Current Topics Developmental Biology (Current Topics in Developmental Biology)

Current Topics in Developmental BioIogy Volume 41

Series Editors Roger A. Pedersen

and

Reproductive Genetics Division Department of Obstetrics, Gynecology, and Reproductive Sciences University of California San Francisco, California 94143

Gerald P. Schatten Departments of Obstetrics-Gynecology and Cell and Developmental Biology Oregon Regional Primate Research Center Oregon Health Sciences University Beaverton, Oregon 97006-3499

Editorial Board Peter Gruss Max Planck Institute of Biophysical Chemistry, Gottingen, Germany

Philip lngham University of Sheffield, United Kingdom

Mary Lou King University of Miami, Florida

Story C. Landis National Institutes of Health/ National Institute of Neurological Disorders and Stroke, Bethesda, Maryland

David R. McClay Duke University, Durham, North Carolina

Yoshitaka Nagahama National Institute for Basic Biology, Okazaki, Japan

Susan Strome Indiana University, Bloomington, Indiana

Virginia Walbot Stanford University, Palo Alto, California

Founding Editors A. A. Moscona Alberto Monroy

Current Topics in Developmental Biology Volume 41 Edited by

Roger A. Pedersen Reproductive Genetics Division Department of Obstetrics, Gynecology, and Reproductive Sciences University of California San Francisco, California

Gerald P. Schatten Departments of Obstetrics-Gynecology and Cell and Developmental Biology Oregon Regional Primate Research Center Oregon Health Sciences University Bea verton, Oregon

Academic Press San Diego

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Front cover photograph: Expression patterns of Tbx5 (left) and Tbx4 (right) in stage 23 chick embryo forelimbs and hindlimbs, respectively.

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on acid-free paper.

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Copyright 0 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher's consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923). for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1999 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0070-2153/99 $25.00

Academic Press a division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com Academic Press 24-28 Oval Road, London NWI 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Book Number: 0-12-153141-4 PRINTED IN THE UNITED STATES OF AMERICA 98 99 0 0 0 1 02 0 3 B B 9 8 7 6

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Contents

Contributors Preface xi

ix

1 Pattern Formation in Zebrafish-Fruitful and Genetics

liaisons between Embryology

L ilianna Solnica -Krezel

I. Establishment of the Dorsoventral Polarity during Zebrafish Cleavage Stages 2 11. Establishment of the Dorsal Blastula Organizer (Nieuwkoop Center) 111. Induction of the Gastrula Organizer by the Blastula Organizing Center IV. Structure and Function of the Dorsal Gastrula Organizer in Zebrafish 22 V. Coordination of Gastrulation Movements VI. Conclusions 28 References 29

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2 Molecular and Cellular Basis of Pattern Formation during Vertebrate limb Development Jennifer K. Ng, Koji Tamura, Dirk Buscher, and Juan Carlos Izpislja-Belmonte

I. Introduction 38 11. The Proximal-Distal Axis 39 111. The Anterior-Posterior Axis 46 52 IV. The Dorsal-Ventral Axis 59 V. Conclusions References 60

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3 Wise, Winsome, or Weird? Mechanisms of Sperm Storage in Female Animals Deborah M. Neubaum and Mariana F. Wolfner

I. 11. 111. IV. V. VI. VII.

Introduction 68 Mechanisms of Sperm Storage 73 80 The Fate of Unstored Sperm and Secretions Sperm inside the Storage Organs 80 85 Molecules Important for Sperm Storage 88 The Adaptive Significance of Sperm Storage 89 Conclusions References 90

4 Developmental Genetics of Caenorhabdifis eregans Sex Determination Patricia E. Ku wabara I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI.

Introduction 100 104 The Role of the X:A Ratio Genetic Analysis of Sex Determination 108 1 11 Molecular Analysis of Sex Determination TRA-I Targets and the Conservation of Sex-Determining Mechanisms How to Count Chromosomes: The X:A Ratio Revisited 117 119 Analysis of Germ-Line Sex Determination 120 The Hermaphrodite Sperm-Oocyte Decision Phylogenetic Comparisons and the Evolution of Sex-Determining Genes Unresolved Questions 124 Future Perspectives 126 References 127

L

5 Petal and Stamen Development Vivian F. Irish

I. 11. 111. IV. V. VI. VII.

Introduction 133 Petal and Stamen Ontogeny 135 Genes Controlling the Specification of Petal and Stamen Identities 143 Differentiation of Petals Differentiation of Stamens 148 Coordination of Gene Expression and Tissue Differentiation 152 Summary 153 References 154

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6 Gonadotropin-Induced Resumption of Oocyte Meiosis and MeiosisActivating Sterols Claus Yding Andersen, Mogens Baltsen, and Anne Grete Byskov

I. Introduction

163

165 111. Possible Signal Transduction Pathways Involved in Resumption of Meiosis 169 IV. Hypothesis of a Role for MAS in Gonadotropin-Induced Resumption 175 of Meiosis 178 V. Possible Implications for Fertility References 179 11. Gonadotropin-Induced Resumption of Meiosis

Index

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Contents of Previous Volumes

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Contributors

Numbers in puretitheses indii.ute the pugrs

011

which the authors' contribrctions begin.

Mogens Baltsen (163), Laboratory of Reproductive Biology, The Juliane Marie Centre for Children, Women, and Reproduction, University Hospital of Copenhagen, DK-2 100 Copenhagen, Denmark Dirk Biischer (37), Gene Expression Laboratory, The Salk Institute, La Jolla, California 92037 Anne Grete Byskov (1 63), Laboratory of Reproductive Biology, The Juliane Marie Centre for Children, Women, and Reproduction, University Hospital of Copenhagen, DK-2 100 Copenhagen, Denmark Vivian F. Irish (133), Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520 Juan Carlos Izpisua-Belmonte (37), Gene Expression Laboratory, The Salk Institute, La Jolla, California 92037 Patricia E. Kuwabara (99), Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom Deborah M. Neubaum" (67), Section of Genetics and Development, Cornell University, Ithaca, New York 14853 Jennifer K. Ng (37), Gene Expression Laboratory, The Salk Institute, La Jolla, California 92037 Lilianna Solnica-Krezel ( 1 ), Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37232 Koji Tamura (37), Gene Expression Laboratory, The Salk Institute, La Jolla, California 92037 Mariana F. Wolfner (67), Section of Genetics and Development, Cornell University, Ithaca, New York 14853 Claw Yding Andersen (163), Laboratory of Reproductive Biology, The Juliane Marie Centre for Children, Women, and Reproduction, University Hospital of Copenhagen, DK-2 100 Copenhagen, Denmark *Current address: Cardiovascular Research Center, Massachusetts General Hospital/Harvard Medical School, Charlestown, Massachusetts 02 129.

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Preface

This volume continues the customs of Current Topics in Developmental Biology in addressing developmental mechanisms in a variety of experimental systems and in having a theme-the molecular basis of pattern formation. In the first chapter, Liliana Solnica-Krezel from Vanderbilt University considers pattern formation in zebrafish. Then, Jennifer K. Ng, Koji Tamura, Dirk Buscher, and Juan Carlos Izpis6a-Belmonte from the Salk Institute discuss vertebrate limb development. In Chapter 5 , Vivian F. Irish from Yale University presents petal and stamen development, another molecular challenge in the broad field of pattern formation. Sex is never far from the minds of developmental biologists and Patricia E. Kuwabara from the MRC in Cambridge considers the developmental genetics of sex determination in Caenorhabditis elegans in Chapter 4. The molecules that trigger the resumption of meiosis in mammals are still being discovered, and Claus Yding Andersen, Mogens Baltsen, and Anne Grete Byskov from the University Hospital of Copenhagen review their novel results on meiosis-activating sterols during gonadotropin-induced resumption of oocyte meiosis in mammals. Weird sex is also considered in Chapter 3 by Deborah M. Neubaum and Manana F. Wolfner from Cornell University in their discussion of the mechanisms of sperm storage in female animals. Together with the other volumes in this series, this volume provides a comprehensive survey of major issues at the forefront of modem developmental biology. These chapters should be valuable to researchers in the fields of plant, invertebrate, and vertebrate development, as well as to students and other professionals who want an introduction to current topics in cellular, molecular, and genetic approaches to both developmental and reproductive biology. This volume in particular will be essential reading for anyone interested in gene regulation of pattern formation, sex determination, genetic controls of development, signaling molecules, cell cycle arrest checkpoints and resumptions, and gamete preservation and viability. This volume has benefited from the ongoing cooperation of a team of participants who are jointly responsible for the content and quality of its material. The authors deserve full credit for their success in covering their subjects in depth, yet with clarity, and for challenging the reader to think about these topics in new ways. We thank the members of the Editorial Board for their suggestions of xi

topics and authors, and Michelle Emme for her exemplary administrative and editorial support. We are grateful for the unwavering support of Craig Panner and Michele Bidwell at Academic Press in San Diego and for the assistance of Kathy Nida. We are also grateful to the scientists who prepared chapters for this volume and to their funding agencies for supporting their research. Gerald P. Schatten Roger A. Pedersen

