of Nicotiana suaoeolens. FIG. 27. A stage (heart-shaped) in the development of a plantlet of Nicotiana in a free cell culture. FIG.28. A plantlet of Nicotiana grown from a free cell culture showing a welldeveloped shoot. FIG.29. A complete plant grown aseptically from a free cell culture showing a flower.

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the authors note that plantlets originated after some 3 months’ growth of the callus without the formation of embryos. In this respect the plantlets originated in a somewhat different manner from those observed in carrot, Coriandrum, and Sium in this laboratory. Arabidopsis thaliuna, a member of the Cruciferae, is a plant which has become popular in genetic and morphogenetic studies because of its short life cycle of a few weeks and its ready growth in culture. This plant has also been brought into free cell culture using cells of embryo origin and, from the free cells, organization into both shoots and roots has been obtained (Figs. 30-32) (Mapes et al., 1965). A tissue culture was established from a minute shoot growing point of an orchid (Cymbidium-sp). This followed from the work of Morel on propagation of orchids by meristem culture. This tissue culture was then converted into a free cell culture, and these cells in turn recapitulated normal development with the intermediary formation of a globular stage (small protocorm) from which shoots arose (Figs. 33-35). Although bulbils readily develop on the leaves of Kalanchoe (or Bryophyllum ) and leaf propagation of Saintpaulia is familiar, neither of these plants have yet proved, in our hands, as prone to develop embryonically from their free cell cultures as might have been expected. Kalanchoe leaf-cell cultures have given rise to roots in this laboratory but not yet to shoots. A somewhat similar result has been described by Wadhi and Mohan Ram (1964). Also, Acer, a currently popular subject for investigation by the use of a clonally cultured strain originally of cambial origin (Lamport and Northcote, 1960), which, like the potato tuber (Steward and Caplin, 1951), was first brought into cultivation by the use of coconut milk and 2,4-D (cf. Goldstein et al., 1962), has also been reestablished by us in free cell culture using embryos as the point of departure. Although these cells grow well and form roots, they have not yet recapitulated embryogeny in the manner now familiar with carrot and some other plants. Therefore, in this laboratory, various examples drawn from different families show that free somatic cells of angiosperms can be made to grow in isolation and, if they do so and receive the correct balance of nutrients and stimuli, they may be brought into the state in which they will organize into plantlets. The effectiveness of coconut milk in promoting this behavior is attributable to its ability to simulate as nearly as possible the nutritional environment of the zygote in the ovule. When these conditions can be met the development from free cells tends to recapitulate-

FIG.30. A culture grown from cells of Arabidopsis thaliana showing abundant roots. FIGS.31-32. Cultures showing organized growth which originated from cells of Arabidopsis to show roots only (31) and roots plus shoot ( 3 2 ) . The tubes shown are those commonly used for the growth of carrot explants in liquid.

144

a

9 M M .J

K

3

n M FIG. 33. Cells of an orchid (Cymbidium sp.). which originated in the shoot apex, as grown in a liquid medium containing coconut milk and 2,4-D. FIGS.34-35. Stages in the development of a plantlet of Cymbidium from free cells showing a cluster of protocorms ( 3 4 ) in a culture tube (cf. Fig. 31-32) and a plantlet (Fig. 35) grown from a single protocorm (cf. Fig. 34).

F

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often quite accurately-the normal development of zygotic embryos. Therefore, those relatively rare cases of natural, nonsexual ( adventive ) embryonic development that are seen as apomictic, polyembryonic, and parthenogenetic development and that have been studied in such plants as Hieracium megatum, Allium odorum, Citrus sp., and Mangifera indica (see Maheshwari and Sachar, 1963), may now, under appropriate physiological conditions, find their counterparts in the freely growing cells of almost any angiosperm. The above events having been described by reference particularly to the work of this laboratory, brief reference may now be made to numerous other contributions of a similar kind. VIII. Other Relevant Studies on Embryogenesis and Morphogenesis

Many other morphogenetic studies on higher plants have been made; particular attention should be paid to the literature on vegetative propagation (Priestly and Swingle, 1929; Boulliene and Went, 1933). Inasmuch as localized morphogenetic events occur in such highly organized structures they are not yet amenable to interpretation in terms of the stimuli that determine the behavior of the single formative cells which, as initials, give rise to root or shoot primordia. Similarly there are many published examples of the growth of substantial tissue explants, or portions of isolated organs, of proliferated callus within which a measure of differentiation and morphogenesis occurs. This may range from the presence of disconnected tracheid-like elements, formed within the callus mass, to the origin of adventitious buds or roots within a proliferation that may itself have originated from cells of a cambial region. Again it is hard to specify what caused the initiating cells to behave differently from the rest of the proliferated, cultured mass of cells. Wetmore has interpreted the development of vascular strands within a callus mass in terms of stimuli supplied by sugar on the one hand and by locally applied auxin on the other; these ideas also received support from grafting experiments performed on masses of callus (Wetmore, 1959). The control of morphogenetic development within a callus mass has been attributed by Skoog (Skoog and Miller, 1957) to a balance of formative substances; these are recognized as auxins ( indoleacetic acid) on the one hand and kinins (of which kinetic or 6-furfurylaminopurine is a synthetic prototype) on the other. Actively growing cultures (e.g.,

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tobacco pith) have responded to different proportions of exogenous auxin and kinin by forming roots under one set of conditions and shoots under others; with the appropriate balance both roots and shoots may occur. Also, in the work of Paulet and Nitsch (1963) the effects of balanced nutrient treatment or stimuli in the formation of buds on a callus of Nicotiana suavedens have been noticed. Paulet and Nitsch (1964) also obtained flowers on tissue explants of Cichorium intybus, although the roots from which the explants were obtained had to be vernalized so that flowers would form. Previously Aghion (1962) had described a case of flower formation on pieces of aseptically cultured tobacco tissue. All such examples in which the formative cells respond while they are present within a large tissue mass which supplies both endogenous nutrients and stimuli are not discussed in this review, which is concerned primarily with the dramatic growth and development which is now traceable to free cells. No account of embryogenesis in free cells should ignore the large literature on the technique of embryo culture and its significance (La Rue, 1936; Rappaport, 1954; Narayanaswami and Norstog, 1964). Embryo culture of orchids (which owed much to the pioneer work of Knudsen in 1922) is a special case of the adaptability of primitive embryos of parasitic or semiparasitic plants to external nutrients. By contrast, the more general experience is that embryos that are still globular are difficult to grow in isolation so that it is noteworthy when this is achieved ( Raghavan and Torrey, 1963). Embryos with preformed cotyledons can usually be cultured. Hence, young cotyledons may have some formative as well as nutritional role (Haccius, 1956). In the light of this the behaviour of isolated free cells is the more remarkable. Embryos which have suspensors (e.g., Capsella) may need them to make use of the nutrients in the ovule; embryos which lack a suspensor must be able to circumvent its normal role. Observations made as early as those of van Tieghem in 1873 and as recently as those of Haccius in 1963 suggest that experimentally injured portions of embryos often respond by forming new embryos. Embryos which are subject in the ovule to inhibiting substances, or which abort for a variety of reasons, or which become dormant, may be particularly responsive to growth factors when they are isolated; it was, in fact, a situation of this sort that led Blakeslee and van Overbeek to their experiments with isolated, immature Datura embryos grown on a medium containing coconut milk (van Overbeek et al., 1941). Tukey (1933, 1944)

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used similar devices to culture immature cherry and peach embryos. Thus, the extensive literature on embryo culture shows that preformed isolated embryos can be grown under proper nutritive conditions, though their response varies with their development. Paradoxically, it is now easier, as shown above, to cause isolated cells of some plants to behave like zygotes than it is to isolate and grow their zygotes. Thus, many different laboratories have now contributed to work of this sort in ways that should be summarized. The ability of nucellar cells to generate nucellar embryos, as in Taraxacum Citrus and other plants, prompted work by Maheshwari and Ranga Swamy (1958) and by Ranga Swamy (1961); “pseudo bulbils” and the eventual formation of embryos from the nucellus of Citrus were described. Later Norstog (1965) described the formation of embryoids in Zamia integrifoh from cells of the megagametophyte and suggested that this might be fostered by a higher phosphate concentration in the medium. The Botanical Laboratory at the University of Delhi, led by Professors Maheshwari and Johri, has pioneered in the establishment of cell and tissue cultures from the reduced embryos of parasitic of semiparasitic plants, and some of these cultures have culminated in embryonic forms (Maheshwari and Baldev, 1961; Johri and Singh Bajaj, 1962, 1963, 1964). This laboratory has also reported the formation of embryonic forms on seedlings of Anethum, another member of the Umbelliferae (Johri and Sehgal, 1965). In another laboratory a callus derived from the more complex embryos of Cichorium endiva has given rise to cell suspensions from which small plants have been produced ( I . K. Vasil et al., 1964). By nutrient imbalance young seedlings of Oenanthe sp. responded to an excess of glycine by the neoformation of embryonic forms as described by Miettinen and Waris (1958) and Waris (1959, 1962) in Finland, Other published accounts of embryogenesis and morphogenesis in cells and tissue explants of the carrot have appeared (Reinert, 1959; Kato and Takeuchi, 1963; Halperin and Wetherell, 1961). Though the experimental details and responses to them differ somewhat from one investigation to another, the essential point remains: Potentially any living cell of the carrot plant can be caused, by appropriate exogenous stimuli, to grow in an organized embryonic way. Tobacco, i.e., N . tabacum (Haccius and Lakshmanan, 1965); Datura anthers (Guha and Maheshwari, 1964); leaf cells of Macleaya (Kohlenbach, 1965); and certain medicinal plants, e.g., Digitalis, Urginea, and

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Pupaver (Staba et al., 1965) have all yielded callus, or isolated cells, which in varying degrees have simulated in culture an embryonic type of development. In addition, proliferations on the cotyledons of Biota orientalis, an ornamental conifer, have also yielded embryo-like structures (Konar and Oberoi, 1965). The work of Konar and Nataraja (1964, 1965a,b) has produced evidence of adventive embryogeny in Ranunculus sceleratus. Embryo-like forms have originated from a callus developed on flower buds (Konar and Nataraja, 1964); free cells derived from this callus (Konar and Nataraja, 1965a); and, repetitively, from epidermal cells on plantlets which in turn had developed from cells (Konar and Nataraja, 1965b). In the last-mentioned case, the development from single, but attached cells quite faithfully recapitulated early embryogeny; almost exactly parallel observations on carrot have been made by Ann E. Kent. Enough has been said, therefore, to show that the conclusions drawn from experiments in this laboratory are substantiated by the work of other investigators in many other centers. IX. Concluding Remarks

One should now assess the significance of this relatively new but actively developing line of work. This should be done with respect to its bearing upon general biology and also upon plant science. The implications for the interpretation of development for embryology, morphogenesis, and differentiation seem obvious. The integrity of the nuclear, genetically determined stock of information is preserved in the living cells of higher plants throughout the complex events of tissue and organ formation. The controls which determine the expression of this information, i.e., the way in which the cytoplasm transcribes the message it receives at various points during development, must therefore be extranuclear, even exogenous to the cells in question, for they are surely inherent in the environment in the plant body where the cell occurs. This is the point of saying that the cell in its responses during development is conditioned by extrinsic factors, whereas in its free state it is able to give full play to its intrinsic potentialities. Cells behave as they do in the plant body by virtue of “where they are,” not only because of “what they are.” It is, of course, a major problem of modern biology to determine how these effects are mediated. Current thinking gives credence to the view, inherited from Jacob and Monod, that a given part of the genetic mechanism transmits its message to the cytoplasm via a specific “messenger”

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RNA which, in turn, activates the synthesis of particular proteins that, as enzymes, give specificity to particular cells or organs. Even though the basic metabolic processes and events may need to be recapitulated in all living cells (e.g., those of glycolysis, pentose pathway, Krebs cycle, etc.), the precise means by which these are achieved may be specific for cells in a given situation; each major enzyme system may, as in the work of Markert (1963) on dehydrogenases, have a specific and different set of isozymes which are peculiar to a given organ. Although there is, as yet, little evidence on this point for plants, work in this laboratory, not here reported in detail (Steward and Barber, 1965), shows that different parts of the tulip plant may contain different complements of electrophoretically separable proteins and that a given class of enzymes (e.g., esterases) may be mediated by proteins that are characteristic of a given morphological setting. It is, however, difficult to believe that the major morphogenetic events are determined solely by such controls of virtually universal metabolic reactions. In the growth of cells to form organs and of free cells to form organisms, the resources at the disposal of these essentially totipotent plant cells are the variations that are possible in the ways by which they may grow by division and by enlargement and, having laid down a cellular pattern in this way, they may be caused to differentiate. I t is because the events of cell division and cell enlargement seem so subject to specific external, non-nuclear, epigenetic controls that the problems of development and morphogenesis in higher plants possess so much intrinsic interest for biologists generally. Morphogenetic stimuli (long and short days, high and low night temperatures, etiolation and light intensity, seasonal cycles, etc.) profoundly determine plant growth in organisms in which the living cells are essentially totipotent. This makes it all the more imperative that the factors by which the variables in question make their impact upon the growing regions in cells, and the means by which they work, should be elucidated. Modem plant cell and tissue cultures in systems in which the full range of developmental events occur under controlled conditions present unique opportunities for these problems to be studied. It is, however, already apparent that-as in all problems of organization-it is a synthesis of the information that accrues when a given system is understood from all points of view (experimental morphology, cell physiology, fine structure, biochemistry, metabolism, etc. ) that leads to understanding.

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It may well be that higher plants and higher animals, which in many ways have evolved different modes of life, may differ here also. There are those who hold that sequential and probably irreversible “aging of nuclei occurs as animal organs progress in their development. Even if this is so for animals (cf. King and Briggs, 1956), it can hardly be so for plants. In plants one should look to the cytoplasm, rather than solely to the nucleus, for the sequential changes that must accompany differentiation; and even these changes might be reversible in response to external stimuli. The differences between plants and animals may, however, be a matter of degree. When enough is known of the factors that limit the behavior of animal cells in culture, the potentially tighter restrictions which seem to determine their organ specificity may be unleashed and a greater measure of totipotency be expressed. In this context it is noteworthy that Stevens (1960) has shown that a mouse testicular tumor has the ability to produce in large number free-floating “embryoid bodies” which closely resemble 5-6 day mouse embryos. Thus, even in animals, situations may exist in which embryonic potency may be retained by cells other than the typical germ cells. The lesson to be learned from culture of free plant cells is, however, that any free cell provided with the appropriate conditions for its growth and with the right balance or sequence of exogenous growth-regulating substances may recapitulate development and, in doing so, retraverse the events of embryogeny, even without benefit of the environment of the ovule in which, as a sort of “nursing organ,” it is wont to occur. REFERENCES Aghion, D. (1962). Compt. Rend. 255, 993. Bidwell, R. G. S., Barr, R. A., and Steward, F. C. (19%). Nature 203, 367. Blakely, L. M. (1963). Ph.D. Thesis, Cornell University, Ithaca, New York. Blakely, L. M. (1964). Am. J. Botany 51, 792. Blakely, L.M., and Steward, F. C . (1961). Am. J. Botany 48, 351. Blakely, L. M., and Steward, F. C. (1984). Am. J. Botany 51, 780. Bouillene, R., and Went, F. W. ( 1933). Ann. Jardin Botan. Buitenzorg 43,25. Caplin, S . M., and Steward, F. C. (1948). Science 108, 655. Caplin, S. M., and Steward, F. C. (1952). Ann. Botany (London) [N.S.] 16, 219. De Ropp, R. S. (1955).Proc. Roy. Soc. B144, 86. Fitting, H. (1909). 2. Botan. 1, 1. Gautheret, R. J. (1938). Compt. Rend. 208, 118. Goldstein, J. L., Swain, T., and Tjhio, K. T. (1962). Arch. Biochem. Biophys. 98, 176. Guha, S., and Maheshwari, S. C. (1964). Nature 204, 497.

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Haberlandt, G . (1902). Sitzber. Akad. Wiss. Wien, Math.-Naturw. KZ. Abt. I. 1 3 1 , p. 69. Haberlandt, G. (1921a). Sitzber. Preuss. Akad. Wiss., Physik.-Math. Kl., p. 861. Haberlandt, G. (1921b). Sitzber. Preuss. Akad. Wiss., Physik.-Math. K1. p. 695. Haberlandt, G. ( 1 9 2 1 ~ )“Wundhormone . als Erreger von Zellteilungen.” Borntraeger, Berlin. Haberlandt, G. (1922). Sitzber. Preuss. Akad. Wiss., Physik.-Math. K1. p. 386. Haocius, B. (1956). Ber. Deut. Botan. Ges. 69, 87. Haccius, B. ( 1963). Phytornorphobgy 13, 107. Haccius, B., and Lakshamanan, K. K. ( 1965). Planta 65, 102. Halperin, W., and Wetherell, D. F. (1964). Am. J. Botany 51, 274. Harris, M. (1964). “Cell Culture and Somatic Variation.” Holt, New York. Israel, H. W., and Steward, F. C. (1966). Ann. Botany (London) [N.S.] (in press). Johri, B. M., and Sehgal, C. B. (1965). Nature 205, 1337. Johri, B. M., and Singh Bajaj, Y. P. (1962). Nature 193, 194. John, B. M., and Singh Bajaj, Y. P. (1963). In “Plant Tissue and Organ Culture-A Symposium” (P. Maheshwari and N. S. Ranga Swamy, eds.), p. 292. Intern. SOC. Plant Morphologists, Univ. of Delhi, India. Johri, B. M., and Singh Bajaj, Y. P. (1964). Nature 204, 1220. Kato, H., and Takeuchi, M. ( 1963). Plant CeU Physiol. (Tokyo) 4 , 243. Kent, A. E., and Steward, F. C. (1965). Am. J. Botany 52, 619. King, T. J., and Briggs, R. (1956). Cold Spring Harbor Symp. Quant. B i d . 2, 271. Knudson, L. (1916). N . Y. Cornell Mem. 9. Knudson, L. (1919). Am. J . Botany 6, 310. Knudson, L. (1922). Botan. Gaz. 73, 1. Kohlenbach, H. W. (1965). Planta 64, 37. Konar, R. N., and Nataraja, K. (1964). Phytomorphology 14, 558. Konar, R. N., and Nataraja, K. (1965a). Phytomorphology 15, 206. Konar, R. N., and Nataraja, K. ( 1985b). Phytomorphology 15, 132. Konar, R. N., and Oberoi, Y. P. (1965). Phytomorphofogy 15, 137. Krikorian, A. D. (1965). Ph.D. Thesis, Cornell University, Ithaca, New York. Lamport, D. T. A,, and Northcote, D. H. (1960). Nature 188, 665. La Rue, C. D. (1936). Bull. Torrey Botan. Club 63, 365. Maheshwari, P., and Baldev, B. (1961). Nature 191, 197. Maheshwari, P., and Ranga Swamy, N. S. (1955). Indian 1. Hort. 15, 275. Maheshwari, P., and Sachar, R. C. (1963). In “Recent Advances in the Embryology of Angiosperms” (P. Maheshwari, ed.), 11. 265. Intern. SOC. Plant Morphologists, Univ. of Delhi, Delhi, India. Mapes, M. O., Steward, F. C., and Kent, A. E. (1965). Plant Physiol. 40, Suppl., IXXVii.

Markert, C. L. ( 1963). In “Cytodifferentiation and Macromolecular Synthesis” ( M . Locke, ed.), p. 65. Academic Press, New York. Miettinen, J. K., and Waris, H. (1958). Physiol. Plantarum 11, 193. Miller, C. O., Skoog, F., Okumura, F. S., von Saltza, M. H., and Strong, F. M. (1955). J . Am. Chem. SOC. 77, 1392. Morel, G., and Wetmore, R. H. ( 1951). Am. J . Botany 38, 138.

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Muir, W. H., Hildebrandt, A. C., and Riker, A. J. (1958). Am. J. Botany 45, 589. Narayanaswami, S., and Norstog, K. (1964). Botan. Reu. 30, 587. NobCcourt, P. (1938). BuU. SOC.Botan. France 85, 1. Norstog, K. (1965). Am. J. Botany 52, 614. Paulet, P., and Nitsch, J. P. (1963). Bull. SOC.Botan. France 110, 361. Paulet, P., and Nitsch, J. P. (1964). Ann. Physiol. Vegetale 6, 333. Pollard, J. K., Shantz, E. M., and Steward, F. C. ( 1961). Plant Physiol. 36, 492. Priestley, J. H., and Swingle, C. (1929). U.S. Dept. Agr., Tech. Bull. 15, 1. Raghavan, V., and Torrey, J. G. (1963). Am. J. Botany 5, 540. Ranga Swamy, N. S. (1961). Phytomorphotogy 11, 109. Rappaport, J. (1954). Botan. Rev. 20,201. Reinert, J. (1959). Planta 53, 318. Robbins, W. J. (1922). Botan. Gaz. 73, 376. Shantz, E. M., and Steward, F. C. (1964). In “Regulateurs naturels de la croissance vegetale,” p. 59. C.N.R.S., Paris. Skoog, F., and Miller, C. 0. (1957). Symp. SOC.Exptl. Biol. 11, 118. Skoog, F., Strong, F. M., and Miller, C. 0. (1965). Science 148, 532. Staba, E. J,, Laursen, P., and Biichner, S. (1965). In “International Conference on Plant Tissue Culture” (P. R. White, ed.), p. 191. McCutchan Publ., Berkeley, California. Stevens, L. C. ( 1960). Deuelop. Biol. 2, 285. Steward, F. C. ( 1961). In “Growth in Living Systems” (M. X. Zarrow, ed.), p. 453. Basic Books, New York. Steward, F. C., and Barber, J. T. (1965). Am. J. Botany 52, 623. Steward, F. C., and Caplin, S. M. (1951 ). Science 113, 518. Steward, F. C., and Mapes, M. 0. (1963). 1. Indian Botan. Soc. 43A, 237. Steward, F. C., and Mohan Ram, H. Y. (1961). Aduan. Morphogenesis 1, 189-265. Steward, F. C., and Shantz, E. M. ( 1956). In “The Chemistry and Mode of Action of Plant Growth Substances” (R. L. Wain and F. Wightman, eds.), p. 165. Academic Press, New York. Steward, F. C., Bidwell, R. G. S., and Yemm, E. W. (1956). Nature 178, 734 and 789. Steward, F. C., Mapes, M. O., and Smith, J. (1958a). Am. J. Botany 49, 693. Steward, F. C., Mapes, M. O., and Mears, K. (195813). Am. J. Botany 45, 705. Steward, F. C., Shantz, E. M., Pollard, J. K., Mapes, M. O., and Mitra, J. (1961 1. In “19th Annual Growth Symposium” (D. Rudnick, ed.), pp. 193-246. Ronald Press, New York. Steward, F. C., Blakely, L. M., Kent, A. E., and Mapes, M. 0. (1963). Brookhaven Symp. Biol. 16, 73. Steward, F. C., Shantz, E. M., Mapes, M. O., Kent, A. E., and Holsten, R. D. (1964a). In “Regulateurs naturels de la croissance vegetale,” pp. 45-58. C. N. R. S., Paris. Steward, F. C., Mapes, M. O., Kent, A. E., and Holsten, R. D. (196413). Science 143, 20. Street, H. E. (1957). Biol. Rev. 32, 117. Svobodova, J. (1964). 10th Intern. Botan. Congr., Edinburgh, 1964 p. 485.

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Tukey, H. B. (1933). J . Heredity 24, 7. Tukey, H. B. (1944). Proc. Am. Soc. Hort. Sci. 45, 211. van Overbeek, J., Conklin, M. E., and Blakeslee, A. F. (1941). Science 94, 350. van Tieghem, P. ( 1873). Ann. Soc. Botan. Lyon 17, 205. Vasil, V., and Hildehrandt, A. C. (1965). Science 150, 889. Vasil, I. K., Hildebrandt, A. C., and Riker, A. J. (1964). Science 146, 76. Wadhi, M., and Mohan Ram, H. Y. (1964). Phyton 21, 143. Waris, H. (1959). Physiol. Plantarum 12, 753. Waris, H. (1962). Physiol. Plantarum 15, 736 Wetmore, R. H. (1959). Am. Scientist 47, 326. White, P. R. (1934). Plant Phydol. 9, 585. White, P. R. (1963). “The Cultivation of Animal and Plant Cells,” 2nd ed. Ronald Press, New York.

CHAPTER 6

GENETIC AND VARIEGATION MOSAICS IN THE EYE OF DROSOPHILA Huns Joachim Becker ZOOLOGIYCHES INSTITUT DEH

UNIVERSITAr

MUNCHEN,

MUNICH, GERMANY

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Genetic Mosaics and the Development of the Eye ...... A. The Mosaics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Development of the Eye . . . . . . . . . . . . . . . . . . . 111. Variegation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Zeste Variegdtion . . . . . . . . . . . . . . . . . . . . . . . . . B. Position-Effect Variegation ..................... C. The Pattern of Defects in an Eye Mutant . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155 156 156

157 161 161 163 168 169

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1. Introduction

Because of their unique advantages for fairly clearcut and simple analysis, the special types of differentiation shown by variegation mosaics in Drosophila are useful for clarifying general problems in differentiation. With the knowledge available concerning the phenotypes, characteristics, and location in the giant chromosomes of the various eye genes one can, from looking at mosaic patterns in adult eyes, deduce some events that have taken place during their embryology. A Drosophilu compound eye consists of roughly 25,000 cells, regularly arranged into about 800 ommatidia. The surface of the eye is an almost oval convex area with a reguIar pattern of hexagonal facets, each one 155

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belonging to one ommatidium. The color of a normal eye is dull red. In a mosaic eye there are regions that differ from one another in their color, or texture, or possession of ommatidia. These regions are arranged in characteristic patterns. The causes for these types of mosaicism can be placed in two categories. In the first category, the genetic mosaics, parts of the eye differ from other parts in their genetic constitution. These differences arise in early developmental stages as gene mutations or chromosome losses or somatic crossing over in cells from which the eye develops. The affected cell then hands down its newly acquired character to all its descendants, and a mosaic spot in the adult eye is the result; the size of the spot depends upon the number of cell generations between the occurrence of the genetic change and the completion of eye development. Even when induced these changes are infrequent enough that a mosaic spot usually represents the lineage of a single cell. In the second category, the variegation mosaics, mosaic areas often exhibit cell lineage patterns identical to those of genetic mosaics, but genetic changes of the types mentioned can safely be excluded. The changes in the cells that lead to variegation are still for the most part unknown and are the basic problem in the studies reported here. Genetic mosaics are one of our tools for investigating variegations; they will be discussed first. This will be followed by the information on the embryology of the eye that was gained from those experiments. Finally, several types of variegation will be described and their usefulness as a tool for the study of differentiation will be discussed. II. Genetic Mosaics and the Development of the Eye A. THE MOSAICS

The development of the fruit fly can be divided into six distinct stages. Inside the egg embryonic development takes pIace. It lasts about I day. The first larval instar, which hatches from the egg, also lasts about 1 day, as does the second larval instar, which moults from the first. The third larval instar moults from the second. It lasts 2 days and then forms a pupa. After about 4 more days the imago emerges. In the experiment to be described use has been made of the eye color gene white ( w ) and one of its alleles, white-coral ( t o c 0 ) . The gene is located close to the tip of the X chromosome. Flies carrying the allele w have white eyes, flies carrying wCohave coral-colored eyes, and flies

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heterozygous for both mutant alleles ( w / w c o )have eyes intermediate in color, i.e., light coral. When larvae with such a heterozygous constitution are treated with X-rays, mosaic spots arise in the eyes of the flies developing from these larvae. Most of these spots are so-called twin spots, one partner of the twin showing the white, the other showing the coral phenotype (Becker, 1956, 1957b). These spots can have arisen only as the result of somatic crossing over, a phenomenon first described by Stem (1936). If in one cell crossing over takes place between the kinetochore of the chromosome and the

W

W

WCO

W

w co

w co

FIG. 1. Schematic representation of somatic crossing over in the X chromosome of Drosophila melanogaster and the twin mosaic spot resulting from it. (From Becker, 1957a.)

white locus, each of the daughter cells will be homozygous for one of the mutant alleles. One will be W / W , the other one W ' ~ / W ~Both ~ . cells will form adjacent parts of the eye, and each part will show the color that corresponds to its genetic constitution (Fig. 1). After treatment of young larvae with X-rays spots are large but few; after treatment of older larvae spots are small but numerous. The size and shape of a spot, its location on the eye surface, and its position with respect to its partner have given some insight into the development of the eye (Becker, 1957a,b). B. THE DEVELOPMENT OF THE EYE Each eye develops as part of a so-called head anlage that first becomes visible toward the end of the embryonic stage. Two anlagen form the

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whole head of the fly, except for the mouth parts. One anlage forms the left half of the head and the other the right half. The mosaic spots found do not cover the whole eye. In the largest ones, a horizontal borderline going through the middle of the eye is rather conspicuous as part of a spot’s margin. In 17 of 23 spots with a size of

FIG. 2. Images of twin spots in the eyes were observed after irradiation of the embryos and after first and second instar larvae. (From Becker, 1957a. )

more than 220 ommatidia, this horizontal line was the upper borderline (in the case of spots in the lower half; Fig. 2 ~ or) the lower borderline of a spot (in the case of spots in the upper half). The other six spots stretch over both halves of the eye. For the most part, therefore, the cell lineages of the upper and the lower halves of the eye seem to be separated.

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In addition to this marked central borderline there are others, especially in the lower half of the eye. The positions of these borderlines on the eye surface become clearest if tracings of the mosaic patterns of lower eye halves are superimposed. For the present purpose the eyes selected were those in which the spots reached from the horizontal midline of the eye to the margin of the lower half of the eye (Fig. 2, u-i). Such a tracing is shown in Fig. 3a. The outlines of mosaic spots are bunched in

FIG.3. ( a ) Tracings of mosaic spots ( i n the lower half of the eye) which reach from the horizontal midline to the lower eye margin. ( 1 2 ) Schematic representation (derived from a ) of commonly found spot sizes, shapes, and predominant positions in the lower half of the eye (soIid lines) and of less commonly found spot limits in the lower and upper parts of the eye (hatched lines). (From Becker, 1957b.)

certain regions, and this bunching has been used for the scheme given in Fig. 3b, in which the sections average 40 ommatidia. Spots of the size of one single section are found predominantly when larvae are treated around the molt from the first to the second larval instar, as shown in Table I. It was estimated, therefore, that late in the first larval instar the presumptive eye area consists of about 20 cells, each giving rise to an eye section of an average of 40 ommatidia (yielding the total adult number of 800 ommatidia). The bunching of borderline suggests that at the end of the first larval instar each cell of the eye area seems to have been

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TABLE I NUMBEROF TWINSPOTSAND SINGLESPOTSFOUND IN FLIES AFTER IRRADIATION OF MOLTING SECOND INSTAR LARVAE ( 2 3Hou~s)a Number

Number of ommatidia per spot 3-4 5-8 9-16 17-32 33-64

2

Twin spots Number: 64 Percent:

-

Single spots Number: 161 Percent :

3 13 1.8 8.1

65-128

129-256 4 6.3

5 7.8

8 12.5

17 26.6

19 29.7

11 17.2

23 14.3

45 28.0

35 21.8

25 15.5

17 10.6

-

-

5 The spots are arranged in classes according to their size. The class limits take the growth of the anlage by cell divisions into consideration. Single spots from experiments in which only one of the two twin partners could be recognized morphologically.

assigned to formation of a definite section of the eye. This is not true for earlier stages. Spots two or more times larger than one of the sections schematically outlined in Fig. 3b can cover any combination of sections (Table 11).Therefore, they have not been assigned definite places within the eye area yet. TABLE I1 THERANDOMLY OVERLAPPING DISTRIBUTION OF 52 SPOTSTHE SIZEOF Two SECTIONS WITHIN SECTIONS I-VIIa Section I

a

/

1 1 1

9

I

111

I 8

IV

1 I

V

I 11

VI

1 I

vll

I

See Fig. 3b.

One can conclude, then, that at the end of the first larval instar the presumptive eye area consists of about 20 cells; at earlier stages, however, the eye area is the equivalent of only 10 cells, 5 cells, and so on. It seems, therefore, that the eye region has been assigned its definite place within the head anlage at this 20-cell stage or, at the latest, one cell division later, i.e., at the beginning of the second instar. This has been discussed in detail earlier ( Becker, 1957b) . The positions of twin spot partners relative to one another reveals the

6.

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orientation of cell divisions in the prospective eye area. Judging from the way the bar- and sector-shaped partners in the lower half of the eye lie next to each other (Fig. 2, u-f) they appear to have arisen from cell divisions oriented predominantly in a general upper-anterior to lowerposterior direction. The stretched shape of each sector in Fig. 3b suggests that afterwards, i.e., by the second and third larval instars, the orientation of cell divisions is roughly at right angles to the previous one. The twin spots in Fig. 2, g-k, obtained after X-irradiation of second instar larvae, show that this is true at least for the first two or three divisions after the change of mitotic orientation. The latter spots demonstrate also a regularly occurring size difference between twin partners. For 34 twin spots similar to those shown in Fig. 2, g and h, the average sizes of the proximal and distal twin partners were 14.0 and 34.5 ommatidia. Such a difference has not been found in the large spots obtained by irradiation of the first larval instar. The end of the first larval instar, then, is distinguished by a change in the orientation of cell divisions and, presumably, by the assignment of the prospective eye area to a definite region within the eye anlage. Along with this, a gradient for mitotic activity seems to be established with its center in the midposterior region of the eye. Spots “induced by irradiation at the end of the first larval instar exhibit characteristic shapes, as shown in Fig. 2, b-f, and Fig. 3. In the anterior region of the eye they are predominantly bar-shaped, on the lower eye margin they tend to be triangular, and in the lower posterior region they often have the shape of a bent bar, as for instance the white twin partner in Fig. 2e. The great significance of the end of the first larval instar in the development of the eye was inferred from the experiments with genetic mosaics. It will find further support in the analysis of different types of variegation. So far, it is not known if some morphological change is associated with the establishment of the eye anlage at the end of the first larval instar. It is possible that the connection which the head anlage makes with the larval brain at this stage is the origin of the changes. 111. Variegation

A. THE Zeste VARIEGATION Also on the X chromosome there is another eye color gene, xeste ( 2 ) . It is located close to the white locus and shows a peculiar interaction with it. Two of the mutant types which have been described as alleles of x,

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HANS JOACHIM BECKEX

zeste-light ( z ' ) , and zeste-mottled ( zm) (Becker, 1959), do not seem to be alleles at all but carry the unchanged z allele and in addition some change at the white locus (Judd, 1965). Since the nature of this change is not quite clear so far we will use the old designations, z1 and z". In these flies mosaic eyes are the rule (Becker, 1960). In zz the eyes are yellow with red areas. This indicates that these flies carry two types of eye cells. They are genetically identical but differ from each other in their ability to synthesize pigment. The red cells synthesize red pigment, the yellow cells do not. The areas differing in their color are identical in position, size, and shape to the mosaic spots described in Section 11 B, as can be secn in Fig. 4. The allele z", which has been used in Fig. 4,

FIG. 4. Left eyes of zfn males; red areas are shown in black. (From Becker, 1960.)

differs from zz only in its shade of yellow. The yellow and red areas are, therefore, derived from single cells, in which the ability or inability to synthesize red pigment has been fixed at a certain stage of development. The size of those parts tells that the stage of fixation is the end of the first larval instar. Whatever the nature of this fixation, it is stable for all subsequent cell divisions: Between the end of the first larval instar and the stage in which the pigments are synthesized each presumptive eye cell undergoes about ten cell divisions; after these no further divisions take place. We assume that until the end of the first larval instar all the cells are still potentially capable of forming the pigment and that many cells lose this potentiality at the critical stage. However, one can restore the pigment-forming ability by making use of a peculiar mutant in this neste series. A normal isoallele za is known.