1 Pattern Formation in Zebrafish-Fruitful Liaisons between Embryology and Genetics L ilia nna Soln ica- Krezel Department of Molecular Biology Vanderbilt University Nashville, Tennessee 37232

I. Establishment of the Dorsoventral Polarity during Zebrafish Cleavage Stages 11. Establishment of the Dorsal Blastula Organizer (Nieuwkoop Center) 111. Induction of the Gastrula Organizer by the Blastula Organizing Center

I v. Structure and Function of the Dorsal Gastrula Organizer in Zebrafish A. Is the Embryonic Shield Equivalent to the Dorsal Gastrula Organizer? B. Molecular Genetics of the Inductive Functions of the Organizer V. Coordination of Gastrulation Movements A. Relationships between Dorsoventral Patterning and Gastrulation Movements VI. Conclusions References

Vertebrate embryos, despite quite diverse early morphologies, appear to employ similar cellular strategies and conserved biochemical pathways in their development (Eyal-Giladi, 1997). In the past decade, a small tropical teleost, zebrafish (Darzio rerio), became an important model system in which to study development (Streisinger er al., 1981). By combining embryology with molecular and classical genetic methods, our understanding of early inductive and morphogenetic events during vertebrate embryogenesis significantly advanced. In zebrafish, dorsal-ventral polarity is established during early cleavage and is dependent on microtubular transport of determinants from the vegetal pole to the blastomeres positioned on top of the yolk cell. The syncytium forming from these marginal blastomeres in the early blastula exhibits dorsal-ventral asymmetry with p-catenin localized to the nuclei on the presumptive dorsal side of the syncytium. The yolk cell is a source of signals that induce and pattern overlying blastoderm. Therefore, the dorsal yolk syncytial layer is equivalent to the Nieuwkoop center of the amphibian embryo. The embryonic shield, a thickening of the dorsal blastoderm margin, exhibits properties similar to the amphibian Spemann organizer. However, certain inductive and patterning signals from the organizer might be produced before the shield forms or might originate outside of the shield. Similar to the amphibian embryo, the key patterning functions of the fish dorsal organizer (Le., dorsalization of mesoderm, ectoderm, and coordination of gastrulation movements) are performed by secreted molecules that antagonize the ventralizing activity of the swirl (zbrnp-2) and zbmp-4 gene products expressed on the ventral side of the embryo. These functions of the dorsal organizer require the activity of the chordino gene (a zebrafish homologue of chordin), bozozok, mercedes and ogon loci. Copyright 0 1999 by Academic Press.

Currenr Topics in Developmenral Sio/ogy, Vol. 41 Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved. 0070.2153/99 $25.00

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1. Establishment of the Dorsoventral Polarity during Zebrafish Cleavage Stages The specification of embryonic polarity is initiated at early stages of zebrafish development, leading to the establishment of a dorsal blastula signaling center, an equivalent of the Nieuwkoop center, by the midblastula stage. In contrast to Xenopus, fertilization of the zebrafish egg is restricted spatially and occurs at a single sperm entry point located at the future animal pole of the embryo (Hart et al., 1992; Hart and Donovan, 1983). Fertilization is followed by a separation of cytoplasm from the yolk such that the zygote exhibits a cytoplasmic blastodisk at the animal pole of a large yolk sphere (Fig. 1A) [for an overview of stages of zebrafish development, see Kimmel et al. (1995)l. Subsequent rapid and synchronous cellular divisions occur in a stereotyped fashion. The planes of the first divisions, however, are not correlated with the future dorsoventral axis of the embryo (Abdelilah et al., 1994; Helde et al., 1994; Wacker et al., 1994). The initial cleavages are meroblastic, so that all early blastomeres maintain large cytoplasmic connections with a thin cytoplasmic layer of the yolk cell. Starting at the 16-cell stage, only the marginal blastomeres remain connected with the yolk cell. The synchrony of the early divisions is lost at the midblastula stage (9-10th division), which marks initiation of the zygotic transcription and cell motility (Kane and Kimmel, 1993). At or soon before the midblastula transition, the marginal blastomeres fuse completely with the yolk cell to form the yolk syncytial layer (YSL) (Kimmel and Law, 1985). Concurrently, the most superficial Fig. 1 Key steps in the establishment of dorsoventral polarity during zebrafish development. (A) In the zebrafish zygote, cytoplasmic streaming leads to separation of the cytoplasmic island located on top of a large yolk cell. Dorsal determinants are thought to reside at the vegetal pole of the teleost zygote. (B) Before the first cleavage, an array of parallel microtubules forms in the vegetal cytoplasmic layer of the yolk cell. The polarity of microtubules anticipates the future DV axis of the embryo. Furthermore, transport of cytoplasmic particles (dorsal determinants) by this microtubule array is necessary for axis formation. (C) During cleavage, a second array of microtubules extends from the marginal blastomeres into the yolk cytoplasmic layer. These microtubules are thought to mediate transport of dorsal determinants to the dorsal marginal blastomeres. (D) Around the midblastula transition, nuclear accumulation of p-catenin is first detected in the prospective dorsal yolk syncytial layer. This distribution of p-catenin, together with the inductive potential of the yolk cell, suggests that this region most likely corresponds to the Nieuwkoop center of zebrafish. Yellow arrows indicate putative dorsalizing signal(s) emanating from the dorsal YSL, and green arrows indicate putative mesoderm-inducing signal emanating from lateral and ventral YSL. (E) At the sphere stage, nuclear localization of p-catenin is maintained in the dorsal YSL, while it can also be detected in the dorsal blastomeres. The functional significance of this aspect of p-catenin distribution is not understood. (F) At the early stages of gastrulation, ingressing mesendoderm can be seen as a germ ring along the circumference of the embryo viewed from the animal pole. Embryonic shield is a dorsal thickening of the germ ring. Expression of zbmp-2/4 is shown schematically in the ventral and lateral regions of the gastrula; expression of the chordino locus is indicated in the dorsal region of the gastrula. Expression and functional requirements for other genes in the dorsal and ventral signaling centers are also indicated.

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1. Pattern Formation in Zebrafish

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blastomeres become an enveloping layer (EVL). The remainder of the blastomeres located between the EVL and YSL, so-called deep cells, will give rise to all embryonic structures (Kimmel et al., 1995). This characteristic three-layered blastula of zebrafish appears radially symmetric, without any obvious dorsoventral asymmetry [reviewed in Solnica-Krezel et al. (19931. Dorsal maternal determinants in teleost embryos are thought to be present in the vegetal mass of the yolk cell soon after fertilization (Fig. 1A) (Mizuno et al., 1997). Studies demonstrate that the specification of future dorsal structures is dependent on microtubular arrays present in the yolk cell, and most likely on microtubule-dependent transport of particles from the vegetal pole to the prospective dorsal marginal blastomeres (Fig. lB,C) (Jesuthasan and Strahle, 1997; Trimble and Fluck, 1995). The zebrafish zygote transiently exhibits a dense array of parallel microtubules at the vegetal pole, whereas microtubules at the equator do not exhibit any clear organization. During cleavage stages, microtubules extend from blastomeres into the yolk cytoplasmic layer (Jesuthasan and Strahle, 1997; Solnica-Krezel and Driever, 1994). Disruption of microtubule arrays in the zygote or during cleavage, prior to the 32-cell stage, interferes with animalward movement of particles from the vegetal hemisphere into marginal blastomeres. These treatments inhibit the subsequent axis formation, as judged by the lack of expression of dorsal specific markers at the blastula stage (nuclear p-catenin) and gastrula stage (gsc, axial). The resulting embryos either do not exhibit any dorsal axis or possess very reduced axes with medially fused somites lacking notochord and head structures [Jesuthasan and Strahle (1997) and references therein]. Furthermore, in medaka fish, the direction of movement of cytoplasmic parcels on cortical microtubules anticipates the future dorsal-ventral axis of the embryo (Trimble and Fluck, 1995). These studies form the basis for an attractive hypothesis that the asymmetric transport of dorsal determinants from the vegetal pole to marginal blastomeres is one of the initial events involved in the establishment of the dorsoventral axis in teleost embryos (Jesuthasan and Strahle, 1997; Trimble and Fluck, 1995). Notably, other work indicates that in amphibians a similar microtubuledependent transport is involved in specification of the dorsoventral axis (Rowning et al., 1997). In a frog embryo, the dorsal-ventral axis is specified during the first cell cycle, when the cortex rotates relative to the cytoplasmic core along parallel microtubules associated with the vegetal core (Elinson and Rowning, 1988). Disruption of microtubules blocks the rotation and results in embryos lacking dorsal structures. On the basis of these observations, it has been thought that the rotation of the cortex relative to the core is effecting the translocation of dorsalizing components and dorsal axis induction (Elinson and Rowning, 1988). However, whereas the dorsalizing components are transported approximately 90" from the vegetal pole, the cortex rotates only 30" during cortical rotation. This apparent contradiction was clarified by the observation that endogenous organelles as well as fluorescent carboxylated beads injected into the vegetal pole are

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transported along the subcortical microtubules at least 60" toward the future dorsal side (Rowning et al., 1997). Hence, rather than transporting the dorsal determinants directly, the cortical rotation might serve to align the subcortical microtubules. The latter in turn would mediate the transport of dorsal determinants to the prospective dorsal equatorial region, a process very reminiscent of that described for teleost embryos. Therefore, despite the distinctly different organization of the frog and fish zygotes, the process of initial polarization in the two embryos might be as similar as the later patterning events seem to be, as discussed in this chapter.

II. Establishment of the Dorsal Blastula Organizer (Nieuwkoop Center) The identities of substances transported on microtubule tracks are mostly unknown. A notable exception is p-catenin, a component of the Wnt signaling pathway, which was shown to colocalize with subcortical microtubules on the dorsal side of the frog egg at the end of cortical rotation (Rowning et al., 1997). Furthermore, an important conserved step in the pathway of dorsal axis formation appears to be a nuclear accumulation of p-catenin on the dorsal sides of both Xenopus and zebrafish blastulae (Funayama et al., 1995; Schneider et al., 1996). This region is thought to constitute the dorsal blastula organizer (Nieuwkoop center), which is responsible for the subsequent induction of the dorsal gastrula (Spemann) organizer in the overlying cells. Peter Nieuwkoop first demonstrated the importance of vegetal cells (presumptive endoderm) in induction and patterning of the mesoderm in a series of recombination experiments involving fragments of amphibian blastulae (Nieuwkoop, 1969). In those experiments, mesodermal tissues, notochord, muscles, kidney, and blood, formed only when vegetal (endodermal) cells were combined with animal cap (future ectoderm) fragments, but not when vegetal or animal cap fragments were cultured alone. Moreover, the dorsoventral polarity of the induced mesoderm was determined by the particular subregion of endoderm used. Specifically, dorsal mesoderm was obtained only when dorsovegetal fragments were the inducer (Nieuwkoop, 1969). The dorsal mesoderm-inducing activity localized to the dorsalmost vegetal cells of the Xenopus blastula was named the Nieuwkoop center (Gerhart et al., 1991). p-Catenin is a multifunctional protein involved in cadherin-dependent cell adhesion as well as in the transduction of receptor-mediated intercellular signals [reviewed in Miller and Moon (1996)l. p-Catenin is a homologue of the Drosophila segment polarity gene armadillo (arm) (Peifer and Wieschaus, 1990). The activity of arm as a signaling molecule is dependent on its nuclear localization, which is regulated by the wingless (wg)signaling pathway (Riggleman et al., 1990). In this pathway, the secreted Wg glycoprotein (Wnt in vertebrates)

1. Pattern Formation in Zebrafish

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binds to its cell surface receptor thought to be encoded by the D - f r i ~ f e d -(fi) 2 gene. The ligand-receptor complex stimulates, in an unknown fashion, the activity of a phosphoprotein Dishevelled (Dsh). Activated Dsh in turn inhibits the activity of zeste white shaggy-3 kinase (the homologue of glycogen synthase kinase-3 in vertebrates), which normally destabilizes and thus inhibits arm (p-catenin) activity. In this manner, Wingless signaling leads to nuclear accumulation of p-catenin. In the nucleus, p-catenin interacts with an architectural transcription factor pangolin (LEF-1 family of proteins in vertebrates). In the vertebrate blastula, this complex is thought to activate the transcription of genes involved in axial specification. The likely central role of the Wnt-like signaling pathway in dorsal axis formation in frog embryos is underscored by the ability of several components of the pathway [i.e., wnt genes (Sokol et al., 1991), dsh (Sokol et al., 1995), dominant negative gsk-3 (Pierce and Kimelman, 1995; He et al., 1995), p-catenin (Funayama et al., 1995; Guger and Gumbiner, 19951 to induce the formation of secondary axes when expressed ectopically in the embryo. Furthermore, treatment of blastula stage embryos with lithium, which is thought to inhibit GSK-3 kinase activity (Hedgepeth et al., 1997; Klein and Melton, 1996), also leads to the induction of dorsal mesoderm in the entire marginal zone (Backstrom, 1954; Kao and Elinson, 1986, 1988). However, only p-catenin (Heasman et al., 1994) and GSK-3 (Pierce and Kimelman, 1995; He et af., 1995) have been demonstrated to be required for the formation of the endogenous axis in frog embryos. Notably, dominant negative mutant forms of Wnt (Hoppler et al., 1996) and Dsh (Sokol, 1996), while able to inhibit the formation of ectopic axes by their wildtype counterparts, do not prevent formation of the endogenous axis. Therefore, either a redundant pathway or a distinct pathway(s) leading to dorsoventral differences in GSK-3 activity and activation of p-catenin during normal development might exist (Yost et af., 1996). An involvement of the Wnt signaling pathway components, other than a p-catenin and GSK-3, in the zebrafish embryogenesis remains to be investigated (Kelly et al., 1995; Stachel et al., 1993). Important conjugation experiments in frog embryos indicate that cells in a dorsal vegetal mass, expressing p-catenin, send a “dorsal signal” to other cells at the midblastula stage, after the onset of zygotic transcription (Wylie et al., 1996). These observations are consistent with p-catenin regulating the inducing activity of the Nieuwkoop center. A systematic analysis of changes in intracellular distribution of P-catenin during Xenopus development demonstrated that p-catenin displays greater cytoplasmic accumulation on the future dorsal side of Xenopus embryo by the two-cell stage (Larabell et al., 1997). This is likely to be effected by the dorsally directed microtubular transport discussed previously (Rowning et al., 1997). The accumulation of p-catenin in dorsal nuclei was reported as early as the 16- to 32-cell stages (Larabell et af., 1997). In another report, nuclear accumulation of p-catenin was first observed shortly after stage 8 and was most prominent at stage 8.5, disappearing before the blastopore lip

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formation at the onset of gastrulation (Schneider et al., 1996). The domain of nuclei positive for p-catenin is located in the dorsal region of the blastula, occupying approximately one-third of the equator. Interestingly, this domain extends from the marginal zone into the vegetal and animal regions and is several cell layers deep (Schneider et al., 1996). If the distribution of p-cateninpositive nuclei reflects the extent of the Nieuwkoop center, it would mean that this organizing center is not located exclusively in vegetal blastomeres but also in marginal and some animal blastomeres. Alternatively, nuclear accumulation of p-catenin might extend beyond the Nieuwkoop center. In the latter case, activities of molecules other than p-catenin need to be implicated in the specification of the Nieuwkoop center. Where is the Nieuwkoop center equivalent located in a teleost embryo? This question was addressed by transplantation experiments in Salmo (Long, 1983) and in zebrafish (Mizuno et al., 1996), as well as by blastoderm isolation experiments in Fundulus (Oppenheimer, 1936b). Results of these experiments revealed the key role of the YSL in the process of mesoderm induction and patterning in teleost embryos. In the blastoderm isolation experiments, relatively normal development occurred in blastoderms deprived of their yolk cell at the 32-cell and later cleavage stages. In contrast, blastoderms isolated earlier did not gastrulate. Oppenheimer hypothesized that the blastoderms removed from the yolk before the 32-cell stage lacked some of the substance that is passed from periblast to blastoderm during or later than the 16-cell stage (Oppenheimer, 1936b). It is intriguing that treatments that inhibit the microtubular transport of particles from the vegetal hemisphere to marginal blastomeres, and thus consequently impair formation of the dorsal axis, also are effective only at or before the 32-cell stage [see Jesuthasan and Strahle (1997)l. These studies support the notion that the microtubules translocate the “axis-inducing” activity from the vegetal pole to the marginal blastomeres by the 32-cell stage. However, yolk cells from midblastula stage embryos, when transplanted to the animal pole of another zebrafish embryo, still exhibited the full potential to induce and pattern the host mesoderm (Mizuno et al., 1996). Thus, the axisinducing activity is present in the yolk cell at the midblastula stage. In zebrafish the YSL forms when the marginal blastomeres collapse onto the yolk cell at about the ninth or tenth cleavage (Kimmel and Law, 1985). Hence, the “axisinducing” material translocated from the vegetal pole to the marginal blastomeres during early cleavage most likely becomes incorporated into the YSL during a fusion of the marginal blastomeres with the yolk cell. Consistent with this view, P-catenin-positive nuclei are detected within the YSL on the prospective dorsal side of the high blastula stage zebrafish embryos about 20-30 min after the formation of the syncytial layer (Fig. 1D) (Schneider et al., 1996). Subsequently, at the sphere stage nuclear localization of p-catenin is observed in the blastoderm cells overlying dorsal YSL (Fig. 1E). The localization of p-catenin to the dorsal YSL, together with the ability of the yolk cell to induce dorsal mesoderm upon