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Flies carrying it have entirely normal eyes. Its mutant character becomes apparent only in heterozygotes with other mutant zeste alleles, e.g., in z'/za. Such flies have yellow eyes with occasional red spots. Such red areas can be made artificially by X-irradiating larvae during the second and third instar. This treatment induces somatic crossing over as described in Section I1 A, which, in turn, leads to homozygous za/za cells. The eye areas derived from them synthesize red pigment ( Becker, 1964). Homozygotization of this zeste allele is enough to restore to these cells the ability which they normally do not have. From this we conclude that the site at which zz cells are fixed in one of two alternative paths is the zeste locus itself or its immediate neighborhood, for instance the white locus. B. POSITION-EFFECT VARIEGATION

When the function of a gene depends upon other genes located near it on the chromosome, the phenomenon is called position effect. If, as in W+

I L Normal a

4

4

7 w258-18 W+

W+

L b

D

FIG. 5. ( a ) Origin of the translocation ~ 9 5 8 - 1 8 ;left, X chromosome; right chromosome IV. Arrows indicate break points. Heterochromatin of X and IV is shown in black. ( b ) Genotype and one characteristic phenotype of flies referred to in text.

our case, a gene is transposed by a chromosomal rearrangement from its normal location in euchromatin into heterochromatin or vice versa, variegation of the phenotype controlled by that gene is often the result. In one chromosome translocation, ZU"""'~, the iv locus is transposed close to the heterochromatin of chromosome IV as shown in Fig. 5a (Demerec and Slizynska, 1937). A fly which carries a mutant ic;-allele W, variegated eyes; besides the transposed w+-allele, i.e., ~ U " " ~ - ' ~ /shows f

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HANS JOACHIM BECKER

some sections of the eye are a shade of red slightly lighter than normal while the other sections are a very light shade of red (Fig. 5 b ) . The eyes of a w + / w fly are normal. Thus, when the w+-allele is adjacent to heterochromatin it does not function normally. Also, in such cases the appearance of the salivary gland chromosome at the locus of the gene can be seen to be changed. This change and the possible increase of nucleic acids at such a locus have been discussed by Schultz (1956). Recent microspectrophotometric studies of chromosomal replication and function under the influence of nearby heterochromatin have been reported by Rudkin ( 1965). The lighter and the darker colored parts in the eyes of w2558-18/w flies are again identical to the X-ray-induced mosaic spots (Section 11) in position, shape, and size. This indicates that again the end of the first larval instar is the stage when a “decision” is made. This is the decision concerning how strongly the w + locus of a cell will be influenced by the neighboring heterochromatin. In one cell it is influenced less, and the eye area derived from it is almost normally pigmented. In an adjacent cell it is influenced more strongly, and out of this cell develops a lightly pigmented eye area. Just as in zeste variegation, position-eff ect variegation depends upon a decision made at the end of the first larval instar. Both types of variegation also show a dependence on the temperature to which developing pupae are exposed (Chen, 1948), with the difference that with increasing temperatures eye colors become darker in position-effect variegation and lighter in zeste variegation. There is another major difference between zeste and position-effect variegations. In zeste a change in temperature changes the overall deepness of pigmentation without changing the numerical relationship between red and yellow eye sections. In positioneffect variegation a change in temperature does change the numerical relationship between the light and dark sections as shown in Table I11 (Becker, 1961). When w258-1a/w flies were raised at 25°C throughout all the developmental stages, 41.0%of all 2400 eye sections investigated (300 eyes with 8 sections each) were light. However, when the larval stages of flies with the same genotype were kept at 25°C and the pupae at 19”C, the frequency of light sections among 2368 sections investigated rose to 50.9%. The characteristic cell lineage pattern is not altered by changing the temperature during the pupal development. That is, it is still at the end of the first larval instar that the decision seems to have been made

6.

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165

whether a cell and its descendants can be light or dark. The descendants of each single cell, therefore, still react as a unit. There are some cells, 9.9% of all cells (50.9 minus 41.0) in the experiments described above, that behave differently from the others. The w + locus of these cells seems to be influenced by the adjacent heterochromatin in such a way that all their progeny can synthesize eye pigments almost normally when exposed to 25°C during the time of synthesis, i.e., during the pupal stage. When the temperature is only 19°C during the pupal stage, however, these same cells do not synthesize the eye pigments normally. TABLE I11 THEEFFECT OF TEMPERATURE DURING PUPALSTAGE ON THE FREQUENCY OF LIGHTSECTIONS IN POSITION-EFFECT VARIEGATION Temperature ( “C) during

Embryo and

Pupal stage

Light sections ( % )

No. of

larval stages

wm-ia/w

25

w658-18/w ys

25 25 25

25 19 25 19

41.0 50.9 20.6 33.0

300 296 546 274

Genotype

eyes

This suggests strongly that within one cell heterochromatin transforms the adjacent w + locus into a stable state which determines the ability of the cell’s descendants to form pigment. However, considering the whole cell population there are not only two alternative states into which the white locus is fixed, as might appear from the two alternative shades of pigmentation in the eye, but probably a continuous gradient of different states. Each state has its temperature threshold, above which a cell’s descendents form a dark section of the eye, and below which they form a light section. In summary, then, heterochromatin impairs the ability of the w + locus to function normally and consistently, but to different degrees. During pigment synthesis differences in temperature create the two alternative groups from the postulated multitude of cell types. The influence of temperature in other cases of position-eff ect variegation during embryonic development has been discussed by Schultz ( 1956). There are two further modifying factors that influence the state of “heterochromatization,” that is, the degree to which the normal function of the gene is impaired by the adjacent heterochromatin. One of them is the amount of heterochromatin present in the genome of a cell. Addi-

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HANS JOACHIM BECKER

tional heterochromatin, e.g., a supernumerary Y chromosome or even any part of it, is known to "suppress" position-effect variegation (Gowen and Gay, 1933). The phenotype of W " * / W / Y females appears to be more normal than that of W " ~ / W females. In our investigation we introduced the short arm of the Y chromosome (Y") only. It was attached to an X chromosome carrying the mutant w allele. The genotype of these flies was ~ 8 . 5 0 - 1/8w . Y y . Again, the cell lineage pattern of variegation was not altered, but when animals were raised at 25°C throughout all stages only 20.6% of their eye sections were light. When pupae were kept at 19"C, the frequency of light eye sections was increased to 33%. This tempera, is, in the abture effect is similar to the one shown on W " ~ * ' ~ / Wthat sence of extra Y heterochromatin. We conclude from these data that the amount of heterochromatin introduced by the Y" chromosome arm reduced the degree of heterochromatization of the W + locus. With the Y"-arm in the genome about an additional 20% (20.6 versus 41.0 and 33.0 versus 50.9) of the cells now reach at the end of the first larval instar the threshold at which almost normal pigment synthesis is possible. The basis of the modifying effect of extra heterochromatin on position-effect variegation is entirely unknown. The other factor that modifies the degree of heterochromatization of a transposed euchromatic gene is the distance of the gene from the nearby heterochromatin. The closer to heterochromatin the gene is located, the greater the probability that it will become strongly heterochromatized. This is demonstrated by a phenomenon known as the "spreading effect" (Schultz, 1941). In a translocation similar to the one discussed above, w158-11, the split gene (spZ+ ), which influences the facet arrangement on the eye surface, lies closer than the W + locus to the heterochromatin of chromosome IV. In the eyes of such flies the lightly pigmented sections always show the split phenotype, i.e., disarranged facets, and in addition some darkly pigmented sections show the split phenotype. That is, when one compares the loci w + and spl+, more sections of the eyes show the results of impairment of the split locus, the one that is located closest to heterochromatin. Since both types of mosaic spots show again the pattern typical for changes induced at the end of the first larval instar, it seems very likely that there has been simultaneous heterochromatization of both loci but to varying degrees. The notion that this phenomenon indicates heterochromatization of the spZ+ and w + locus at subsequent cell divisions (Schultz, 1958) seems less

6.

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167

likely. If this were so one would expect, for instance, in different chromosomal rearrangements the appearance of variegation types with all the different spot sizes, depending on the distance of the white locus from heterochromatin, The patterns of several stocks with different chromosomal rearrangements have been examined, and to all appearances there is no such variety of spot sizes. Besides the variegation with the cell lineage pattern, i.e., with spots of the sizes given in Fig. 3b, only one other kind of variegation is known, the so-called salt-and-pepper type. This type suggests that there is one other stage very late in development that is decisive for the function of the W + locus within one particular cell. This late stage might be identical to the period in the pupa during which temperature is known to exert its influence. In any case, in salt-and-pepper variegation the spots are too small to allow any cell lineage studies. TABLE IV THEPERCENTAGE OF C E L L LINEAGESPOTS I N Zm ( D A R K S P O T S ) , W8 5 8 - 1 8 /W (LIGHTSPOTS),AND THE EYEDEFECT MUTANTFOUNDWITHIN THE EIGHTSECTlONS OF THE LOWER HALFO F THE EYEf SEE F I G . 3h)

I1

111

IV

v

VI

VII

Total sectors covered by a VIII spot

12.3 16.7 17.5

11.7 17.5 15.3

13.3 15.1 12.6

11.1 12.5 11.2

11.7 10.2 8.4

15.8 6.5 7.9

11.7 5.0 7.2

Sector

Type of variegation p L , S ~ ~ - I S / ~

Lobe-like

I 13.3 16.6 19.7

316 1206 2100

There is another characteristic feature that distinguishes the type of position-effect variegation discussed above from the zeste-variegation. In both cases any of the eight sections of the lower half of the eye can show one or the other of the two alternative phenotypes. However, in z1 flies the choice between red and yellow is made randomly among the prospective eye cells at the end of the first larval instar, while in W ” ~ - ~ ~ flies / W slightly pigmented sections are not randomly distributed (Table IV ) but are more often found in the anterior region of the eye than in the posterior. Whether in this case W + loci in prospective anterior cells become more strongly heterochromatized than in prospective posterior cells, or whether the threshold for normal pigment synthesis is lower in the posterior region of the eye of the pupa than in the anterior, is an open question.

168

HANS JOACHIM BECKER

EYEMUTANT A dominant mutant which' changes the size and the shape of the eyes has been found and analyzed (Becker, 1957a,b). The genetic data available make it most probable that the mutant is an allele of Lobe, located on the right arm of chromosome 11. Only heterozygotes were investigated, since the mutant allele is linked with the lethal wing mutation CurZy (Cy). The expression of the mutant phenotype depends on the temperature at which the flies are reared. At 25°C eye and head size are strongly reduced. At 18°C the size of the head is normal, and 35% of the flies have normal eyes. In the other 65% only the upper halves of the eyes are normal, whereas the lower halves show reductions of varying degrees (Fig. 6 ) . The gaps in the eyes that do not carry ommatidia are filled with C. THE PATTERNOF DEFECTS IN

AN

FIG. 6. Left eyes of mutants with the Lobe-like eye defect, raised at 18°C. (From Becker, 1957b. )

apparently normal head cuticle. In the most extreme cases of reduction the whole lower half of the eye is absent (Fig. 6 e ) . The shape of this and all smaller gaps is strikingly similar to that of eye sections covered by mosaic spots. The smallest gaps are similar in shape and size to one of the eight sections of Fig. 3b. This indicates that in this mutant prospective eye cells at the end of the first larval instar are also different from one another, this time with respect to their ability to form ommatidia. All cells seem to multiply at a normal mitotic rate during the second and early third larval instars. Then the descendants of some go ahead with normal cell divisions that lead to the formation of ommatidia; the descendents of others, although in the eye region, behave like other epidermal cells and form head cuticle, often with interspersed bristles. Here, again, a difference between prospective eye cells becomes apparent. The difference most probably sets in at the same stage of development as the differences discussed in the two preceding sections, i.e., at the end of the first larval instar, and the character of each cell is handed down to all its descendants in a stable form. A common feature of these ~is the~ nonrandom ~ eye defects and the position-effect variegation of w

~

~

6.

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169

distribution of cells with different characters (Table IV). It is difficult to imagine that at the end of the first larval instars of normal flies eye cells become differently endowed with ommatidia-forming capabilities, a character which becomes apparent in the present eye mutant. It seems more reasonable to assume that it is the function of the mutant to reduce the ommatidia-forming capacity of late first instar cells. As discussed elsewhere (Becker, 1957b) an attempt has been made to explain this mutant action as influencing the size of the eye anlage which supposedly becomes established at the end of the first larval instar. IV. Conclusions

All three types of variegation discussed have several features in common. A differentiation takes place at a specific stage during development. It affects one specific character in the prospective eye cells, with respect to which all cells are normally alike. This differentiation is fairly stable. The character conferred upon a cell is “inherited by all its descendants. It becomes realized only after all mitotic activity has ceased, i.e., after about 10 cell divisions in the pupa at the time of pigment synthesis. This is the case in variegations where eye color genes were involved. Or the character becomes realized in the middle of the third larval instar, i.e., after about 5 cell divisions, when the subsequent divisions that lead to the formation of ommatidia set in. This is the case in the eye-reducing mutant. In spite of the stability of the cells’ characters, genetic changes (i.e., changes in the base sequence of DNA) as causes for the differentiations can safely be ruled out both in position effect variegation and in the eye-reducing mutant. The evidence against somatic mutations in the case of position-effect variegation has been surveyed in detail by Baker (1963). Only brief mention of each piece of evidence will be made here: (1) Extra Y chromosomes suppress variegation in some cases and enhance it in others. In cases where one additional Y chromosome suppresses variegation, a second one can enhance it again (Cooper, 1956). ( 2 ) Temperature influences the degree of variegation at a stage long after the mutational event would have to be assumed. ( 3 ) No changes caused by position effect are found in the germ line. ( 4 ) Certain pteridines, precursors of the red eye pigment, are found in excess when compared with amounts in normal flies or any of the known mutant alleles at the white locus (Baker and Spofford, 1959; Baker and Rein, 1962). ( 5 ) There is an effect of the parental genotype on variegation (Spofford, 1959, 1961), even of com-

170

HANS JOACHIM BECKER

ponents not passed to the fly being studied. ( 6 ) The nonrandom distribution of eye areas carrying one of the characters would call for a pattern of differential mutability in the anlage. Points ( 3 ) and ( 6 ) also can be applied to the eye-reducing mutant and are sufficient evidence against somatic mutations in their case. In the zeste variegation it is not possible to exclude genetic changes on the evidence given above. Points ( l ) ,( 2 ) , ( 3 ) (see Section IV), and ( 6 ) (see Table IV) do not apply; points (4)and ( 5 ) have not been investigated. The only evidence against somatic mutations in zeste variegation is the high frequency at which mutational events would have to occur. The question arises, then, as to ( a ) the site and ( b ) the nature of the differentiation. In the zeste variegation we concluded that the site is probably the chromosome section on which the gene is located. In the case of positioneffect variegation we have no evidence, but assumed also, that it is the white locus which is heterochromatized to various degrees, a state that remains stable from the time it has been created. There are numerous observations in other groups of organisms which support such an assumption; for instance the heterochromatization of a haploid chromosome set at the blastoderm stage in mealy bugs (Brown and Nur, 1964), or the heterochromatization of all but one X chromosome in early embryonic stages of mammals (Morishima et al., 1962). In both cases heterochromatization sets in at a definite stage during development; thereafter the euchromatic or heterochromatic state of a chromosome or chromosome section remains the same throughout the life of the individual. In both the zeste and the position-effect variegation, then, it is reasonable to suggest that the sites of differentiation are on the chromosomes. In the eye-reducing mutant we have no information on this problem. The nature of the determination of cells in the cases of variegation discussed is entirely unknown. The attempt to unravel it is particularly fascinating since all three types of variegation share with normal cellular differentiation the specific timing of stable changes in the states of genes that determines their functional potentialities. Most important, however, is that in variegation such changes apparently affect only single genes. In a normal process of differentiation,even the most simple one, where one type of cell changes into another type under the influence of only a single inducing stimulus, it seems certain that in most cases numerous genes are involved in the whole process. To the investigator of cellular differentiation this situation presents the same difficulty as a high rate of

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simultaneous or interdependent mutations would to someone wanting to discover the laws of heredity. Only the singleness and independence of mutations made the discovery of those laws possible. An analogous situation seems to be available in the phenomenon of variegation. The cells under discussion are alike with respect to all characteristics but one, and this one character goes back, possibly, to the functional state of one gene. An immense help for analyzing the differences in genetic states in the differing cell types would be a technique for effective somatic cell genetics. Even the propagation of somatic cells in insects still is not worked out sufficiently well. Until this becomes practical there are other promising ways to attack the problem, such as more quantitative studies on factors affecting variegation; studies on the simultaneous variegation of more than one gene; and the investigation of a case in which there seem to be germ line changes similar to the ones in prospective eye cells. REFERENCES Baker, W. K. (1963). Am. Zoologist 3, 57. Baker, W. K., and Rein, A. (1962). Genetics 47, 1399. Baker, W. K., and Spofford, J. B. (1959). Texas, Unio., Publ. 5914, 135. Becker, H. J. ( 1956). Drosophila Inform. Sew. 30, 101. Becker, H. J. (1957a). Verhandl. Deut. Zool. Ges., Hamburg, 1956, p. 256. Becker, H. J. ( 1957b). 2. Induktive Abstammungs-Vererbzcngslchre 88, 333. Becker, H. J. (1959). Drosophila Inform. Seru. 33, 82. Becker, H. J. (1960). Genetics 45, 519. Becker, H. J. (1961). Verhandl. Dtrrt. Zool. Ges., Bonn, 1960, p. 283. Becker, H. J. (1964). Naturwisscnschaften 51, 230. Brown, S. W., and Nur, U . (1964). Science 145, 130 Chen, S. Y. (1948). Bull. Biol. France Belg. 82, 114. Cooper, K. W. (1956). Genetics 41, 242. Demerec, M., and Slizynska, H. (1937). Genetics 22, 641. Gowen, J. W., and Gay, E. H. (1933). Proc. Natl. Acad. Sci. U.S. 19, 122. Judd, B. H. (1965). Proc. 11th Intern. Congr. Genet., The Hague, 1963 Vol. 1, p. 3. Pergamon Press, Oxford. Morishima, A., Grumbach, M. M., and Taylor, J. H. (1962). PTOC. Natl. Acad. Sci. U . S. 48, 756. Rudkin, G . T. (1965). Proc. 11th Intern. Congr. Genet. The Hague, 1963 Vol. 2, p. 359. Pergamon Press, Oxford. Schultz, J . ( 1941 ). Proc. 7th Intern. Congr. Genet., 1939, J. Genet. ( S u p p l . V o l . ) p. 257. Schultz, J . ( 1956). Cold Spring Harbor Synzp. Quant. Biol. 21, 307. Schultz, J. (1958). Ann. N . Y. Acad. Sci. 71, 994. Spofford, J. B. (1959). Proc. Natl. Acad. Sci. U . S. 45, 1003. Spofford, J. B. (1961). Genetim 46, 1151. Stem, C. (1936). Genetics 21, 625.

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CHAPTER 7

BIOCHEMICAL CONTROL

OF ERYTHROID CELL DEVELOPMENT Eugene Goldwasser ARGONNE CANCER RESEARCH HOSPITAL A N D DEPARTMENT O F BIOCHEMISTRY, UNIVERSITY OF CHICAGO, CHICAGO, ILLINOIS

I. Introduction . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . , . 11. Systeni under Study . . . . . . . . . . . . . . . . . . . . . . , . . A. Hornional Control of Erythropoiesis . . . . . . . B. The Nature of Erythropoietin . . . . . . . . . . . . . . . . . . 111. The Role of Other Hormones in Control of Erythropoiesis A. Pituitary Hormones . . . . . . . , . . . . . . . . . . . . . . . . . . B. Steroid Horniones . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Thyroid Hormones . .. . . . . . . . . . . . . . . . . . . . . .. .. IV. The Role of Nonhormonal Substances in Erythropoiesis A. Batyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . B. Cmobalt Salts . . . . . . . . . . . . . ...... . . . . . . . . . . . . .. V. Erythropoietin as Inducer of Red Cell Differentiation . . A. Regulation of Normal Erythropoiesis . . . . . . . . . . . . B. The Nature of the Erythropoietin Target Cell . . . . C. The Mode of Action of Erythropoietin . . . . . . . . . . VI. Models of Erythroid Differentiation . . . . . . . . . . . . . . . . VII. Summary . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. References . . . . . . . . . . . . . _.......... ......

173 174 175 177 182 182 182 183 184 184 184 185 185 187 188 200 206 206

1. Introduction

The task of the biochemist studying the process of differentiation is to determine the detailed molecuIar mechanisms that account for the appearance and disappearance of specific groups of synthetic capabilities 173

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and properties in differentiated cells. In general, the material of choice for this type of investigation has been the developing embryo, or cellular systems derived from it. Another approach to this problem is through the study of stem cell differentiation in mature animals, of which the development of hemopoietic cells is an outstanding example. The processes of formation of recognizably distinct circulating blood cells from more primitive precursor cells are, in a formal sense, the same as those in embryonic development. There is, in addition, one significant difference: the selfrenewal property of the stem cell. For this reason, any complete description of the mechanism of stem cell differentiation must eventually explain both differentiation and the steady-state maintenance of the undifferentiated stem cell population. It is not known whether all types of circulating blood cells derive from a single type or from separate types of precursor cells that are already differentiated, but not recognizably so. Still another difficulty is our almost complete ignorance of the morphological or biochemical properties of the precursor cells. Where the term “stem cell” is used in this article, it can be understood to mean only the ultimate primitive hemopoietic precursor cell or cells. I propose to discuss what is now known about the biochemistry of the control of erythrocyte formation. A closely related subject, the synthesis of hemoglobin in erythroid cells, is the topic of Marks’ chapter, in this volume. Although information on the biochemical properties of the red cell is immense, considerably less is known about the molecular mechanisms for the formation of erythrocytes from precursors. This is the problem with which our work is concerned. 11. System under Study

The numerous biochemical events that accompany the change from stem cells to erythrocytes are understood only in broad outline (Thorell, 1947; Grasso et al., 1963); the morphological changes during this process are well documented. Induction of erythroid differentiation in a stem cell results in its descendants’ eventually acquiring the ability to synthesize and store hemoglobin as their predominant product. During the same period of time the cells lose functions such as the ability to divide, to synthesize DNA and RNA, to maintain mitochondria and their complement of enzymes, and to synthesize proteins. The end product of the process is the erythrocyte with its long life span, designed specifically for oxygen transport.

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There are a number of reasons for choosing red blood cell formation as a model for the biochemical investigation of differentiation. Some of

them may be summarized as follows: a. There is a large body of data on the composition of erythrocytes, including various enzymes. b. Information about the physical and chemical properties of hemoglobin, the major component of the red cell, is abundant, as is knowledge of the biogenesis of both heme and globin. c. The genetic control of globin structure is fairly well understood. d. There is some understanding of the mechanisms involved in the regulation of erythropoiesis in the adult animal. This last point will be the focus of this article, within the larger context of how erythrocytes develop from nonerythrocytic precursors. CONTROL OF ERYTHROPOIESIS A. HORMONAL The basic assumption in the following account is that the hormone, erythropoietin, is the primary inducer of erythroid differentiation. Before examining the evidence for this assumption it might be helpful to outline the physiology and biochemistry of erythropoietin. The original description of a plasma-borne factor that is formed in response to anemic or anoxic stress and that causes increased red cell formation dates back some 60 years (Carnot and Deflandre, 1906). After a long period of dormancy the concept is now widely accepted and ampIy documented. Background information on erythropoietin may be found in a number of reviews and symposia (Grant and Root, 1952; Gordon, 1959; Jacobson et al., 1960; Jacobson and Doyle, 1962). Although the role of erythropoietin in stimulating red cell formation as a response to anemic or anoxic stress is clear, its possible regulatory action in the normal, unstressed animals is still not completely established. The assumption that erythropoietin does regulate normal erythropoiesis is supported by the following considerations: The regulatory mechanism proposed by Fried et al. (1957) states that, in situations where the oxygen requirement of the animal is greater than its capacity to provide oxyhemoglobin to the responsive tissue, erythropoietin production is increased. Conversely, when the ability to carry oxygen in the circulating blood exceeds the need of the responsive tissue, erythropoietin production decreases. In polycythemic, fasted, and hyperoxic animals the rate of erythropoiesis is appreciably lower than in the normal animal, in which, in turn, it is lower than in anemic or hypoxic

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animals. Because of the great difficulty in the quantitative estimation of small amounts of erythropoietin, determinations of erythropoietin titers lower than in normal plasma under conditions of suppressed erythropoiesis have not yet been reported. Qualitative information shows less erythropoietic activity in plasma from polycythemic rats than in plasma from normal rats ( Reichlin and Harrington, 1960). There is also evidence of an indirect nature which suggests that erythropoiesis in the normal animal is under the control of erythropoietin. In mice made polycythemic by injection of red cells, the rate of erythropoiesis falls to an extremely low level and there is no morphological evidence of red cell formation (Jacobson et al., 1957) or appreciable incorporation of labeled iron into circulating cells (DeGowin et al., 1962). Such plethoric mice, whose erythropoietin production is assumed to be negligible owing to absence of erythropoietin, can respond to small amounts of exogenously administered erythropoietin by increased formation of hemoglobin and reticulocytes. Three lines of evidence indicate that erythropoietin is present in normal plasma: ( 1 ) Polycythemic mice injected with normal mouse plasma show a definite increase in circulating reticulocytes compared to controls injected with saline solution (Jacobson et al., 1957). In addition, polycythemic rats show increased iron uptake into circulating red cells when given normal plasma in place of saline solution ( Reichlin and Harrington, 1960). ( 2 ) Rabbits immunized to human erythropoietin develop an anemia, which suggests that their own erythropoietin is being neutralized by the antibody (Garcia and Schooley, 1963). Similarly, mice given rabbit antierythropoietin develop an anemia (Schooley and Garcia, 1962). The conclusion drawn from these experiments with the antierythropoietin is that anemia develops probably because the antibody neutralizes the endogenous erythropoietin of the recipient animals. (3) Normal plasma samples from nonanemic rabbits were concentrated by a heat denaturation method based on that used by Borsook et d.( 1954). The data from three experiments (Goldwasser, Kung, and Shin, unpublished) given in Table I show that normal rabbit serum contains approximately 0.02 units of erythropoietin per milliliter.* This figure is in fair agreement with the value (0.04-0.14 units/ml ) calculated from indirect immunological measurements by

* The definition of a unit of erythropoietin used here is that of Goldwasser and White (1959) and is equivalent, in biological effect, to 5 bmoles of CoCI2. Assay methods, in uiuo, are usually based on measurement of incorporation of labeled iron into circulating red cells of fasted or polycythemic animals.

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Schooley and Garcia ( 1965). These conclusions must be considered tentative since we do not have sufficient data to rule out all influences on erythropoiesis, other than erythropoietin. TABLE I ERYTHROPOIETIN CONTENT OF NORMAL RABBITSERU& Experiment A B C

D

Preparation Normal Anemic Anemic Normal Anemic

rabbit serum concentrate rat serum rat serum concentrate rabbit serum rat serum Concentrate

Volume of original, ml

Total units found

175 10 10 175 10

4.5 12.0 10.2

12.6

,a The data from experiments B and C show 85% recovery in the concentration process. Correction of the amount found in A by 0.85 yields a total of 5.3 units/ 175 ml or 0.03 units/ml. Correction of experiment D by 0.85 yields a total of 14.8 units, of which 12 were added as anemic serum so that the remainder, 2.8 units, was due to the 175 ml of normal serum or 0.016 units/mI. The mean of these values is 0.02 units/ml of normal rabbit serum.

Recently Finne (1965) has reported that normal human urine contained a measurable amount of erythropoietin. Calculations from his data indicate a daily excretion of about 3 units. Although the role of erythropoietin in the regulation of normal erythropoiesis cannot be yet considered firmly established, the suggestive evidence is good enough to be accepted as a working hypothesis.

B. THE NATUREOF ERYTHHOPOIETIN Studies of the chemical nature of erythropoietin have been hampered by two major difficulties: inadequate methods of assay and inadequate sources of material for purification of the hormone. A discussion of problems of assay would be beyond the scope of this review. One should indicate, however, that the methods of assay in vivo, including that used in this laboratory (Fried et al., 1957), require too much material or too much time or are inaccurate. 1 . Sources of Erythropoietin Although erythropoietin appears to be formed in the kidney, there seems to be too little present in normal kidney tissue or in the kidneys of stimulated animals to make it a practical source of the hormone. The data of Contrera et al. (1965a) show that there is about 0.4 units/gm of kidney from anemic rats and none in kidneys from norma1 animals; the

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data of Kurnick, Dukes, and Goldwasser (unpublished) show that normal rat kidney contains approximately 0.25 units/gm. Kuratowska ( 1965) fractionated kidneys from normal, anemic, and cobalt-treated rabbits and found erythropoietic activity predominantly in the large particle (nuclear) fraction that was sedimented at 600 g. The activity was slight, unless the preparation was incubated with an aglobulin fraction from normal plasma. Somewhat similar experiments were reported by Contrera et al. (1965b) in which the activity was found in the “light mitochondria” fraction of kidney. This activity increased upon incubation with normal plasma. In contrast to the minor amounts of the hormone detectable in its apparent tissue of origin, the plasma of sufficiently anemic laboratory animals contains appreciable quantities. Under the proper conditions plasma erythropoietin titers may reach 10 or more units/ml. Some years ago, we developed methods for the large-scale preparation of partially purified erythropoietin from the plasma of sheep made anemic by treatment with phenylhydrazine (White et al., 1960). We obtained preparations with potencies in the range 5-20 units/mg of protein. That became the widely used standard A, which was the first standardized erythropoietin in general use since the discovery of the hormone. These preparations have been further purified ( Goldwasser et al., 1962a,b), and more recently we obtained fractions with potencies of about 3000 units/mg of protein, albeit in quite low yield (Goldwasser and Kung, unpublished). Since the starting plasma had a potency of 0.007 units/mg of protein, this represents a purification factor of approximately 400,000. Not enough of this high-potency erythropoietin has yet been accumulated for even the simplest tests for homogeneity. However, such a test might well be premature since on occasion we have achieved a still higher potency. The minute quantities of very highly purified erythropoietin that are at present obtainable leave the investigator almost no recourse but to depend on the potency, or specific activity, as an indication of heterogeneity. That is to say, only a preparation that cannot be further purified to a higher potency should demand any effort to determine whether it is homogeneous and, if so, what the chemical and physical properties are. Assuming that the pure hormone has a potency of 3000 units/mg of protein, and if we assume a 1% over-all yield from the original plasma, in order to get 10 mg (which would be enough for the partial physical and chemical characterization of the hormone), we would require plasma

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containing 3 million units. With present methods, this would correspond to approximately the yield from 300-400 liters of high-activity plasma. Erythropoietin can also be found in substantial quantities in urine of some severely anemic patients. However, it should be noted that methods of fractionation that have worked for plasma have not yielded equally good results when applied to urine concentrates. Experience in this laboratory has confirmed that of Graham et al. (1963), who found that urinary erythropoietin fractions appear to be less stable than plasma fractions with similar potencies. 2. Properties of Erythropoietin

The chemical properties of the hormone from the two sources appear to be different, but these differences may be more artifactual than intrinsic. The activity found in urine is almost certainly derived from the plasma, and since there is evidence that the activity in plasma does exist in more than one form (Goldwasser et al., 1962b), possibly as complexes with normally occurring proteins, the differences observed may reflect differences in the nature of the materials that can form a complex with erythropoietin. In the absence of pure erythropoietin for the determination of its physical and chemical properties, some information can be gleaned from the study of specific reagents upon the biological activity of the hormone. Erythropoietin activity in plasma is resistant for short times to temperatures as high as 100°C (Borsook et al., 1954). The activity is also stable at 0°C to 0.5 N perchloric acid for short periods of time (Goldwasser et al., 1957). Both of these observations provide a method for the preparation of small quantities of plasma erythropoietin concentrates, since they result in the removal of large amounts of the inactive proteins. Lowy and Borsook (1962) have shown that partially purified erythropoietin from rabbit plasma is inactivated by reaction with fluorescein isothiocyanate, which substitutes on phenolic hydroxyl groups ( tyrosine residues) and on free amino groups (lysine and/or the N-terminal residue of a polypeptide chain). They also showed that esterification with anhydrous formic acid (reaction with serine or threonine hydroxyl groups) caused no loss of activity. Substitutions with iodine, acetic anhydride, formaldehyde, and methanol, however, inactivated rabbit plasma erythropoietin (Lowy et al., 1960). W e have found that p-chloromercuribenzoate did not inactivate erythropoietin ( Goldwasser and Kung, unpublished observations ) . Although the presence of serine, threo-

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nine, or cysteine in erythropoietin is not ruled out, if they are present they either are not required for biological activity or are buried in the tertiary structure, Trypsin treatment causes loss of activity, indicating that erythropoietin is composed of a protein or polypeptide-containing lysine and/or arginine (Slaunwhite et al., 1957; Lowy et al., 1958). Erythropoietin also appears to contain a carbohydrate portion containing sialic acid. Rambach et al. (1958) showed that mild acid hydrolysis, under conditions known to remove sialic acid from glycoproteins, inactivated erythropoietin. More specific data from the experiments of Lowy et al. (1960) showed that sialidase inactivated both urinary and plasma erythropoietin preparations. Recent studies of inactivation by sialidase treatment ( Dukes and Goldwasser, unpublished) have raised new questions concerning the role of sialic acid in erythropoietin activity. The enzyme inactivates erythropoietin only when the hormone is assayed in vivo. If the assay is done in vitro by the marrow cell culture method, there is no loss of activity when either hemoglobin synthesis (Krantz et al., 1963) or stroma synthesis (Dukes et al., 1964) is measured. This is true whether the sialic acid is removed enzymatically or by acid hydrolysis. When tryptic hydrolysis is used, both in vitro methods show the loss of activity. These results might be interpreted as indicating that the in vitro assay methods respond not to erythropoietin but to some adventitious impurity. This seems very unlikely, since the in vitro responses agree quantitatively with the in vivo assay for preparations that differ by a factor of several hundred thousand in potency. It would seem highly improbable that the fractionation would concentrate two entirely different activities in parallel. There are two possible explanations for the divergence of results with sialidase-treated erythropoietin. Erythropoietin may be held in a complex form in which the sialic acid is a part of a hypothetical binding component. Removal of sialic acid dissociates the complex (or shifts the equilibrium toward dissociation). The resulting “free” erythropoietin is excreted or inactivated in the assay animal, while in vitro it remains in the culture medium and can act on its target cells. To test this we attempted to “revive” the activity of sialidase-treated erythropoietin by addition of possible binding components. None of the large number of plasma fractions tested had a “reviving effect. Alternatively, sialic acid may be an intrinsic part of the erythropoietin molecule and is required in the animal only for transport or for protection

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against excretion or inactivation; it is not needed for interaction with the cells or for its effect upon them. In this case, there would be no possibility of “reviving” the in vivo activity short of replacing the sialic acid on the hormone molecule. There is some indirect support for this view ( Schooley and Garcia, 1965). In the absence of the pure hormone, the only feasible methods for determining molecular weight are those that utilize measurements of biological activity. Inactivation by ionizing radiation has been used (Rosse et al., 1963) with somewhat conflicting results. With X-ray inactivation of crude human erythropoietin they found an apparent molecular weight of 66,000, but with high energy electron inactivation it was 27,000. They assumed that the difference was due to an asymmetric target with an axial ratio of 10, and that the molecular weight was 27,000. In work that is as yet unpublished, Hodgson (personal communication) used 6oCo y-irradiation of rabbit plasma erythropoietin and found a target molecular weight of about 68,000. In this laboratory (Goldwasser and Kung, unpublished) we determined the sedimentation coefficient of highly purified sheep plasma erythropoietin by the ultracentrifugal separation-cell method ( Yphantis and Waugh, 1956) and by sucrose-gradient centrifugation (Martin and Ames, 1961). Values obtained by the two methods agree fairly well at approximately 5 S. If we use the molecular weight of 27,000 we can calculate, from it and from the sedimentation coefficient, a frictional ratio of 0.6, which is a physical impossibility. The frictional ratio of an ellipsoid with an axial ratio of 10 would be 1.54 (Scheraga, 1961). A similar calculation with the molecular weight of 66,000 yields a frictional ratio of 1.01, indicating a nearly spherical molecule. All of these data are much too inaccurate to yield more than a rough estimate of the actual molecular size of the hormone, but at present it would appear to be of the order of 60,000-70,000. The fact that there is little or no species specificity in erythropoietin action suggests that the hormone is a poor antigen. When we attempted to prepare an antiserum to sheep plasma erythropoietin (Goldwasser et al., 1962a), the antibody activity we found was directed against some of the impurities in the antigen mixture, not against the hormone. On the other hand, Schooley and Garcia (1962), using crude human urinary erythropoietin as the antigen, did succeed in producing a rabbit antiserum capable of neutralizing the biological activity. Antiserum to human erythropoietin, they found, can neutralize rat, rabbit, mouse, sheep, and human erythropoietin (Garcia and Schooley, 1963). Although it

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might seem desirable to use the specific antibody as an aid in purifying the hormone, in developing an immunoassay for erythropoietin, and in localizing the site of erythropoietin production, the fact that the original immunizing antigen was highly impure would severely limit its use for these purposes. I doubt whether the use of absorption techniques to purify the antibody would overcome this disadvantage. The implicit assumption that the only new antigenic component arising because 01 the anemia is erythropoietin has never been put to a test. The antibody however, has been used to great advantage in the study of erythropoietin action (Schooley and Garcia, 1962, 1965; Schooley, 1965) and also to show that angiotensin (Fisher and Crook, 1962) and ceruloplasmin (Hatta et ul., 1962, 1963) are not identical with erythropoietin. Ill. The Role of Other Hormones in Control of Erythropoiesis

A. PITUITARY HORMONES There is now fairly wide agreement that there is no specific pituitary hormone that regulates erythropoiesis. The actions of such hormones as corticotropin in increasing the red cell mass (Garcia et al., 1951) are understood to be indirect, probably via the erythropoietin mechanism. The apparent anemia resulting from hypophysectomy is considered to reflect the lowered metabolic activities of an animal without a pituitary, not the absence of a pituitary erythropoietic factor. The recently reported effect of prolactin in increasing the rate of erythropoiesis in nonlactating mice (Jepson and Lowenstein, 1965) may also be an indirect effect, although experimental tests of this interpretation have not yet been published.

B. STEROIDHORMONES It has long been known (Crafts, 1941; Volmer and Gordon, 1941) that testosterone causes an increase in red cells. Recent evidence indicates that this effect is probably mediated through the erythropoietin mechanism. Fairly large amounts of testosterone cause increased erythropoiesis in plethoric mice (Fried et ul., 1964; Naets and Wittek, 1964), and a similar effect was found with the synthetic androgen, nandrolone phenylpropionate (Gurney and Fried, 1965a). Although it first appeared that the androgen acted to potentiate the effect of erythropoietin on its target cells (Naets and Wittek, 1964; Gurney and Fried, 1965a), more recent experiments indicate that androgen treatment increases a plasma eryth-

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ropoietic activity that may be erythropoietin (Fried and Gurney, 1965a,b). Janda et al. (1965) have shown that cobaltous ions (see below) and testosterone act synergistically when the androgen is given to mice 2-3 days before the cobalt and suggest that, while both may cause an increase in circulating erythropoietin, this increase may arise by way of different mechanisms. Since testosterone is known to increase kidney growth (Selye, 1939), possibly by increasing the mitotic rate of some kidney cells, it may well be that the synergism is the result of more potential erythropoietin-producing cells that have been stimulated to synthesize erythropoietin by cobalt. If this were the mechanism, one would expect to see the synergism abolished by a mitotic inhibitor such as colchicine. In contrast to androgens, estrogens cause a decrease in erythropoiesis. As little as 2 pg of estradiol given to a male rat can significantly lower the rate of hemoglobin synthesis (Dukes and Goldwasser, 1961). The data suggested that estradiol can act on the target tissue of erythropoietin to interfere with erythroid digerentiation; however, the possibility still exists that estrogens inhibit the formation of erythropoietin in a manner reciprocal to the effect of androgens. Studies of the effect of adrenal cortical steroids (Fruhman and Gordon, 1956) have shown that hydrocortisone increases erythropoiesis. Fisher and Crook (1962) found that small amounts of corticosterone, ll-dehydrocorticosterone, and hydrocortisone given to hypophysectomized rats stimulated the incorporation of radioiron into circulating red cells, whereas aldosterone and large amounts of hydrocortisone had no effect. It is not yet certain whether those cortical steroids that stimulate erythropoiesis do so by directly increasing the erythropoietin level, since they also increase the metabolic rate of hypophysectomized animals. C. THYROID HORMONES Information on how thyroxin and triiodothyronine increase erythropoiesis (Fried et al., 1957; Meineke and Crafts, 1959; Fisher and Crook, 1962) is also incomplete. There are indications that thyroid hormones may act indirectly by increasing the metabolic rate, as does dinitrophenol (Fried et al., 1957): There has been, as yet, no direct demonstration of increased circulating erythropoietin in the treated animals. It would be of interest to determine whether antibody against erythropoietin would abolish the effects of testosterone, cortisone, and thy-

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EUGENE GOLDWASSER

roxin, in which case the direct participation of erythropoietin in the action of these hormones would be on a firmer basis. IV. The Role of Nonhormonal Substances in Erythropoiesis

A. BATYLALCOHOL There have been reports of the effect of nonhormone lipids, particularly batyl alcohoI, on erythropoiesis (reviewed by Linman and Bethell, 1960; Linman and Pierre, 1962) that suggested the following conclusions: There are two erythropoietin factors, a thermolabile protein that influences hemoglobin synthesis, and a thermostable lipid that influences the rate of erythroid cell division, The lipid factor administered to animals by itself caused an increased number of circulating erythrocytes, smaller than normal and with a shorter life span, but no increase in the amount of circulating hemoglobin. These contentions, however, have not been confirmed, and there is a brief published account of a failure to reproduce the results (Evenstein et al., 1958). Furthermore, lipid-free erythropoietin concentrates can cause an increase in the total amount of circulating hemoglobin as well as increases in the number of reticulocytes and red cells (Gurney et al., 1961). In addition, Gurney (personal communication) failed to find a reticulocytosis in polycythemic mice treated with batyl alcohol.

B. COBALTSALTS It has been known (Waltner and Waltner, 1929) that cobalt salts can cause increased red cell formation, but the detailed mechanism of this action is not completely established. Cobaltous ions are thought to induce the formation of erythropoietin (Goldwasser et d.,1957, 1958); however, this point will not be definitely settled until the hormone is purified and shown to be the same whether from plasma of anemic or of cobalt-treated animals. Evidence concerning the reactivity of active plasma from cobalt-treated animals with erythropoietin antibody would be helpful in this context. Assuming that erythropoietin production is stimulated by cobalt, another interesting problem arises. What is the mechanism by which cobaltous ions act on the site of erythropoietin production? Cobalt has been shown to inhibit glycine incorporation into heme (Laforet and Thomas, 1956) and to dissociate heme and globin synthesis in the marrow by inhibiting the former (Morel1 et al., 1958). It is possible that the effect of cobalt on erythropoiesis is due to a specifically localized anoxia that

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causes increased erythropoietin formation. The possible role of cobalt as a constituent of erythropoietin has not been entirely excluded, but we failed to find any label in active fractions of plasma from animals injected with 60C0C12.Other data show that vitamin B12 does not increase erythropoiesis in fasted rats, making it unlikely that cobalt salts act via this compound. V. Erythropoietin as Inducer of Red Cell Differentiation

A. REGULATIONOF NORMAL ERYTHROPOIESIS The suggestion that erythropoietin is directly concerned with initiation of normal red cell differentiation was first made by Alpen and Cranmore (1959) and by Erslev (1959). Data in support of this suggestion have come from several other laboratories. Among the more compelling arguments for assigning to erythropoietin the role of primary inducer of erythroid differentiation are those derived from the experiments of Jacobson et al. ( 1957, 1961). As noted earlier, artificially polycythemic mice can be kept devoid of any recognizable erythroid cells in their hemopoietic tissues, and of circulating reticulocytes, for as long as they are kept plethoric. Regardless of the duration of the plethoric state, the maximum number of circulating reticulocytes, after erythropoietin administration, appears at the same time ( 3 days). These observations suggest that in the absence of red cell formation, after all previously induced cells have progressed to the erythrocyte stage, the stem cells can be kept in a quiescent state. In this state they remain capable of responding in a normal fashion to the presence of erythropoietin by eventually giving rise to erythrocytes. It is true that the hemopoietic tissues in the plethorasuppressed mouse contain a number of different types of cells that are not identifiably erythroid and that there are no morphological criteria for stem cells, so that the “quiescent” cells may represent an already differentiated population, capable of becoming erythroid cells only when erythropoietin is present. Bruce and McCulloch (1964), in fact, have suggested that this is the caye after studying the time course of endogenous erythropoietin action on the number of colony-forming cells in the spleen and marrow of mice. This problem will be discussed in a later section; for the present it will suffice to stress our ignorance regarding the nature of the target cell for erythropoietin. In an operational sense there is little difference between an unrecognizable “erythroid cell becoming a recognizable erythroid cell under the influence of erythropoietin, and an

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unrecognizable “stem” cell becoming a recognizable erythroid cell under the same influence. The question of the nature of the stem cell is, of course, an important one, and will have to await the application of new techniques before it can be answered. The presence of erythropoietin in animals making red cells under normal conditions also provides supporting evidence for its roIe as a primary inducer. Other information provides negative evidence. Erythropoietin does not appear to act upon nucleated erythroid cells or upon reticulocytes ( Erslev, 1964). Data from this laboratory (Goldwasser et al., unpublished), seen in Table 11, show clearly that, while rat reticulocytes can take up labeled TABLE I1 LACKOF EFFECTOF ERYTHROPOIETIN ON RETICIJLCCYTES~ Control Incorporation of glycine-2-14C into heme Incorporation of glucosamine-lJ4C into cells

Erythropoietinb

14,300 (+- 3400) 15,400 (+- 1200) 1890 ( & 150) 2200 (+- 215)

a Cell suspension consisted of 2.2 x 109 total cells per ml, from phenylhydrazinetreated rats, containing 7.7 x 108 reticulocytes per ml. Medium was 50% newborn calf serum, 50% NCTC 109. Cells were preincubated for 2.5 hours without erythropoietin or label, washed, resuspended, then incubated 21 hours with the hormone and labeled precursor. Numbers in parentheses are standard deviations of the mean. b Measured in 0.2 units.

glucosamine and glycine readily, erythropoietin has no effect on these processes. In marrow and spleen cells, however, the same processes are markedly affected by erythropoietin, as will be discussed in a later section. Other studies in vitro also support the concept of erythropoietin as primary inducer. Marrow cells in culture lose their capacity for heme synthesis in the absence of added erythropoietin (Krantz et al., 1963); in its presence this synthetic capacity is maintained for some time. This, too, will be discussed subsequently. Whether red cells newly formed under the influence of erythropoietin are normal erythrocytes is still not entirely settled. There have been reports (Stohlman, 1961a,b; Brecher and Stohlman, 1961; Borsook et al., 1962; Borsook, 1964) that red cells and reticulocytes formed in response to severe anemia (large amounts of erythropoietin) have a greater diameter and are shorter-lived than normal. This suggested that erythrocyte development is more rapid under conditions of high erythropoietin concentration and, possibly because one or more mitoses are by-passed, the

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resulting cells have not had time to attain their final (smaller) size. Changes in the red cell membrane also accompany anemic stress (Hillman and Giblett, 1965). Whether this is also true with physiological concentrations of erythropoietin has not been determined. If so, then obviously, erythropoietin cannot be the agent for physiological regulation: Conversely, if it does act in the normal process, the resulting red cells must be of normal size and life span. Some recent data (Ito and Reissmann, 1965) show that protein-depleted rats can be maintained in normal steady state erythropoiesis with 1.8 units of erythropoietin per day and that the erythrocytes have a normal life span. For the purposes of this essay, I will continue to assume that the hormone does regulate normal red cell formation and will avoid the complications introduced by nonphysiological amounts of the hormone. B. THE NATUREOF

THE

ERYTHROPOLETIN TARGET CELL

As was indicated earlier, an important problem in the study of erythropoietin-induced differentiation centers about the target cell for the hormone. It is outside the scope of this article to discuss the enormous (and often contradictory) body of work on the nature and behavior of stem cells, but since our postulate has been that erythropoietin acts upon stem cells, whatever they may be, some mention must be made of the problem. There have been only a few experimental approaches to determine what type of cell is acted on by erythropoietin. Takaku et al. (1964) found that some degree of cell specificity was involved in the erythropoietin stimulation of stroma synthesis (see below), Mixed populations of rat marrow cells and polycythemic mouse spleen cells respond to the hormone, but peritoneal granulocytes do not. Marrow cells separated into five crude fractions in a polyvinyl pyrollidone gradient showed the greatest erythropoietin effect in the fraction that contained the highest proportion of immature cell types. This method of cell fractionation has too little resolving power to accomplish the task of separating out the target cells, but the small degree of success obtained with it, and the development of newer techniques such as those involving continuous gradient centrifugation (Anderson, 1962), stable flow electrophoresis and sedimentation (Mel, 1964), and separation methods based upon volume difference ( Fulwyler, 1965), make it clear that the problem is potentially soluble. The indirect experimental approach, such as that used by Bruce and

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McCulloch (1964), based on the study of competing pressures for differentiation and self-renewal, has suggested that the cells acted upon by endogenous erythropoietin are already differentiated and not kinetically identical with what are generally assumed to be stem cells (i.e., cells capable of both differentiation and self-renewal). On the other hand, Liron and Feldman (1965) found that when polycythemic mice were used for the assay of colony-forming cells (stem cells) by the method of Till and McCulloch (1961) there was a reduction in the number of erythroid colonies and a concomitant increase in the number of granuloid colonies. When erythropoietin (as anemic plasma) was given, the suppression of erythroid colony formation was reversed (Bleiberg et al., 1965). These findings suggested that the same cells gave rise to one or the other type of colony depending on the stimuli that were present in the animal. Unfortunately, the change in the number of colonies of the granuloid type was too small to allow an unequivocal interpretation. C. THE MODEOF ACTION

OF

ERYTHROPOIETLN

1. Biochemical Actions in vivo Several studies of biochemical effects of erythropoietin in vivo have been reported. In general, these have all demonstrated an increase, in the hematopoietic tissues, of one or more indicators of cellular activity such as DNA synthesis (Rambach et al., 1957; Linkenheimer et al., 1959; Kurtides et al., 1963), RNA synthesis (Pieber-Perretta et al., 1965), RNA polymerase, DNA polymerase, thymidylate kinase, and 8-aminolevulinic acid ( ALA) dehydrase (Fischer, 1962). The increase in ALA dehydrase was shown not to be due to an activation of a previously formed proenzyme (Cooper and Gordon, 1964).

2. Biochemical Actions in vitro Because of the complexity of the erythropoietic system in vivo, interpretations of data concerning, for example, the effects of erythropoietin in elevating the levels of enzyme activity are not without hazard. There are no simple methods of dissociating possibly indirect and nonspecific effects of the hormone from what might be direct and specific ones. This difficulty may be exacerbated by the necessary use of impure preparations of the hormone. For these reasons (and other obvious ones) much effort has been spent in the development of a system that can respond to erythropoietin in vitro. (The phrase in vitro is used here in the sense of culture outside the whole animal, not “cell-free.”)

7.

ERYTHROD CELL DEVELOPMENT

189

Some marrow cell culture methods have used morphological and/or autoradiographic techniques to evaluate the intactness of erythroid function and effect of erythropoietin (Lajtha and Suit, 1955; Suit et al., 1957; Astaldi and Cardinali, 1959; Rosse and Gurney, 1959; Berman and Powsner, 1959; Matoth and Kaufmann, 1962). Others have measured the uptake of labeled iron into marrow cells (Erslev and Hughes, 1960; Erslev, 1962, 1964) or incorporation of glycine (Powsner and Berman, 1959) or iron (Korst et al., 1962) into the heme fraction of marrow cells. Most of these studies showed that erythropoietin could stimulate increased iron uptake and heme synthesis, although one such study (Thomas et al., 1960) showed no effect of the hormone. We could demonstrate an erythropoietin effect on marrow cells in vitro (Krantz et al., 1963) by measuring the rates of heme synthesis at intervals after erythropoietin introduction, and with different hormone concentrations. Under these conditions there was a dose-response curve similar to that found with the in vivo assay performed with erythropoietin (in the form of anemic rat plasma or of a highly purified fraction). With partially purified fractions of relatively low specific activities the situation was different, as indicated in Fig. 1. In this experiment, a fraction with a potency of about 2 unitslmg caused a marked inhibition of heme synthesis at levels of hormone above 0.5 units/ml. This depression must have been due to some nonspecific inhibitory substance in the crude erythropoietin fractions which was lost on further purification. A similar inhibition was found when incorporation of glucosamine (see below) was studied (Dukes et al., 1964). Two observations (Krantz et al., 1963) with this type of marrow cell culture suggest that the cells can act in a nearly physiological manner under the conditions we use: (1) There was increased heme synthesis when the amount of added erythropoietin was approximately equal to that in normal plasma. ( 2 ) The amount of heme synthesized per day by rat marrow in vitro, as calculated from the initial rate of iron incorporation into heme, is of the same order of magnitude as that calculated , about 1.4 for the system in vivo. The rate of heme synthesis, in u i t r ~is mg/day/lO1° nucleated rat marrow cells, while the corresponding estimate in vivo (assuming that there are 8 x lo9 total nucleated marrow cells in the adult male rat) (Cohrs et nl., 1958) is 10 mg/day/lO1° cells (Bozzini, 1965). While the discrepancy between these amounts appears large, the approximations are so uncertain that only an order of magnitude is really pertinent.

190

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We evaluated marrow cell heme synthesis by extracting the newly formed heme, which had been labeled with a suitable precursor, into a nonaqueous solvent after acidification. The need for acidification suggested that the heme was protein-bound and not free (Krantz et al., 1963). Subsequent studies ( Gallien-Lartigue and Goldwasser, 1964) showed that at least 93% of the labeled heme was derived from hemoglobin. These data provided a basis for using this technique with the

0.01

0.1

1.0

5

UNITS PER ml FIG. 1. Effect of impure erythropoietin on heme synthesis. Cultures started with 2 x 106 nucleated marrow cells/ml from 3-day-fasted rats. Medium: NCTC 109-rat plasma (1:l) preincubated 47 hours with step I11 erythropoietin (lot 192A), 2 units/mg protein. Heme synthesis assessed for the next 8 hours with "Fe-labeled rat plasma.

marrow cell system in uitro in studying differentiation as indicated by erythropoietin-induced hemoglobin synthesis. Another functional aspect of erythrocyte development studied in this laboratory is the erythropoietin-stimulated incorporation of labeled glucosamine into cell stroma. By using fluorescein-labeled antibody against human erythrocyte stroma, Yunis and Yunis (1963) showed that even the earliest erythroid cells already had some of the specific antigen on their surfaces, whereas nonerythroid cells did not. Dukes et al. (1963, 1964) found that erythropoietin increased glucosamine incorporation

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191

into a stroma-like fraction; this stimulation was dependent upon the amount of hormone in the medium, and the stimulated increment was linear with time of incubation. The biochemistry of this process is still obscure, since the details of biosynthesis and the structure of stroma have not been worked out. In the marrow cell system, about one-third of the incorporated glucosamine was found, after acid hydrolysis of the stroma, in N-acetyl and N-glycolyl neuraminic acids. None of the label appeared in free glucosamine and the remaining two-thirds has not yet been identified. The increase in hemoglobin or stroma synthesis in marrow cells in response to erythropoietin has been further studied in order to learn more about the action of the hormone (Krantz and Goldwasser, 1965a). The cells were removed at intervals up to 15 hours after incubation in erythropoietin-containing medium, and the amount of erythropoietin remaining in the medium was determined by addition of marrow cells (preincubated in erythropoietin-free medium ) . There was no detectable depletion of the hormone from the medium by the cells, although by this method we should have detected a fall of 20% or more. Nevertheless, cells exposed to erythropoietin for 15 hours, then transferred to erythropoietin-free medium, continued to synthesize hemoglobin for a number of hours. [In these experiments, preincubated media were used to control against the possible effects of medium conditioning (Parker, 1961).] The results suggest that a small fraction of the hormone to which the marrow cells are exposed is sufficient to initiate and maintain, for some time, the process of hemoglobin synthesis in culture. In a somewhat similar experiment ( Dukes and Goldwasser, 1965), the marrow cells were exposed to erythropoietin for 1hour in the presence of labeled glucosamine. The medium was then replaced with preincubated medium containing labeled glucosamine but no erythropoietin. During the next 6 hours, the rate of glucosamine incorporation into these cells was the same as that of cells exposed continuously to erythropoietin. The rate then declined to the control level seen in cells that had not had contact with erythropoietin at all. The l-hour exposure to erythropoietin was sufficient to induce a period of stimulated glucosamine incorporation which lasted another 6 hours, suggesting that induction of differentiation (as measured by this particular function) may occur in waves or bursts. The effect of cell number on erythropoietin stimulation of hemoglobin synthesis, at several levels of hormone, showed a sigmoid rather than the linear relationship expected from a simple model involving the in-

192

EUGENE GOLDWASSER

teraction of hormone molecules with susceptible cells. The curves in

Fig. 2, derived from the original data (Krantz and Goldwasser, 1965a), show the effect of cell number on hemoglobin synthesis per cell. Since there is no detectable depletion of erythropoietin from the medium, the fact that hemoglobin formation is greater per cell as cell number increases cannot be explained by a “multihit” kind of mechanism. The number of cells used in these experiments, even at the low levels, makes 45 I B

0

40-

X -I J

35-

5a

30-

W V

D

y

’

25-

LL

W

5I

20-

150.085 UNITS

2 - I 50

I

2

3

4

5

0 6

7

8

9

0.0210 UNITS 0 . 0 UNITS

l

NUCLEATED CELL NUMBER X

FIG.2. Relationship between rate of heme synthesis per cell and number of cells. (Data from Krantz and Goldwasser, 1965b.)

unlikely an explanation based on simple nutritional support among the cells (Eagle and Piez, 1962). There would seem, then, to be a cooperative interaction between the marrow cells in these cultures, and the following speculation is offered to explain this cooperation. Rat marrow cells have a tendency to aggregate even when care is taken to start the cultures with singly dispersed cells. The hypothesis postulates that there is an aggregate size optimal for erythropoietin-stimulated hemoglobin synthesis. As the total cell number increases, the probability that such optimal clusters will occur would increase according to Poisson statistics. If the rate of hemoglobin synthesis were a direct function of the number of such clusters, an increase in cell number would result in a sigmoid

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193

increase in the rate of hemoglobin formation. The curve would tend to flatten at the higher cell numbers, possibly because of limited entry of erythropoietin and/or nutrients needed for hemoglobin synthesis when the cluster became too large. A rationale for the optimal-cluster idea can be derived from the phenomenon of contact inhibition ( Abercrombie, 1962; Levine et al., 1965). It is likely that cells in the smaller-thanoptimal clusters will be dividing more actively than those in larger clusters. Assuming that a cell (potentially erythroid) cannot be induced by erythropoietin when it is dividing, the probability would be greater for the larger, optimally sized aggregates to contain inducible cells. When erythropoietin-stimulated hemoglobin synthesis was measured, the relationship to cell number discussed above was found, but when glucosamine incorporation was measured, we found a linear relationship between stimulated incorporation and cell number. Another instance of a difference between these two methods of study of erythropoietin action comes from evaluating the media. We found maximum hemoglobin formation in marrow cell cultures when the medium contained serum of either fetal or newborn calves. However, glucosamine incorporation was substantially greater when normal calf serum (heated to 56°C for 30 minutes) was used. We have no explanation for these differences. The mode of action of erythropoietin was further analyzed by studying the effect of specific inhibitors on the hormone-induced changes. Actinomycin D, which inhibits DNA-dependent RNA synthesis, interfered with the action of erythropoietin in mice (Gurney and Hofstra, 1963), and we have found that it also inhibits erythropoietin-induced changes in vitro. These observations suggest that erythropoietin action may be mediated through DNA-dependent RNA synthesis, and raise the possibility that the hormone acts on genetic transcription. In culture the effect of erythropoietin on hemoglobin synthesis became actinomycin resistant if an interval of 24 hours elapsed between addition of hormone and inhibitor ( Gallien-Lartigue and Goldwasser, 1965). These experiments demonstrated that whatever RNA syntheses were involved in mediating erythropoietin action had ceased by 24 hours, and that the RNA species formed had a rather long life span. A stable messenger RNA for globin synthesis by recticulocytes has been known to exist for some time (Nathans et al., 1962) and has also been suggested to occur in fetal liver cells (Grasso et al., 1963). Erythropoietin-dependent glucosamine incorporation is also sensitive to actinomycin and to puromycin (Dukes and Goldwasser, 1965), im-

194

EUGENE GOLDWASSER

plicating both RNA and protein synthesis in this process too. We have used actinomycin inhibition to evaluate the mean life span of those mRNA’s involved in the complex process of stroma synthesis. Marrow cells preincubated for 15 hours with erythropoietin, before addition of adinomycin, continued to incorporate glucosamine at an undiminished rate for 3.5 hours after addition of the inhibitor. After this time, increased incorporation ceased. This short life span (3.5 hours) is appreciably shorter than that of the hemoglobin mRNA. When the culture contained 0.25 pg of actinomycin per million cells, there was complete abolition of any erythropoietin effect; when the ratio was 0.156 pg per million cells, the rate of glucosamine incorporation was reduced to about 60% of the control level (Dukes and Goldwasser, 1965). This reduced rate persisted for about 8 hours, after which increased incorporation due to erythropoietin could not be detected. These observations suggest that two or more distinct processes may be inhibited by actinomycin, one of which is only slightly affected when the ratio of inhibitor to cells is low. The relationship between degree of inhibition of glucosamine incorporation and concentration of actinomycin is expressed by a curve with at least two distinct slopes (Fig. 3 ) . While this might suggest two or more sensitive processes that are dependent upon DNAdirected RNA synthesis, a comparable study of the inhibition of purified RNA polymerase in the presence of a DNA primer shows a similar curve (Reich, 1964). It might also be interpreted as indicating that there are guanines in the DNA that can react differentially with the inhibitor. Further interpretations of these data on inhibition of glucosamine incorporation will be discussed in a later section. These data on inhibition of erythropoietin-induced changes by actinomycin prompted us to examine the possibility that the hormone acted directly on RNA synthesis. Some years ago Perretta and Thomson (1961) showed that crude urinary erythropoietin stimulated the incorporation of labeled formate into the nucleic acid (RNA and DNA) purines of spleen and liver slices but not of marrow cell suspensions. These observations were not supported by our subsequent findings, which suggested that most of the effects described in their earlier paper may have been nonspecific and unrelated to erythropoietin action (Dukes and Goldwasser, 1962). More recently, we have shown that highly purified erythropoietin does cause an increase, in vitro, of labeled uridine incorporation into marrow cell RNA (Krantz and Goldwasser, 1965b). After incubation of the cells with erythropoietin for 6 hours we detected an increased rate

7.

195

ERYTHROID CELL DEVELOPMENT

of total cellular RNA synthesis, as measured by a 20-minute uridine pulse. When the RNA was fractionated by sucrose gradient centrifugation, a small but significant increase in incorporation could be detected 15 minutes after addition of the hormone, using a 10-minute pulse. The RNA whose synthesis was stimulated was found in the 12-20 S region of the gradient, while both larger and smaller RNA species did not show

140

X

0.010 UNITS ERYTHROPOIETIN

0

CONTROL

130

0 U

I

0 0

LL 0 W 0

U

W

a

50-

30

-

20

-

------c_ _

10 -

I

I

0.1

X

1

I

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

pg ACTINOMYCIN/ml

FIG. 3. Effect of increasing amounts of Actinomycin D on incorporation of glucosamine by marrow cells. Cultures started with 5.2 x 10" nucleated rat marrow cells per ml in culture medium that consisted of NCTC 109-calf serum ( 1:l); incubated for 24 hours. Erythropoietin used as 0.1 unit per rnl.

any signilkant increase in label due to erythropoietin under these conditions. The stimulated RNA synthesis was abolished in the presence of actinomycin and was not seen when the erythropoietin had been pretreated with trypsin. In addition, the stimulation was probably not due to some general stimulation of RNA synthesis unrelated to erythropoiesis, since we found that the hormone had no effect on RNA formation by suspensions of Murphy-Sturm tumor cells under the same conditions. Incorporation of formate into rat marrow nucleic acids is also increased

196

EUGENE GOLDWASSEH

by erythropoietin ( Pieber-Perretta et al., 1965). When RNA synthesis was increased by erythropoietin, we found that incorporation of thymidine into DNA for as long as 9 hours was not affected by the hormone (Table 111) (Krantz and Goldwasser, unpublished). TABLE I11 INCORPORATION OF THYMIDWE INTO DNA

BY

MARROW CELLS~

cpm/mg DNA

Time (minutes)

Control

Erythropoietin

0 30 60 180 360 570

0 6.6 12.4 58.6 124 194

0 5.3 13.0 58.4 129 196

a Cultures had 2 x 107 nucleated cells/ml from rats starved for 3 days; erythropoietin at 0.67 units/ml; total volnme of' each dish 0.9 ml; 0.04 pmoles of thymidine ( 1 . 3 kc) added per dish.

The data so far accumulated with this system are all consistent with a model, discussed below, of erythropoietin-induced differentiation derived from the Jacob-Monod model for the regulation of gene activity (Jacob and Monod, 1961, 1963), but a large number of gaps remain to be filled. The exact nature of the RNA that is formed rapidly in response to erythropoietin needs to be determined. Although the sucrose density experiments show clearly that it is neither 28 S ribosomal RNA nor 4-5 S RNA (tRNA), it remains possible that the population of newly formed RNA molecules includes 18 S ribosomal RNA as well as other species of RNA, among which might be specific messengers. We do not know yet whether the rapidly synthesized RNA can act as a template for protein synthesis, or, if it is a messenger, for which particular proteins it contains the code. 3. Control of Hemoglobin Synthesis

Investigation of the primary steps in erythropoietin induction of erythroid differentiation will be hampered for some time, no doubt, because of our ignorance concerning the controlling reactions in the various processes involved. * There are data indicating that the rate-limiting reaction An excellent summary, including many provocative speculations, of this aspect o€ erythroid differentiation may be found in the review article by Granick and

Levere ( 1964 ) .

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ERYTHROID (3ELL DEVELOPMENT

197

in heme synthesis is the one catalyzed by 6-aminolevulinic acid ( ALA) synthetase, i.e., the condensation of glycine and succinyl coenzyme A to form ALA. The experiments, which show that the overall rate of porphyrin synthesis is determined by the activity of ALA synthetase, were done with mammalian liver preparations (Granick and Urata, 1962; Urata and Granick, 1963), photosynthetic bacteria ( Burnham and Lascelles, 1963; Higuchi et al., 1965), and chick embryo cells (Levere and Granick, 1965), but the same considerations may hold for marrow cells. Another important observation is that heme added to reticulocytes can increase the formation of soluble protein (presumably hemoglobin) as measured by incorporation of labeled amino acid (Bruns and London, 1965). These experiments, and those on ALA synthetase, lead to a plausible model suggesting that a part of the developmental sequence initiated by erythropoietin is as follows: The hormone can react with the specific repressor which keeps the structural gene (or, if there are such in the mammalian systems, the operator gene) for ALA synthetase from being expressed. Such a derepression results in the formation of mRNA specific for ALA synthetase, followed by enzyme synthesis. Since the other enzymes in the biosynthetic chain leading to heme are present in nonratelimiting amounts, the rate of heme synthesis would increase with an increase in synthetase. As heme accumulates in the cell it stimulates globin formation so that hemoglobin formation is controlled, at least in part, by the level of a single enzyme. If the rate of globin synthesis cannot keep pace with heme synthesis owing, for example, to a deficiency of a- and P-chain messengers or of free ribosomes, there is feedback inhibition of ALA synthetase by heme (Burnham and Lascelles, 1963; Karibian and London, 1965) so that no appreciable accumulation of porphyrins, which may be deleterious to the cell, can occur. We have attempted to test these concepts by determining the level of ALA synthetase shortly after introduction of erythropoietin into the culture medium. There is a low but detectable level of the enzyme in marrow cells when they are taken immediately from the animal, but after a short period in zjitro we could find no enzyme at all. Quite evidently the cells are capable of heme synthesis, and the fault must lie in the method of estimation of the enzyme. There is fairly widespread agreement that this particular enzyme is one of the least stable and most difficult to work with. If the regulatory reaction actually were the synthesis of ALA, addition of it to cells making heme should result in a bypass of the enzyme and

198

EUGENE GOLDWASSER

an accelerated heme synthesis. Levere and Granick (1965) have shown this to be the case for chick embryo cells in culture. There are, however, some other observations which are not consistent in detail with the mechanisms proposed. The paper of Bruns and London (1965) that showed the stimulation of globin synthesis by heme also showed that this occurred only in reticulocytes obtained from iron-deficient animals; if they were not iron deficient, no effect was seen. Winterhalter and Huehns (1963) have detected a small amount of free globin in mature human erythrocytes. If confirmed, this must mean that the formation of hemoglobin cannot be regulated solely by the availability of heme. If it were, no free globin would remain at the end of the developmental process. In addition, Gribble and Schwartz (1965) found that protoporphyrin increased the amount of soluble protein formed by a cellfree system derived from reticulocytes without any change in the total amount of amino acid incorporated. These data suggest that the stimulatory role of heme seen by Bruns and London may have been confined to the release of polypeptide chains from the particulate protein-synthesizing system. The rate of formation of soluble globin would then be dependent upon heme only in an apparent fashion; the actual incorporation of amino acid into polypeptide would be independent of the presence of heme. In chick blastoderm cultures, a protein that reacts with antiglobin appears to be formed before heme is detected (Wilt, 1962), suggesting that globin synthesis is dependent upon prior heme formation. The data of Schwartz and his collaborators (Schwartz et al., 1959, 1961) showing that heme synthetase, the enzyme that adds iron to protoporphyrin, is stimulated by native globin also suggest that protein participates in the regulatory processes. We have recently found that heme synthesis in marrow cells is inhibited shortly after an inhibitor of protein synthesis ( puromycin or Actidione ) is added to the culture medium (Hrinda and Goldwasser, 1966). The rate at which heme synthesis is reduced by these inhibitors suggests either that the enzymes involved in heme synthesis by marrow cells have a very short life span or that heme synthesis is regulated by some sort of protein synthesis. Some recent observations (Tschudy et al., 1965) indicate that ALA synthetase in the livers of animals treated with allylisopropylacetamide (to induce ALA synthetase formation) has a half-life of about 70 minutes, as does its mRNA. The situation in erythroid cells, however, must be different. The half-

7.

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199

life of the enzyme in those cells forming hemoglobin is probably considerably longer. From the data of Karibian and London (1965) the estimated half-life of the system synthesizing heme from glycine is 3 4 hours in reticulocytes. Our data indicating a long life span for the hemoglobin-forming system ( GaIlen-Lartigue and Goldwasser, 1965) are in qualitative agreement with those of Grasso et al. (1963) and of Wilt (1965). This latter author showed that, in the chick embryo culture system, hemoglobin synthesis, in the head-fold stage, occurs 8 hours after inhibition with actinomycin. If the inhibition was complete, this observation would be consonant with a long life span for the entire system that catalyzes the synthesis of heme and globin. If ALA synthetase and the other enzymes in the sequence have a relatively long life span in erythroid cells, it may be that globin synthesis is required for heme formation (Granick and Levere, 1964) and that the RNA formed rapidly in response to erythropoietin is composed of globin chain messengers. It may also suggest a dual control of hemoglobin synthesis. 4. Other Possible Primary Efects

Whether the primary steps of erythropoietin action are, in fact, concerned with hemoglobin formation is still an unanswered question. Some recent experiments (Hrinda and Goldwasser, 1966) now suggest that one of the early effects of erythropoietin is facilitation of the entry of iron into some cells of the marrow. This effect is discernible before the stimulation of hemoglobin synthesis can be detected, indicating that it is not due to the displacement of the equilibrium distribution of iron caused by sequestration of the iron as heme. Puromycin inhibits this erythropoietin-effected iron uptake, suggesting that the hormone has induced the synthesis of some new protein, or proteins, required for the facilitated entry of iron into the cells. This may be a membrane component that has a direct influence on iron transport, or it may be an intracellular component involved in transfer of iron from the membrane to the heme-synthesizing system. In other studies, we examined the proposed role of erythropoietin as a mitotic stimulant ( Matoth and Kaufmann, 1962). As mentioned above, erythropoietin did not increase DNA synthesis when there was already increased RNA, hemoglobin, and stroma synthesis; this indicates that cell duplication was not required prior to initiation of differentiation. The

200

EUGENE GOLDWASSER

mitotic inhibitor colchicine at 5 x lops M did not completely inhibit erythropoietin-stimulated hemoglobin synthesis; about 14% of the hormone effect remained ( Gallien-Lartigue and Goldwasser, 1965). Erslev and Hughes (1960) found earlier that colchicine (2.5 X M ) caused a small inhibition (3040%) of iron uptake by marrow cells in vitro. We interpret our data as suggesting that the induced cells could maintain hemoglobin synthesis in the absence of mitoses; by blocking cell divisions, colchicine decreased the amplification of hemoglobin synthesis due to the few cell doublings that would normally have occurred during erythropoiesis. When the effect of colchicine on erythropoietin-stimulated glucosamine incorporation was studied, quite different results were found (Dukes and Goldwasser, 1965). The inhibitor had no effect on the process. We can reconcile these findings with those on hemoglobin synthesis by referring back to the suggestion that stimulated glucosamine incorporation occurs in short bursts during some part of the cell cycle, while hemoglobin synthesis occurs continuously. Even if cell division were completely inhibited, the entire erythropoietin-induced stimulation of glucosamine incorporation would be seen, because of the continuous generation of cells in the proper phase of the cell cycle. We might expect inhibition by colchicine if the experiment were greatly prolonged, but this experiment has not yet been performed. Alternatively, it may be that the stimulated glucosamine incorporation occurs only in those digerentiated cells incapable of further division. This is doubtful in view of our finding that increased glucosamine incorporation can be seen as earIy as 4 hours after introduction of the hormone. It would seem improbable that the whole process of stimulation of primitive cells through the stage of nondividing erythroid cells could occur so rapidly. VI. Models of Erythroid Differentiation

Our observations on the mode of action of erythropoietin on marrow cells in culture can be fitted into the highly speculative models for induction of erythroid differentiation described in Figs. 4 and 5. In Fig. 4, the process of erythroid differentiation is divided into three distinct stages: sensitization, induction, and specialization. Sensitization is the process by which stem cells, potentially capable of being converted to erythroid cells, become inducible or competent. * We

* In Fig. 4 the position of sensitization in the cell cycle is not specified; it could be at any stage.

7.