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transplantation, strongly suggests that the dorsal YSL in the zebrafish blastula corresponds to the Nieuwkoop center located in the vegetal dorsal blastomeres of the frog blastula (Schneider et al., 1996). It is noteworthy that the accumulation of p-catenin in the YSL shortly precedes the onset of zygotic transcription at the midblastula transition. Therefore, the initial specification of the dorsoventral polarity and establishment of the inductive center in the dorsal YSL are dependent on the maternal program.

111. Induction of the Gastrula Organizer by the Blastula Organizing Center A key question is what are the downstream targets of the P-catenin-LEF-1 complex in the nuclei of the Nieuwkoop center? Moreover, what is the molecular nature of the “dorsal signal” that establishes the dorsal gastrula organizer in the overlying blastomeres (Wylie et al., 1996)? One potential candidate that might function in both frog and fish embryos is Vgl. This secreted, TGF-P-related molecule was described originally in frogs as maternal mRNA localized to the vegetal blastomeres (Rebagliati et al., 1985; Weeks and Melton, 1985). Ectopic expression of mRNA encoding Vgl fails to induce axis formation. However, injections of mRNA encoding a chimeric BMP-2-Vgl fusion protein that allows the production of the mature form of the Vgl ligand in vivo can both restore axis formation in UV-ventralized embryos and induce ectopic axes. In addition, the blastomeres expressing BVgl appear to act as a Nieuwkoop center (Thomsen and Melton, 1993). Interestingly, the zebrafish vgl orthologue (ZDVR-1) mRNA is found to be distributed uniformly in the egg and later in all embryonic cells (Helde and Grunwald, 1993). ZDVR-1 mRNA fails to induce axis formation when overexpressed in fish embryo. However, the normal zDVR-1 precursor does appear to be processed to mature protein when expressed in Xenopus and can act as a potent inducer of axial mesoderm in this system. These observations form the basis for a proposal that localized posttranslational processing of Vgl precursor protein on the prospective dorsal side plays a regulatory role in the development of the dorsal axis in frog and fish embryos (Dohrmann et al., 1996). How is the proposed activation of Vgl related to the p-catenin pathway? Because mature Vgl ligand has not been detected during normal development, the timing of its presumably proteolytic activation in vivo is not clear. However, BVgl mRNA injections can rescue the effects of depleting maternally encoded p-catenin in frog embryos, suggesting that Vgl acts downstream of p-catenin and thus around the midblastula transition (Wylie et al., 1996). These experiments cannot, however, exclude the possibility that Vgl acts in a pathway parallel to p-catenin. Indeed, additional evidence from Xenopus indicates that Vgl acts synergisticallywith a maternal Wnt-like signal to specify dorsal fates in both mesoderm and endoderm (Cui ef al., 1996).

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An important finding has been that a downstream target of p-catenin in Xenopus embryos appears to be the siamois gene, which encodes a paired-class homeodomain protein (Lemaire et al., 1995). Injections of RNA encoding Siamois into frog embryos lead to the induction of complete secondary axes, but the progeny of injected cells to not participate in the secondary axes. siamois is expressed after the midblastula transition and is present most abundantly in the dorsal endoderm of early gastrulae, indicating that it might be an important component of the Nieuwkoop center (Lemaire et al., 1995). Interestingly, whereas p-catenin and other components of the Wnt pathway highly activate expression of members of the siamois, TGF-fl pathway (such as Vgl) were shown to activate siamois only weakly (Brannon and Kimelman, 1996). This observation is consistent with Vgl acting in a parallel rather than the same pathway as p-catenin (see previous discussion). The involvement of TGF-p signaling in the formation of the zebrafish gastrula organizer is further supported by the activity of nodal and TARAM-A genes. The murine nodal gene is a TGF-P-related ligand that is essential for the formation of the node, the mouse equivalent of the Spemann organizer (Zhou et al., 1993). Injections of mouse nodal mRNA into zebrafish embryos lead to ectopic expression of the organizer-specific genes gsc and Ziml and formation of ectopic axes containing notochord and somites (Toyama et al., 1995). TARAM-A is a serinethreonine kinase type I receptor related to TGF-f3 and activin receptors (Renucci et al., 1996). Whereas ectopic expression of RNA encoding wild-type TARAMA leads only to an expansion of the axial mesoderm, injections of RNA encoding a constitutively active receptor kinase, TARAM-A-D, result in a massive induction of the dorsal and panmesodermal markers, gsc and ntl, shortly after the midblastula stage as well as numerous dorsal mesoderm genes in the late blastula stage (50% epiboly). Furthermore, injections of TARAM-A-D RNA induce the formation of complete secondary axes in which cells that inherited injected RNA mostly contribute to the anterior dorsal mesoderm (hatching gland and head mesoderm) (Peyrieras et al., 1996; Renucci et al., 1996). Notably, TARAM-A mRNA is found to be distributed uniformly in the zygote and during cleavage stages and disappears at the 500-cell stage from the marginal blastomeres that are fated to form the YSL. In the mid- and late blastula, TARAM-A mRNA is detected at the blastoderm margin around the circumference of the embryo, with its expression increasing on the dorsal side starting at 40% epiboly (Renucci et al., 1996). Both the activity and the expression pattern of TARAM-A make it a good candidate for a transducer of signals from the Nieuwkoop center involved in formation of the Spemann organizer. Another link between the Nieuwkoop center, p-catenin, TGF-P pathway, and the Spemann organizer in zebrafish has been revealed by the zebrafish zygotic bozozok mutation. Homozygous bozm’68mutants are characterized by the lack of the main derivatives of the gastrula organizer, prechordal plate and notochord, and also exhibit defects in the neuroectoderm (Solnica-Krezel et al., 1996).

1. Pattern Formation in Zebrafish

9

Notably, an embryonic shield cannot be detected morphologically in boz mutants at the early gastrula stage, and expression of all tested organizer-specific genes is reduced or absent, indicating that boz function is required for the proper formation of the dorsal gastrula organizer (L. Solnica-Krezel and K. Fekany, unpublished observations). The boz phenotype is reminiscent of defects observed in embryos in which microtubules were disrupted during cleavage stages, a treatment that prevented nuclear accumulation of p-catenin and inhibited dorsal axis formation ( Jesuthasan and Strahle, 1997). Several additional observations indicate that boz acts during the blastula stage within or downstream of the Nieuwkoop center in the pathway specifying axial mesoderm. First, expression of organizer-specific genes (gsc, JEh, TARAM-A) is already reduced at the late blastula stage. Second, whereas ectopic expression of p-catenin leads to the induction of full secondary axes in wild-type embryos (Kelly et al., 1993, the axes induced in boz embryos lack notochord and other midline cell types. Thus, p-catenin is unable to suppress the boz phenotype. Finally, the lack of notochord and prechordal plate, the hallmarks of the boz phenotype, is fully suppressed by ectopically expressed mRNAs encoding mouse nodal and the activated form of TARAM-A-D (Renucci et al., 1996), but not by wild-type TARAM-A (L. Solnica-Krezel, A. Renucci, and K. Fekany, unpublished observations). These studies indicate that the boz locus is required in a TGF-p signaling pathway that establishes the dorsal gastrula organizer in zebrafish acting downstream or in parallel with dorsalizing signals like p-catenin.

IV. Structure and Function of the Dorsal Gastrula Organizer in Zebrafish Transplantationexperiments performed in the 1920s by Hilde Mangold and Hans Spemann identified the dorsal blastopore lip of the amphibian gastrula as a tissue that, upon transplantation to the ventral side of the gastrula, will induce formation of the secondary body axis. The organizer tissue, while contributing mostly to chordamesoderm and to a lesser extent to paraxial mesoderm and the floor plate of the neural tube of the secondary axes, was able to induce and pattern the ectopic neuraxis as well as coordinate gastrulation movements (Spemann, 1938).

A. Is the Embryonic Shield Equivalent to the Dorsal Gastrula Organizer?

All vertebrates exhibit an equivalent dorsal gastrula organizer region, as defined by its inductive properties and expression of an array of organizer-specific genes, including gsc, HNF-3p, Xlim-1, Xnot, noggin, chordin, siamois, ADMP, and others [reviewed in Lemaire and Kodjabachian (1996)l. In zebrafish, the embryonic shield, a dorsal thickening along the blastoderm margin of the early gastrula,

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Lilianna Solnica-Krezel

is thought to correspond to the Spemann organizer (Fig. 1F). During normal development, this region gives rise to axial mesoderm, notochord and prechordal plate, and to some neural tissue, predominantly floor plate (Kimmel et al., 1990; Shih and Fraser, 1995). Transplantations of the shield to the ventral blastoderm margin lead to the formation of ectopic axes, with the donor tissue forming notochord, prechordal plate, and floor plate of the neural tube (Ho, 1992; Oppenheimer, 1936a,c; Shih and Fraser, 1996; Driever et al., 1997). Interestingly, the ectopic axes resulting from the shield transplantations are not complete and usually lack the anterior head structures (Shih and Fraser, 1996). In addition, the reciprocal experiments-extirpations of the shield-yield embryos without notochord, but with a relatively normal anterior-posterior (AP)pattern of the neural tube (Shih and Fraser, 1996). One explanation is that in these experiments the extirpated and/or transplanted tissue did not contain the entire organizer. This could be due to experimental difficulties with extracting the entire shield, leading to residual organizer activity remaining in place. Indeed, the expression of organizer-specific genes was not monitored in the operated embryos (Shih and Fraser, 1996). It is noteworthy that a different technique, glass pipet versus eyelash knife, can be used to perform the shield extirpationtransplantation experiments, allowing one to remove most of the region expressing organizer-specific genes, such as gsc andJlh (D. Stemple, National Institute for Medical Research, London, UK, personal communication; Driever et al., 1997). The secondary axes formed upon transplantations of shields removed with this technique can be complete. The shield extirpations performed with a glass capillary result in embryos with the axial mesoderm reduced or missing completely and severe deficiencies in the anterior neural structures, including cyclopia and/or eye reduction (Fig. 2B). However, neural keel still forms in these experimental embryos, indicating that a significant level of organizer activity might reside beyond the shield region in the zebrafish embryo. Alternatively, some inductive events happen before the shield stage. Consistent with this notion, the expression of a number of genes like gsc, liml, andJlh can be detected on the dorsal side of the embryo before the embryonic shield manifests itself (Stachel et al., 1993; Toyama et al., 1995; Talbot et al., 1995). Support for the notion that the organizer in fact extends beyond the shield comes from the observation that one of the key organizer-specific genes, chordino (zebrafish chordin homologue), is expressed in a broad dorsal marginal domain extending outside the shield (Miller-Bertoglio et al., 1997; Schulte-Merker et al., 1997).

B. Molecular Genetics of the inductive Functions of the Organizer

The dorsal gastrula organizer is thought to carry out a number of inductive and patterning functions: induction and patterning of the neuroectoderm, dorsalization of paraxial mesoderm, coordination of gastrulation movements, and finally

Fig. 2 Phenotypes of mutations affecting dorsoventral patterning and their phenocopies. (A) Wildtype embryo at one day of development. (B) Embryo resulting from shield extirpation. (C) bozozok. (D-G) Phenocopies resulting from injections of increasing amounts of RNA encoding BMP-4. (H) dino mutant phenotype. (I) Phenocopy obtained after injection of CSKA plasmid expressing zmbp-4. (J) snailhouse mutant phenotype. Phenocopy of snh mutants is obtained by injections with RNA encoding Noggin (K) or dominant-negative BMP-4 receptor (L). [(B) Obtained from D. Stemple, London, UK. (D, E, F, G, K, L) Reprinted from Mechanisms of Development 62; Neave, B., Holder, N., and Patient, R. A graded response to BMP-4 spatially coordinates patterning of the mesoderm and ectoderm in zebrafish. pp. 183-195 (1997), with kind permission from Elsevier Science Ireland Ltd., Bay 15K,Shannon Industrial Estate, Co. Clare, Ireland. (H) Reprinted from Developmenr, Hammerschmidt et al. (1996b). with permission from the Company of Biologists Limited. (I) From Development, Hammerschmidt et al. (1996a), with permission from the Company of Biologists Limited. (J) Reprinted from Developmenr, Mullins et a/. (1996), with permission from the Company of Biologists Limited.]

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self-differentiation into midline structures (Spemann, 1938). Discoveries in a number of animal systems, most notably expression cloning in Xenopus, identified several molecules that might fulfill these distinct functions of the gastrula organizer [reviewed in Lemaire and Kodjabachian (1996)l. Identification of numerous mutations affecting organizer function in zebrafish provides genetic confirmation of the presumed functions for some of these molecules, as well as identification of novel genetic components. Furthermore, the analysis of the zebrafish mutants reveals genetic hierarchies that govern pattern formation in the gastrula (Hammerschmidt et al., 1996b; Mullins et al., 1996; Solnica-Krezel et al., 1996).

1. The Organizer Dorsalizes Mesoderm and Ectoderm by Antagonizing the Ventral Morphogen BMP2/4 Numerous molecular biological and genetic studies support the notion that the two main activities of the dorsal gastrula organizer, dorsalization of mesoderm and neural induction (which could be viewed as dorsalization of ectoderm), are accomplished by secreted factors that antagonize ventralizing signals derived from the ventral organizing center in the gastrula [Piccolo et al., 1996; Zimmerman et al., 1996; reviewed in Hemmati-Brivanlou and Melton (1997)l. This interaction between bone morphogenetic proteins 2 and 4 (BMP-2/4), secreted ligands of the transforming growth factor+ superfamily, and their antagonists appears to be a mechanism for embryonic dorsoventral patterning that is conserved from the fruit fly to vertebrates (Hogan, 1996; Holley and Fergusson, 1997; Holley et al., 1995). The realization that the formation of ventral fates in the embryo is not a default pathway, but rather depends on specific signaling events, was suggested initially by experiments showing that ectopic expression of BMP-4 induced ventral and posterior cell types in animal cap explants and whole Xenopus embryos (Dale et al., 1992; Jones et al., 1992, 1996). Transcripts encoding BMP-4 are present in Xenopus unfertilized eggs and initially are distributed uniformly throughout frog blastula. In contrast to Xenopus (Hemmati-Brivanlou and Thomsen, 1995), transcripts for the zebrafish homologues (zbmp-2 and zbmp-4) are not found in the maternal pool of RNAs. zbmp-2 transcripts are detected first at the sphere stage and are distributed uniformly throughout the blastoderm (Nikaido et al., 1997). However, as the yolk domes at the onset of epiboly, zbmp-2 transcripts disappear from the presumptive dorsal side. At 50% epiboly, just before the onset of the involution/ingression movements that will create the germ layers, zbmp-4 transcripts become detectable. Similar to the Xenopus embryo, at the early (shield) stages of gastrulation, both zbmp-2 and -4 exhibit high levels of transcripts in the presumptive ventrolateral blastoderm in a domain extending from the margin up through the animal pole and are absent on the dorsal side. An interesting and not yet fully understood exception is a small