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ERYTHROID CELL DEVELOPMENT

postulate that sensitization requires the new synthesis of a species of RNA and would, therefore, be inhibited by actinomycin. This inhibition requires less of the inhibitor than do other stages, possibly because of a relative paucity of guanine in the region of the DNA being transcribed for the purpose of sensitization. When sensitization has been inhibited by an amount of actinomycin too small to affect other cellular processes materially, those sequential events initiated by erythropoietin can continue until the supply of sensitized cells is exhausted, after which the SPECIALIZATION

I

I

'

DESENSITIZATION

E'

E"

E"'

RI

RBC

FIG.4.

Proposed model of stem cell differentiation. S represents stem cells; S', sensitized stem cell; E, first stage of erythroid differentiation; E', E", E"', later stages of erythroid differentiation; Rt, reticulocyte; RBC, erythrocyte; e, erythropoietin. The stippled area on S' represents the postulated attachment site for erythropoietin.

erythropoietin effect will cease. This mechanism can explain our observations of the inhibition of the rate of glucosamine incorporation by actinomycin (p. 194). The nature of the sensitization step, if it exists, is even more speculative. It could involve the formation of a messenger RNA with a very short life span concerned with the formation of a specific attachment site (also with a very short life span) on the cell surface for interaction with erythropoietin. While the attachment site for erythropoietin existed, the cell could be "hit" by the hormone. After it was lost, erythropoietin would have no effect. * * The concept of a limited period of sensitivity is formally equivalent to the

202

EUGENE GOLDWASSER

If the concept of specific attachment site has validity, it could account for the specificity of erythropoietin action. We would assume that completely undifferentiated cells would be the only cells capable of becoming sensitized to erythropoietin; all other cells, in various states of differentiation, would have lost (or had repressed) the capability of forming an erythropoietin attachment site, so that, for example, muscle cells could not be induced by erythropoietin to form hemoglobin eventually. Once erythropoietin is within the cell, the second stage, induction, can occur. Induction, in this context, refers to the primary effect of the relatively few hormone molecules inside the ce1l.t If there were a large number of hormone molecules in the cell, the hormone might act simply as a metabolite. Emphasis is put on the primary effect in order to dissociate the immediate result of erythropoietin action from the subsequent secondary events. It is the process of induction that involves the actual mechanism of the hormone action, and it is about this process that we know virtually nothing. After the primary event has occurred, the series of secondary, tertiary, etc. events that may derive from it constitute the third stage, specialization. In this stage the easily recognizable special biochemical and morphological characteristics of the erythroid cell arise. This view of how differentiation of stem cells leads finally to erythrocytes contains no provision for maintenance of the stem cell pool, a condition that obtains in vivo and has been the subject of several kinetic models of stem cell differentiation (Osgood, 1959; Lijtha and Oliver, 1961; Lajtha et al., 1962; Stohlman et nl., 1962; Cronkite, 1964; Till et al., 1964). I would like to propose still another speculative mechanism to explain this phenomenon. Stem cell division may be regulated by a process similar to contact inhibition of cells in uitm. When cells are induced toward erythroid differentiation by erythropoietin, one of two changes may occur. Either the surface properties of the induced cells ( E in Fig. 4) suggestion of Lajtha and Oliver (1901) that there is a part of the cell cycle in which the cells are refractory to the action of a differentiation stimulus. f A calculation based on the concentration of erythropoietin in normal blood, and assuming the following: target cell diameter, 12 p; 5000 units per mg for pure erythropoietin, erythropoietin molecular weight 66.000, and equilibrium distribution of the hormone inside and outside the cell, shows that there would be 10-20 molecules of erythropoietin inside the cell. If there were a concentration mechanism for erythropoietin in the cell, the number of molecules inside would, of course, be considerably greater.

7.

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change SO that they are no longer recognized as neighboring cells by the uninduced stem cells, or the induced cells migrate some distance and are removed from physical contact with their former neighbors. In both of these cases the loss of recognizable like neighbors would be the signal for cell division to fill the gaps. Gurney and Fried (196513) suggest that the signal for stem cell replication may involve a decrease in the amount of a mitosis-inhibiting factor. If the period of sensitization is short with respect to the cell cycle, and if the biological life span of erythropoietin is not long, protection of the stem cell pool from depletion by high levels of the hormone will be provided. A more detailed description of a basically similar type of regulatory mechanism for stem cells is contained in the model proposed by Kretchmar ( 1966) from his analog computer simulation of erythropoietin action. In this model the integrity of stem cell number is maintained by restricting the effective action of erythropoietin to a part of the GI phase of the stem cell cycle and by making GI variable and dependent upon the negative feedback of cell division. As G1 is lengthened owing to reduced stem cell proliferation, the time the cell spends in GI is longer than the effective life span of intracellular erythropoietin, so that the hormone does not persist until S phase where it is supposed to act as a derepressor. Under conditions of maximal stem cell division, on the other hand, the GI period is so short that the probability of effective interactions between stem cells and the inducing hormone is reduced to a value SO IOW that depletion of the stem cell pool does not ensue. Further speculation on how induction can lead to specialization derives from the Jacob-Monod model. If we adopt the view that normal cellular constituents may have roles in gene regulation, as well as their more accustomed functions as enzymes, co-factors, and structural components, a model can be made in which there is no need for specific regulator genes (Waddington, 1962), which have not been described in mammalian cells, and without introducing any new genetic entities ( Barr, 1962). In this model, as schematized in Fig. 5, the primary product in induction, in addition to its other role in the economy of the cell, may also act as a repressor, a derepressor, or both. In model I, erythropoietin ( e ) by combining with the repressor x acts to initiate the synthesis of a gene product i which is a derepressor (combining with t o ) for some other repressed function H and/or a repressor for another function J which will eventually be lost by the differentiated cell. The new derepressor i has the same possibilities of action as did the original one, erythropoietin.

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It can cause the formation of a new functional component of the cell which has the same options (with differing specificities) as i did. The second product h can repress, derepress, or both. This type of process can then lead to a cascade of newly formed properties of the cell and a loss of some preexisting properties. This third stage, specialization, is the one that eventually provides the “constellation of synthesis and properties”

I L

--i

-

ex

U

F

QU

V

G

hv

W

H

iw ex

+

I --i J

--k --I

U

U

V

V

I

e +

-I

--i

-

-i

-

-f

-g --h -i i

K --k

h

-L

9

--I

-f

ug hv -h

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--i

-

-9 --h e

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FIG. 5. Proposed model for speciaEzation. Capital letters inside the vertical lines represent structural genes; Iower case letters to left of lines indicate repressors; to right of arrows indicate gene products. Erythropoietin is indicated as “e”. In model I. e is a derepressor; in model 11, e is a repressor.

mentioned earlier. It also provides for the other aspect of cytodifferentiation, which is often overlooked, the loss of specific functions. A possible alternative, but basically similar, role for erythropoietin is indicated in model I1 of Fig. 5. In this proposal the hormone ( e ) acts as a repressor of a gene I, the product of which, i, is the repressor of one of the functions ( H ) of the erythroid cell, and also has another function in the cell. The inhibition of formation of i can in turn lead to similar cas-

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cading processes, which result in gain of new functions and loss of existing ones. While neither of these models specifically requires the existence of genes that are coordinately repressed or derepressed, such operons can, of course, easily be accommodated by them. The only new concept introduced is the overlapping identities of regulator and structural genes. A similar suggestion, in a somewhat different context, has been made by Stent ( 1964). From this type of model, a prediction about the mechanism of loss of the nucleus during erythroid differentiation can be made. Nuclear loss and cessation of DNA and RNA synthesis may be the result of the repression of the citric acid cycle enzymes by some new constituent of the differentiating cell. This new constituent may be hemoglobin. Granick and Levere (1964) have already suggested that hemoglobin, because of its positive charge, may interact with DNA and act as a repressor substance. The function repressed by hemoglobin may well be the synthesis of one or more key enzymes involved in formation of stored energy from oxidized substrates. Among the enzymes conspicuously absent in the erythrocyte are those of the citric acid cycle, and if they are lost by decay after their synthesis has been repressed, the supply of deoxyribosidetriphosphates will decline due to an inadequate store of ATP for their formation. In the absence of precursors, DNA and RNA synthesis will stop, If the integrity of the DNA requires some repair of adventitious breaks in the polynucleotide, and if such repair requires the input of energy or of deoxyribosidetriphosphates, then the existing DNA will be gradually broken down within the nucleus. At some time, the presence of badly damaged and unrepairable DNA may be detrimental enough to the cell to lead to the extrusion of the nucleus by some completely unknown mechanisms. The study of biochemical mechanisms in erythroid differentiation is still in its infancy; yet the experimental approaches, data, and hypotheses now available can provide a useful point of departure for more sophisticated studies. At present, investigation of the mode of action of erythropoietin is greatly hampered by the lack of both a pure hormone and a homogeneous population of target cells, but these difficulties cannot be assumed to be everlasting. When they have been overcome, it may be possible to test some of the speculative conjectures made here, and to develop others that may come closer to accurate descriptions of the fas-

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cinating developmental process by which specialized cells are formed in metazoans. VII. Summary

Evidence has been presented indicating that the hormone erythropoietin is the direct regulator of red blood cell formation in mammals and is also the primary inducing factor for erythroid differentiation. Erythropoietin can be obtained from the plasma or urine of anemic animals and, although not yet purified, appears to be a glycoprotein of fairly large molecular size. Marrow cells can respond, in vitro, to erythropoietin by increase in hemoglobin, stroma, and RNA synthesis. These increases occur during the time when D N A synthesis is unaffected by the hormone. Synthesis of RNA, not identical with transfer or ribosomal RNA, occurs a very short time after exposure of the cells to the hormone. The data suggest that erythropoietin acts on transcription to induce specific syntheses characteristic of erythroid cells. Nothing is known of the nature of the erythropoietin target cell, but some evidence indicates a cooperative relationship among cells induced to synthesize hemoglobin. Erythroid differentiation has been arbitrarily divided into distinct phases and some speculative models have been proposed for the molecular and cytological mechanisms underlying the processes. ACKNOWLEDGMENT I am indebted to A. Kretchmar, J. Schooley, J. Lewis, and G. Hodgson for permitting me to see and use unpublished experiments even though I did not use all the material available. Thanks are also due to D. Steiner, C. W. Gurney, and M. Doyle for helpful criticisms of this manuscript and to P. P. Dukes, S. B. Krantz, M. Hrinda, M. Gross, A. Dahlberg, and C. Kung for stiniulating discussions of the problem of erythroid differentiation. REFERENCES Abercrombie, M. (1962). Cold Spring Harbor Syntp. Quant. Biol. 27, 427. Alpen, E. L., and Cranmore, D. (1959). In “The Kinetics of Cellular Proliferation” (F. Stohlnian, Jr., ed.), p. 290. Grune & Stratton, New York. Anderson, N. G. (1962). J . Phys. Chem. 66. 1984. Astaldi, G., and Cardinali, G. ( 1959). Arch. Intern. Eniatol. Sper. Clin. 2 , 17. Barr, H. J. (1962). J . Theoret. B i d . 3, 514. Berman, L., and Powsner, E. R. (1959). Blood 14, 1194. Bleiberg, I., Liron, M., and Feldman, M. (1965). Transplantation 3, 706. Borsook, H. (1964). Ann. N . Y. A d . Sci. 119, 523.

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Borsook, H., Graybiel, A., Keighley. G. (1954). Blood 9, 735. Borsook, H., Lingrel, J . B., Scaro, J . L., and Millette, R. L. (1962). Natnre 196, 347. Bozzini, C. E. (1965). Endocrinology 77, 977. Brecher, G., and Stohlman, F., Jr. (1961). PTOC.Soc. Exptl. Biol. Med. 107, 887. Bruce, W. R., and McCulloch, E. A. (1964). Blood 23, 216. Bruns, G. P., and London, I. M. (1965). Biochem. Biophys. Res. Commun. 18, 236. Burnham, B. F., and Lascelles, J. (1963). Biochem. 1. 87, 462. Carnot, P., and Deflandre, C. (1906). Compt. Rend. 143, 432. Cohrs, P., Jaffh, R , and Meesen, H. ( 1958). “Pathologie der Laboratoriumstiere,” Vol. 1, pp. 258-261. Springer, Berlin. Contrera, J. F., Camiscoli, J. F., Weintraub, A. H., and Gordon, A. S. (1965a). Blood 25, 809. Contrera, J. F.. Gordon, A. S., and Weintraub, A. H. ( 196513). Blood 26, 885. Cooper, G. W., and Gordon, A. S. (1964). Proc. Soc. Exptl. Biol. Med. 117, 314. Crafts, R. C. (1941). Endocrinology 29, 596. Cronkite, E. P. (1964). Federation Proc. 23, 649. DeGowin, R., Hofstra, D., and Gurney, C. W. (1962). PTOC.Soc. Exptl. B i d . Med. 110, 48. Dukes, P. P., and Goldwasser, E. (1961). Endocrinology 69, 21. Dukes, P. P., and Goldwasser, E. (1962). Nature 195, 1222. Dukes, P. P., and Goldwasser, E. ( 1965). Biochim. Biophys. Acta 108, 447. Dukes, P. P., Takakn. F., and Goldwasser, E. (1963). Biochem. Biophys. Res. Commun. 13, 223. Dukes, P. P., Takaku, F., and Goldwasser, E. (1964). Endocrinology 74, 960. Eagle, H., and Piez, K. (1962). J. Exptl. Med. 116, 29. Erslev, A. J. (1959). Blood 14, 386. Erslev, A. J. (1962). In “Erythropoiesis” ( L . 0. Jacobson and M. Doyle, eds.), p. 275. Grune & Stratton, New York. Erslev, A. J. ( 1964). Blood 24, 331. Erslev, A. J., and Hughes, J. B. (1960). Brit. J. Haeniatol. 6 , 414. Evenstein, D., Gordon, A. S., and Eisler, M. (1958). Anat. Record 132, 435. Finne, P. H. ( 1965). Brit. Med. J. I, 697. Fischer, S. (1962). In “Erythropoiesis” ( L . 0. Jacobson and M. Doyle, eds.), p. 204. Grune & Stratton, New York. Fisher, J. W., and Crook, J. J. (1962). Blood 19, 557. Fried, W., and Gurney, C. W. (1965a). Nature 206, 1160. Fried, W., and Gurney, C. W. (196513). Proc. SOC. Exptl. Bid. Med. 120, 519. Fried, W., Plzak. L. F., Jacobson, L. O., and Goldwasser, E. (1957). Proc. SOC. Exptl. Biol. Med. 94, 237. Fried, W., DeGowin, R., and Gurney, C. W. (1964). Proc. Soc. Exptl. Biol. Med. 117, 839. Fruhman, 0. J., and Gordon, A. S. (1956). Acta Haematol. 15, 249. Fulwyler, M. J. (1985). Science 150, 910.

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Gallien-Lartigue. O., and Goldwasser, E. ( 1964). Science 145, 277. Gallien-Lartigue, 0.. and Goldwasser, E. ( 1965). Biochim. Biophys. Acta 103, 319. Garcia, J. F., and Schooley, J. C. (1963). Proc. Soc. Exptl. Biol. Med. 112, 712. Garcia, J. F.. van Dyke, D. C., Huff. R. L., Elmlinger, P. J., and Oda, J. M. (1951). Proc. SOC. Exptl. Biol. Med. 76, 707. Goldwasser, E., and White, W. F. (1959). Federation PTOC. 18, 236. Goldwasser, E., Jacobson, L. O., Fried, W., and Plzak, L. F. (1957). Science 125, 1085. Goldwasser, E., Jacobson, L. O., Fried, W., and Plzak, L. F. (1958). Blood 13, 55. Golclwasser, E., White, W. F., and Taylor, K. B. (1962a). Biochim. Biophys. Acta 64, 487. Goldwasser, E., White, W. F.. and Taylor, K. B. (1962b). In “Erythropoiesis” (L. 0. Jacobson and M. Doyle, eds.), p. 43. Grune & Stratton, New York. Gordon, A. S. (1959). Physiol. Rev. 39, 1. Graham, L. A., Winzler, R. J., and Charles, H. E. (1963). Endocrinology 73, 475. Granick, S., and Levere, R. D. (1964). In “Progress in Hematology” (C. V. Moore and E. B. Brown, eds.), p. 1. Grune & Stratton, New York. Granick, S . . and Urata, G. (1962). Federation Proc. 21, 156. Grant, W. C., and Root, W. S. (1952). Physiol. Reu. 32. 449. Grasso, J. A., Woodward, J. W., and Swift, H. (1963). Proc. Natl. Acad. Sci. U.S. 50, 134. Gribble, T. J., and Schwartz, H. C. (1965). Biochim. Biophys. Acta 103, 333. Gurney, C. W., and Fried, W. (1965a). 1. Lab. Clin. Med. 65, 775. Gurney, C. W., and Fried, W. (198513). Proc. Natl. Acad. Sci. U.S. 54, 1148. Gurney, C. W., and Hofstra, D. (1963). Radiation Res. 19, 599. Gurney, C. W., Wacknian, N., and Filmanowicz, E. (1961). Bbod 17, 531. Hatta, Y., Maruyama, Y., Tsuruoka, N., Yamaguchi. A., Kukita, M., Sho, C. T., Sugata, F., and Shimizu, M. (1962). Actu Haematol. Japon. 25, 682. Hatta, Y., Maruyama, Y., Tsuruoka, N., Yamaguchi. A., Ando, M., Veno, T., and Shimizu, M. (1983). Acta Haematol. Jupon. 26, 174. Higuchi, M., Goto, K., Fujimoto, M., Naniiki, O., and Kikuchi, G. (1965). Biochim. Biophys. Acta 95, 94. Hillman, R. S., and Giblett, E. R. (1965). J. Clin. Invest. 44, 1730. Hrinda, M., and Goldwasser, E. (1966). Federation Proc. 25. 284. Ito, K., and Reissmann, K. R. (1965). Blood 26, 831. Jacob, F., and Monod, J. (1961). Cold Spring Harbor Symp. Quant. Biol. 26, 193. Jacob, F., and Monod, J. (1963). In “Cytodifferentiation and Macromolecular Synthesis” (M. Locke, ed.), p. 30. Academic Press, New York. Jacobson, L. O., and Doyle, M., eds. (1962). “Erythropoiesis.” Grune & Stratton, New York. Jacobson, L. O., Goldwasser, E., Plzak, L. F., and Fried, W. (1957). Proc. SOC. Exptl. Biol. Med. 94, 243.

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Jacobson, L. O., Gurney, C. W., and Coldwasser, E. ( 1960). Adoan. Internal Med. 10, 297. Jacobson, L. O., Coldwasser, E., and Gurney, C. W. (1961). Ciba Found. Symp. Haemopoiesis: Cell Prod. Regulation p. 423. Janda, W., Fried, W., and Gurney, C. W. (1965). Proc. SOC. Exptl. Biol. Med. 120, 443. Jepson, J. H., and Lowenstein, L. (1965). Proc. SOC. Exptl. Biol. Med. 120, 500. Karibian, D., and London, I. M. (1965). Biochem. Biophys. Res. Commun. 18, 243. Korst, D. R., Frenkel, E. P., and Wilhelm, J. E. (1962). In “Erythropoiesis ( L . 0. Jacobson and M. Doyle, eds.), p. 310. Grune & Stratton, New York. Krantz, S. B., and Coldwasser. E. (1965a). Biochim. Biophys. Acta 108, 455. Krantz, S. B., and Goldwasser, E. (1965b). Biochim. Biophys. Actu 103, 325. Krantz, S. B., Gallien-Lartigue, O., and Coldwasser, E. (1963). J. Biol. Chem. 238, 4085. Kretchman, A. L. (1968). Science 152, 367. Kuratowska, Z. (1965). Bull. Acad. Polon. Sci., Ser. Sci. Biol. 13, 385. Kurtides, E. S., Rambach, W. A., Alt, H. L., and Wurster, J. C. (1963). J. Lab. Clin. Med. 61, 23. Laforet, M. T., and Thomas, E. D. (1956). J. Biol. Chem. 218, 595. Lajtha, L. G.. and Oliver, R. (1961). Ciba Found. Symp. Haemopoiesis: Cell Prod. Regulation p. 289. Lajtha, L. C., and Suit, H. D. (1955). Brit. 1. Haematol. 1, 55. Lajtha, L. G., Oliver, R., and Gurney, C. W. (1962). Brit. 1. Haematol. 8, 442. Levere, R. D., and Granick, S. (1965). Proc. Natl. Acad. Sci. U.S. 54, 134. Levine, E. M., Becker, Y.. Boone, C. W., and Eagle, H. (1965). Proc. Natl. Acad. Sci. U.S. 53, 350. Linkenheimer, W. H.. Grant, W. C., Berger, H., and Hall, R. H. (1959). R o c . SOC. Erptl. Biol. Med. 100, 225. Linnian. J, W., and Bethell, F. H. ( 1960). “Factors Controlling Erythropoiesis.” Thomas, Springfield, Illinois. Linman, J. W., and Pierre, R. S. (1962). I n “Erythropoiesis” ( L . 0. Jacobson and M. Doyle, eds.), p. 228. Grune & Stratton, New York. Liron. M., and Feldman, M. (1965). Israel J. Med. Sci. 1, 86. Lowy, P. H., and Borsook, H. (1962). In “Erythropoiesis” ( L . 0. Jacobson and M. Doyle. eds.), p. 33. Grune & Stratton, New York. Lowy, P. H., Keighley, G.. and Borsook, H. (1958). Nature 181, 1802. Lowy, P. H., Keighley, G., and Borsook, H. (1960). Nature 185, 102. Martin, R. C., and Anies, B. N. (1961). 1. Biol. Chem. 236, 1369. Matoth. Y.. and Kanfmann, L. (1962). Blood 20, 165. Meineke, H. A., and Crafts, R. C. (1959). Proc. SOC. Exptl. Biol. Med. 102. 121. Mel, H. C . (1964). J. Theoret. Biol. 6, 159. Morell, H., Savoie, J. C., and London, I. M. (1958). J. Biol. Chem. 233, 923.

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CHAPTER 8

DEVELOPMENT OF MAMMALIAN €RYTHROlD CELLS Paul A. Marks and John S. Kovach DEPARTMENT O F MEDICINE, COLUMBIA UNIVERSITY, COLLEGE O F PHYSICIANS AND SURGEONS, N E W YORK CITY

Introduction . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . Sites of Erythroid Cell Development . . . . . . . . . . . . . . . . . Stem Cells . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . Structural and Biochemical Aspects of Erythroid Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cell Size . . . . . . ...... B. DNA and Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Ribosomes . . . ......................... E. Protein Synthesis . , . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Messenger RNA . . . ... ............... A. Stability . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . B. Association with Polyribosomes . . . . . VI. Hemoglobins . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . A. Types of Hemoglobins . . . . . . . . . . . . . . . .. . . .. .. . B. Control Mechanisms in the Synthesis of Hemoglobin VII. Concluding Comment . . . . . . . .................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. 11. 111. IV.

1.

213 215 216 220 22 1 221 225 226 230 233 233 233 235 235 237 245 245

Introduction

Development of mammalian erythroid cells involves a continuous process from a primitive progenitor cell without detectable hemoglobin to a mature non-nucleated erythrocyte in which more than 95% of the total 213

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protein is hemoglobin (Thorell, 1947). It is a most striking feature of mammalian erythroid cell differentiation that a cell line evolves which is uniquely capable of synthesizing hemoglobin and which develops to a non-nucleated stage providing an “envelope” to transport and maintain in a functional state this respiratory pigment. The recent achievements at the molecular level with bacteria and viruses have provided great impetus to studies of cellular differentiation in general, primarily by providing a model for considering the mechanism underlying this fundamental process-namely, that cellular differentiation is ultimately a result of variable gene activity (Jacob and Monod, 1961; Monod, 1961; Davis, 1964; Sonneborn, 1965). Developing erythroid cells are an attractive system for those interested in studying differentiation. Erythroid cell differentiation is characterized by a series of morphologically recognizable stages.* Globin and hemoglobin can be employed as highly specific markers for the course of differentiation. A large body of knowledge has accumulated about the genetic, structural, and biochemical characteristics of erythroid cells and, in particular, hemoglobin. Suitable laboratory animals with genetically determined variants in the patterns of erythroid cell differentiation and hemoglobin synthesis are available for study. Evidence is accumulating concerning the nature of specific factors which induce erythroid cell differentiation and affect its course, The problem of erythroid cell differentiation should be considered with respect to (1)the external forces which act upon the primitive progenitor cell, or stem cell, to initiate the chain of events resulting in differentiation along the erythroid cell line; ( 2 ) the mechanism of action of the external factors which induce stem cell differentiation; ( 3 ) the molecular differences which characterize the differentiated cell from the stem cell; and ( 4 ) whether the process of differentiation is irreversible. At the present time, we can make only beginnings in considering these aspects of erythroid cell differentiation. Within the scope of the present consideration, attention will be directed primarily toward the molecular differences between erythroid cells at various stages in the differentiation. The nature of the external inducer and its mechanism of action is considered in detail by Goldwasser elsewhere in this volume. There are very few data relevant to the question of the reversibility of the process of erythroid cell differentiation. * No references are cited here because these points are developed in detail in the course of this review.

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215

In considering erythroid cell development, emphasis will be placed on studies of the erythroid cell system in mouse, rat, rabbit, and man. This selection reflects largely the interest of the authors and the fact that more information is available about these systems than about those of other mammals. In general, however, the principles, and even many of the details, of erythroid cell differentiation which derive from studies in these species appear to be broadly applicable to erythroid cell development. There is no standard system of nomenclature for the various stages of erythroid cell differentiation ( Bloom and Fawcett, 1962; Wintrobe, 1961; Diggs et al., 1954; Andrew, 1965; Downey, 1938). In the present consideration, the following terms will be used to describe the various stages of erythroid cell development without any implication that this terminology can be justified to the exclusion of any other: stem cell + proerythroblast + basophilic erythroblast + polychromatophilic erythroblast --f orthochromic erythroblast --f reticulocyte 3 erythrocyte. II. Sites of Erythroid Cell Development

In mammals, the sites of erythroid cell differentiation change as fetal development proceeds. While there are temporal differences between species, in most mammals studied to date the initial site is detected in the yolk sac; subsequently the liver, the spleen, and, finally, the bone marrow become primary sites of erythropoietic activity. In the human embryo, blood cells are first formed in the numerous blood islands of the yolk sac (Bloom and Bartelmez, 1940; deAberle, 1927). Hematopoiesis also appears in areas of mesoblastic tissue, but in these sites, blood island development never reaches any considerable proportions (Halbrecht and Klibanski, 1959). The cells in blood islands differentiate into hemoglobin-containing primitive erythroblasts (Jones, 1938). The exact duration of this first, or mesoblastic, period of hematopoiesis is not known. The blood islands are demonstrable in the 2.25-mm embryo and are absent at the 5-mm stage (Knoll, 1949). Throughout this period no blood-forming organ is present, and most of the erythroid cells develop outside the embryo. Hematopoiesis in the liver begins in the embryo of the 5-7-mm period (Knoll, 1949). Erythroblasts, which are morphologically distinct in their appearance from those of the blood islands, can be identified in the extravascular areas of the developing hepatic tissue. These hepatic erythroblasts are smaller in size than the blood island erythroblasts. Erythropoiesis in the spleen begins between

216

P A U L A. MARKS AND JOHN S. KOVACH

the second and third months and continues through about the fifth month of fetal development. Erythropoiesis in the bone marrow commences about the fifth month but does not become the primary center of erythroid cell development until the latter part of fetal development, namely, the seventh through the tenth months. In normal human adults, the bone marrow is the only site of erythropoiesis. Under certain pathological conditions in which there is a failure in hematopoiesis in the bone marrow or a marked stimulus to erythropoiesis, such as severe hemolytic anemias, the spleen and liver may again become centers of erythroid cell development during adult life (Wintrobe, 1961). In fetal mice, active erythroid cell differentiation proceeds in blood islands in the yolk sac approximately at the eighth day of gestation ( d e Aberle, 1927; Russell and Bernstein, 1966). Erythroblasts in yolk sac blood islands are four to five times larger than adult red cells. These yolk sac erythroid cells enter the fetal circulation, but the number of these cells in the peripheral blood appears to remain constant from the twelfth to the fifteenth day of gestation and then decreases rapidly (Craig and Russell, 1963b, 1964). Erythroid cell differentiation is detectable in fetal liver of mice on the twelfth day and continues through the sixteenth day of gestation ( Borghese, 1959; Russell and Bernstein, 1966). The spleen, which is recognizable at the thirteenth day, has detectable erythroid cell development on the fifteenth day of gestation (Borghese, 1959). There is no hematopoietic activity in bone marrow until the sixteenth or seventeenth day of gestation, and it remains almost exclusively leukopoietic until birth (Borghese, 1959). After birth, the bone marrow becomes the primary site of erythropoiesis. In normal adult animals the spleen is not a site of erythroid cell development. However, under conditions of erythropoietic stimulation such as hypoxia (Prentice and Mirand, 1957; Brecher and Stohlman, 1959; Russell and Bernstein, 1966), phenylhydrazine administration, phlebotomy, or erythropoietin administration ( Jacobson and Doyle, 1962), the spleen, as well as bone marrow, exhibit active erythropoiesis. 111. Stem Cells

Erythroid cell differentiation is a process which proceeds at all stages of development of the mammalian organism. In man, during adult life the rate of production of red cells is approximately 2.0 x 10" cells per day (Lajtha, 1964). This is based on an average total red cell mass of 25 x lo1' and an average red cell survival time in the peripheral circu-

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lation of 110 days. This rate of red cell production per day represents cells during an average human life. It is generally approximately 5 x assumed that this impressive rate of red cell production implies that mechanisms exist for the maintenance of a precursor pool of “undifferentiated” cells, stem cells, capable of differentiating into erythroid cells. In the course of erythroid cell differentiation, cell division can occur through the basophilic erythroblast stage. However, there is evidence to indicate that, upon differentiation from the stem cell to the proerythroblast stage, the erythroid cells are not self-maintaining (Alpen and Cranmore, 1959; Bond et al., 1962; Lajtha, 1964; Lajtha et al., 1964). Rather, they are involved in a process of differentiation which results in a unidirectional progression toward mature erythrocytes. Accordingly, the present presumption is that the stem cells represent the undifferentiated cell, capable of maintaining the population of erythroid cells as well as the population of the stem cell (i.e., self-maintenance). At the present time there is no direct method of identifying the stem cells by cytological or biochemical techniques. On the basis of studies in the mouse (Cudkowicz et al., 1964) and in the dog (Fliedner et d., 1964) it has been suggested that a small bone marrow round cell, morphologically indistinguishable from small blood lymphocytes, has stem cell characteristics. However, neither study provides adequate criteria for stem cells, such as a direct demonstration of a capacity of these cells to differentiate to erythroid cells. The best available method of evaluating the presence of stem cells in a particular cell population is the ability of progenitor cells when injected into heavily irradiated mice to give rise to hematopoietic cell colonies (Till and McCulloch, 1961; McCulloch and Till, 1962). In brief, this assay for colony-forming cells involves the intravenous injection into irradiated mice (900 to 1000 rad ) of a suspension of a known number of nucleated cells from an appropriate source, e.g., bone marrow, fetal liver, or spleen. Nine to 11 days later, spleens are removed and fixed and the colonies per spleen are determined and generally expressed as the number of colony-forming units per lo5 cells injected. Over a limited range, there is a linear relationship between the average colony count per spleen and the number of cells injected. In this range, the number of spleen colonies per cells injected from a normal bone marrow is only about 5 to 15 spleen colonies per lo4 cells injected. The spleen colonyforming cells satisfy two basic criteria for being considered at least part of the stem cell pool: namely, they are capable of differentiating to

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PAUL A. MARKS AND JOHN S . KOVACH

erythroid cells, granulocytes, and/or megakaryocytes; and they are capable of self-maintenance (Till and McCulloch, 1961; McCulloch and Till, 1962; Siminovitch et al., 1963; Lewis and Trobaugh, 1964). Colonyforming cells that successfully seed in the spleen of irradiated hosts grow predominantly along one of the three hematopoietic cell lines, with a small proportion of the colonies containing mixtures of all these cell types. In addition, an increase in colony-forming cells can be demonstrated in spleen colonies between 5 and 11 days after injection. By use of donor cells in which chromosome aberrations induced by radiation are used as markers to identify the cells, evidence was obtained to suggest that each spleen colony represents a cell clone derived from a single progenitor cell (Becker et al., 1963). Identification of stem cells by the functional test of capacity to form spleen colonies has limitations which include the possibility that, for a variety of reasons, not all stem cells may be capable of dividing under the experimental conditions and, therefore, not all will be detected. The two criteria, namely, capacity to differentiate and capacity for self-maintenance, do not establish an equivalence between colony-forming cells and stem cells. Another functional test presumed to reflect stem cell activity has been developed by Jacobson et al. (1961) and Gurney et al. ( 1961, 1962). This test is based on the response of the erythropoietic system to erythropoietin. Following injection of erythropoietin into plethoric mice, an increase is measured in the number of cells capable of incorporating iron into hemoglobin or in the number of reticulocytes in the circulating blood. Cells incorporating iron and reticulocytes are differentiated cells and their number is used to infer the activity of ancestor cells stimulated to differentiate by the action of erythropoietin. This approach to measuring stem cell activity has several limitations, including the fact that the site of action of erythropoietin is not defined and may be on differentiated as well as on stem cells (Jacobson and Doyle, 1962; Cronkite, 1964; Goldwasser, this volume). As might be anticipated from the above consideration, there is little direct experimental evidence bearing on the question of the dynamics of the stem cell population. The stem cell population must be capable of maintaining itself as well as producing differentiated cells. This raises the problem of the nature of the cell division which the stem cells undergo. Three kinds of cell division may be considered (Osgood, 1957; Lajtha, 1964) : (1) the stem cell may divide into two daughter cells in an asymmetric fashion, one daughter cell remaining a stem cell, the other be-

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coming a differentiated cell; ( 2 ) the stem cell may divide into two daughter stem cells which remain as stem cells; or ( 3 ) under other conditions the stem cell may divide into two daughter cells both of which are differentiated cells. Results of studies with spleen colony-forming cells are consistent with the concept that stem cells may follow one of these pathways of cell division (Till et al., 1964). It has been proposed that the process of differentiation may be regulated by exposure to a specific inducer substance, or substances, while proliferation is controlled by a population “size” feed-back mechanism (Jacobson and Doyle, 1962; Lajtha, 1964; Till et al., 1964). In this model, at any given time one would anticipate that a certain fraction of the stem cells would be in a resting, nondividing state. There is evidence that stem cells may exist in a resting state. The fraction of the hemopoietic colony-forming progenitor cells which are actively synthesizing DNA in vivo was estimated by determining the ability of tritiated thymidine to inhibit the capacity of cells to form colonies (Becker et al., 1965). This is based on the fact that cells in the phase of DNA synthesis in the cell mitotic cycle incorporate large amounts of the tritiated nucleoside and are prevented from further division. This method was applied to transplanted colony-forming cells proliferating in spleens of heavily irradiated recipients, as well as the cells from normal fetal liver, normal bone marrow, and normal spleen. In situations where the hemopoietic system is expanding (fetal liver and regenerating transplants) a large fraction, 40-6570 of the stem cells, are synthesizing DNA. By contrast, in the steady-state situations, such as in adult marrow and spleen, the fraction of cells synthesizing DNA was imperceptible by this method. These studies suggest that control mechanisms which govern the rate of hemopoiesis operate, at least in part, by altering the generative cycle of stem cells. There is evidence that genetically controlled factors play a role in regulating stem cell proliferation. In mice, two mutations exist, designated W / W v and S l / S l d , which are characterized by severe macrocytic anemias (Russell, 1963). Employing the spleen colony-forming technique, evidence was obtained to indicate that W-genes affect either the number of colony-forming cells or their capacity to form spleen colonies ( McCulloch et al., 1964). On the other hand, the SZ locus was found to affect the capacity of the body to support the growth of spleen colonies, but not the nature of colony-forming ceIls themseIves ( McCulloch et al., 1965).