1. Pattern Formation in Zebrafish

13

distinct domain of zbmp-4 expression in the shield region itself, marking the precursors of the anterior axial mesoderm (Fig. IF) (Chin et al., 1997; Hammerschmidt et al., 1996b; Nikaido et al., 1997). Overexpression of zbmp-2 and zbmp-4 in zebrafish embryos supports the conclusions drawn from studies of frog embryos (Hemmati-Brivanlou and Thomsen, 1995; Jones et al., 1991; Wilson and Hemmati-Brivanlou, 1995) that these factors respecify dorsal mesoderm to ventral mesoderm and expand the prospective epidermis at the expense of neuroectoderm (Hammerschmidt et al., 1996b; Nikaido et al., 1997; Neave et al., 1997). However, the exact phenotypes reported differ in an interesting way. Neave and colleagues performed the most systematic analysis by injecting varying amounts of Xenopus bmp-4 RNA (20-220pg/ embryo) into 1-4-cell-stage zebrafish embryos (Neave et al., 1997). Progressively ventralized embryos were observed in a dose-dependent manner (Fig. 2D-G). The lowest bmp-4 dose led to “ventralized I” class embryos, which lacked head and notochord structures, whereas the somites lost their chevron shape and became fused in the midline. In the “ventralized 11” class of embryos, besides the lack of a head and notochord, the yolk extension failed to form. More ventralized embryos (classes I11 and IV) were observed only after the injection of 100 pg or more RNA. Embryos of class I11 exhibited radial symmetry; cells accumulated at two opposite poles of the round yolk cell with somitic tissue forming at one of the ends. Finally, embryos of class IV likewise were radially symmetric; however, cells accumulated only at one end, suggesting that epiboly also was affected. A similar loss of axial structures and often severe reduction of the hypoblast was observed in embryos injected with 100 pg of zbmp-2 RNA (Nikaido et al., 1997). Analysis of axial mesoderm markers (gsc and ntl) revealed that expression of these genes was normal in bmp-4 RNA-injected embryos at the onset of gastrulation and that reduction or absence of gsc and ntl expression began to be observed by 65-70% epiboly (Neave et al., 1997). These results indicate that even high doses of BMP-2/4 signaling do not prevent the initial formation of the main component of the dorsal gastrula organizer, axial mesoderm. However, exclusion of BMP-2/4 signaling from the dorsal side of the gastrula might be important for maintenance and further development of axial mesoderm, as has been reported earlier for Xenopus (Jones et al., 1996). Interestingly, a distinct phenotype was caused by ectopically expressing BMP-4 at later stages of development by using a plasmid with bmp-4 cDNA under the control of the cytoskeletal actin (CSKA) promoter from Xenopus borealis, which directs BMP-4 expression only after the midblastula transition. Whereas there was expansion of ventral markers and reduction of neuroectodermal markers in injected embryos during gastrulation, at the end of embryogenesis only the posterior part of the notochord was reduced and embryos exhibited multiple ventral fin folds and an enlarged blood island (Fig. 21) (Hammerschmidt et al., 1996~).It is possible that the phenotype obtained with DNA injections represents the mildest ventralization and thus could reflect the lowest dose of BMP-4

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signaling obtained with injections of 20 pg of RNA, as reported by Hammerschmidt et al. (1996b). Overexpression of antagonists of BMP-2/4 signaling in zebrafish embryos leads to a reduction of ventral fates and to an expansion of dorsal fates including neural tissue (Hammerschmidt et al., 1996c; Neave et al., 1997), as reported previously for frog embryos (Graff et al., 1994; Suzuki et al., 1994; Zimmerman et al., 1996). Injections of synthetic mRNA encoding a truncated, dominant negative form of the Xenopus BMP receptor (tBR) led to ectopic domains of expression of the prechordal plate marker, the gsc gene, whereas overexpression of Xenopus noggin mRNA expanded the dorsal expression domain of gsc (Neave et al., 1997). Overexpression of both types of BMP antagonists resulted in enlargement of the notochordal rudiment as judged by the midline expression domain of ntl or axial genes (Hammerschmidt et al., 1996c; Neave et al., 1997). The expansion of axial mesoderm was associated with a reduction of intermediate mesoderm, as monitored by expression of the pax2 gene in the kidney primordia (Neave et al., 1997), as well as with the reduction of ventral mesodermal markers such as gatal (Hammerschmidt et al., 1996~). Within the ectoderm, ectopic expression of both tBR and noggin mRNAs resulted in the expansion of neuroectoderm precursors expressingfkd3 at the expense of nonneural ectoderm, as marked by reduced expression of gta3, d1x3, and eve1 (Hammerschmidt et al., 1996c; Neave et al., 1997). Importantly, the ventral but not dorsoanterior expression domain of BMP-4 also was reduced or absent in these dorsalized embryos. This is consistent with positive feedback loop regulation of bmp-4 expression in the ventral region of the zebrafish embryo, which has been postulated previously in Xenopus (Jones et al., 1992). A consistent picture of dorsoventral patterning in the vertebrate gastrula emerges from the preceding observations (Neave et al., 1997; Wilson et al., 1997). A concentration gradient of a BMP-214 morphogen (as either homo- or heterodimer) provides an instructive signal determining the range of dorsoventral fates in the mesoderm and ectoderm. This ventrodorsal gradient of BMP-2/4 signaling arises as a combination of the expression of BMP-2/4 genes, which is maintained by a positive feedback loop, and the expression of BMP antagonists that bind to the BMP ligands and block signaling through their receptors. 2. Genetic Evidence for Dorsal-Ventral Patterning via a BMP-2/4 and Its Antagonists The preceding model is now supported by analysis of mutations from large-scale genetic screens for mutations affecting zebrafish embryonic development (Driever et al., 1996; Hafter et al., 1996). Mutations defining at least 15 genes affect patterning during gastrulation (Mullins et al., 1996; Solnica-Krezel et al., 1996; Fisher et al., 1997; Halpern et al., 1993; Hammerschmidt et al., 1996b; Talbot et al., 1995). An initial characterization of mutant phenotypes led to the classification of these mutations into two groups, based on their effects on the formation of

15 cell fates in the embryo (Table I). One class of mutants exhibits a loss of cell fates derived from the dorsal part of the gastrula, including neuroectoderm, whereas the second class of mutants has deficiencies in ventral and posterior structures, in some cases accompanied by an expansion of neuroectoderm (Hammerschmidt et al., 1996b; Mullins et al., 1996; Solnica-Krezel et al., 1996). Within the first class, mutations in six loci, including cyclops (cyc) (Hatta et al., 1991),Jloating head (Talbot et al., 1995), bozozok (boz), one-eyed pinhead (oep), and schmalspur (sur; also previously named uncle freddy or unf), result predominantly in deficiencies of mesodermal and neuroectodermal cell fates derived from the dorsal region of the gastrula (axial mesoderm and anterior-ventral neuroectoderm), without an obvious increase in ventrally derived fates (Fig. 2) (SolnicaKrezel et al., 1996; Brand et al., 1996; Schier et al., 1997; Strahle et al., 1997). 1. Pattern Formation in Zebrafish

Table I Mutations Affecting Dorsal and Ventral Organizing Centers Phenotypic class

Locus

Dorsal bozozok organizer (bflz) class Ia; dorsoanterior fates decreased one-eyed pinhead (oepl

cyclops

joating head

UW no tail

Molecule encoded

Alleles m168; i2

References Solnica-Krezel et al., 1996; Blagden et al., 1997

Phenotype Chordamesoderm and prechordal mesoderm missing or reduced; reduced anterior and ventral neural fates

m134. Hammerschmidt et Derivatives of prechoral., 1996a; dal plate missing; tz257, ICRFI, X50 Schier et al., cyclopia and deficiencies in ventral 1997; Strahle et al., 1997 aspects of CNS b16, m101, Brand et al., 1996; Reduced prechordal plate; cyclopia and m122, Hatta et al., 1991; Solnicadeficiencies in venm294, Krezel et al., tral aspects of CNS tj2 19: te262c 1996 m768; Brand et al., 1996; Reduced prechordal ty68b Solnica-Krezel plate; cyclopia and deficiencies in venet al., 1996 tral aspects of CNS n l ; b327; Masai et al., 1997; Lack of notochord and Transcription neurogenesis in epiStemple et al., rm229, factor physis 1996; Talbot et tk249, al., 1995 m614 Abnormal notochord b160: b195; Halpern et al., T-box differentiation in the 1993; Odenthal m149, transcription trunk and lack of nom550, et ul., 1996; factor tochord in the tail; Stemple et al., tb244e, tail reduction 1996 tc41, ts260 (continues)

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Table I (Continued) Phenotypic class Dorsal organizer class Ib; axial mesoderm present ventral posterior fates increased

Locus

chodino (din)

Molecule encoded Chordin

Alleles

tm84; Fisher et al., tt2.50; m52; 1997; m 70; Hammerschmidt m282; et ul., 1996b; m346; Solnica-Krezel m.586; c4, et a[., 1996 b30.5; b386;

m60

Ventral organizer class 11; decreased ventral and posterior fates in some increased neuroectod e m and somites

mercedes (mes) swirl (swr) zbmp-2

rz209; tm309 tc300, ta72

somitabun (sbn)

dct24

snailhouse (snh) piggytail (pgy)

mini jin In4

References

dty40, dti216. re22 7u, tni124a, 10206, tx223 tmllOb, mlOO tv9b,

tc263a, t1203a, tyl30u, tb24/c, lj21 lC1, tfz 1%.