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PAUL A. MARKS AND JOHN S. KOVACH

It is well established that erythroid cell differentiation can be affected by environmental factors, such as hypoxia (D. R. Allen and Jandl, 1960; Thomas et al., 1960; Gruneberg, 1939; Prentice and Mirand, 1957; Brecker and Stohlman, 1959; Bruce and McCulloch, 1964), erythropoietin (Jacobson and Doyle, 1962; Goldwasser, this volume), blood loss, and increased rates of red cell destruction. The evidence concerning the mechanism by which these stimuli affect erythropoiesis is reviewed by Goldwasser in this volume. It should be emphasized that there is no method of identifying stem cells which is adequate to facilitate a direct experimental approach to the fundamental question concerning the nature and mechanism of action of the factors regulating differentiation to erythroid cells. This remains a major objective of current research in this area. IV. Structural and Biochemical Aspects of Erythroid Cell Differentiation

Differentiation of the erythroid cell from the proerythroblast stage to the circulating erythrocyte proceeds through a series of recognizable morphological changes. These structural changes are associated with striking biochemical alterations, the characteristics of which are broadly similar in erythroid cell development in fetal hepatic tissue ( Ackerman, 1962; Grasso et al., 1963; Russell and Bernstein, 1966; Kovach et al., 1966a) and in adult bone marrow and peripheral blood (Thorell, 1947; Bond et al., 1962; London, 1960-1961; Marks, 1962). As indicated above, the erythroid cells from the proerythroblast stage are not a self-maintaining population. Several attempts have been made to construct a model to describe the kinetics of erythroid cell differentiation (Alpen and Cranmore, 1959; Lajtha, 1964; Stohlman et al., 1964). Such models are based on data on the level of tritiated thymidine incorporation as assayed by radioautographic methods, the amount of hemoglobin per cell as determined largely by spectrophotometric and radioautographic techniques, and the total erythroid cell population and the proportion of the population represented by each stage. In the present state of our knowledge, these models can be considered largely speculative, but they provide a framework for considering the dynamic aspects of erythroid cell development from stem cell to proerythroblast to basophilic erythroblast to polychromatophilic erythroblast to orthochromic erythroblast to reticulocyte to erythrocyte. As might be anticipated from the fact that

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differentiation is a continuous process, the transition from one morphologically identifiable cell stage to another is gradual. Accordingly, it is not possible to delineate sharply various stages of differentiating erythroid cells-or even between cells still capable or incapable of cell divisionby either morphological or biochemical criteria. The various stages of erythroid cell development are identified largely on the basis of morphological criteria (Diggs et al., 1954; Wintrobe, 1961; Bloom and Fawcett, 1962). As discussed in detail below, the sequence of the stages of erythroid cell development is based on a variety of evidence. In general, it is interpreted that immature cells have large nucleus-to-cytoplasm ratios, incorporate thymidine into DNA rapidly, and have no hemoglobin, while decreasing ribosome content and increasing hemoglobin concentration are associated with later stages of development.

A. CELL SIZE In studies with human bone marrow, the first recognizable cell in the erythropoietic series, the proerythroblast, has an average diameter of 12 p ( Thorell, 1947). Each successive erythroblast has a reduced diameter and is reported to have about half the volume of its predecessor (Thorell, 1947; Lehman and Huntsman, 1961; Borsook, 1964). The average diameters of cells in the erythroid series are 10 p, 8 p, and 6 p for basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromic erythroblasts respectively. The diameter of nucleated erythroid cells is the same under conditions of normal and increased rates of erythropoiesis, but that of reticulocytes is not. In anemic mice, rabbits, or human subjects, or following administration of erythropoietin to animals, larger reticulocytes develop with average diameters of 8 rather than the 6 p in normal animals (based on studies in mouse, rabbit, and man). In addition to the size difference, these reticulocytes appear to have biochemical characteristics (see Section IV E ) , such as rates of hemoglobin synthesis, which suggest that, under conditions of stimulated erythropoiesis, reticulocytes may develop directly from polychromatophilic erythroblasts, skipping the orthochromic erythroblast stage ( Lajtha and Suit, 1955; Suit et al., 1957; Lajtha and Oliver, 1961; Lehman and Huntsman, 1961; Brecher and Stohlman, 1961; Borsook, 1964). B. DNA

AND

NUCLEUS

In fetal hepatic and adult bone marrow erythroid cell development in rat and man, as well as other species, the nucleus-to-cytoplasm ratio is

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MARKS AND JOHN S . KOVACH

highest at the proerythroblast stage and decreases progressively to the orthochromic erythroblast stage ( Thorell, 1947; Grasso et al., 1963). On the basis of radioautographic studies in human bone marrow and rat fetal liver erythroid cells, DNA synthesis is active in proerythroblasts and basophilic erythroblasts. Cell division does not occur beyond the polychromatophilic erythroblast stage (Lajtha, 1964; Bond et al., 1959; Cronkite et al., 1959; Grasso, 1963; Cronkite, 1961; Borsook, 1964). There are probably at least three cell divisions beyond the stem cell stage in the course of erythroid cell differentiation. It is estimated that the dividing cells have a cycle of at least 12 hours (Bond et al., 1959; Cronkite, 1964) and perhaps as long as 20 hours (Lajtha, 1964). The duration of the mitotic phase in erythroblasts has been estimated as 30 to 40 minutes (Bond et al., 1962). Proerythroblasts have a diploid chromosome number, and with successive stages there may be a loss of chromosomal material (Weicker and Tenvey, 1958). No absolute values for DNA content at each cell stage are available, but relative estimates of DNA content, based on cytochemical methods, have been reported as 19.8, 19.3, 17.5-16.3, and 11.8-10.8 for proerythroblast, basophilic erythroblast, polychromatophilic erythroblast, and orthochromic erythroblast nuclei respectively ( Korson, 1951) . Under conditions of normal erythroid cell development in bone marrow and fetal liver, the nucleus is lost predominantly at the orthochromic erythroblast stage, most likely by extrusion ( Albrecht, 1951; Bessis, 1955; Bro-Rasmussen and Henriksen, 1964). The maturation times for the later, nondividing stages of erythroid cell development are estimated as 40-50 hours for orthochromic erythroblasts and 48-72 hours for reticulocytes ( Finch, 1959; Stohlman et al., 1964). It has been shown that the nucleus may be expelled in the polychromatophilic erythroblast stage under conditions of anemic stress and, apparently, occasionally in the normal course of development (see Section IV, A ) (Borsook et al., 1962; Brecher and Stohlman, 1959). Hemoglobin has been reported to be present in the nuclear annuli and to extend into the interchromatic regions during the later stages of fetal hepatic erythroid development in rats and rabbits (Grasso et al., 1963). The appearance of hemoglobin in the nucleus is characteristic of erythroid cell development in lower forms ( OBrien, 1961a,b; Tooze and Davis, 1963). The significance of nuclear hemoglobin is not clear. It could be synthesized in nuclei. Alternatively, it has been suggested that hemoglobin enters the nucleus from the cytoplasm through nuclear cyto-

FIG. 1. A, A circulating yolk sac erythrocyte of the fetal mouse (11 days’ gestation) with finely granular chromatin and many polyribosomes. B, Yolk sac erythrocyte (15 days’ gestation) with densely clumped chromatin and no ribosomal material in cytoplasm ( x 25,000). All electron micrography performed with Dr. Richard Rifkind and Mrs. Hazel Epler. 223

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PAUL A. MAHKS AND JOHN S. KOVACH

plasmic communications which are readily demonstrable in erythroblasts (Fig. 1). It is conceivable that hemoglobin may have a role in the inactivation of nuclear chromatin material as development proceeds. The pattern of development of yolk sac erythroid cells, based on studies in the mouse (gestation time of 21 days) (Craig and Russell, 1964; Craig and Southard, 1966; Kovach et al., 1966a), has certain differences from that of fetal hepatic or adult bone marrow erythropoiesis. Different developmental stages of yolk sac erythroblasts are not as well defined as those of fetal liver or adult bone marrow. There is a progression in nuclear changes; early erythroblasts contain large nuclei with nucleoli and coarsely granular chromatin and may undergo mitosis in the peripheral blood as late as the eleventh or twelfth day of gestation (Craig and Russell, 1964) (Fig. 1).Later stages of yolk sac erythroblast development are characterized by reduction in volume of the nucleus and an increasingly pycnotic appearance of the chromatin ( Craig and Russell, 1964; Kovach et al., 1966a). By the thirteenth day of gestation, most of the nuclei are pycnotic (Fig. 1 ) and do not incorporate thymidine into DNA (Kovach et aZ., 1966b). The number of yolk sac erythroid cells per unit volume of peripheral blood remains constant from the twelfth to the TABLE I ERYTHROKI CELLSIN PEHIPHERAL CIHCULATION OF FETAL MICE~

Fetal ageh

Total erythroid cells/mm3c

12 13 14 15 16

4.6 5.4 9.8 16.7 26.2

a b 0

d

Yolk sac erythroid Nucleated cellsd cells/m~~ (70)

3.4 3.0 3.6 3.6 2.1

94-100 45-78 23-37 6-8 -

Fetal hemoglobin

(%I 86-90 63-73 19-40 9-26 -

Adapted from Craig and Russell (1964). Days’ gestation. Mean x 106. Range of litter means.

fifteenth day of gestation and then decreases rapidly (Table I ) (Craig and Russell, 1961). The pycnotic nuclei appear to be retained for the life span of these cells. In addition to nuclear changes, yolk sac erythroid cell development differs from that of fetal liver and bone marrow in the pattern of alterations in ribosomes and in hemoglobin synthesis (see Section IV, D ) (Craig and Russell, 1964; Craig and Southard, 1966; Kovach et al., 1966a ) .

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C. RNA In fetal liver and bone marrow, RNA concentration decreases progressively as proerythroblasts differentiate to polychromatophilic erythroblasts, and there is then little change through the orthochromic erythroblast stage ( Thorell, 1947; Grasso et ul., 1963). These estimates are based on microspectrophotometric and radioautographic techniques. In both fetal rat liver erythroid cells (Grasso et al., 1963) and rabbit bone marrow (Borsook et ul., 1962; DeBellis et al., 1964), RNA synthesis occurs primarily during and prior to the basophilic erythroblast stage. The ribosomal RNA synthesized in developing erythroid cells has a base composition characterized by a high content of guanosine and cytosine (DeBellis et al., 1964). The 28 S and 18 S ribosomal RNA differ in base composition, as do the two types of ribosomal RNA of bacteria (Yankofsky and Spiegelman, 1963) and other animal cells, such as the developing chick embryo (Lerner et al., 1963). The functional signscance of these two types of RNA is unknown. Reticulocytes and probably orthochromic erythroblasts of normal or anemic rabbits or man do not synthesize RNA (Marks et al., 1962a,b; Nathans et al., 1962; Borsook et al., 1962). In the fetal rat or mouse, there is even little detectable RNA synthesis at the polychromatophilic erythroblast stage of hepatic erythroid cell development (Grasso et al., 1963; Kovach et al., 1966b). Mouse yolk sac erythroblasts in the peripheral blood at 11 days of gestation actively synthesize RNA (Kovach et d., 1966b ) . During erythroid cell development, messenger RNA is presumably synthesized primarily during and prior to the basophilic erythroblast stage and remains in a stable, functional form through the reticulocyte stage (see Section V ) (Marks et al., 1962b). There are no specific studies on synthesis of transfer RNA during erythroid cell development. RNA is lost from erythroid cells by degradation within the cell to nucleosides and pentose (Bertles and Beck, 1962; Burka et al., 1964). The mechanism by which RNA is degraded during the course of erythroid cell development is not known. A soluble ribonuclease has been purified from reticulocytes which can degrade reticulocyte messenger, ribosomal, and soluble RNA (Farkas et al., 1964; Adachi et al., 1964). This nuclease is inhibited by relatively low concentrations of M g + + ( 3 x lo-: M ) . Its physiological role, if any, in erythroid cell differentiation is yet to be elucidated. Nuclease may have an important function

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PAUL A. MARKS AND JOHN S. KOVACH

in determining the rate of degradation of template, ribosomal, or tRNA and, in turn, the course of erythroid cell development during the later stages when the capacity for RNA synthesis is lost.

D. RIBOSOMES 1. Organization

Ribosomes of erythroid cells are formed from two subunits, 60 S and 40 S particles ( Ts’O and Vinograd, 1961; Watson, 1964). Erythroid cell ribosomes are composed of approximately 50% protein and 50% RNA. The 60 S subunit contains RNA with a sedimentation coefficient of 28 S. The 40 S subunit contains 18 S RNA. A 60 S and a 40 S ribosomal subunit combine to form a single 80 S ribosome. In intact erythroid cells, clusters of two or more 80 S ribosomes associated with messenger RNA, called polyribosomes, are the primary site of protein synthesis (Marks et al., 1962a; Warner et al., 1963; Gierer, 1963). 2. Alterations with Cell Development Ribosomes in nucleated erythroblasts of fetal mouse liver (Fig. 2 ) and of rabbit bone marrow are present predominantly as polyribosomes (Rifkind et al., 1964a; Kovach et al., 1966a). The total ribosome content of erythroblasts is considerably greater than that of reticulocytes ( Lingrel and Borsook, 1963; Rifkind et aZ., 1964a) even when correction is made for the larger cytoplasmic volumes of erythroblasts (Table 11). For the present discussion, the reticulocyte is defined as a non-nucleated cell containing ribosomes. In rabbit bone marrow erythroblasts, 99% of the ribosomes may be present as polyribosomes (Table 11) (Rifkind et al., 1964a). In the later stages of erythroblast development and during reticulocyte maturation, total ribosome content decreases and the proportion of ribosomes which are present as polyribosomes decreases (Marks et al., 1963b; Rifkind et aZ., 1964a; Rowley, 1965; Danon et al., 1965) (Table 11, Fig. 3). A cell stage may be reached where only single ribosomes are present, which subsequently disappear as erythrocytes are formed. In fetal hepatic erythroid cell development in the mouse, the progression of changes in polyribosome and ribosome content appears to be analogous to that described above for erythroid cell differentiation in rabbit bone marrow. However, quantitative analyses of electron micro-

FIG. 2 . A, An erythroblast from fetal mouse liver ( 15 days’ gestation) with finely granular and clumped nuclear material and many polyribosomes ( x 35,000). B, A circulating liver reticulocyte, anucleated but with numerous polyribosomes ( x 29,000). 227

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graphs or physicochemical data such as are available for bone marrow erythroid cells have not been obtained for the fetal hepatic system. In mice, erythroid cells of fetal hepatic origin are released into the circulation by the tweIfth day of gestation (Craig and Russell, 1964). These cells enter the circulation predominantly, if not entirely, after the nucleus has been lost. At this stage, these cells contain many ribosomes, with TABLE I1 THEDISTRIBUTION OF RIBOSOMES AS SINGLES AND POLYRIBOSOMES DURING MATURATION O F RABBITEHYTHHOlD CELLSn

Cell type Erythroblast Early reticulocyte" Intermediate reticulocyte" Late reticulocytec

Total number ribosomes" Per standard Singles area in ___ 50 cells 1

6730 2775 1050 350

1 22 62 58

% Ribosomes Polyribosomes 2

3

4

5

fi

11 25 24

22 29 14 3

43 18 0

16 4 0

7 3 0

39

Adapted from Rifkind et ul. ( 1964a). Counted in standard area, l-inch square on electron micrographs at final magnification of 25,000 X . Count corrected for the error introduced by the transectioning of polyribosomes during sectioning ( Perl, 19fi4). These stages of reticulocyte development are expressed as a function of the duration of incubation in an in vitro system for maturation of these cells (Rifkind et ul., 1964a). a b

over 70-80% being polyribosomes (Fig. 2 ) . As these cells develop in the circulation, ribosomes disappear. Yolk sac erythroid cell development, again, appears to differ from that of fetal hepatic or bone marrow tissue. As noted above, as yolk sac erythroid cells develop the nucleus becomes pycnotic, but is usually retained. At 11 days of gestation, the yolk sac erythroid cells in the circulation contain many ribosomes and polyribosomes (Fig. 1).By the fourteenth or fifteenth day of gestation there may be no detectable ribosomes in the cytoplasm of these cells (Kovach et al., 1966). 3. Formation

Polyribosome formation has been examined in intact rabbit erythroid cells. Studies of rabbit bone marrow erythroid cell development indicate

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FIG. 3. A. A young rabbit reticnlocyte with numerous polyribosomes. B. More mature rabbit reticulocytes; at the left, with moderate numbers of single and double ribosomes; at the right, with few scattered single ribosomes ( x 30,000).

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PAUL A. MARKS AND JOHN S. KOVACH

that the protein as well as RNA of ribosomal subunits are synthesized in early erythroblast stages (Lingrel and Borsook, 1963; DeBellis et al., 1964). Evidence from several systems, including erythroid cells, is consistent with the conclusion that 60 S and 40 S subunits are precursors of polyribosomes which are active in protein synthesis (Girard et al., 1965; Joklik and Becker, 1965; Marks et al., 1966). The nature of the binding between 80 S ribosomes and mRNA is unknown. The dynamic aspects of polyribosome function will be considered in relation to control mechanisms in protein synthesis (Section VI, B). The pool of 80 S ribosomes in reticuIocytes is probably derived largely from dissociation of polyribosomes and not by association of 60 S plus 40 S subunits. In intact cells 80 S ribosomes may not dissociate to ribosomal subunits (Marks et al., 1966). The evidence for this is as follows: Incubation of rabbit or human reticulocytes with NaF is associated with a complete dissociation of polyribosomes to 80 S ribosomes without detectable breakdown of 80 S ribosomes to ribosomal subunits. During reticulocyte maturation, a marked decrease in polyribosome content can occur without a significant alteration in the amount of ribosomal subunits. Analogous observations on polyribosome dissociation have been made with other mammalian cells, such as Hela cells (Joklik and Becker, 1965). Thus, it is likely that polyribosome dissociation and ribosome degradation in the later stages of erythroid cell development does not involve a breakdown of 80 S ribosomes to ribosomal subunits. This may have implications with respect to the stability of the mRNA in erythroid cells during the later stages when there is no RNA synthesis, but active protein synthesis. Evidence from other systems indicates that mRNA is bound to the 40 S subunit (Okamoto and Takanami, 1963). However, in erythroid cells after cessation of RNA synthesis, e.g., in late polychromatophilic erythroblasts or orthochromic erythroblasts, there may be little or no net polyribosome formation and mRNA is maintained in a stable form in association with polyribosomes (see Section V ) .

E. PROTEINSYNTHESIS As erythroid cells develop there are changes in the overall rate of protein synthesis and in the concentration of protein which are characteristic of the different morphologically identifiable cell stages. Early studies, employing microspectrophotometric techniques, indicated that hemoglobin synthesis did not begin until the polychromatophilic erythroblast stage (Thorell, 1947). Later studies have confirmed this conclusion,

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again employing techniques of microspectrophotometry ( Sondhaus and Thorell, 1960; Ackerman, 1962; Grasso et al., 1963) based on Soret band measurements which detect the porphyrin-globin complex. There are no precise data on when globin synthesis is initiated in the course of erythroid cell differentiation. It is possible that globin synthesis begins at an earlier stage than hemoglobin formation ( Ackerman, 1962; Wilt, 1965). The total cytoplasmic protein decreases between the proerythroblast and early polychromatophilic erythroblast, increasing sharply thereafter to the reticulocyte stage as hemoglobin is formed (Thorell, 1947; Ackerman, 1962; Grasso et al., 1963). The decline in total protein in early stages of development is associated with a decrease in cytoplasmic area and probably does not reflect the relative rates of protein synthesis. It appears that the rates of total protein synthesis change little during the early erythroblast stage of development prior to the polychromatophilic erythroblast stage (Grasso et al., 1963; Borsook, 1964). Heme and globin synthesis proceed at parallel rates in later stages of erythroblasts and in reticulocytes (Kruh and Borsook, 1956; Nizet, 1957; Morell et al., 1958). Heme affects the rate of globin synthesis and may have a role in the control of the rate of hemoglobin formation (Bruns and London, 1965; Waxman and Rabinowitz, 1965). Heme and globin synthesis can be dissociated so that globin formation may be enhanced under conditions which depress heme synthesis (Morell et al., 1958; Richmond et al., 1951). In ll-day mouse embryos, protein synthesis in yolk sac erythroid cells is active in erythroblasts, which contain polyribosomes ( Craig and Southard, 1966; Kovach et aZ., 1966a). By the fourteenth day of gestation, these cells, as indicated above, have lost all ribosomes, though retaining a pycnotic nucleus. At this stage of development, there is no detectable protein synthesis in these cells. When the site of erythroid cell development has shifted to the liver or bone marrow, the pattern of development is strikingly altered. As summarized above, fetal hepatic or adult bone marrow erythroid cells lose nuclei when the cytoplasm contains polyribosomes and these cells actively synthesize protein. Indeed, fetal hepatic erythroid cells synthesize hemoglobin at a greater rate at the non-nucleated reticulocyte stage than in precursor erythroblasts ( Table 111) (Kovach et al., 1966a). In bone marrow erythroid cell development, the rate of hemoglobin synthesis is maximal at the polychromatophilic and/ or orthochromic erythroblast stages (Thorell, 1947; Borsook et al., 1962). There is a correlation between the loss of capacity to synthesize protein

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PAUL A. M A R K S AND JOHN S. KOVACH

and the loss in polyribosome content as reticulocytes develop to erythrocytes (Marks et al., 1963b; Rifkind et d.,1964a). However, factors other than a disappearance of polyribosomes are involved in the loss in capacity to synthesize protein as reticulocytes mature (see Section V I ) (Marks et al., 1962a; Mathias et al., 1964; Glowacki and Millette, 1965). TABLE I11 HEMOGLOHIN SYNTHESIS IN FET.4L MOUSEEHYTHHOIE CELLS Hemoglobin Day

Cell type

Type'L

Synthetic rateb

11

Circulating yolk sac erythroblast

Fetal

-c

14

15

Circulating yolk sac erythroblast Liver erythroblast Circulating liver reticulocyte Circulating yolk sac erythroblast Liver erythrohlast Circulating liver reticulocyte

Fetal Aclult

0 7

Adult

24

Fetal Adult

0 34

Adult

67

a Hemoglobins characterized by standard polyacrylamide gel. (Disc electrophoresis Newsletter No. 3 Hemoglobin Analysis, Canalco, 1963.) b cpm of valine-14C incorporated into protein (Burka and Marks, 1963). Rate is measured by ( cpni/OD,,j, Ribosome) x 104. c At 11 clays the cells are too few to permit quantitation of ribosomes for the purpose of comparing the rate of hemoglobin synthesis in these cells with that in cells at later stages of gestation; however, relating incorporation of amino acid to protein concentration indicates active hemoglobin synthesis at this stage.

As erythrocytes age, there is no loss of hemoglobin (London, 19601961; Marks, 1962). The maturation of reticulocytes to erythrocytes is associated with a loss of mitochondria and a loss of the activity of enzymes, including enzymes of the cytochrome system, the tricarboxylic acid cycle, and lipid synthesis (Rubenstein et al., 1956; Marks et al., 1958; London, 1960-1961; Marks, 1962). There is a series of biochemical and structural alterations which occur as erythrocytes age in the circulation. Consideration of these changes is not within the scope of present review (London, 1960-1961; Marks, 1962, 1965; Pennell, 1965; Van Deenen and DeGier, 1965; D. W. Allen, 1965; Jaffe, 1965).

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V. Messenger RNA

A. STABILITY The concept of a template nucleic acid which contains the information to direct the assembly of amino acids into a peptide chain has been considered for many years (Brachet, 1955; Singer and Leder, 1966), but the hypothesis of messenger RNA provided great impetus to a consideration of the regulation of protein synthesis (Jacob and Monod, 1961). In the transfer of information from D N A to the site of protein synthesis, at least two alternatives may be considered for programming polyribosomes during the course of development of erythroid cells (o r any other cells). DNA may be continuously transcribed, the messenger RNA synthesized having a short life span, with the formation of specific proteins dependent on the continued synthesis of specific messenger RNA's. Alternatively, regions of D N A may be transcribed during a period early in the course of cell development, the polyribosomes becoming programmed with messenger RNA, which is relatively stable, permitting them to serve as a template for the synthesis of specific proteins at later developmental stages. In mammalian erythroid cell development in fetal hepatic tissue and in bone marrow, messenger RNA directing hemoglobin synthesis seems to be stable throughout the stages of development during which most of the hemoglobin is synthesized. This does not imply that all messenger RNA in erythroid cells is stable. There is no information on the stability of nonhemoglobin messenger RNA, but there is accumulating evidence that most cells may produce both stable and unstable messenger RNA (Singer and Leder, 1966). The primary evidence for the stability of messenger RNA for hemoglobin are the studies summarized in the preceding discussion which indicate that, in fetal hepatic erythroid cell development, RNA synthesis ceases by the polychromatophilic erythroblast stage, while hemoglobin synthesis appears to begin at this stage and continue through the reticulocyte stage. Similarly, in bone marrow erythroid cell development, there is no RNA synthesis beyond the polychromatophilic erythroblast stage, while hemoglobin is synthesized in reticulocytes. B. ASSOCIATION WITH POLYRIBOSOMES It is the current concept of protein synthesis that functional messenger RNA is bound to polyribosomes, which are the predominent site of hemo-

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PAUL A. MARKS AND JOHN S. KOVACH

globin synthesis in intact cells (Marks et al., 1962a; Warner et al., 1963; Gierer, 1963). In the cell-free system, while polyribosomes are considerably more active than 80 S ribosomes, single or monoribosomes can be isolated which are capable of hemoglobin synthesis (Lamfrom and Knopf, 1965; Luzzatto et al., 1965). Presumably, such 80 S ribosomes are also associated with messenger RNA. Electron microscopic studies have demonstrated thin filamentous structures between reticulocyte ribosomes which have a diameter consistent with their being messenger RNA (Rich et al., 1963; Rifkind et al., 1964b; Mathias et al., 1964). Other lines of evidence have been interpreted as consistent with the concept that messenger RNA is a component of polyribosomes. A high proportion of reticulocyte polyribosomes appear to contain four to six ribosomes, which has led to the suggestion that this is an appropriate number of ribosomes to fill a messenger RNA strand of a size suitable to code for a globin peptide (Rich et al., 1963). However, larger polyribosomes have been demonstrated in reticulocytes ( Rifkind et al., 1964b) , and labeling reticulocytes with amino-I4C acids indicates that globin is synthesized on polyribosomes containing two to as many as six to ten ribosomes (Burka and Marks, 1964b). Numerous attempts have been made to isolate messenger RNA from reticulocytes and from polyribosomes prepared from reticulocytes. RNA has been recovered from these sources which can stimulate amino acid incorporation in a cell-free system prepared from reticulocyte or nonreticulocyte sources ( Arnstein et al., 1964,1965; Drach and Lingrel, 1964; Kruh et al., 1964a,b; Weisberger et al., 1964; Brawennan et al., 1965). In none of these studies has globin synthesis been demonstrated, though formation of globin-like material or globin peptides has been reported. Recently, evidence has been obtained that a soluble protein-containing fraction prepared from reticulocyte ribosomes may be important for globin synthesis in the cell-free system (Miller et aZ., 1965). It is apparent that our knowledge of the characteristics of messenger RNA for hemoglobin, including the properties which contribute to its relative stability and the factors which determine its function at different stages of erythroid cell development, is rather incomplete. Further insight into the possible role of messenger RNA in the control of hemoglobin synthesis in Iater stages of erythroid cell development, when RNA synthesis has ceased, may be obtained from an analysis of studies on the formation of specific types of hemoglobin (see Section VI, B ) .

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ERYTHROID CELLS

235

Hemoglobins

Much information is available about the structure, properties, and function of hemoglobins, and the genetic control and mechanisms of synthesis of these proteins have been studied extensively (Itano, 1956; Schroeder, 1963; Jonxis, 1963; Ingram, 1963; Baglioni, 1963; Gerald, 1964; Weatherall, 1965; Keil, 1965; Kraut, 1965). In this discussion, only those aspects of hemoglobin structure, function, and synthesis will be reviewed which provide a basis for considering mechanisms which may regulate erythroid cell development. A. TYPESOF HEMOGLOBINS Hemoglobins are a structurally heterogeneous group of proteins. Three types of heterogeneity can be distinguished (Schroeder, 1963); heterogeneity associated with development; genetically determined heterogeneity; and so-called minor-component heterogeneity. Many mammalian species have hemoglobins during fetal development which differ from those present during adult life. This has been demonstrated for man, mouse, rat, rabbit, monkeys, apes, sheep, goat, and cattle (Chernoff, 1953; White and Beaven, 1959; Gilmour, 1941; Walker and Turnbull, 1955; Popp, 1965a; Barrowman and Roberts, 1960; Ackerman, 1962; Ingram, 1963; Weatherall, 1965). The hemoglobins of man have been studied more extensively than those of the mouse. There are mutations associated with hemoglobin variants which represent structural alterations in the amino acid sequence of one or another of the globin chains. In addition, there are hemoglobins normally present in small amounts which differ lrom the major component in amino acid sequence of one of the globin chains. I . Human Hemoglobins In adult subjects, hemoglobin comprises hemoglobin A, the major component in normal subjects, hemoglobin AZ, normally accounting for 1-3% of the total hemoglobins, and hemoglobin As?. Hemoglobin A, (Kunkel and Wallenius, 1955) is present in adult erythrocytes and appears to be a derivative of hemoglobin A, which develops with cell aging. Hemoglobin A, differs from hemoglobin A by having a glutathione group bound to it ( Muller, 1961). All normally occurring human hemoglobins consist of two pairs of identical polypeptide chains, one pair with the terminal amino acid structure valyl-leucyl, called a chains, and the other pair with the terminal structure valyl-histidyl, the @-type chains. In

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PAUL A. MAHKS AND JOHN S. KOVACH

hemoglobin A, these chains are termed (3, in hemoglobin AB, 6, and in hemoglobin F, y. The complete amino acid sequence of the a, P, 6, and y chains has been reported (Konigsberg et al., 1961, 1963; Braunitzer et al., 1961; Muller and Jonxis, 1960; Schroeder et al., 1963). The 6 chains differ from the P chains by about eight amino acids in a total of 146. The y and fl chains differ by 17 amino acids. There is also evidence for an “embryonic” hemoglobin in early human fetal development. Hemoglobins Gower 1 and Gower 2 have been detected in human fetuses with a crown-rump measurement of less than 8.5 cm, and the proportion of these hemoglobins is highest in the youngest embryos (Huehns et al., 1964a,b). It is believed that hemoglobin Gower 2 has normal a chains. The non-a chains, called E chains, differ in several tryptic peptides from the y chains of hemoglobin F, while hemoglobin Gower 1 consists entirely of one chain type (Huehns, 1964a). The peptide structures of human hemoglobin types are as shown in the following tabulation: Hemoglobin Hemoglobin Hemoglobin Hemoglobin

Gower 2 F A A,

In man, there are at least thirty hemoglobins which have been characterized as having a genetically determined alteration in structure which in each instance involves a single amino acid change in one or the other peptide chain. Studies of the mode of inheritance of these structurally abnormal hemoglobins has provided important information concerning the genetic control of hemoglobin synthesis (Ingram, 1963; Baglioni, 1963; Marks and Gerald, 1964; Gerald, 1964). The present evidence indicates that four separate genetic loci determine the structure of a, p, y, and 6 chains respectively. The genetic loci controlling a and p chain synthesis are not linked (Smith and Torbert, 1958; Atwater et al., 1960; Pugh et al., 1964). There is evidence that the genetic loci controlling [3 and 6 chain synthesis are closely linked. This is based on (1) family studies of subjects heterozygous for both a p chain and a 6 chain hemoglobin variant (Ceppellini, 1959; Horton et al., 1961; Ranney et al., 1963; Boyer et al., 1963; Horton and Huisman, 1963), and ( 2 ) the demonstration that a genetically determined abnormal hemoglobin, hemoglobin Lepore, has an amino acid sequence which suggests a fusion between the

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NHZ-terminal portion of the h chain and COOH-terminal portion of a chain ( Baglioni, 1962).

p

2. Mouse Hemoglohim In the mouse, as in man, adult hemoglobin contains two pairs of polypeptide chains ( also called (x and p). On electrophoresis, the hemoglobins of all strains of mice studied have either a single band or diffuse pattern composed of a major band with one or more minor bands (Ranney and Gluecksohn-Waelsch, 1955; Russell and Gerald, 1958; Popp and St. Armand, 1960). This electrophoretic difference in hemoglobins has been related to differences in 13 chain tryptic peptides (Hutton et ul., 1962a,b; Yopp, 1962a, 1965a). Differences in tryptic digests of a chains of certain strains have also been noted (Hutton et ul., 1962a, 1963; Popp, 1962b). A large portion of the amino acid sequence of the a chain of C57B1/6J mouse hemoglobin is known. Like human a, it consists of 141 amino acids and has a valine NH2-termiiial. It is estimated that it differs from human a chain in a minimum of 16 amino acids. The 1-3 chain is reported to contain 146 amino acids, as does human p chain, but its sequence is not yet known (Riggs, 1963; Popp, 1965a,b). Mouse fetal hemoglobin has been reported to have three or four electrophoretically distinct components ( Barrowman and Roberts, 1960; Barrowman and Craig, 1961; Craig and Russell, 1963a, 1964, 1966; Kovach et al., 1966a). These bands show characteristic quantitative changes during development ( Craig and Russell, 1966). Nothing is known of the structure of fetal hemoglobins. The fetal hemoglobins from strains of adults with hemoglobin of either electrophoretic pattern-the single band or the diffuse type-appear to be identical (Craig and Russell, 1963a). B. CONTROL MECHANISMS IN

THE

SYNTHESIS OF HEMOGLOBIN

A fundamental question in cell differentiation relates to the nature of the mechanisms regulating protein synthesis. Our present knowledge of the mechanisms of protein synthesis, a good portion of which has been derived from studies of reticulocyte hemoglobin formation, has been reviewed elsewhere (Crick, 1963; Lipmann, 1963; Watson, 1964, 1965; Schweet, 1964; Marks et al., 1964; Ingram 1965; Moldave, 1965; Singer and Leder, 1966). Factors which operate at the level of gene transcription are well recognized (Jacob and Monod, 1961; Stent, 1965; Watson, 1965). Regulation of protein synthesis at the level of translation is less well

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PAUL A. MARKS AND JOHN S . KOVACH

established but may be particularly important in cells with relatively stable messenger RNA. Hemoglobin synthesis during erythroid cell development presents several specific examples of control. One has to seek explanations for the striking preponderance of hemoglobin synthesis relative to that of other proteins; the apparently parallel synthesis of a and (3 type chains during the course of erythroid cell development; the conversion from synthesis of fetal hemoglobin to adult hemoglobin, which appears to be associated with changing sites, perhaps even structural patterns, of erythropoiesis; the relative amounts of major and minor hemoglobins synthesized. 1. Hemoglobin F and Hemoglobin A Synthesis

a. Hemoglobin Formation in Human Fetuses. In man at birth the level of hemoglobin F is generally about 85% of the total hemoglobin present, but this value is variable (White and Beaven, 1959; Armstrong et al., 1963). Synthesis of hemoglobin A has been demonstrated in erythroid cells of human fetuses aged 9 and 17 weeks (Thomas et al., 1960). In these studies, hemoglobin A and hemoglobin F synthesis was found in hematopoietic tissue obtained from the spleen, liver, and bone marrow of the human fetuses, although in different ratios. More hemoglobin A was synthesized in bone marrow cells than in spleen or liver erythroid cells. At birth, erythroid cell synthesis of hemoglobin A relative to that of hemoglobin F is threefold to fourfold greater than the ratio of hemoglobin A to hemoglobin F in the peripheral blood (Burka and Marks, 1964a). This is compatible with the concept that the erythroid cell synthesis of hemoglobin F decreases during late fetal development while the capacity for hemoglobin A synthesis is increasing. At birth, the major portion of the hemoglobin present in peripheral blood was formed at an earlier stage in fetal development. By the sixth month of life, the level of hemoglobin F has generally decreased to that of normal adults, namely, less than 2% of the total hemoglobin. b. Hemoglobin Formation in Adult man. In normal human adult subjects, hemoglobin F synthesis is very limited. However, in adults with certain types of thalassemia, hemoglobin F may again comprise as much as 90% of the total hemoglobin (Rich, 1952). The thalassemia syndromes are a group of inherited diseases which are characterized by a marked decrease in the capacity of the erythroid cells to synthesize hemoglobin A (Marks and Burka, 1964). In the group of thalassemic subjects with

8.

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239

elevated hemoglobin F, the defect in hemoglobin A formation appears to reflect a selective impairment of (3 globin chain synthesis (Weatherall et al., 1965). This defect has been localized to the polyribosomes (Bank and Marks, 1966). The capacity of the ribosomes of thalassemic cells to respond to polyU-directed phenylalanine incorporation is comparable to that of ribosomes from normal cells. This suggests that the primary defect in thalassemia major involves a genetically determined decrease or alteration in messenger RNA for (3 chains. c. Hereditary Persistence of Hemoglobin F . Further insight into the mechanisms that may be involved in control of hemoglobin synthesis has been derived from studies of a condition known as hereditary persistence of hemoglobin F, which is characterized by the persistence of high levels of hemoglobin F into adult life (Edington and Lehman, 1955; Jacob and Raper, 1958; Weatherall, 1965) . Hereditary persistence of hemoglobin F has been described in the homozygous state, in which no hemoglobin A or hemoglobin A2 is synthesized. In the heterozygous state, there is evidence that there is lack of function of one p and one 6 chain locus. Thus, in subjects heterozygous both for persistent hemoglobin F and for a structurally abnormal adult hemoglobin, such as hemoglobin S or C, there is no detectable hemoglobin A, suggesting that there is no functioning normal P-chain locus (Jacob and Raper, 1958; Went and MacIver, 1958). This condition of hereditary persistence of hemoglobin F may provide information about the genetic mechanisms regulating the conversion from the synthesis of hemoglobin F to that of hemoglobin A during the course of erythroid cell differentiation in the developing fetus. The failure of function of the p and 6 loci in this condition could arise by a deletion of the genetic loci determining the synthesis of these globin chains. Such a hypothesis, however, does not explain the persistence of hemoglobin F synthesis, since the amount of hemoglobin F is not related to that of hemoglobin A. It is possible that complete lack of function of the (3 and 6 loci permits the continued function of the y locus in the cis position. By analogy to bacterial genetic systems (Jacob and Monod, 1961), it is difficult to visualize how such a repression mechanism would act only on the y-locus in the cis position if it were not closely linked to it. There is no evidence of linkage between the y locus and the loci for (3 and 6 chains. Several workers (Gerald, 1960; Ceppellini, 1961; Neel, 1961; Motulsky, 1962) have proposed an alternative mechanism to account for the change from hemoglobin F to A, that is, from y chain to (3 chain synthesis. The function of the 0 and 6 loci may be under the control of a

240

PAUL A. MARKS AND JOHN S. KOVACH

closely linked regulator gene, again analogous to the operator gene in bacteria. If this regulator gene fails to function, the synthesis of P and 6 chains does not occur. This hypothesis also does not explain the continued function of the y locus in hereditary persistence of hemoglobin F, unless it is assumed that y chain synthesis is repressed only when the and 6 loci are activated. d. Cell Distribution of Hemoglobins A and F . Another aspect of the control of synthesis of fetal and adult hemoglobin is whether both proteins are synthesized in the same cell. A method is available which permits analysis of the presence of hemoglobin F within single cells based on washing the cells with solutions that selectively remove hemoglobin A from the erythrocytes (Kleihauer et al., 1957). In human subjects with hereditary persistence of hemoglobin F, this hemoglobin is found uniformly distributed throughout the cells of subjects heterozygous for this trait (Sheperd et al., 1962). This would indicate that in hereditary persistence of hemoglobin F the genetic defect is such that both y and 13 chains are formed in all cells and at similar relative rates. In adults with thalassemia and other anemias associated with increased levels of hemoglobin F, hemoglobin F is not distributed uniformly in the erythrocyte population (Bradley et al., 1961) . This has been interpreted ( Baglioni, 1963; Ingram, 1963) as suggesting that hemoglobin F synthesis in adult anemic subjects reflects a clonal selection of differentiating erythroid cells which favors synthesis of hemoglobin F relative to that of hemoglobin A. e. Hormonal Factor and Hemoglobin A and F Synthesis. Hormonal iactors (Rucknagel and Chernoff, 1955; Bromberg et al., 1957) may play a role in affecting the rate of hemoglobin F synthesis. Increased levels of hemoglobin F were detected in pregnant women and in women with hydatid moles, conditions associated with elevated levels of chorionic gonadotropins. Recently, it has been demonstrated that the thyroid hormone effects a specific induction of adult hemoglobin synthesis during amphibian metamorphosis (Moss and Ingram, 1965). The effect of the hormone erythropoietin on hemoglobin formation has been most extensively studied and is reviewed by Goldwasser in this volume. It is conceivable that the site of action of hormonal factors or other environmental influences which affect the relative rates of hemoglobin A and F synthesis may be at the level of regulator gene or genes controlling the activity of the structural genes for globin chains. f . Fetal Hemoglobin Formation in Mice. In fetal mice the synthesis of fetal and adult hemoglobins in different sites of erythropoiesis is easier

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to study than in human fetuses. In mice, it appears that yolk sac erythroid cells exclusively make fetal hemoglobin. This was first suggested by the finding of a correlation between the disappearance of fetal hemoglobin and the loss of yolk sac erythroid cells from the circulation (Table I ) (Barrowman and Craig, 1961; Craig and Russell, 1964). Recently, a cell separation technique was developed which permits examination of the type of hemoglobin synthesized in isolated yolk sac erythroid cells and hepatic erythroid cells. Little, if any, synthesis of fetal hemoglobin was detected in circulating liver erythroid cells or their precursors in hepatic tissue. Fetal hemoglobin synthesis was found in yolk sac erythroid cells (Kovacli et al., 1966a). The mechanisms regulating the synthesis of fetal and adult hemoglobin in mouse fetal hematopoietic tissue are not known. It is possible that environmental factors play an important role. The development of erythroid cells in yolk sac and in hepatic tissue occurs in sequential fashion, the activity in one site declining as that in the other increases. It appears that erythroid cell development in the yolk sac may differ in numerous aspects from that in hepatic fetal tissue. There are no data with respect to the differences in gene activation which underlie these different patterns of cell development. One may speculate that changes in the activity of multiple genes are involved in the different lines of erythroid cell development. Such a concept does not exclude the possibility that there is a single gene locus which has a primary or initiator function in effecting the sequence of gene actitvation which, in turn, determines the patterns of erythroid cell development. This might be analogous to the apparent mechanism of action of Ecdysone on the pattern of chromosomal puffing in giant chromosomes of flies (Beerman, 1962). 2. Control at the Lezjel of Translation

In the course of erythroid cell development, the synthesis of hemoglobin largely proceeds in cells in which there is no RNA synthesis. The control of the total amount and of the amounts of different types of hemoglobin synthesized probably involves, in part, factors which operate at the level of polyribosomes. A considerable body of data supports a dynamic model of polyribosome function in intact erythroid cells (Rich et al., 1963; Hardesty et al., 1963; Slayter et al., 1963; Marks et al., 1963a, 1965; Williamson and Schweet, 1964, 1965; Lamfrom and Knopf, 1964; Knopf and Lamfrom, 1965). Polyribosomes are a complex of two or more ribo-

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PAUL A. MARKS AND JOHN S. KOVACH

somes and messenger RNA. Polypeptide synthesis on polyribosomes proceeds sequentially from the NH2-terminal amino acid end by addition of amino acids carried by soluble RNA (Bishop et al., 1960; Dintzis, 1961). a. Polyribosome and Ribosome Function. In reticulocytes pretreated with puromycin (Williamson and Schweet, 1965) or NaF (Marks et al., 1965; Conconi et al., 1966) to inhibit protein synthesis and dissociate polyribosomes to 80 S ribosomes, the relationship between polyribosome formation and hemoglobin synthesis may be studied when the cells are then washed free of inhibitor and reincubated under appropriate conditions. Under these experimental conditions, polyribosomes form by a mechanism consistent with a sequential addition of 80 S ribosomes to messenger RNA as hemoglobin synthesis proceeds. The attachment of 80 S ribosomes to messenger RNA is at a position corresponding to the NHa-terminal amino acid of the polypeptide chain, or at a point before it. Release of globin chains from ribosomes occurs only after addition of the COOH terminal amino acid. The nature of the movement of ribosomes and messenger RNA relative to each other is not known. The recognition mechanism on polyribosomes for initiation and release of the globin chain is also not resolved. There has been recent progress in this area ( Adams and Capecchi, 1966) in studying bacterial protein synthesis which suggests that N-formylmethionine may constitute a start signal for the initiation of the polypeptide chains. The 80 S ribosomes in reticulocytes appear to be a heterogeneous population with respect to their activity in protein synthesis (Luzzatto et al., 1965). The majority of 80 S ribosomes which accumulate as erythroid cells develop to reticulocytes and mature erythrocytes have little or no endogenous activity and are incapable of combining with messenger RNA. These ribosomes probably reflect the process of erythroid cell development and are a step in the path of disappearance of the ribosomes. Some 80 S ribosomes also exist in reticulocytes which appear not to be associated with messenger RNA, but which are capable of attaching to preexisting polyribosomes with messenger RNA (Luzzatto et al., 1965; Joklik and Becker, 1965; Rich et al., 1963; Hardesty et al., 1963). These 80 S ribosomes are probably derived from polyribosomes and represent a minor portion of the total 80 S population in developing reticulocytes. In addition, there may be 80 S ribosomes associated with more or less intact messenger RNA, as evidenced by the capacity of a portion of 80 S ribosomes to synthesize hemoglobin in a cell-free system (Lamfrom and Knopf, 1965; Luzzatto et al., 1965). The proportion of these various forms

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243

of 80 S ribosomes, e.g., 80 S ribosome with attached endogenous messenger RNA, 80 S ribosome without messenger RNA but capable of attaching to messenger RNA; 80 S ribosomes without messenger RNA and not capable of attaching to messenger RNA, is probably determined by a variety of, as yet, undetermined factors. The amount of each form of 80 S ribosomes may be a factor determining the rate of hemoglobin synthesis during the course of erythroid cell development. Studies with the NaF- or puromycin-treated reticulocytes (Marks et al., 1965; Williamson and Schweet, 1965), as well as with developing erythroid cells (Marks et al., 1964; Glowacki and Millette, 1965) and with erythroid cells of thalassemic subjects (Burka and Marks, 1963), indicate that polyribosomes, per se, are not the rate-limiting factor in globin synthesis. Such studies indicate that the activity of polyribosomes in protein synthesis may vary considerably. It appears that there may be polyribosomes in intact erythroid cells which are inactive in protein synthesis (Marks et al., 1965). The regulation of synthesis rates and perhaps even of types of globins synthesized could involve conversion of polyribosomes from active to inactive forms and vice versa. b. Synthetic Time of Globin Chains. The speed of globin chain synthesis on a polyribosome can change during the course of protein synthesis in intact reticulocytes. For example, as polyribosomes re-form in cells pretreated with NaF, the synthetic time for globin chain synthesis may increase more than twofold (about 17 seconds to 42 seconds in reticulocytes incubated at 37°C) (Conconi et al., 1966). More information on the control of specific globin chain synthesis at the level of polyribosomes was obtained in studies of the rates of formation of hemoglobins A and A, (Ingram, 1964; Reider and Weatherall, 1965; Winslow and Ingram, 1966). Hemoglobin A is synthesized at about forty times the rate of hemoglobin A,. The synthetic time for 6 chain synthesis appears to be considerably longer than that for a or p chain. There are several factors which may explain different synthetic times for different globin chains. The assembly time for a given globin chain may be controlled by the requirement for a specific sRNA ( Ames and Hartman, 1963). Such a control mechanism would predict that on either side of the site of the “modulating” sRNA the rate of growth of the peptide chain would be similar (Englander and Page, 1965; Winslow and Ingram, 1966). This does not seem to be the case for globin chain synthesis. Analysis of the assembly of (3 chains in rabbit reticulocytes (Dintzis, 1961)

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PAUL A. MARKS AND JOHN S. KOVACH

and of [3 and 6 chains in human reticulocytes (Winslow and Ingram, 1966) indicates a nonuniform rate of peptide chain growth in each instance, with a region of rapid rate of assembly followed by a region of slower assembly. Rate-controlling factors which could account for this pattern of globin chain synthesis include an effect of folding of the growing peptide chain, or a structural factor in the polyribosome. It has also been suggested that heme insertion in the growing globin chain may be a factor controlling its synthetic rate ( Winslow and Ingram, 1966). There is no evidence that heme is inserted in globin on the ribosomes. Heme and iron do increase polyribosome size and the rate of globin synthesis by a mechanism which is not elucidated (Waxman and Rabinowitz, 1965; Grayzel et al., 1965; Conconi and Marks, 1966). c. Globin Chain Complementation. Regulation at the polyribosome level probably is important in maintaining the balance between a chain and 0-type chain synthesis in the course of erythroid cell development. Evidence has been obtained which is interpreted as indicating a requirement for P chain in the release of a chain from ribosomes (Baglioni and Colombo, 1964), supporting the concept that chain complementation may play a role in regulating globin synthesis. In thalassemia, the selective impairment in P-chain synthesis is associated with a marked decrease in hemoglobin A (Marks and Burka, 1964). In thalassemia, there is an excess of a chains synthesized relative to fl chains ( Bank and Marks, 1966). These excess a chains are released from the ribosomes. These data suggest that there is no absolute requirement of fi chains for the release of a chains from ribosomes. d. Template Decay. The increase in synthesis of hemoglobin F in cells from adult subjects with certain anemias may also reflect control at the level of polyribosomes. It has been suggested that messenger RNA for different globins may decay at different rates during the course of erythroid cell development ( Burka and Marks, 1964a). There is no direct evidence bearing on rates of decay of specific messenger RNA in developing erythroid cells. Such an hypothesis could explain certain findings with respect to relative rates of globin chain synthesis. Thus, the increased levels of hemoglobin F under certain conditions in which erythroid cell development is stimulated might reflect a slower rate of decay of messenger RNA for y chains than occurs during the course of normal erythropoiesis. In normal bone marrow cells the rate of hemoglobin F synthesis relative to that of hemoglobin A exceeds that in circulating reticulocytes (Necheles et al., 1964, 1965). This observation is compatible with a more

8.

DEVELOPMENT OF M A M M A L I A N ERYTHROID CELLS

2 15

rapid rate of decay for y messenger RNA than for p messenger RNA under normal conditions. Further, it appears that the ratio of synthesis of hemoglobins A2 to A is also greater in the more immature cells of human bone marrow than in reticulocytes (Reider and Weatherall, 1965; Winslow and Ingram, 1966). These findings, too, can be explained by a more rapid rate of decay of 6 messenger than of p messenger in the course of normal erythroid cell development. In summary, control at the level of polyribosomes may be exerted by many factors, including (1) polyribosome content, ( 2 ) specific sRNA's, ( 3 ) peptide chain folding, ( 4 ) globin chain complementation, ( 5 ) availability of iron or heme, ( 6 ) energy, and ( 7 ) rate of decay of messenger RNA. VII. Concluding Comment

Erythroid cell differentiation is an attractive system for studying a variety of fundamental problems in differentiation. The great strides in techniques and knowledge in molecular biology, genetics, and biochemistry are making approaches to these problems increasingly feasible. Particular emphasis must be placed on the need to characterize better the stem cell precursor of erythroid cells. It is apparent from this review that studies in bacterial and viral systems have had a profound impact on the approach to problems of development. It seems reasonable to expect that the spectrum of mechanisms involved in regulation of erythroid cell differentiation include factors which are not apparent in studies of bacterial and viral systems. ACKNOWLEDGMENTS The authors are indebted to their colleagues who have permitted them to quote from their unpublished manuscripts. We are grateful to Miss Lila Manor and Mrs. Barbara Pappin for their expert assistance in preparing the manuscript. W e are deeply grateful to Dr. Vernon Ingram for reading this manuscript during its preparation. The investigations summarized in this review from the laboratory of the authors were supported, in part, by U.S. Public Health Service Grant GM 07368 and CA 02332 and National Science Foundation Grant NSF-GB 1817 (4631 ). John S. Kovacli is a Trainee in hematology and i\ supported by U S . Public Health Service Grant TI-AM 5231-06. REFERENCES Ackerman, G. A. (19G2). Anat. Record 144, 239. Adachi, K., Negnno, K., Nakao, T., and Nakao, M. (19G4). Biochim. Biophys. Acta 91, 513.

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CHAPTER 9

GENETIC ASPECTS OF

SKIN AND LIMB DEVELOPMENT* P . F . Goetinck DEPARTMENT OF ANIMAL GENETICS, STOFIRS AGRICULTURAL EXPERIMENT STATION, UNIVERSITY OF CONNECTICUT, STORRS, CONNECTICUT

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Differentiation of the Embryonic Chick Skin . . . . . . . . . . A. Description of Normal Feather and Scale Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Role of Dennis and Epidermis in Feather and Scale Differentiation .......................... C. Dermal-Epidermal Relationships in Mutants ...... D. Protein and RNA Synthesis during Feather Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Differentiation of the Embryonic Limb . . . . . . . . . . . . . . A. Description of the Early Stages of Limb Development in the Chick Embryo .................... B. The Role of the Ectoderin in Limb Outgrowth . . . C. Mesoderm-Ectoderm Interactions in Normal and Mutant Limb Development .................... IV. Summary and Concluding Remarks ................. References ......................................

253 254 254 255 258 261 263 263 264 270 277 281

1. Introduction

The differentiation of a number of embryonic structures is dependent on an interaction between the epithelial and mesenchymal components Scientific contribution No. 171 of the Agricultural Experiment Station, University of Connecticut. Contribution No. 129 of the Institute of Cellular Biology.

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of the particular system. Such interactions have been described in the development of the kidney (Grobstein, 1955), salivary gland (Grobstein, 1953), thymus ( Auerbach, 1960), pancreas ( Golosow and Grobstein, 1962), skin and feathers (Sengel, 1957; Saunders, 1958; Wessells, 1962; Rawles, 1963), and limbs (Zwilling, 1961; Amprino, 1963, 1965). The availability of a number of mutations which affect skin and limb development in the chick embryo makes these two systems particularly suitable for investigations of the genetic control of developmental processes. The present review will be limited to the effects of mutations on epitheliomesenchymal interactions in skin and limb development. The studies on mutant long-bone development in vitro (Wolff and Kieny, 1957, 1963; Kieny, 1962; Kieny and Abbott, 1962; Elmer and Pierro, 1964) will not be covered. II. Differentiation of the Embryonic Chick Skin

A. DESCRIPTION OF NORMALFEATHER AND SCALE DEVELOPMENT The purpose of the brief accounts in this and the following section is to provide the background for describing the genetic studies. Embryologically the skin of birds arises from two layers, the mesoderm and the ectoderm, and gives rise to a variety of regionally specific integumentary derivatives such as feathers, scales, beak, spurs, combs, wattles, and glands. The feathers are arranged in tracts which are separated by featherless areas. They first appear in the chick embryo anteriorly on the back between the sixth and seventh days of incubation. By the eleventh day all feather tracts are completed. Scales are first visible on the anterior part of the foot at about 11 days of incubation and at 12 days on the posterior part of the limb. Histologically, one of the first indications of feather and scale formation is seen as a condensation of the loosely arranged mesodermal cells into localized groupings immediately under the ectoderm (Fig. 1 ) . At the same time, or slightly before these dermal condensations become evident, the ectodermal cells associated with them elongate and slant inward at their distal end. Wessells (1965) has recently studied DNA synthesis in relation to feather germ formation. Cell density measurements and quantitative pulse-labeling experiments with tritiated thymidine in carefully staged skin suggest that the dermal condensations arise as a result of differential mitotic activity in the mesoderm. Before this stage, cells exhibiting mitotic activity and uptake of tritiated thymidine are

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distributed at random throughout the mesoderm. Once the dermal condensations have formed, their cells show no uptake of tritiated thymidine and no mitotic activity for about 20-30 hours. After this period of replicative inactivity, DNA synthesis starts again as outgrowth of the feather germ begins. During the formation of the feather one also observes differences in the distribution of several macromolecules. Thus, alkaline phosphatase appears in each dermal condensation of feathers and scales soon after this structure is formed, and at the same time the RNA concen-

FIG. 1. Transverse sections through middorsal skin of embryos ranging from 6% ( 1 ) to 8 days ( 4 ) of development. x 300.

tration increases rapidly in the basal cytoplasm of the epidermis overlying the dermal papilla (Hamilton, 1965; Thomson, 1964). Similarly, sulfated mucopolysaccharides are distributed uniformly throughout the dermis before germ formation and later become concentrated in distinct parts of the dermal condensations and the outgrowing feather (Sengel et al., 1962).

B. THEROLE OF DERMISAND EPIDERMIS IN FEATHER AND SCALE DIFFERENTIATION Interactions between the mesoderm and the epidennis have been shown to take place in skin differentiation by separating the embryonic skin into these component parts and recombining them with dermis or

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epidermis from other areas. The separation of the embryonic skin into its mesodermal and epidermal components can be achieved by incubating the skin in a dilute trypsin solution, After the appropriate recombinations, the composite skins are cultured either in vitro or on the chorioallantoic membrane of a chick embryo (Sengel, 1957, 1958; Rawles, 1963; Wessells, 1962, 1965). From these studies it is clear that the outcome of interactions between the dermis and epidermis differs depending on the age and site of origin of the particular components. For example, the epidermis in the tarsometatarsal region of the leg develops scales in response to interaction with the dermis of this area; it can be shown that tarsometatarsal dermis acquires the ability to induce scales only in later development, for, when tarsometatarsal dermis, derived from embryos after the thirteenth day of incubation, is combined with back epidermis from 5-82pday embryos, scales develop; however, when tarsometatarsal dermis from earlier embryos is used, it induces feather formation in back epidermis. On the other hand, the mesoderm of the spur primordium (located distally on the posterior part of the foot) already has a spur-inducing capacity in the 9-day embryo, since when combined with 5-8Q-day dorsal epidermis it causes it to form a spur. The dermis of the middorsum of =+-day embryos induces feathers when combined with tarsometatarsal epidermis from 9-12-day embryos. Beak dermis is strongly inductive, for it is capable of inducing beak formation in the middorsal epidermis of 8-8J-day embryos. On the basis of these results Rawles (1963) has classified the various sources of mesoderm according to "strength" of inductive activity, tarsometatarsal dermis being the weakest inducer, followed in increasing strength by middorsal and beak and spur mesoderm. The responsiveness of dorsal epidermis to mesodermal induction changes around 8-84 days of development ( Rawles, 1963). When this epidermis is recombined with tarsometatarsal mesoderm from 13-15-day embryos, it no longer forms scales, as it does when younger, but continues to differentiate in the feather direction. Presumably it has become by that time induced by its own dermis and hence is no longer responsive to the weak stimulus of the tarsometatarsal dermis. This restriction in response does not pertain to all mesodermal stimuli, however; as mentioned above, the developmental course of this epidermis can still be altered at this stage by combination with beak mesoderm, resulting in the formation of a beak. Further evidence for developmental lability of the epidermis and for the significance of the mesoderm in epidermal differentiation has been

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presented by McLoughlin ( 1961a,b). Recombinations of epidermis from 5-day embryonic limb buds with mesenchyme from proventriculus, gizzard, and heart of the same age resulted in each case in the formation of epithelial structures representative of the type of mesenchyme with which the epidermis was in contact. The nature of the interactions between the dermis and the epidermis in skin differentiation is not known. However, some of the dermal functions are indicated by experiments in which isolated epidermis was cultured in uiit~o.Under such conditions, the nuclei in the epidermis of 5-day limb buds show few mitotic figures ( McLoughlin, 1961a). Similarly, nuclei in isolated and cultured shank epidermis of ll-day-old chick embryos do not divide, as evidenced by failure to incorporate tritiated thymidine, and the cells in the basal layer lose their characteristic columnar shape (Dodson, 1963; Wessells, 1963). It is from this normally mitotically activr basal cell layer that the outermost cells are derived which synthesize keratin and become cornified. Both thymidine incorporation and the histodifferentiation of the epidermis can be restored by recombining it with dermis. Attempts at characterizing the conditions in the dermis responsible for maintaining the epidermis as an organized structure have met with some success. Dodson (1963) reported that dermis (either killed by repeated freezing and thawing or dissociated into single cells by trypsin) or collagen gels could sustain the orientation of the basal cells in epidermis cultured on an embryo extract-plasma clot. On the other hand, epidermis did not survive very long when supplied with dermis that was killed by freezing and thawing and subsequently trypsinized, or with heat-killed dermis. These results were confirmed by Wessells ( 1964), who used similar culture conditions. However, when a chemically defined medium was used, frozen-thawed dermis and tropocollagen failed to duplicate the effects obtained in the cultures on plasmaembryo extract medium. This led Wessells (1964) to investigate the effects of supplementing the defined medium with chicken plasma, chick embryo extract, and other large molecular substances. Of these only the embryo extract or a particulate fraction therefrom provided an effective supplement to the defined medium for maintaining basal cell orientation in the isolated epidermis cultured on a substratum of tropocollagen, frozen-thawed dermis, or millipore filter. The macromolecular fraction has been partially characterized and found to be nondializable, heat labile, and sensitive to proteolytic enzymes ( Wessells, 1964). Outgrowth of feather germs usually does not take place in culture

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when embryonic skin is explanted before 6-6i days of incubation (Sengel, 1958; Bell, 1964). After this stage normal feather development will take place in explants in culture. It may be significant that this stage corresponds in time to the formation of the dermal condensations. In fact, Wessells ( 1965 ) has shown by reciprocally exchanging epidermis and dermis from skin of predermal and postdermal condensation stages that only one of the two layers of the composite skin must have reached the dermal condensation stage in order for feather formation to take place in vitro. The percentage of feathers that develop in skin explants from embryos before the sixth day of incubation can be greatly increased by supplementing the defined medium with various proteins. Serum albumin is particularly suitable for this purpose (Bell, 1964). Sengel (1958) reported that early skin, which normally would not form feathers in vitro, will do so when cultured in association with neural tissue or when the medium is supplemented with a fraction of chicken brain. This active fraction was dialyzable, heat stable, and resistant to acid and alkaline hydrolysis (Sengel, 1964). Similarly, feathers failed to develop on the back of operated embryos from which the spinal cord had been removed very early in development. Therefore, the suggestion arises that neural tissue may play some role in feather differentiation and that its action may take place before the onset of the mesodermal-epidermal interactions described above. The nature of these neural-tissue or brain extract effects and their possible relationship to the action of the serum proteins mentioned above is not known. C. DERMAL-EPIDERMAL RELATIONSHIPSIN MUTANTS

Studies with normal embryonic skin have revealed that differentiation of the epidermis can be altered by recombining it with mesenchyme with different inductive capacities. That both induction and the response to it are under genetic control was shown by the analysis of a mutant in which ectodermal differentiation is absent and another in which the normal developmental pathway is altered. Birds which are homozygous for an autosomal recessive gene, “scaleless,” lack feathers in all but a few specific areas of the body. They also lack scales, footpads, and spurs ( Abbott and Asmundson, 1957). Reciprocal exchanges between leg-bud components of 39 -day scaleless and normal embryos resulted in expression of the scaleless phenotype only in composite limbs made up of scaleless epidermis and normal mesodermal

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constituents (Fig. 2). Leg buds composed of scaleless mesoderm and normal epidermis gave rise to normally scaled limbs (Goetinck and Abbott, 1960, 1963). These results were confirmed in in uitm experiments and extended to interchanges involving epidermis and dermis from areas which normally form feathers ( Sengel and Abbott, 1962, 1963). Thus the

FIG. 2 . Composite limbs which developed as flank-grafts. Left: limb composed of scaleless ectoderni and normal mesoderm. Although this limb is covered with some feathers, no scales are evident. Right: limb composed of scaleless mesoderm and normal ectoderni. x 2.8. (From Goetinck and Abbott, 1963. )

scaleless mutation affects only the epidermis in the interacting system in such a way that it is not competent to respond to the dermal induction. The scaleless mesoderm from both the shank and back areas reacts completely normally. Recently I have investigated the dermal-epidermal interactions in the limb buds of embryo of the Brahma breed by means of exchanges and recombinations of limb components ( Goetinck, 1966). This particular breed of chickens is characterized by having one to three rows of feathers

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along the fourth tarsometatarsus and on the fourth digit, leg areas which in other breeds are covered entirely by scales (Fig. 3 ) . This condition, known as ptilopody, is inherited as an autosomal dominant trait. In the embryo, feather germs appear on these leg areas first on the tenth day of development, scales on the eleventh day on the anterior part of the foot and on the twelfth day posteriorly. For the recombination experiments

FIG.3. Legs of 12-day-old embryos. Left: normal. Right: ptilopody. Note feathers along the fourth tarso-metatarsus in the mutant. x 3.5.

white leghorn embryos were used as a source of components of normally scaled limbs. Recombinations were made between 3-34-day-old hind limb bud parts of Brahma and of white leghorn embryos and the composite limb buds were grafted into the flanks of embryos and allowed to develop. Normal anteroposterior and dorsoventral relationships of the two components were maintained in recombining them into composite limb buds. It was first found that limb buds composed of Brahma mesoderm and of white leghorn epidermis gave rise to limbs with feathers along the fourth tarsometatarsus and on digit IV. However, when the reciprocal recombination-Brahma epidermis with white leghorn mesoderm-was

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analyzed, it too resulted in the formation of limbs with feathers distributed as in the intact Brahma. Therefore, unlike the scaleless mutation which affects only the epidermis, the ptilopody genotype affects both the limb mesoderm and limb epidermis, endowing both with feather-forming capacity in response to the white leghorn counterpart. In order to establish whether in the Brahma mutant the epidermis or the mesoderm determines the characteristic topographical localization of the feathers along the fourth tarsometatarsus, Brahma limb epidermis was recombined with a Brahma limb mesoderm with its anteroposterior axis reversed. The composite limbs had feathers along the fourth tarsometatarsus and on digit IV and the resulting feather pattern clearly demonstrated that it is the mesoderm which determines the localization of the feathers. These results are analogous to those presented by Zwilling (1959) from his studies on chicken-duck limb chimeras produced by combination of limb components. In this case duck-like interdigital webbing developed in limbs composed of chicken mesoderm and duck epidermis as well as in those made up of duck mesoderm and chicken epidermis. Zwilling suggested that a reinforcing mechanism for the development of webbing in the duck limb may have evolved through natural selection. It is possible that a similar reinforcing mechanism may have been brought about by artificial selection for ptilopody. The Brahma is a fancy breed of poultry selected continuously for specific breed characteristics; feathered shanks is one of the “desirable” characteristics in the Brahma breed. D. PROTEINAND RNA SYNTHESIS DURING FEATHER DEVELOPMENT The concept of differential gene activation as a fundamental aspect of histodifferentiation was formulated by Morgan (1934). We have since learned that genes determine the sequence of amino acids in proteins and that this step is mediated through RNA. Since different tissues of the same genotype contain a different array of proteins which appear at various times during development, the presence or absence of any protein in a cell is a reflection of the activated or repressed state of specific genes. With respect to embryonic skin we have already mentioned that alkaline phosphatase appears in the dermal condensation immediately after this structure is formed. Hamilton and his associates (see Hamilton, 1965, for a review) have stressed the importance of this enzyme for the continued growth and differentiation of the feather. The addition to the culture medium of compounds which inhibit alkaline phosphatase has

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been shown to arrest feather development. Furthermore, the addition of the enzyme to culture medium will stimulate feather outgrowth and increase the RNA in the basal parts of the cells in the epidermis. Hamilton has reported that this RNA is located in the mitochondria and that respiratory and mitochondria1 inhibitors will inhibit feather formation with a concomitant reduction in RNA content in the basal parts of the epidermaI cells. Various analogs of naturally occurring bases of RNA have also been shown to inhibit feather formation, and the results of their action may be noticed either in the epidermis or in the mesoderm. Other methods for the investigation of biochemical events which take place at the time of induction and during feather development have been used by Bell and his associates. Using double diffusion tests in agar, they detected three new antigens in 6-day-old skin ( Ben-Or and Bell, 1965). Of these three, one is skin-specific and two are stage-specific in that their presence could be established in other tissues by absorbing the antiserum with extracts from other organs. Since the time at which these antigens are first detected corresponds to the developmental stage at which formation of the dermal condensations is observed, it would be of interest to determine their localization, and particularly that of the skin-specific antigen, in the developing feather germ. Two additional stage-specific antigens become apparent at about 11 to 12 days of incubation, and another skin-specific antigen is found in 13-day embryos. None of these antigens has been identified; presumably they are new proteins which are being synthesized at the time of detection and their appearance therefore reflects the genetic activity of these tissues at a specific time of development. However, the requirement for messenger RNA as an intermediate between DNA and a protein to be synthesized does not permit one to use the detection of new proteins as a criterion for establishing the exact time of gene activation. Indeed, control mechanisms at the translation level have been postulated in the developing feather. The optical density profile of polysomes from embryonic skin and feathers in a sucrose density gradient remains unchanged between 5+ and 13 days of incubation, and it is different from that of other chick tissues examined in that it shows a sharp polysome peak which consists of four ribosomes and which has a sedimentation constant of 158 S (Bell, 1964; Scott and Bell, 1964). In skin from 9-13-day-old embryos this 158 S polysome population is characterized by and different from others in the skin in its resistance to ribonuclease. Of particular significance is the fact that no protein synthesis can be associated with the 158 S fraction up to 14 days. Beginning with

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14 days, however, the 158 S material becomes sensitive to ribonuclease and it is now activated to make protein for the first time. Electron micrographs of these inactive ribonuclease-sensitive polysomes show them to be arranged in tightly packed symmetrical squares, in contrast to the 158 S polysomes of 14-day feathers, which are strung out in a chain (Bell et al., 1965). At 14 days the polysome profile also changes and polysomes consisting of five and six ribosomes become evident in addition to the four-ribosome polysomes. It has been suggested that the inactive 158 S polysomes, whose messenger RNA is somehow protected against ribonuclease, are precursors of the five- and six-ribosome polysomes and that the appearance of the latter may indicate either that there is random attachment of ribosomes during translation of a single species of messenger RNA or, alternatively, that several species of messenger RNA may be found in the inactive 158 S material (Humphreys et aZ., 1964). It seems therefore that as early as 9 days of incubation messenger RNA is being synthesized in the embryonic skin which is being translated into protein (or proteins) only at 14 days. It should be stressed that a number of events take place at this stage of development; as mentioned earlier a new skin-specific antigen becomes apparent and X-ray diffraction studies of embryonic skin first show the adult pattern of p-keratin at about 14 days of incubation (Bell and Thathachari, 1963). Although it is very possible that all events which take place around 13 and 14 days are causally related, this very important question remains to be established. 111. Differentiation of the Embryonic Limb

A. DESCRIPTION OF THE EARLYSTAGESOF LIMB DEVELOPMENT IN THE CHICKEMBRYO The two main components of the embryonic limb are an internal mass of mesodermal cells, the mesoblast, which progressively gives rise to the skeleton, the muscles, and the dermis; and the epidermal jacket from which develops the integument of the limb with its differentiations (feathers, scales, spurs, etc.). The first indication of a limb bud is the persistent thickening of a longitudinal fold of the body wall lateral to the somites. As the mesoderm thickens at the limb sites, the overlying epidermis changes distally from a cuboidal to a columnar type of epithelium. At stage 18 (stages of Hamburger and Hamilton, 1951) the epithelium covering the distal tip of the limb bud forms a nipple-like crest or ridge of columnar cells, the apical epidermal ridge ( AER). Viewed in a

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cross section the cells of the AER are radially oriented toward a small sinus which lies at the immediate base of the AER (Fig. 4). Between stages 19 and 20 the limb becomes asymmetrical owing to a more rapid development of the postaxial side. The dorsal side of the bud is more convex than the ventral side, and the AER is located more ventrally to the midline. From stages 21 to 22, the asymmetries become even more pronounced and the limb bud develops most rapidly postaxially. Asso-

FIG.4. Cross section of a limb bud from a normal embryo (stage 19) showing the apical epidermal ridge. x 200.

ciated with the postaxial growth is a much thicker AER. At stage 24 the outline of the foot-paddle becomes asymmetrical, being more developed posteriorly than anteriorly. By stage 27 the digits may be distinguished and the major components of the leg are clearly recognizable. The AER, though somewhat flattened, is still present, whereas the rest of the epidermis remains cuboidal. B. THE ROLEOF

THE

ECTODERM IN LIMBOUTGROWTH

The importance of the ectoderm and more specifically the AER in the development of the limb of the chick embryo was first shown by Saunders

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(1948), who found that after the surgical removal of the AER the limb developed with its distal parts missing. The younger the embryos at the time of AER removal the greater was the deficiency of the terminal parts. Using similar surgical methods for the removal of the AER, Amprino and Camosso (1955a,b) confirmed these results. Of particular interest are the studies on wing development in a wingless mutation in the chicken ( Zwilling, 1919). In embryos homozygous for the recessive “wingless” gene wing buds develop through the third day of incubation, at which time their further development ceases and no distal parts are formed. Histological examinations showed degeneration of the AER of the wings. The suggested requirement for AER in the outgrowth and development of the distal limb parts received support from further studies. Zwilling (1955) removed the AER from extirpated limb buds by incubating them in a solution of the chelating compound disodium ethylenediaminetetraacetate (EDTA), He obtained no distal outgrowth following grafting of the denuded mesoblasts into the flank of embryos. Distal outgrowth is obtained from EDTA-denuded mesoblasts only if they are recovered with an AER (Zwilling, 1955; Gasseling and Saunders, 1961) or if the mesoblasts are deliberately (Bell et al., 1962) or accidentally contaminated with ectoderm. Goetinck and Abbott (1963) investigated the possibility of incomplete removal of ectoderm from limb buds of normally scaled stock after EDTA incubation by transplanting the mesoblasts into the flank of scaleless hosts. Since the development of the phenotype of scaleless ectoderm is autonomous even when it is in contact with normal mesoderm, this combination of phenotypically different grafts and hosts made it possible to ascertain the source of ectoderm in case distal structures developed from the presumably denuded mesoblasts. The results from these studies were clear. Of the grafts which could be recovered, 92y0 developed no distal structures. These grafts were covered with host (scaleless) ectoderm. The other 8% developed distal structures, but in each case the terminal parts of the grafts were covered with scales (Fig. 5). The use of scaleless ectoderm as a marker therefore clearly showed that the outgrowth in the exceptional cases was associated with contamination of the mesoblasts with donor ectoderm. HampC (1956, 1959) studied the development of limb buds from which the prospective area (mesoblast and AER) of the tarsometatarsals was excised. The most distal structure which developed in such limbs was the tibia; however, when an AER was placed back on the cut surface of the stump, limbs complete with metatarsals and phalanges developed. It ap-

FIG.5 ( a ) A limb which developed from an EDTA-denuded mesoblast. The transplant stock. The host embryo is scaleless. Incomplete removal of the epidermis from the transplanted on the terminal part of the limb. The arrow points to the line of demarcation of scaled and view of the grafted limb at the line of demarcation of scaled and scaleless tissues (arrow). 1963.)

was derived from normally scaled mesoblast is indicated by the scales scaleless tissues. x 3. ( b ) Enlarged x 20. (From Goetinck and Abbott,

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peared as if the AER induced the formation of the distal structures from more proximal parts of the mesoblast. Additional evidence for an inductive role of the AER epidermis came from the laboratory of Saunders (Saunders et al., 1955, 1957, 1959b). Blocks of mesoblast were transplanted from the prospective thigh region (i.e., proximal part of the leg bud) to various regions of the wing bud. Only when grafted in contact with the AER of the wing bud did this prospective thigh tissue give rise to distal leg structures, indicating that the AER influenced the development of the grafts. Using focused ultrasound to remove the AER, Bell et al. (1959) in an early report stated that 19% of the denuded mesoblasts developed distal structures following grafting to the flank or in the coelomic cavity of 3day host embryos. In a more recent publication (Bell et al., 1962) only 2% of the limb buds denuded by ultrasonication gave rise to distal structures, presumably owing to a more effective removal of the AER. However, another possibility had previously been raised; Bell et ul., (1959) made the observation that not only the epidermis but in some cases also the epidermal basement membrane breaks down as a result of ultrasonication and suggested that the absence of this structure may be a prerequisite for distal outgrowth of mesoblasts denuded of the AER. While this explanation may hold for mesoblasts deprived of basement membrane by ultrasonication, it is not more generally applicable. When the basement membrane of EDTA-denuded limb buds is removed with collagenase, the denuded mesoblasts do not develop distal structures when grown as flank grafts (Goetinck and Abbott, 1963). However, when such collagenase-treated mesoblasts are recovered with limb epidermis normal Iimbs develop. A different point of view toward the role of AER has been advanced by Amprino and Camosso (1955a,b). They too found that no distal outgrowth takes place after the surgical excision of the AER. They found, however, that distal outgrowth can take place if, in addition to the AER, a few cell layers of the underlying mesoderm are also removed. These authors proposed that the amount of distal outgrowth obtained is inversely related to the amount of cell death caused in the distal mesoderm. Although they do not view the epidermis as an entirely passive component of the limb, they do not ascribe an inductive role to the AER (Amprino, 1965). Amprino and Camosso (195%) have done carbon marking experiments which indicate that the epidermis grows distally more rapidly relative to the mesoderm and they have suggested that the AER

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may simply be an area where the ectodermal cells pile up, and not necessarily a site of specific induction. More recently, Searls and Zwilling (1964) found that a new AER may regenerate from residual epidermis; they cautioned that a variety of previous experiments in which distal limb parts developed following removal of the AER and presumably in

FIG. 6. Right leg of a 9G-day polydactylous embryo. The duplication involves only digit I (arrow). x 8.

its absence, may have to be reconsidered in the light of this finding. For a complete analysis of the two points of view on the role of the AER in limb development the reader is referred to the reviews by Zwilling (1961) and by Amprino ( 1965). While in the absence of AER distal limb parts fail to develop, grafting of an AER on each lateral surface of an EDTA-denuded mesoblast results in the formation of distal limb parts in two different planes (Zwilling, 1956b). A correspondence between AER and distal limb development

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is abundantly suggested by descriptive studies in four mutants which affect the normal morphology of the limb. Thus, embryos heterozygous

or homozygous for “dominant polydactyly” (Fig. 6 ) or its allele “duplicate” have a more extensive AER in the preaxial part of the limb, i.e., the site of supernumerary digit formation (Zwilling and Hansborough, 1956). Embryos homozygous for two autosomal recessive genes of different origin, “talpid“’ (Abbott et al., 1960) and “talpid”” (Ede and Kelly,

FIG.7. Left leg of dactylous foot. x 10.

it

B%-cIay talpid embryo. Seven digits are evident in this syn-

1964), have seven to eight syndactylous digits per foot. Of these, none can be identified as normal digits (Fig. 7 ) . During development the talpid limb buds have a very extensive AER covering the greatly enlarged distal area of the entire limb paddle. The area covered by the AER in talpid’ leg buds increases significantly over that of normal embryos and by stage 25 is 50% more extensive than in normal siblings ( Goetinck and Abbott, 1964). As mentioned earlier, embryos homozygous for two recessive wingless mutations of different origin have been examined (Fig. 8); in these the AER flattens out on the third day of development with a concomitant cessation of distal limb outgrowth ( Zwilling, 1949, 1956c) . The autosomal recessive trait, “eudiplopodia”

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(Rosenblatt et al., 1959), is characterized by having the supernumerary digits situated in a second plane above the normal toe complement, which is always present (Fig. 9 ) . The outgrowth of these digits is preceded by the formation of a secondary AER (Fig. 10) on the dorsal surface of the limb bud (Goetinck, 1964). The int,eractions between mesoblast and epidermis in these mutants will be the subject of the following section.