tn2 17b

Phenotype Variable degrees of multiplication of caudal fin, increased ventral and posterior fates (blood, pronephros); decreased neuroectoderm and somitic mesoderm, shorter body axis

Solnica-Krezel et al., 1996 Hammerschmidt el al., 1996b Mullins et al., Variable degrees of re1996 duction of ventral and posterior fates, lack of caudal fin, progressive deletions of tail and trunk; reduction of ventral Mullins et al., and mesodermal 1996 fates, blood, and pronephros; expansion of somites and neuroectodem Mullins et al., Enlongated gastrula 1996 shape Mullins et al., 1996

Mullins el al.. 1996; SolnicaKrezel et al., 1996 Mullins et al., I996

1. Pattern Formation in Zebrafish 17 Mutations in three loci, chordino (din) (Hammerschmidt et al., 1996c; SchulteMerker et al., 1997), mercedes (mes) (Hammerschmidt et al., 1996b), and ogon (ogo) (Solnica-Krezel et al., 1996) lead to an increase in ventral fates and concomitant decrease in dorsolateral fates, mostly somites and neuroectoderm. The phenotypes of mutants in each of the preceding classes can be strikingly phenocopied either by ectopic expression of BMP-2/4 from RNA or DNA constructs or by injections of RNAs encoding BMP-2/4 antagonists, as described earlier (Fig. 2). Specifically, the phenotypes resulting from mutations at din, mes, and ogo loci are characterized by multiple ventral fin folds (Fig. 3), increased numbers of blood cells, and increased expression levels of ventral markers like eve1 (Fisher et al., 1997; HammerSchmidt et al., 1996b,c; Solnica-Krezel et al., 1996). Expansion of ventral fates is accompanied by a reduction of neuroectoderm, somitic, and chordamesoderm in din mutants. All of the preceding defects also were observed in embryos after injections with bmp-4 cDNA on a CSKA plasmid. In addition, the phenotype of the din mutation can be suppressed by ectopic expression of tBR and noggin, further supporting the notion that the defect in this mutant is in the inhibition of BMP-2/4 signaling (Hammerschmidt et al., 1996~). On the other hand, the phenotypes of the "dorsalized" class of mutants at the swirl (swr), somitabun (sbn), snailhouse (snh), lost-a-jin (laf ), piggytail (pgy), and mini j n (mfn) (Mullins et al., 1996; Solnica-Krezel et al., 1996) closely

Fig. 3 Effects of dorsalizing-ventralizing mutations and treatments on caudal fin development. (A, B) Lateral view of the caudal fin at day I of development in wild-type (A) and luf mutant (B) embryos. (C-G) Posterior view of the caudal fin in wild-type (C), mes (D), din (E), and ogo (C) mutant embryos and in embryos injected with 20 pg of bmp-4 RNA (F). [(A, B) Reprinted from Development, Solnica-Krezel et al. (1996). (C-F) Reprinted from Development, Hammerschmidt ef al. (1996b) with permission from the Company of Biologists Limited.]

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resemble defects obtained by ectopic expression of the BMP-2/4 antagonists (Neave et al., 1997) (Hammerschmidt et al., 1996~). Moreover, analysis of swrdin double mutants demonstrated that swr is epistatic to din (Hammerschmidt et al., 1996~).These observations gave support to the hypothesis that din encodes an inhibitor of swr function, with din and swr affecting the components of the morphogenetic system itself, chordin and BMP-2/4, respectively (Hammerschmidt et al., 1996b; Fisher et al., 1997; Holley and Fergusson, 1997). It is very satisfying that this hypothesis has been confirmed by evidence that din alleles affect the zebrafish chordin homologue (hence the proposed new name of the locus, chordino; Schulte-Merker et al., 1997). First, din mutant allele and the zebrafish chordin gene were shown to be closely linked (<0.25 cM). Second, the dinff250mutation was demonstrated to introduce a deletion of 104 base pairs in the chordino ORF that led to a frame shift. The resulting, truncated dintf250 mutant protein had only the first 42 amino acids of the wild-type form, thereby lacking the conserved cysteine-rich repeats. Third, injections of Xenopus chordin mFWA into dinff25O mutants led to a full rescue of the mutant phenotype (Schulte-Merker et al., 1997). These observations provide genetic evidence for the requirement of Chordin function as a BMP-2/4 antagonist in the Spemann organizer of a vertebrate embryo (Fisher et al., 1997; Hammerschmidt et al., 1996b; Holley and Fergusson, 1997). Notably, two groups have demonstrated that, as hypothesized, the swr locus encodes the morphogen itself, BMP-2 (Kishimoto et al., 1997; M. Mullins, personal communication). Single strand conformation polymorphism (SSCP) was used to show linkage between zbmp-2 and the swr locus. Furthermore, cloning and sequencing of the two swr alleles, S W and~ swrACIOO, ~ ~ revealed ~ that each exhibited a point mutation in the zbmp-2 ORE Upon RNA injection, the swrAC‘O0 mRNA was demonstrated to be inactive. Interestingly, mRNA encoded by the swrA72 mutant allele was weakly dorsalizing, suggesting that swrA72 might be an antimorph. In addition, injections of wild-type zbmp-2 RNA can rescue the swirl mutant phenotype. Embryos from a cross of a rescued swirl homozygous female with a wild-type male appear completely normal, indicating that zbmp-2 has no strict maternal function. This is consistent with the lack of maternal stores in the zbmp-2 mRNA in zebrafish eggs (Nikaido et al., 1997). It will be crucial to determine whether the remaining mutations from the “dorsalized” group affect known components of the BMP-2/4 signaling pathway, like type I and type I1 receptors or MAD proteins, or whether they will identify new components so far undetected by nongenetic analyses. Interestingly, the phenotype of boz mutants-lack of a notochord and prechordal plate as well as deficiencies in the ventral neural fates-is phenocopied by injections of low doses of BMP-214 mRNAs (Neave et al., 1997; Solnica-Krezel et al., 1996). Furthermore, these aspects of the boz mutant phenotype also are phenocopied by inhibition of microtubules at early cleavage stages as well as by shield extirpations, as discussed previously (Fig. 2) (Jesuthasan and Strahle,

1. Pattern Formation in Zebrafish

19 1997; D. Stemple, personal communication). Therefore, this class of phenotype might identify processes and genes involved in the specification, development, and maintenance of axial mesoderm in zebrafish. Indeed, there are differences in the timing at which development of axial mesoderm is affected in these different experimental situations. In boz mutants, expression of axial mesoderm markers (gsc, axial, liml) is affected even before the onset of gastrulation, as early as 40% epiboly (K. Fekany and L. Solnica-Krezel, in preparation). In contrast, in embryos injected with mRNA encoding BMP-4, expression of gsc is normal at the beginning of gastrulation, becomes reduced, and finally disappears by midgastrulation (Neave et al., 1997). Therefore, whereas inhibition of microtubules at early cleavage stages and the boz mutation both seem to affect specification of axial mesoderm or its maintenance before the onset of gastrulation, overexpression of bmp-4 RNA affects the maintenance of axial mesoderm derivatives (notochord and prechordal plate) during gastrulation. The initial expression of prechordal plate markers and their later disappearance or reduction in the course of gastrulation also is observed in cyc (Thisse et al., 1994), oep (Schier et al., 1997; Strahle et al., 1997), and sur mutants (Brand et al., 1996). Therefore, these genes might be required for the maintenance or further development of prechordal plate derivatives. It is intriguing that injections of plasmid encoding BMP-4, as well as the mutations in the din locus, lead only to gaps in the posterior notochord (Hammerschmidt et al., 1996c) or do not affect notochord formation (Fisher et al., 1997). Furthermore, boz mutants do not exhibit a very characteristic feature of din, mes, and ogo mutants: multiple caudal fin folds. Nevertheless, could both classes of phenotypes, boz and din, result from ectopic BMP-2/4 signaling caused by decreased inhibition of BMP-4 action? One possibility is that the two phenotypes reflect different amounts of BMP-4 signaling, with the din phenotype representing low levels and the boz-like phenotype higher levels of ectopic BMP-4 activity. Another possibility is that the phenotypic differences observed in these various experimental situations are not caused solely by different levels of BMP-4 signaling, but rather by the differences in the timing of the BMP-4 ectopic activity. Indeed, injections of RNA would lead to an earlier accumulation of BMP ligands, even in early blastula, than in the case of plasmid injections, which would bring about expression of the protein only after the midblastula transition. It is also tempting to speculate that not only would the onset of ectopic activity be different in these two cases, but so would its duration. If chordino functions during late gastrulation and somitogenesis, it is likely that the multiple ventral fins might reflect the ectopic activity of BMP-4 at late stages of gastrulation or even during segmentation stages. Indeed, BMP-4 is expressed at the edges of developing caudal fins during segmentation (Nikaido et al., 1997; Chin et al., 1997). Hence, it is likely that its ectopic activity at these stages might affect fin development. In contrast, shield extirpations and boz mutations might not affect BMP-2/4 activity at these later stages of development. It will be very informative

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Lilianna Solnica-Krezel

to compare carefully the timing and levels of expression of zbmp-2/4 and chordino between these different phenotypes.