FIG. 8. Cross section through a 4-day wingless limb bud. In contrast to normal limb buds (see Fig. 4) the apical epidermal ridge is lacking in the mutant. x 300.

Finally, it might be pointed out that the morphological and functional differences between the AER and the rest of the limb epidermis are paralleled by sharply delineated metabolic activities in the AER, as revealed in histochemical studies. Thus, Hinrichsen (1956) reported a high concentration of ribonucleic acid in the AER of the mouse embryo, and has interpreted this to be indicative of active protein synthesis in this structure. Furthermore McAlpine (1956) and Milaire (1956, 1963, 1965) have demonstrated a high level of alkaline phosphatase in the AER in several mammalian species as well as in the chick embryo. C. MESODERM-E~ODERM INTERACTIONS IN NORMAL AND MUTANT LIMB DEVELOPMENT

In the preceding section results from several laboratories have been presented which have led to a wide, although not unanimous, acceptance of the inductive role of the AER. Some of the most cogent evidence bear-

FIG.9 ( a ) Left leg of a 9%-day eudiplopod embryo. The duplications of the digits are in two planes. of a 9%-day normal embryo. x 10. (From Goetinck, 1964.)

x

10. ( b ) Left leg

FIG. 10. Cross sections of eudiplopod hind limb buds, ( a ) Left limb, stage 24; the arrow points to the second epidermal ridge in the dorsal ectoderm. x 200. ( b ) Right limb, stage 27; the dorsal outgrowth seen in this section would have formed the supernumerary digits. The normal toe complement would have formed from the ventral outgrowth. Both areas of outgrowth are covered by an apical epidermal ridge. x 120. (From Goetinck, 1964.)

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ing on this problem comes from tissue recombination experiments using normal and mutant limb components. The first investigations on the interaction between mesoblast and limb epidermis in mutants were carried out by Zwilling ( 1 9 5 6 ~ and ) by Zwilling and Hansborough (1956). From these studies they advanced a hypothesis which postulated that the function of the AER, on which proximodistal outgrowth of the limb is dependent, is not autonomous but that it, in turn, depends for its continued activity on a factor in the mesoblast; they referred to the latter as the apical ectoderm maintenance factor ( AEMF). The hypothesis was based on an analysis of the results obtained in reciprocal recombinations between mutant and normal limb-bud components. Limbs combined from dominant polydactylous and normal components gave rise to the polydactylous phenotype only when the mesoblast was of polydactylous origin. The normal epidermis in this combination developed a more extensive AER in the preaxial area in addition to that found normally in the postaxial region. From these results it was proposed that the excessive preaxial AER developed in response to stimulation of the epidermis in this area by a mesodermal factor, the AEMF; it was further suggested that in the normal mesoblast AEMF activity is distributed only postaxially, i.e., in the direction of the normal AER, but that in the polydactylous mutation its distribution is extended also into the preaxial region, resulting in excessive AER formation. Alternatively, it may be that the cells in preaxial part of the mesoblast of polydactylous, but not of normal embryos, are capable of synthesizing the hypothetical AEMF. While the end result of these two possibilities would be the same, the mechanisms at the cellular level would be completely different. As we shall see later on, under certain experimental conditions the preaxial portion of the normal mesoblast may acquire and stabilize the AEMF (Saunders and Gasseling, 1963). The asymmetrical distribution of the AEMF activity is further supported by experiments in which the thicker portions of several AERs are placed in tandem along the distal edge of an EDTA-denuded mesoblast. In such experiments no supernumerary digits were obtained; rather, the ectoderm conformed to the normal asymmetrical pattern imposed by the mesoderm (Zwilling, 1956b). A situation anaIogous to that described for dominant polydactyly has been seen in the talpid' mutant (Goetinck and Abbott, 1964). In recombinations between limb-bud components of this mutant and of normal

274

P. F. GOETINCK

embryos, development of the talpid phenotype is associated with the talpid mesoblast, regardless of the genotype of the limb epidermis with which it is covered. In these two mutants, therefore, the genotype of the mesoblast determines the formation of the polydactylous limbs, and when normal epidermis is recombined with the mutant mesoblast a more extensive AER develops in the recombinant. On the other hand, the epidermis of these two mutants combined with normal mesoblast conforms to the normal pattern, although if left on the mutant mesoblasts the epidermis would have formed extra AER preaxially in the dominant pdydactylous limb and an extended AER over the whole limb paddle in talpid2 embryos. To account for the abnormal development of the limb in talpid2 it was proposed that the distribution of AEMF activity was affected in these embryos (Goetinck and Abbott, 1964); however, the situation in this mutant is probably different from and much more complex than in dominant polydactyly. As already mentioned, the entire talpid embryo is extremely abnormal; no digits can be recognized as normal, whereas in the dominant polydactylous limbs only the preaxial part of the foot is affected. It is therefore conceivable that an abnormal distribution of the hypothesized AEMF in talpid? embryos is a secondary aspect of a much more general developmental abnormality. That the postulated AEMF activity can also be lost as the result of mutations has been concluded from mesoblast-epidermis recombinations of limb-bud parts of “wingless” mutants (Zwilling, 1 9 5 6 ~ )Limbs . composed of mesoblasts of wingless embryos and of normal epidermis grow distally only somewhat more than intact limbs of wingless embryos. In combination with the mutant mesoblast the normal AER was not maintained in an active state; it later regressed and correspondingly limb outgrowth stopped. The combination of wingless epidermis and normal mesoblast also did not result in distal outgrowth, contrary to expectations that the postulated AEMF of the normal mesoblast might stimulate an AER in the wingless epidermis. Zwilling ( 1 9 5 6 ~ )proposed that either the epidermis at the stage tested had been irreversibIy affected by its association with the mutant mesoblast or that the wingless mutation affects both components of the developing limb bud. Recombinations with wingless epidermis from younger embryos could clarify this point. On the basis of the original work it was thought that only the epidermis of the distal edge of the limb bud, i.e., the normal AER, was capable of participation in distal limb outgrowth. Several cases are now known in which epidermis from other sites enables the formation of distal limb

9.

GENETIC ASPECTS OF SKIN AND LIMB DEVELOPMENT

275

elements. The first was reported by Kieny (1960), who showed that the mesoblast provides the initial stimulus for the subsequent interactions between the two limb components in the chick, as it does in amphibians (Tschumi, 1957). Presumptive limb mesoderm from very young embryos (stages 15 to 18) was grafted under the flank epidermis of early embryos (stages 12 to 14) in an area which normally forms no limbs; it stimulated the formation of an ectopic AER and subsequently a complete limb developed. Recently Searls and Zwilling (1964) reported that terminal parts can develop when fragmented limb mesoblast is placed in the epidermal pouch of a tail bud. Again distal outgrowth was associated with the presence of an AER. The third case of exceptional limb outgrowth is found in the eudiplopodia mutant (Goetinck, 1964). In this mutant a supernumerary AER develops on the dorsal epidermis of the leg buds. In normal limbs this dorsal epidermis never develops an AER even when brought experimentally in contact with the distal tip of a normal mesoblast (Zwilling, 1956a). When the eudiplopod epidermis is combined with a normal mesoblast it forms a secondary AER and under its influence extra digits develop, but the combination of mutant mesoblast with normal epidermis results in a completely normal limb. It seems, therefore, that in this mutant in addition to the norma1 AER some of the dorsal leg epidermis becomes responsive at a specific stage of development to the stimulus for AER formation, resulting in the appearance of an additional AER and subsequent duplications of the distal leg parts. This mutant therefore lends strong support to the view that the AER acts as a distal limb outgrowth inductor. How this inductive interaction takes place in the eudiplopod limb or for that matter in normal development is not known. However, one might consider the dorsal ectoderm, which in normal limb buds as a rule does not form an AER, as being repressed for this event, without specifying at which of the many possible activity levels this repression could take place. Is it at the genomic level, or may we consider the basement membrane as a barrier to the interactions between the epidermis and the mesoblast? In the latter case, we would postulate a breakdown of the basement membrane in the dorsal surface of the eudiplopod limb bud at the site of AER formation. Such changes in the basement membrane were proposed by Balinsky (1956), who found in Amblystoma that the disappearance of the epidermal basement membrane precedes the development of supernumerary limbs.

276

P. F. GOETINCK

Amprino and Camosso (1958a,c, 1959; Amprino, 1965) and Saunders (Saunders et al., 1958, 1959a; Saunders and Gasseling, 1959, 1963; Gasseling and Saunders, 1960a,b) reported that duplicate hand parts developed when the apical zone ( AER with the mesoblast adjacent to it) of a 3-4-day wing is severed and replaced in reversed anteroposterior orientation. Amprino and Camosso have interpreted these results in terms of differences in the developmental states of the different regions of the mesoblast as indicating that the development of the less advanced distal parts is governed by the already more developed proximal tissues. Saunders, on the other hand, interprets the duplication of the hand parts in terms of the asymmetrical distribution of the AEMF. The reversal of the anteroposterior axis of the apical zone would result in two centers of AEMF activity: one preaxially in the reoriented distal part and one postaxially in the stump. Both centers of activity would have their effect through the AER, which in turn would induce the formation of duplicate parts. Indeed a more extensive AER becomes evident in the preaxial part of the reversed apical zone. Saunders and Gasseling (1963) have also shown that direct cell contact is not necessary for the transmission of AEMF activity, for duplicate hand parts will form when a strip of a millipore filter is inserted between the postaxial part of the stump and the preaxial part of the limb in a mediolateral direction but not, as judged from the spatial restrictions of the AEMF activity in the normal limb, in an anteroposterior direction. With respect to transmission of the postulated AEMF activity, normal preaxial mesoblast resembles wingless mesoblast in the following sense: When the latter is covered with a normal AER and grafted to the dorsal surface of a normal wing bud, the mutant mesoblast will elongate and maintain the AER as long as the graft is situated distally (Zwilling, cited in Saunders and Gasseling, 1963). However, as the graft shifts more proximally, owing to the elongation of the host wing, elongation of the mutant mesoderm ceases; this was attributed to the absence of AEMF activity in the more proximal areas of the older limb buds (Saunders et al., 1959a; Saunders and Gasseling, 1963). Whereas both wingless and normal preaxial mesoderm can transmit AEMF activity, the necessity of a continuous supply of this activity for the outgrowth of wingless mesoderm makes this mesoderm different from the preaxial mesoderm of the normal limb. AEMF activity can become stabilized in the latter in less than 12 hours, as shown from the duplications obtained when the normal anteroposterior position of the reversed apical zone is reestablished after

9.

GENETIC ASPECTS OF SKIN AND LIMB DEVELOPMENT

277

10-12 hours (Saunders and Gasseling, 1963). The ability to transmit AEMF activity, however, is a unique property of limb mesoderm, whether from normal preaxial limb regions or from wingless wing buds, in that a normal AER quickly degenerates in association with nonlimb mesoderm even when the latter is backed by normal wing mesoblast ( Zwilling, 1964 ) . IV. Summary and Concluding Remarks

Skin and limb development depend on interactions between the epithelial and mesenchymal components which make up these structures. These two developmental systems are unique in that several mutations are known which upset the normal interactions and thus cause these structures to deviate from their normal developmental pathways. The mutations, their description, and the results of recombination experiments between mutant and normal tissues are summarized in Table I. The analyses of the mutants to date have shown that in some-ptilopody, interdigital webbing, and possibly wingless-both the mesoderm and the ectoderm can be affected through mutation; in others-scaleless, dominant polydactyly, talpid", and eudiplopodia-the effects of the mutations are observed only in one of the two components. Furthermore, the effects of the mutations can be observed through either a loss or a gain of properties which lead to abnormal development. While one would like to describe the action of these mutations in terms of specific macromolecules, the information presently available limits us to the detection of their effects at the tissue level. With the rapid advances in biochemistry it may be expected that we will soon be able to describe these developmental pathways in molecular terms and that the use of mutants will continue to be useful in the pursuit of these endeavors. It must be stressed, however, that the manifestation of a certain mutant phenotype is not determined by the mere presence or absence of a mutant gene or a pair of mutant genes but depends on an integrated action of the particular genes with the whole genotype of the developing individual. This is demonstrated by the variation observed in the expression of the phenotype in mutant individuals. For example, scaleless birds show a small amount of feathers in the later-appearing feather tracts, and the eudiplopod phenotype may range in its expression from a single duplicated toenail to a complete extra foot located on top of the normal toe complement. Furthermore, a gene or pair of mutant genes may express itself in one way in one genetic background and differently in another.

to

SUMMARY OF RECOMBINATIONS BETWEEN

TABLE I NORMAL AND MUTANT COMPONENTS OF EMBRYONIC SKINAND LIMB Source of

Description

Mesoderm

Ectoderm

Phenotype of composite organ

Scaleless

Autosomal recessive trait; scaleless birds lack scales, feathers, footpads, and spurs

Normal Scaleless

Scaleless Normal

Scaleless Normal

Ptilopody

Autosomal dominant trait; one to three rows of feathers along the 4th tarsometatarus and on the 4th digit replace the scales normally found

Normal Ptilopody

Ptilopody Normal

Ptilopody Ptilopody

Interdigital webbing characteristic of ducks

Duck Chick

Chick Duck

Webbed feet Webbedfeet

Autosomal dominant trait; duplications of the preaxial parts of the limbs; extension of the normal AER preaxially

Normal Polydactyly

Polydactyly Normal

Normal Polydactyly

Wingless

Autosomal recessive trait; wings and often the legs cease to develop; AER dcgencrates

Normal Wingless

Wingless Normal

Wingless Wingless

Talpid2

Autosomal recessive trait; of the 7 or 8 syndactylous digits per foot, none can be recognized as normal; the enlarged distal end of the limbs is covered with an extensive AER

Normal Ta1pid2

Talpidz Normal

Normal Talpidp

Phenotype

3

Skin

Webbing Limb Dominant polydactyly and its allele duplicate

td ?1 0

0 2

TABLE 1 (Continued) z.

v,

Source of Phenotype Eudiplopodia

Description Autosomal recessive trait; the development of the supernumerary digits, which are located in a second plane above the normal toe complement, is preceded by the formation of a secondary AER in the dorsal ectodenn

Mesoderm

Ectoderm

Normal Eudiplopodia

Eudiplopodia Normal

Phenotype of composite organ Eudiplopodia Normal

;4

9 i Z 4

8 Y

km

280

P. F. COETINCK

Landauer ( 1948) has reported differences in the phenotypic expression of dominant polydactyly by selection. Similarly, Taylor et al. (1959) observed, after outcrossing carriers of the recessive lethal gene diplopodia (Taylor and Gunns, 1947) to an unrelated stock, that the offspring no longer segregated in a normal 3:l phenotypic ratio. Through selection they were successful in establishing three lines which differed significantly in their segregation ratios. One line was restored to the 3:l ratio characteristic of the original stock, and this level could not be exceeded by further selection. The line selected for low incidence of diplopodia gave rise to individuals producing only 2% of diplopod phenotypes in their offspring. The third line, intermediate in penetrance, fluctuated between 17 and 8% of mutant embryos in their offspring. Furthermore, as penetrance was reduced in the low-incidence line the expressivity of affected embryos was attenuated ( Abbott, 1959). From all considerations it seems that what is observed in the low-incidence line is a complete masking of the diplopodia phenotype in individuals homozygous for the once lethal gene. Selection has also been successful in increasing the number of feathers in homozygous scaleless chickens, but the selection for high feather number has had no effect on scale formation (Abbott, 1965). Several examples are known in which mutations present in a latent form in certain breeds have been discovered by outcrossing these breeds to stocks with different genetic histories (Landauer, 1965). These observations have been explained in terms of Waddington’s (1957) concept which formulates that the uniformity in the phenotypic expression of individuals in a population is the result of a buffering or canalization by natural selection of the many interacting developmental pathways, in spite of the genetic variability between the individuals of that population. The masking of mutants by selection is then visualized as restoring the canalization of the original or other related developmental pathways, the unmasking of mutants by outcrossing as a breaking up of the “epigenetic armor” (Landauer, 1965), which most certainly had been brought about by artificial selection in the specific breeds examined. While observations of this kind will by themselves surely not lead to an understanding of the primary gene events which control normal development, they have been presented here to emphasize the complexity of this control and to stress the importance of establishing and identifying the overall genotype in embryological and biochemical analyses in which specific mutants are used for studying the genetic control of developmental processes.

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281

ACKNOWLEDGMENT The author wishes to express his appreciation to Dr. Louis J. Pierro for the reading of a preliminary draft of the manuscript and for his valuable suggestions and criticism. REFERENCES Abbott, U. K. (1959). J . Genet. 56. 197. Abbott, U. K. (1965). Poultry Sci. 44, 1347. Abbott, U. K., and Asmundson, V. S. (1957). J. Heredity 48, 63. Abbott, U. K., Taylor, L. W., and Abplanalp, H. (1960). 1. Heredity 51, 195. Amprino, R. (1963). Monit. Zool. Ital. Suppl. 70-71, 7. Amprino, R. ( 1965). In “Organogenesis” ( R . L. DeHaan and H. Ursprung, eds.1, pp. 255-281. Holt, New York. Amprino, R., and Camosso, M. ( 1955a). 1. Erptl. Zool. 129, 453. Amprino, R., and Carnosso, M. (1955b). Compt. Rend. Assoc. Anat. 42, 197. Amprino, R., and Camosso, M. (1958a). Erperientiu 14, 241. Amprino, R., and Camosso, M. (195813). Arch. Entwicklungsmech. Organ. 150, 509. Amprino, R., and Camosso, M. ( 1 9 5 8 ~ )Compt. . Rend. Assoc. Anat. 45, 93. Amprino, R., and Camosso, M. (1959). Arch. Anat. Microscop. Morphol. Exptl. 48, 261. Auerbach, R. (1960). Deuelop. Biol. 2, 271. Balinsky, B. I. (1956). Proc. Natl. Acad. Sci. U. S. 42, 781. Bell, E. (1964). Cancer Res. 24, 28. Bell. E., and Thathachari, Y. T. (1963). J. Cell Biol. 16, 215. Bell, E., Kaighn, M. E., and Fessenden, L. M. (1959). Deuelop. Biol. 1, 101. Bell, E., Gasseling, M. T., Saunders, J. W., Jr., and ZwilIing, E. (1962). Deuelop. Biol. 4, 177. Bell, E., Hinnphreys, T., Slayter, H. S., and Hall, C. E. (1965). Science 148, 1739. Ben-Or, S., and Bell, E. (1965). Deuelop. Biol. 11, 184. Dodson, J. W. (1963). Erptl. Cell Res. 31, 233. Ede, D. A., and KelIy, W. A. (1964). J . Emhryol. Exptl. Morphol. 12, 339. Elmer, W. A., and Pierro, L. J. (1964). Am. Zool. 4, 381. Gasseling, M. T., and Saunders, J. W., Jr. (1960a). Anat. Record 136, 195. Gasseling, M. T., and Saunders, J. W.. Jr. (1960b). Anat. Record 138, 350. Gasseling, M. T., and Saunders, J. W., Jr. (1961). Deuelop. Biol. 3, 1. Goetinck, P. F. (1904). Deuelop. Biol. LO, 71. Goetinck, P. F. ( 1966). In preparation. Goetinck, P. F., and Abbott, U. K. (1960). Poultry Sci. 39, 1252. Goetinck, P. F., and Abbott, U. K. (1963). J. Exptl. Zool. 154, 7. Goetinck, P. F., and Abbott, U. K. (1964). 1. Exptl. Zool. 155, 161. Golosow, N., and Crobstein, C. (1962). Deoelop. Biol. 4, 242. Grobstein, C. (1953). Nature 172, 869. Grobstein, C. (1955). J. Exptl. Zool. 130, 319. Hamburger, V., and Hamilton, H. L. (1951). 1. Morphol. 88, 49. Hamilton, H. L. (1965). In “Biology of the Skin and Hair Growth” (A. G. Lyne and B. F. Short, eds.), pp. 313-328. American Elsevier, New York. HampB, A. (1956). Compt. Rend. SOC. Biol. 150, 1671.

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HampB, A. (1959). Arch. Anat. Microscop. Morphol. Exptl. 48, 345. Hinrichsen, K. ( 1956). Z. Anat. Entzoicklungsgeschichte 119, 350. Humphreys, T., Penman, S., and Bell, E. (1984). Biochem. Biophys. Res. Commun. 17, 618. Kieny, M. (1960). J. Embryol. Exptl. Morphol. 8, 457. Kieny, M. (1962). Arch. Anat. Microscop. Morphol. Exptl. 51, 577. Kieny, M., and Abbott, U. K. (1962). Deuelop. Bwl. 4, 473. Landauer, W. (1948). Genetics 33, 133. Landauer, W. (1965). 3. Heredity 56, 131. McAlpine, R. J. (1956). Anat. Record 126, 81. McLaughlin, C. B. (1961a). J. Embryol. Exptl. Morphol. 9, 370. McLoughlin, C. B. (1961b). 1. Embryol. Exptl. Morphol. 9, 385. Milaire, J. (1956). Arch. Biol. (Liege) 67, 297. Milaire, J. ( 1963). Arch. Biol. (Liege) 74, 129. Milaire, J. (1965). In “Organogenesis” ( R . L. DeHaan and H. Ursprung, eds.), pp. 283-300. Holt, New York. Morgan, T. H. (1934). “Embryology and Genetics.” Columbia Univ. Press, New York. Rawles, M. E. (1963). 3. Embryol. Exptl. Morphol. 11, 765. Rosenblatt, L. S., Kreutziger, G. O., and Taylor, L. W. (1959). Poultry Sci. 38, 1242. Saunders, J. W., Jr. (1948). J. Exptl. Zool. 108, 363. Saunders, J. W., Jr. (1958). In “Symposium of the Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), pp. 239-253. Johns Hopkins Univ. Press, Baltimore, Maryland. Saunders, J. W., Jr., and Gasseling, M. T. (1959). J. Exptl. Zool. 142, 553. Saunders, J. W., Jr., and Gasseling, M. T. (1963). Deuelop. Bio2. 7, 64. Saunders, J. W., Jr., Gasseling, M. T., and Cairns, J. M. (1955). Nature 175, 673. Saunders, J. W., Jr., Carins, J. M., and Gasseling, M. T. (1957). J. Morphol. 101, 57. Saunders, J. W., Jr., Gasseling, M. T., and Gfeller, M. D., Sr. (1958). J. Exptl. Zool. 137, 39. Saunders, J. W., Jr., Gasseling, M. T., and Bartizal, J. (1959a). Anat. Record 133, 332. Saunders, J. W., Jr., Gasseling, M. T., and Cairns, J. M. (195913). Deuelop. BioE. 1, 281. Scott, R. B., and Bell, E. (1964). Science 145, 711. Searls, R. L., and Zwilling, E. (1964). Deuelop. Biol. 9, 38. Sengel, P. (1957). Experientia 13, 177. Sengel, P. (1958). Ann. Sci. Nut. Zool. 20, 431. Sengel, P. (1964). In “The Epidermis” (W. Montagna and W. C. Lobitz, Jr., eds.), pp. 15-34. Academic Press, New York Sengel, P., and Abbott, U. K. (1962). Compt. Rend. 255, 1999. Sengel, P., and Abbott, U. K. (1963). J. Heredity 54, 254. Sengel, P., Bescol-Liversac, J,, and Guillam, C. (1962). Deuelop. Biol. 4, 274. Taylor, L. W., and Gunns, C. A. (1947). J. Heredity 38, 67. Taylor, L. W., Abbott, U. K., and Gunns, C. A. (1959). J. Genet. 56, 161. Thomson, J. L. (1984). I . Morphol. 115, 207. Tschumi, P. A. (1957). J. Anat. 91, 149.

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Waddington, C . II. (1957). “The Strategy of the Genes.” Allen & Unwin, London. Wessells, N. K. (1962). Develop. B i d . 4, 87. Wessells, N. K. (1963). Exptl. Cell Res. 30, 36. Wessells, N. K. (1964). PTOC. Natl. Acad. Sci. U . S. 52, 252. Wessells, N. K. (1965). Develop. B i d . 12, 131. Wolff, E., and Kieny, M. (1957). Compt. Rend. 244, 1089. Wolff, E., and Kieny, M. (1963). Develop. BioE. 7, 324. Zwilling, E. (1949). J. Exptl. 2001.111, 175. Zwilling, E. (1955). I. Exptl. 2001.128, 423. ZwiUing, E. (1956a). J. Exptl. 2001.132, 157. Zwilling, E. (195613). J. Exptl. Zool. 132, 173. Zwilling, E. ( 1 9 5 6 ~ )J.. Exptl. 2002.132, 241. Zwilling, E. (1959). 1. Exptl. 2001.142, 521. Zwilling, E. ( 1961). Advan. Morphogenesis 1, 301. Zwilling, E. (1964). Develop. B i d . 9, 20. Zwilling, E., and Hansborough, L. (1956). J. Exptl. Zool. 132, 219.

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AUTHOR INDEX Numbers in italics refer to pages on which the complete references are listed.

A

B

Abbott, U. K., 254. 258, 259, 265, 266, 267, 269, 273, 274, 280, 281, 282 Abercrombie, M., 193, 206 Abrahamov. A., 240, 247 Ackerman, G. A,, 220, 231, 235, 245 Adachi, K., 225, 245 Adams, J., 242, 245 Aghion. D., 147, 151 Ahplanalp, H., 269, 281 Ajtkhozhin, M. A,, 12, 13, 14, 16, 19, 36, 38 Albrecht, M., 222, 246 Alexanian, R., 217, 249 Alfert, M., 47, 58 Allen, D. W., 220, 232, 246 Allen, M. J., 45, 47 Allende, J., 8, 36 Allfrey, V. G . , 53, 54, 57, 58 Alpen, E. L., 185, 206, 217, 220, 246 Alt, H. L., 180, 188, 209, 210 Altman, K. I., 231, 251 Ames, B. N., 181, 209, 243, 246 Amprino, R., 254, 265, 267, 268, 276, 281 Anderson, M. L., 65, 83 Anderson, N. G., 187, 206 Ando, M., 182, 208 Andrew, W., 215, 246 Arlinghaus, R., 241, 242, 248 Armentrout, S., 234, 252 Armstrong, D. H., 238, 246 Arnstein, H. R. V., 234, 246 Aronson, A. I., 10, 38 Askonas, B. A., 11, 14, 37 Asmundson, V. S., 258, 281 Assrian, I. S., 12, 27, 38 Astaldi, G., 189, 206 Atwater, J., 236, 246 Auerhach, R., 254, 281

Baglioni, C., 235, 236, 237, 240, 246 Baker, W. K., 169, 171 Baldev, B., 148, 152 Baldini, M., 244, 250 Balinsky, B. I., 275, 281 Bank, A., 239, 243, 246, 247 Banks. J., 230, 234, 242, 249, 250 Barber, J. T., 125, 150, 153 Bard, S. C., 4, 5, 38 Barner, H. D., 82, 82 Barr, B. A., 125, 151, Barr, H. J., 203, 206 Barrowman, J., 235, 237, 241, 246 Bartelmez, G. W., 215, 246 Barth, L. G., 99, 110 Bartizal, J., 276, 282 Basilio, C., 8, 36 Beaven, C. H., 235, 236, 238, 248, 252 Beck, W. S . , 225, 246 Becker, A. J., 218, 219, 246 Becker, H. J., 157, 158, 159, 160, 162, 163, 164, 168, 169, 171 Becker, U., 87, 88, 89, 96, 99, 110, 112 Becker, Y., 12, 20, 21, 22, 25, 37, 193, 209, 230, 242, 248 Beerman, W., 26, 36, 45, 58, 106, 1 1 1 , 241, 246 Belitsina, N. V., 12, 13, 14, 16, 19, 24, 26, 36, 38 Bell, A., 215, 221, 247 Bell, E., 31,;37, 258, 262, 263, 265, 267, 281, 282 Bennett, M., 217, 247 Ben-Or, S., 262, 281 Berger, H.. 188, 209 Bergstrand, A., 4, 37 Berman, G. R., 226, 247 Berman, L., 189, 206, 210 Bernstein, S . E., 216, 219, 220, 249, 251 Berry, R. J.. 181, 210

285

286

AUTHOR INDEX

Bertles, J. F., 225, 246 Bescol-Liversac, J., 255, 282 Bessis, M., 222, 246 Bethell, F. H., 184, 209 Betke, K., 240, 249 Bidwell, R. G. S., 125, 151, 153 Biezunski, N . , 234, 247 Bishop, J., 237, 242, 246, 248 Blakely, L. M . , 123, 124, 125, 128, 129, 134, 151, 153 Blakeslee, A. F., 147, 154 Bleiberg, I., 188, 207 Bloom, W., 215, 221, 246 Bond, V. P., 217, 220, 222, 246, 247 Bonner, J. T., 62, 64, 82 Boone, C. W., 193, 209 Borghese, E., 216, 246 Born, J., 89, 104, 112 Borsook, H., 176, 179, 180, 186, 207, 209, 221, 222, 225, 226, 230, 231, 246, 249 Bouillene, R., 146, 151 Boveri, T., 105, 111 Boyer, S. H., 236, 246 Bozzini, C . E., 189, 207 Brachet, J., 2, 3, 5, 8, 37, 38, 98, 101, 108, 111, 233, 246 Bradley, T. B., Jr., 236, 240, 246, 250 Braun, H., 240, 249 Braunitzer, G., 236, 247 Bravo, M., 8, 36 Brawerman, G., 234, 247 Brawner, J. N., 240, 246 Brecher, C., 186, 202, 207, 210, 216, 220, 221, 222, 247 Bridges, M. T., 236, 248 Briggs, R., 8, 37, 151, 152 Bromberg, Y. M., 240, 247 Bronsweig, R., 63, 82 Bro-Rasmussen, F., 222, 247 Brown, D. D., 4, 7, 37, 102, 104, 105, 111 Brown, S. W., 170, 171 Bruce, W. R., 185, 188, 207, 220, 247 Bruns, G. P., 197, 198, 207, 231, 247 Buchner, S., 149, 153

Burka, E. R., 225, 226, 232, 234, 237, 238, 241, 242, 243, 244, 247, 249, 250 Burnham, B. F., 197, 207

C Cairns, J. M., 267, 282 Camiscoli, J. F., 177, 178, 207 Camosso, M., 265, 267, 276, 281 Capecchi, M., 242, 246 Caplin, S. M., 119, 120, 143, 151, 153 Cardinali, G., 189, 206 Carnot, P., 175, 207 Cartwright, G. E., 198, 210 Caspersson, T., 101, 111 Catalano, C., 4, 37 Cather, J. N., 41, 58 Ceccarini, A., 65, 82 Ceppellini, R., 236, 239, 247 Charles, H. E., 179, 208 Chaung, H., 96, 111 Chen, S. Y., 164, 171 ChernoE, A. I., 235, 240, 247, 251 Clegg, J. B., 239, 252 Clegg, V. S., 65, 82 Cohen, S. S., 82, 82 Cohrs, P., 189, 207 Collier, 1. R., 41, 42, 48, 53, 54, 55, 58 Collins, A., 198, 210 Colombo, B., 244, 246 Conconi, F., 230, 232, 241, 242, 243, 244, 247, 250 Conklin, M . E., 147, 154 Conley, C. L., 240, 246, 251 Contrera, J. F., 177, 178, 207 Cooper, G. W., 188, 207 Cooper, J. A. D., 180, 188, 210 Cooper, K. W., 169, 171 Cordova, F. A., 236, 250 Cormick, J., 236, 251 Cottier, H., 217, 220, 222, 246 Cousineau, G. H . , 3, 4, 8, 37 Cox, R. A., 234, 246 Crafts, R. C., 182, 183, 207, 209 Craig, M., 224, 237, 240, 246

AUTHOR INDEX

Craig, M. L., 216, 224, 228, 231, 241, 247 Cranmore, D., 185, 206, 217, 220, 246 Crick, F. H . C., 237, 247 Crocco, R. M., 32, 37 Cronkite, E. P., 202, 222, 207, 216, 217, 218, 220, 246, 247, 248 Crook, J. J., 182, 183, 207 Crowell, J., 54, 58 Cudkowicz, G., 217, 247

D Dalcq, A,, 8, 37 Dameshek, W., 244, 250 Dance, N., 236, 248 Danon, D., 226, 231, 247, 250, 251 Darnell, J. E., 25, 37, 225, 230, 248, 249 Das, N . K., 47, 58 Davenport, J. C., 48, 58 Davenport, R., 48, 58 Davidson, E. H., 44, 53, 54, 57, 58 Davis, B. D., 214, 247 Davis, H. G., 222, 251 de Aberle, S . B., 215, 216, 247 De Bellis, R. H., 9, 37, 225, 230, 247 Decroly, M., 5, 37 Deflandre, C., 175, 207 DeGier, J., 232, 251 DeGowin, R., 176, 182, 207 DeHaan, R. L., 64,82 Demerec, M., 163, 171 Denis, H., 3, 8, 37, 101, 111 Denny, P. C., 3, 37 Denstedt, 0. F., 232, 251 De Ropp, R. S., 121, 151 de Vitry, F., 8, 37 Diggs, L. W., 215, 221, 247 Dintzis, H. M., 242, 243, 247 Dodson, J. W., 257, 281 Downey, H., 215, 247 Doyle, M., 175, 208, 216, 218, 219, 220, 248 Drach, J. C., 234, 247 Dreyfus, J. C., 234, 249 Dukes, P. P., 178, 180, 183, 187, 189,

287

190, 191, 193, 194, 200, 207, 209, 210 Dure, L., 9, 37

E Eagle, H., 192, 193, 207, 209 Ede, D. A., 269, 281 Edington, G. M., 239, 247 Edstrom, J. E., 45, 58 Eisenstadt, J., 234, 247 Eider, M., 184, 207 Ellis, F., 189, 210, 221, 251 Elmer, W. A., 254, 281 Elmlinger, P. J., 182, 208 Englaender, H., 98, 111 Englander, S. W., 243, 247 Ennis, H. L., 65, 82 Erickson, R., 234, 250 Erslev, A. J., 185, 186, 189, 200, 207 Evenstein, D. 184, 207 F Fantoni, A., 224, 225, 249 Farkas, W., 225, 247 Fawcett, D., 215, 221, 246 Feeley, J., 8, 38 Feigenbaum, L., 43, 58 Feldman, M., 188, 207, 209 Fennell, D., 65, 82 Fenninger, W. D., 238, 246 Fessenden, L. M., 267, 281 Ficq, A., 5, 37 Filmanowicz, E., 184,208,218,248 Filosa, M., 65, 82 Finch, C. A., 222, 248 Finne, P. H., 177, 207 Finney, R. J., 53, 54, 57, 58 Fischer, S., 188, 207 Fisher, J. W., 182, 183, 207 Fitting, H., 119, 151 Fliedner, T. M., 217, 222, 246, 247, 248 Franklin, R. M., 73, 82 Frenkel, E. P., 189, 209 Fried, W., 175, 176, 177, 179, 182, 183, 184, 185, 203, 207, 208, 209 Fruhman, 0. J., 183, 207

288 Fujimoto, M., 197, 208 Fukuhara, H., 81, 82 Fulwyler, M. J., 187, 207

AUTHOR INDEX

Golosow, N., 254, 281 Gothie, S., 98, 111 Goto, K., 197, 208 Goudsmit, R., 198, 210 0 Could, H., 234, 246 Gowen, J. W., 166, 171 Gall, J. G., 45, 58 Gallien-Lartigue, O., 180, 186, 189, 190, Graham, L. A., 179, 208 Granick, S., 196, 197, 198, 199, 205, 208, 193, 199, 200, 208, 209 209, 210 Garcia, J. F., 176, 177, 181, 182, 208, Grant, W. C., 175, 188, 208, 209 210 Grasso, J. A., 174, 193, 199, 208, 220, Garren, L. D., 32, 37 222, 225, 231, 248 Gasseling, M. T., 265, 267, 273, 276, 277, Graybiel, A., 176, 179, 207 281, 282 Grayzel, A. I., 244, 248 Gautheret, R. J., 118, 151 Green, E., 8, 37 Gavrilova, L. P., 12, 13, 17, 36, 37 Greenough, W. B., 111, 220, 238, 251 Gay, E . H., 166, 171 Geinendenger, L. E., 217, 220, 222, 246 Gregg, J. H., 63, 64, 65, 82 Gribble, T. J., 198, 208 Gendon, Yu. Z., 24, 38 Grobstein, C., 208, 254, 281 Georgiev, G. P., 12, 25, 27, 37, 38 Gerald, P. S., 235, 236, 237, 239, 248, Gros, F., 225, 250 Gross, P. R., 3, 4, 8, 37, 55, 58 249, 251 Groves, B., 64, 83 Gerisch, G., 64, 82 Grumbach, M. M., 170, 171 Gezelius, K., 65, 82 Gruneberg, H., 220, 248 Gfeller, M. D., Sr., 276, 282 Guha, S., 148, 151 Giblett, E. R., 187, 208 Guidotti, G., 236, 249 Gierer, A., 226, 234, 248 Guillam, C., 255, 282 Gilbert, C. W., 217, 249 Gunns, C. A., 280, 282 Gilmour, J. R., 235, 248 Gurdon, J. A., 102, 105, 111 Girad, M., 25, 37, 230, 248 Gurney, C. W., 175, 176, 178, 182, 183, Glowacki, E. R., 232, 243, 248 184, 185, 189, 193, 202, 203, 207, Gluck, N., 9, 37, 225, 230, 247 208, 209, 210, 211, 218, 248 Gluecksohn-Waelsch, S., 237, 250 Goetinck, P. F., 259, 265, 266, 267, 269, H 270, 271, 272, 273, 274, 275, 281 Haberlandt, G., 116, 119, 152 Goldberg, E., 41, 43, 58 Haccius, B., 147, 148, 152 Goldberg, I. H., 56, 59 Halbrecht, I., 215, 248 Goldstein, J., 236, 249 Hall, C. E., 31, 37, 241, 251, 263, 281 Goldstein, J. L., 143, 151 Goldwasser, E., 175, 176, 177, 178, 179, Hall, R. H., 188, 209 180, 181, 183, 184, 185, 186, 187, Halperin, W., 148, 152 189, 190, 191, 192, 193, 194, 196, Hamburger, V., 263, 281 198, 199, 200, 207, 208, 209, 210, Hamilton, H. L., 255, 261, 263, 281 HampB, A., 265, 281, 282 211, 218, 248 Golov, V. F., 17, 37 Hansborough, L., 269, 273, 283 Hardesty, B., 241, 242, 248 Goodman, H. M., 234, 241, 242, 251 Gordon, A. S., 175, 177, 178, 182, 183, Harrington, W. J., 176, 210 184, 188, 207, 208, 210 Harris, H., 10, 38

289

AUTHOR INDEX

Harris, M., 115, 152 Hartman, D. E., 243, 246 Harvey, E. B., 3, 8, 37 Haslett, G. W., 53, 54, 57, 58 Hatta, Y., 182, 208 Hausmann, E., 234, 250 Hayashi, M., 55, 59 Hayashi, Y.,98, 111 Hayer, B. H., 106, 111 Hecht, F., 236, 248 Heftmann, E., 64, 82 Henriksen, O., 222, 247 Henshaw, E. C., 12, 21, 23, 25, 27, 37 Hiatt, H. H., 12, 21, 23, 25, 27, 37 Higuchi, M., 197, 208 Hildebrandt, A. C., 129, 141, 148, 153, 154 Hill, R. J., 236, 249 Hill, R. L., 198, 210 Hillnian, R. S., 187, 208 Hilschmann, N., 236, 247 Hike, K., 236, 247 Hinrichsen, K., 270, 282 Hirschberg, E., 249 Hoagland, M. B., 11, 14, 37 Hodgson, G., 181, 188, 196, 210 Horchner, P., 244, 248 Hofstra, D., 176, 193, 207, 208 Holfreter, J., 87, 99, 106, 109, 111, 112 Holsten, R. D., 121, 134, 153 Honig, G . R., 56, 58 Hopkins, J. W., 12, 23, 25, 37 Horton, B., 236, 248 Howard, D., 220, 222, 251 Howell, R. R., 32, 37 Hrinda, 198, 199, 208 Hubbard, M., 3, 4, 37 Huehns, E. R., 198, 211, 236, 248 Huff, R. L., 182, 208 Hughes, J. B., 189, 200, 207 Huisman, T. J. H., 236, 248 Hultin, T., 3, 4, 5, 37, 38 Humphreys, T., 31, 37, 64, 82, 109, 111, 263, 281, 282 Hunt, J. A., 234, 246 Huntsman, R. G., 221, 249 Hutton, J. J., 237, 248 Huxley, H. E., 232, 234, 250

I Infante, A. A., 15, 16 Ingram, V. M., 235, 236, 237, 240, 243, 244, 245, 248, 250, 252 Israel, H. W., 124, 152 Itano, H. A., 235, 248 Ito, K., 187, 208 Ivanov, D. A., 17, 37 Iverson, R. U., 29, 38 Iyeiri, J., 89, 90, 111

J Jacob, F., 2, 37, 38, 196, 208, 214, 233, 237, 239, 248 Jacobs, A. S., 236, 250 Jacobson, L. O., 175, 176, 177, 178, 179, 183, 184, 185, 207, 208, 209, 211, 216, 218, 219, 220, 248 Jaffe, E. R., 232, 248 J&b, R., 189, 207, 248 Janda, W., 183, 209 Jandl, J. H., 220, 246 Jepson, J. H., 182, 209 Johnen, A. C., 98, 111 Johnson, A. B., 249 Johnson, E. M., 65, 82 Johri, B. M.,148, 152 Joklik,, W. K., 12, 20, 21, 22, 25, 37, 230, 242, 248 Jones, 0. P., 215, 248 Jones, R. T., 235, 251 Jonxis, J. H. P., 235, 236, 249, 250 Judd, B. H., 162, 171

K Kahn, A. J., 65, 82 Kaighn, M. E., 267, 281 Karibian, D., 197, 199, 209 Kato, H., 148, 152 Kaufmann, L., 189, 199, 209 Kawakami, I., 89, 90, 111 Keighley, C., 176, 179, 180, 207 Keighley, G., 179, 209 Keil, J. V., 235, 236, 248, 249 Kelly, W. A., 269, 281 Kent, A. E., 121, 131, 134, 143, 152, 153 Kerr, N. S., 64, 83

290

AUTHOR INDEX

Kesselring, K., 89, 96, 98, 112 Kieny, M., 254, 275, 282, 283 Kikuchi, G., 197, 208 King, T. J., 8, 37, 151, 152 Kirby, K. S., 89, 111 Kleihauer, E., 240, 249 Klibanski, C., 215, 248 Knoll, W., 215, 249 Knopf, P. M., 226, 234, 241, 242, 252 Knudson, L., 118, 147, 152 Kocher-Becker, U., 89, 96, 98, 99, 106, 107, 112 Kohlenbach, H. W., 148, 152 Konar, R. N., 149, 152 Konigsberg, W., 236, 249 Korson, R., 222, 249 Korst, D. R., 189, 209 Kovach, J. S., 220, 224, 225, 226, 231, 237, 241, 249 Krantz, S. B., 180, 186, 189, 190, 192, 194, 196, 209 Kraut, J., 235, 249 Kretchmar, 203, 209 Kreutziger, G. O., 270, 282 Krichevskaya, A. A., 12, 27, 38 Krikorian, A. D., 124, 152 Kruh, J., 225, 231, 234, 249, 250 Kukita, M., 182, 208 Kullyev, P., 9, 38 Kung, 176, 178, 179, 181, 186 Kunkel, H. G., 235, 249 Kuratowska, Z., 178, 209 Kurnick, 178, 209 Kurtides, E. S., 188, 209 Kuusi, T., 98, 111

249,

104,

228, 191,

1 Laforet, M. T., 184, 209 Lajtha, L. C., 189, 202, 209, 210, 216, 217, 218, 219, 220, 221, 222, 248, 249, 251 Lakshamanan, K. K., 148, 152 Lamfrom, H., 234, 241, 242, 249 Lamport, D. T . A., 143, 152 Landauer, W., 280, 282 Lankester, E. R., 40, 58

Lareau, J., 234, 249 La Rue, C. D., 147, 152 Lascelles, J,, 197, 207 Latham, H., 25, 37, 230, 248 Laursen, P., 148, 153 Leahy, J., 242, 246 Leder, P., 233, 237, 251 Lee, F., 64, 66, 83 Lehman, H., 221, 239, 247, 249 Lerman, M . I., 16, 17, 25, 26, 37, 38 Lerner, A., 225, 249 Leventhal, B., 220, 222, 251 Levere, R. D., 196, 197, 198, 199, 205, 208, 209 Levine, E. M., 193, 209 Levine, L., 63, 64, 65, 82, 83 Lewis, J. P., 218, 249 Liddel, G. U., 64, 82 Liebold, B., 236, 247 Lingrel, J. B., 186, 207, 222, 225, 226, 230, 231, 234, 246, 247, 249 Linkenheimer, W. H., 188, 209 Linman, J. W., 184, 209 Lipmann, F., 193, 210, 225, 237, 249, 250 Liron, M., 188, 206, 209 Littna, E., 4, 7, 37, 104, 111 Lochte, H. L., Jr., 210, 220, 238, 251 London, I. M., 184, 197, 198, 199, 207, 209, 220, 231, 232, 244, 247, 248, 249, 250 Lovgren, N., 62, 68, 83 Lowenstein, L., 182, 209 Lowy, P. H., 179, 180, 209 Lucarelli, G., 220, 222, 251 Luykx, P., 47, 58 Luzzatto, L., 232, 234, 242, 249, 251

M McAlpine, R. J., 270, 282 McAuslan, B. R., 31, 33, 37 McCarthy, B. S., 106, 111 McConkey, E. H., 12, 23, 25, 37 McCulloch, E. A., 185, 188, 202, 207, 210, 217, 218, 219, 220, 246, 247, 249, 251 MacIver, J. E., 239, 252

291

AUTHOR INDEX

McLoughlin, C. B., 257, 282 Maggio, R., 3, 4, 5, 6, 7, 29, 37, 38 Maheshwari, S. C., 146, 148, 151, 152 Malkin, L. I., 3, 4, 8, 37 Mangold, O., 86, 88, 111 Mapes, M. O., 121, 123, 125, 130, 131, 134, 141, 143, 152, 153 Marcus, A., 8, 38 Markert, C. L., 150, 152 Marks, P. A., 9, 37, 220, 224, 225, 226, 228, 230, 231, 232, 234, 236, 237, 238, 241, 242, 243, 244, 246, 247, 249, 250, 251 Martin, E. B., 225, 249 Martin, R. G., 181, 209 Maruyama, Y., 182, 208 Marver, H. S., 198, 210 Masui, Y., 99, 111 Mathias, A. P., 232, 234, 250 Matoth, Y.,189, 199, 209 Matsumoto, A., 89, 111 Mears, K., 123, 130, 153 Meesen, H., 189, 207 Meineke, H. A., 183, 209 Mel, H. C., 187, 209 Meyer, B., 105, 111 Meyer, L. M., 217, 248 Miettinen, J. K., 148, I52 Milaire, J., 270, 282 Miller, C. O., 119, 146, 152, 153 Miller, R., 234, 236, 247, 250 Millette, R. L., 186, 207, 222, 225, 231, 232, 243, 246, 248 Minnich, V., 236, 250 Mirand, E. A., 180, 210, 216, 220, 250 Mirsky, A. E., 53, 54, 57, 58 Mitra, J., 130, 153 Mo, H., 96, 112 Mohan Ram, H . Y., 116, 143, 153, 154 Moldave, K., 237, 250 Monical, T. V., 236, 250 Monod, J,, 2, 37, 38, 196, 208, 214, 232, 237, 239, 248, 250 Monro, R., 193, 210 Monroy, A., 3, 4, 5, 6, 7, 29, 38, 43, 55, 59

Moores, R. R., 202, 210

Morel, G., 120, 152 Morell, H., 184, 209, 231, 250 Morgan, T. H., 261, 282 Morishima, A., 170, 171 Morrill, J. B., 41, 42, 43, 59 Morse, B., 220, 222, 251 Moscona, A., 64, 82, 108, 109, 111 Moscona, H., 64, 82, 108, 111 Moss, B. M., 240, 250 Motulsky, A. G., 236, 239, 248, 250 Moyer, W . A., 3, 8, 37 Muhlethaler, K., 65, 82 Muir, W . H., 129, 153 Muller, C. J., 235, 236, 250 Munro, R., 225, 250

N Naets, J. B., 182, 210 Nakano, E., 4, 38 Nakao, M., 225, 245 Nakao, T., 225, 245 Namiki, O., 197, 208 Narayanaswami, S., 147, 153 Nataraja, K., 149, 152 Nathans, D., 193, 210, 225, 250 Naughton, M. A., 239, 252 Necheles, T. F., 244, 250 Needham, J., 2, 38 Neel, J. V., 239, 250 Negano, K., 225, 245 Nerner, M., 4, 5, 7, 14, 15, 16, 29, 30, 38 Neyfakh, A. A., 8, 38 Nitsch, J. P., 141, 147, 153 Niu, M. C., 98, 111 Nizet, A., 231, 250 Nobbcourt, P., 118, 153 Norris, E., 41, 59 Norstog, K., 147, 148, 153 Northcote, D. H., 143, 152 Nur, U., 170, 171

0 Oberoi, Y. P., 149, 152 OBrien, B. R. A., 222, 250 Oda, J. M., 182, 208 Odartchenko, N., 217, 220, 222, 246 Okamoto, T., 21, 25, 38, 230, 250

292

AUTHOR INDEX

Okumura, F. S., 119, 152 Oliver, R., 189, 202, 209, 210, 218, 221, 248, 249, 251 Osborn, M. J,, 65, 66, 67, 83 Osgood, E. E., 202, 210, 218, 250 Ottolenghi, P., 232, 251 Ovchinnikov, L. P., 24, 38 Ozaki, H., 8, 38

P Page, L. A., 232, 243, 247 Page, S., 234, 250 Parker, R. C., 191, 210 Paulet, P., 141, 147, 153 Payne, R. A,, 236, 248 Penman, S., 25, 31, 37, 230, 248, 263, 282 Pennell, R. B., 232, 250 Perl, W., 228, 232, 241, 242, 243, 250 Perretta, M. A., 188, 194, 196, 210 Pieber-Perretta, M. P., 188, 196, 210 Pierre, R. S., 184, 209 Pierro, L. J., 254, 281 Piez, K., 192, 207 Plzak, L. F., 175, 176, 177, 179, 183, 184, 185, 207, 208 Pollard, J . K., 121, 130, 153 Popp, R. A., 235, 237, 250 Porteous, D. D., 217, 249 Powsner, E. R., 189, 206, 210 Prentice, T. C.,180, 210, 216, 220, 250 Priestley, J. H., 146, 153 Pugh, R. P., 236, 250

Q Quertier, J., 5, 37

R Rabinowitz, M., 56, 58, 231, 244, 252 Rafael, D. E., 65, 82 Raghavan, V., 147, 153 Rambach, W. A., 180, 188, 209, 210 Ranby, B. G., 65, 82 Ranga Swamy, N . S., 148, 152, 153 Ranney, H . M., 236, 237, 250 Raper, A. B., 239, 248 Raper, K. B., 62, 65, 82

Rappaport, J., 147, 153 Rawles, M. E., 254, 256, 282 Reich, E., 56, 59, 194, 210 Reichlin, M., 176, 210 Reider, R. F., 243, 245, 250 Rein, A., 169, 171 Reinert, J., 148, 153 Reissmann, K. R., 187, 208 Revel, M., 12, 21, 23, 25, 27, 37 Rich, A., 226, 234, 238, 241, 242, 251, 252 Richmond, J. E., 231, 251 Rifkind, R. A., 224, 225, 226, 232, 234. 237, 241, 242, 243, 249, 250, 251 Riggs, A., 237, 251 Riker, A. J., 129, 148, 153, 154 Rinaldi, A. M., 3, 4, 5, 6, 7, 29, 38 Robbins, W. J., 118, 153 Roberts, K. B., 235, 246 Robertson, J. S., 222, 246 Rollhauser, J., 99, 111 Root, W. S., 175, 208 Rosas del Valle, M.: 10, 38 Rosenblatt, L. S., 270, 282 Rosse, W. F., 181, 189, 210 Roth, R., 65, 82 Rowley, P., 226, 251 Rubenstein, D., 232, 251 Rubini, J. R., 222, 246, 247 Rucknagel, D. L., 236, 240, 246, 251 Rudkin, G. T., 164, 171 Rudloff, V., 236, 247 Rudolph, W., 188, 196, 210 Runyon, E. H., 64, 82 Russell, E. S., 216, 219, 220, 224, 228, 237, 241, 247, 248, 249, 250

S Sachar, R. C., 146, 152 Salomon, K., 231, 251 Salzberger, M., 240, 247 Salzman, N. P., 12, 20, 38 Samarina, 0. P., 12, 27, 38 Saunders, J. W., Jr., 254, 265, 267, 273, 276, 277, 281, 282 Savoie, J. C., 184, 209, 231, 250 Saxbn, L., 102, 110, 111, 112

293

AUTHOR INDEX

Scaro, J. L., 186, 207, 222, 225, 231, 246 Schapira, C . , 234, 249 Scheraga, H. A,, 181, 210 Schlessinger, D., 225, 226, 232, 234, 249 Schooley, J. C., 176, 177, 181, 182, 208, 210 Schramm, G., 89, 111 Schroeder, W. A., 235, 238, 246, 251 Schultz, J., 164, 165, 166, 171 Schuster, H., 89, 111 Schwartz, H. C., 198, 208, 210 Schwartz, I . R., 236, 246 Schweet, R., 234, 237, 241, 242, 243, 246, 248, 250, 251, 252 Scott, R. B., 262, 282 Searls, R. L., 268, 275, 282 Sebring, E. D., 12, 20, 38 Sehgal, C. B., 148, 152 Sekiguchi, M., 82, 82 Selye, H., 183, 210 Sengel, P., 254, 255, 256, 258, 259, 282 Shaeffer, J., 241, 242, 248 Shaffer, B. M., 64, 82 Shafiz, A., 56, 59 Shantz, E. M., 121, 130, 134, 153 Shatkin, A. J., 12, 20, 38 Shaw, R. A., 180, 210 Shearer, G. M., 217, 247 Shelton, J. B., 236, 251 Shelton, R., 235, 251 Shen, S., 96, 112 Shepherd, M. K., 240, 251 Shimizu, M., 182, 208 Shin, 176, 186 Sho, C. T., 208 Siminovitch, L., 202, 210, 218, 219, 246, 249, 251 Simon, S., 8, 37 Singer, M., 225, 247 Singer, M. F., 233, 237, 251 Singh Bajaj, Y. P., 148, 152 Skoog, F., 119, 146, 152, 153 Slaunwhite, W . R., 180, 210 Slayter, H. S., 31, 37, 241, 251, 263, 281 Slizynska, H., 163, 171 Smirnov, V. N., 9, 38 Smith, E. W., 238, 251

Smith, J., 125, 141, 153 Smith, S. D., 41, 59 Sofer, W. H., 29, 38 Solomon, E., 65, 82 Sondhaus, C. A., 231, 251 Sonneborn, D. R., 63, 64,65, 66, 82, 83 Sonnebom, T. M., 214, 251 Southard, J. L., 224, 231 Spemann, H., 85, 86, 105, 111 Spencer, T., 10, 38 Spiegel, M., 8, 38 Spiegelman, S., 55, 59, 225, 252 Spirin, A. S., 7, 9, 12, 13, 14, 15, 16, 17, 19, 24, 26, 29, 30, 36, 37, 38 Spofford, J . B., 169, 171 Staba, E. J., 149, 153 Stafford, D. W., 29, 38 St. Arnand, W., 237, 250 Steiner, M., 244, 250 Stent, G. S., 205, 210, 237, 251 Stern, C., 157, 171 Stem, J. L., 82, 82 Stevens. L. C., 141, 153 Steward, F. C., 116, 119, 120, 121, 123, 124, 125, 130, 131, 134, 141, 143, 150, 151, 152, 153 Stohlman, F., Jr., 186, 202, 207, 210, 216, 220, 221, 222, 247, 251 Street, H . E., 118, 153 Strong, F. M., 119, 152, 153 Sturm, D., 215, 221, 247 Sugata, F., 182, 208 Suit, H. D., 189, 209, 210, 251 Suit, H . F., 221, 249 Sussman, M., 62, 63, 64, 65, 66, 67, 68, 69, 70, 73, 77, 80, 81, 82, 83 Sussman, R. R., 65, 70, 73, 83 Suzuki, A., 89, 111 Svobodova, J., 136, 153 Swain, T., 143, 151 Swift, H., 174, 193, 199, 208, 220, 222, 225, 231, 248 Swingle, C., 146, 153

T Takaku, F., 180, 187, 189, 190, 207, 210 Takanami, M., 21, 25, 38, 230, 250

294

AUTHOR INDEX

Takata, C., 87, 96, 111, 112 Takeuchi, I., 64, 65, 83 Takeuchi, M., 148, 152 Taylor, J. H., 170, 171 Taylor, K. B., 178, 179, 181, 208 Taylor, L. W., 259, 270, 280, 281, 282 Tchemos, V. I., 24, 38 Tencer, R., 5, 37 Terskikh, V. V., 25, 37 Terwey, K. H., 222, 252 Thathachari, Y. T., 263, 281 Thomas, E. D., 184, 209, 210, 217, 220, 238, 248, 251 Thomson, J. L., 255, 282 Thomson, R. Y., 194, 210 Thorell, B., 174, 210, 214, 220, 221, 222, 225, 230, 231, 251 Tiedemann, H., 87, 88, 89, 97, 98, 99, 101, 104, 106, 107, 110, 110, 111 Till, J . E., 188, 202, 210, 217, 218, 219, 246, 249, 251 Tjhio, K . T., 143, 151 Tocantins, L. M., 236, 246 Toivonen, S., 88, 102, 110, 111, 112 Tomkins, G. M., 32, 37 Tooze, J., 222, 251 Torbert, J. V., 236, 251 Torrey, J. G., 147, 153 Tomes, P. I., 109, 112 Trinkhaus, J. P., 64, 83 Trobaugh, F. E., Jr., 218, 249 Tschudy, D. P., 198, 210 Tschumi, P. A., 275, 282 Ts’O, P. O., 226, 251 Tsuruoka, N., 182, 208 Tukey, H. B., 147, 154 Turnbull, E. P. N., 235, 251 Tweedell, K., 47, 59 Tyler, A., 3, 4, 5, 8, 29, 37, 38, 43, 55, 59

U Urata, G., 197, 208, 210

V Vahs, W., 88, 98, 111, 112 Vainio, T., 102, 112 Van Deenen, L. L. M.,232, 251

van Dyke, D. C., 182, 208 van Overbeek, J., 147, 154 van Tieghem, P., 147, 154 Vasil, I. K., 148, 154 Vasil, V., 141, 154 Veno, T., 182, 208 Vinograd, J., 226, 251 Vittorelli, M. L., 3, 4, 5, 37 Vladimirzeva, E. A., 25, 37 Volmer, E. P., 182, 210 von Ehrenstein, G., 193, 210, 225, 250 von Saltza, M . H., 119, 152

W Wackman, N., 184, 208, 218,248 Waddington, C. H., 2, 38, 203, 211, 280, 283 Wadhi, M., 143, 154 Waldman, T. A., 181, 210 Wales, M., 220, 238, 251 Walker, J., 235, 251 Wallenius, G., 235, 249 Waltner, K., 184, 211 Wang, Y., 96, 112 Ward, C., 64, 65, 83 Waris, H., 148, 152, 154 Warner, J. R., 226, 234, 241, 242, 251, 252 Waters, L., 9, 37 Watson, J. D., 226, 237, 252 Watson-Williams, E. J., 236, 246 Waugh, D. F., 181, 211 Waxman, H. S., 231, 244, 252 Weatherall, D. J., 235, 236, 239, 240, 243, 245, 246, 250, 251, 252 Weicker, H., 222, 252 Weintraub, A. H., 177, 178, 207 Weisberger, A. S., 234, 252 Weisner, R., 56, 59 Went, F. W., 146, 151 Went, L. N., 239, 252 Wessells, N. K., 254, 256, 257, 258, 283 Wessels, N., 9, 38 Wetherell, D. F., 148, 152 Wetmore, R. H., 120, 146, 152, 154 White, G. J., 64, 65, 66, 68, 69, 82, 83 White, J. C., 235, 238, 252

295

AUTHOR INDEX

White, P. R., 117, 118, 154 White, W. F., 178, 178, 179, 181, 208, 211 Wilhelm, J. E., 189, 209 Williamson, R., 232, 234, 241, 242, 243, 250 Willson, C., 225, 250 Wilson, E. B., 105, 112 Wilt, F. H., 4, 5, 7, 9, 38, 198, 199, 211, 231, 252 Winslow, R. M., 243, 244, 245, 252 Winterhalter, K. H., 198, 211 Wintrobe, M. M., 198, 210, 215, 216, 221, 252 Winder, R. J., 179, 208 Wittek, M., 182, 210 Wolfe, H. G., 237, 248 Wolfe, S., 234, 237, 252 Wolf€, E., 254, 283 Woodward, J. W., 174, 193, 199, 208, 220, 222, 225, 231, 248

Wright, B. E., 64,85, 82, 83 Wurster, J. C., 188, 209

Y Yamada, T., 87, 98, 98, 111, 112 Yamaguchi, A., 182, 208 Yanagisawa, K., 70, 83 Yankofsky, S. A., 225, 252 Yehuda, M., 240, 247 Yemm, E. W., 125, 153 Yphantis, D. A., 181, 211 Yunis, E., 190, 211 Yunis, J. J., 190, 211

Z Zbarsky, I. B., 9, 38 Zehavi-Willner, T., 228, 247 Zellig, W., 89, 111 Zwilling, E., 254, 201, 265, 267, 288, 289, 273, 274, 275, 277, 281, 282, 283

of erythroid cells, 185-200, 200-206, 220-232 RNA metabolism and, 101-105 DNA, in erythroid cell development, 221-224 DrosophiZu, 155 ff. genetic mosaics in, 156-161 variegation mosaics in, 161-171

A Actinomycin D in slime molds, 73-76 in spiralian embryo, 42-43, 48-50 Adrenal cortical steroids, erythropoiesis control by, 183 Amphibian embryos, differentiation in early development of, 85 ff. Androgens, erythropoiesis control by, 182-183 Animal cells, mRNA-carrying postribosoma1 particles in, 11-28 Arabidopsis thaliana, 143, 144

B Batyl alcohol, erythropoiesis control by, 184 C Callus, 141 Carrot plant, growth from free cells, 129-136 Cell affinities, 106-109 Chick embryo limb development in, 263-280 skin development in, 254-263, 277-280 Cleavage, in spiralian embryo, 42 Cobalt salts, erythropoiesis control by, 184-185 Coriandrum, 136 ff. Corticotropin, erythropoiesis control by, 182 Cycloheximide, genetic transcription and, 77 ff. Cymbidium, 143, 145

D Dermis, in chick embryo, 255 ff. Dictyostelium discoideum, 62 ff. Differentiation in amphibian embryos, 85 ff. of embryonic chick limb, 263-280 of embryonic chick skin, 254-263, 277-280

E Ectoderm, in chick embryo limb, 264270, 270-277 Embryos amphibian, 85 ff. chick limb development in, 263-280 skin development in, 254-263, 277280 masked RNA in, 7-11 spiralian, 39 ff. Epidermis, in chick embryo, 255 ff. Erythroblasts, 215, 221 Erythroid cell development biochemical control of, 173 ff. hemoglobin synthesis during, 238-245 in mammals, 213 ff. differentiation, 220-232 messenger RNA, 233-234 sites of, 215-216 models of differentiation, 200-206 Erythropoiesis, 215-216 hormonal control of, 176177, 182-184 nonhormonal control of, 184-185 Erythropoietin, 175 ff. biochemical actions in uitro, 188-196 biochemical actions in uiuo, 188 hemoglobin synthesis control, 196-199 as inducer of red cell differentiation, 185-200 properties of, 179-182 sonrces of, 177-179 Estrogens, erythropoiesis control by, 183 Eye development of in Drosophila, 157-161

296

297

SUBJECT INDEX

mosaics in Drosophila, 156-157, 161171 pattern of defects in Brasophila mutant, 168-169

F Feather development, in embryonic chick, 254 ff. 45 S particles, 23-26 FR-17, 66-67, 70-76 Free plant cell culture, 113-115, 117-123 Haberlandt, 115-1 16 morphology and division, 123-129 Fruit fly. See Drosophilu

G Genetic mosaics, 156-161 Genetic transcription in slime molds, protein synthesis during, 76-82 in spiralian embryo, 44 ff. morphogenesis and, 48-53 precocious segregation and, 53-55 Globins, 243-244 synthesis of, 231 Growth, in free plant cells, 116-117

H Haberlandt, concepts of, 115-116 HeLa cells, mRNA-containing ribosomal particles in virus-infected cells, 20-23 Hematopoiesis, 215 Hemoglobin A synthesis, 238-241 Hemoglobin F synthesis, 238-241 Hemoglobins, types, 235-237 Hemoglobin synthesis control mechanisms, 237-245 erythropoietin, 196-199 in mammals, 231-232 Heterochromatization, 165-166, 170 Hormones controlling erythropoiesis erythropoietin, 175-182 pituitary hormones, 182

steroid hormones, 182-183 thyroid hormones, 183-184 Humans hemoglobin formation in, 238-240 hemoglobins in, 235-237 Hypophysectomy, 182

I Ilyanassa embryo, 41 ff. Inducing factors, 86, 96 intracellular distribution of, 105-106 mechanism of action, 98-101 test methods for, 87-88 Induction, 202 Informosomes, 13-20

1 Limb development, in embryonic chick, 263-280 Loach, sucrose density gradient centrifugation, 13-20

M Mammals, erythroid cell development in, 213 ff. Masked mRNA in early developing embryos, 7-11 in postribosomal particles, 11-28 in unfertilized eggs, 3-7 Masked polyribosomes, experimental data on existence of, 28-33 Mesoderm, in embryonic chick limb development, 270-277 Mesoderm-inducing factor characteristics of, 96-98 isolation and properties of, 88-96 Messenger RNA association with polyribosomes, 233234 forms of, 33 ff. in mammalian erythroid cell development, 233-234 masked. See Masked mRNA Mice hemoglobin formation in, 240-241 hemoglobins in, 237

298

SUBJECT INDEX

Morphogenesis in free plant cells, 11f3-117, 123-129, 148 ff. genetic transcription in spiralian embryo and, 48-53 Mosaics genetic, 158-181 variegation, 181-171

N Neural-inducing factor, 88 Nicotianu, 141, 142 Nuclear particles, mRNA-containing, 2 6 27 Nucleic acid synthesis in slime molds, 70-76 in spiralian embryo, 40-42 Nucleus, in erythroid cell development, 221-224

during feather development in embryonic chick, 261-283 hemoglobins, 237 ff. in slime molds, 70-78 during genetic transcription, 7 6 8 2 in spiralian embryo, 40-42

R Red blood cells. See Erythroid cell development Ribosomes in erythroid cell development, 226230 hemoglobin synthesis and, 242-243 RNA, ervthroid cell develoDment and. 22s-228 RNA metabolism, differentiation and, 101-105 RNA synthesis, during feather development in embryonic chick, 261-263

0

S

Oligoribosomes, stored, in early embryos, 28-31 Oogenesis, in spiralian embryo, 44 Orchid, 143, 145 Organs, formation of complex, 109-110

Scale development, in embryonic chick, 254 ff. Sea urchin, inactive oligoribosomes in, 28-31 Sensitization, 200 Sialic acid, erythropoietin activity and, 180-181 Siuni, 138 ff. Skin development, in embryonic chick, 254-263, 277-280 Slime molds, 82-65 FR-17, 88-67 mutant strains, 85 ff. regulatory program for development of, 81 ff. Specialization, 202-204 Spiralian embryo, transcription of genetic information in, 39 ff. Stem cells, 174, 200 ff., 214 in mammals, 216222 Steroid hormones, erythropoiesis control by, 182-183

P Pituitary hormones, erythropoiesis control by, 182 Plants culture of free cells. See Free plant cell culture growth from free cells, 129-148 Polyribosomes hemoglobin synthesis and, 242-243 messenger RNA and, 233-234 “repression” of, 31-33 Position-effect variegation, 183-167 Postribosomal particles, masked mRNA in, 11-28 Precocious segregation, 40 gene transcription and, 53-55 Proerythroblasts, 221 Progenitor cells. See Stem cells Prolactin, 182 Protein synthesis in erythroid cells, 230-232

T Testosterone, erythropoiesis control by, 182-183 Thalassemia, 244

SUbJECT INDEX

Thymidine kinase, 31-32 Thyroid hormones, erythropoiesis control by, 183-184 Transferase, 87-70

299

v

U

Variegation mosaics, 181-171 position effect, 183-187 zeste, 181-183, 170 Virus-infected cells, mRNA-containing postribosomal particles in, 20-23-

UDP-Gal polysaccharide transferase, 8770

Z

Tryptophan pyrrolase, 32

Unfertilized egg, masked mRNA in, 3-7

Zeste variegation, 181-183, 170

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