3. Anterior-Posterior and Dorsoventral Neural Patterning Our understanding of the mechanisms underlying the anterior-posterior (AP) patterning of the CNS is still very rudimentary (Doniach, 1995; Kelly, 1995). In all existing models of AP neural patterning, the involuting axial mesoderm is a key player (Doniach, 1995; Ruiz i Altaba, 1994). According to one model, distinct regions of axial mesoderm directly induce specific regions of the CNS along the AP axis of the overlying ectoderm (Mangold, 1933). Alternatively, AP patterning is a two-step process, with all of the initially induced neuroectoderm being specified as forebrain by a so-called activator. The more posterior neural structures, midbrain, hindbrain, and spinal cord, then result from the anterior neuroectoderm being influenced by a second signal, a transformer (Nieuwkoop et al., 1952; Saxen and Toivonen, 1961). Other experiments have demonstrated that fragments of the lateral margin of zebrafish gastrula posteriorized forebrain when transplanted into the animal pole region, resulting in ectopic expression of hindbrain markers (Woo and Fraser, 1997). Therefore, the transformer signals might be emanating from the lateral blastoderm margin of the zebrafish gastrula, which indeed underlies future hindbrain progenitors (Woo and Fraser, 1995). Noggin and Chordin are thought to induce neural tissue of anterior character, thus acting as activators in the two-signal model of Nieuwkoop (Lamb et al., 1993). Upon the influence of transformer signals, including Xwnt3A, retinoic acid, and fibroblast growth factor (FGF) (Doniach, 1995), more posterior neural fates would arise. How does this model hold up in view of the phenotypes obtained with overexpression of BMP-2/4 and its antagonists and mutations affecting the dorsoventral pattern in zebrafish embryos? Consistent with observations from frog embryos, overexpression of BMP-2/4 in zebrafish, or the increased BMP-214 activity in the din mutants, leads to dorsal expansion of nonneuronal ectoderm at the expense of neural tissue. An opposite effect is observed in experiments in which embryonic BMP-2/4 signaling was compromised by ectopic expression of BMP antagonists, as well as in dorsalized mutants like swr (Mullins et al., 1996). Indeed, in swr mutants, neural tissue seems to be present around the entire circumference of the embryo, as evidenced by a dramatic circular expression of the pax2 midbrain marker. A similar phenomenon is observed forJkd3 in swirl mutants, as well as in noggin- and tBR-injected wild-type embryos (Hammerschmidt et al., 1996c; Mullins et al., 1996; Neave et al., 1997). What is the dorsoventral (DV) and anterior-posterior patterning of the diminished and expanded neural tube in "ventralized" and "dorsalized" mutants, respectively? In embryos injected with BMP-4 encoding mRNA, the decreased neural tube shows a reduction of ventral fates, cells expressing shh as well as primary motoneurons. However, the loss of ventral neural fates in these embryos

1. Pattern Formation in Zebrafish

21

might be a secondary defect due to axial mesoderm reduction or loss that takes place during gastrulation (see previous discussion). Interestingly, embryos injected with bmp-4 mRNA exhibited an excess of dorsal neural fates, specifically isll-expressing cells in trigeminal ganglia and the presumptive Rohon Beard sensory neurons (Neave et al., 1997). This effect might be due to an ability of BMP-4 to promote differentiation of neurons in the lateral neural plate, demonstrated previously in chick embryos (Liem et al., 1995). The ectopic Rohon Beard and trigeminal ganglion neurons were positioned normally, limited to the dorsal aspect of the neural tube (Neave et al., 1997). This indicates that certain aspects of DV pattern were maintained in the neural tube of these embryos. Although the neural plate of dinc4 mutants is reduced, these mutants possess axial mesoderm and they also exhibit ventral neural fates like floor plate and primary motoneurons (Fisher et al., 1997). This would support the notion that the absence of these cell types observed after the injection of bmp-4 RNA is a secondary effect due to the loss of midline mesoderm rather than a direct influence of dorsal expansion of BMP-4 activity. Interestingly, the din mutants were not reported to exhibit increased numbers of dorsal-specific sensory neurons. Rather, the overall number of primary neurons was decreased (Fisher et al., 1997; Hammerschmidt et al., 1996b). The underlying cause of this difference between dinc4 mutants and embryos injected with bmp-4 RNA needs to be investigated. Still, little is known about the AP pattern in the reduced neural plates of “ventralized’ mutants and their induced phenocopies. dinc4 mutants show reduced expression of the anterior neural marker otx2 at late stages of gastrulation (80% epiboly) and reduction of otxl expression during the segmentation period (20 somites). Hindbrain expression of Krox20 is present during early segmentation (4-6 somites). However, the expression domains of Krox20 in rhombomeres 3 and 5 appear reduced in the mediolateral and AP directions. The proportional decrease of otxl, otx2, and Krox20 expression domains suggests a mediolateral reduction of neural plate along the entire AP axis, while the AP pattern remains relatively normal (Fisher et al., 1997). Similarly, in 22-somite-stage din mutants all expression domains of pax2 are present along the AP axis of the neural tube, such as prospective optic stalks, midbrain, and otic vesicle anlagen (Hammerschmidt et al., 1996b). In contrast, the boz mutants show not only a more narrow neural plate but also a clear abnormality in its AP neural pattern (Driever et al., 1997). At day 1 of development, severe reduction of the forebrain and midbrain regions and lack of eyes are observed. The nature of defects in the AP patterning is revealed by expression of a sekl (previously rtkl; Xu et al., 1994) gene. In wild-type embryos, sekl is expressed in the forebrain region and in rhombomeres 1, 3, and 5 of the hindbrain (Macdonald et al., 1994). Remarkably, in the boz mutant embryos the forebrain expression domains are missing. This is accompanied by a longitudinal expansion along the AP axis of the three rhombomeric domains of sekl expression. It will be important to further compare neural patterning defects between boz and din mutants and their phenocopies

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Lilianna Solnica-Krezel

obtained by BMP-2/4 overexpression. It is likely that the timing at which the reduction of dorsal-specific genes occurs in these distinct embryos might play an important role.

V. Coordination of Gastrulation Movements Another remarkable property of the dorsal gastrula organizer is its ability to induce and orchestrate gastrulation movements characteristic of the dorsal side of the gastrula. Similar to other vertebrate embryos, in teleosts, the main components of the organizer, prospective prechordal and chordamesoderm involute/ingress and move toward the future head region below the prospective neuroectoderm (Trinkaus, 1996; Warga and Kimmel, 1990). Furthermore, cells of mesendoderm and ectoderm precursors converge toward the organizer. While in the dorsal region of the gastrula, these cells as well as ectodermal cells participate in mediolateral intercalation, effectively leading to extension of the embryonic body along the anterior-posterior axis (Trinkaus et al., 1992). These convergent extension and involution/ingression movements are preceded and accompanied by epibolic movements during which all the cell types of the zebrafish embryo, YSL, all germ layers, and the enveloping layer expand vegetally to cover completely the yolk cell by 10 hpf (Kimmel et al., 1995; Solnica-Krezel et al., 1995). It is noteworthy that practically every single cell of the zebrafish gastrula is engaged simultaneously in these three types of movements, epiboly, involution/ingression, and convergent extension, as well as in inductive interactions that pattern germ layers. Identification of the cues that these cells use to navigate through the embryo and make decisions about their prospective fates presents a formidable challenge. Gastrulation mutants identified in the genetic screens, as well as experiments with expression of BMP-2/4 and its antagonists, reveal important links between the inductive events in the embryo and gastrulation movements. Furthermore, the mutant phenotypes uncover interdependencies or the lack thereof between distinct morphogenetic movements. A. Relationships between Dorsoventral Patterning and Gastrulation Movements

1. Epiboly The first important lesson we learn from the mutant phenotypes is that the process of epiboly appears to be independent of DV patterning. Indeed, epiboly occurs relatively normally in all the mutants discussed previously in which DV defects are observed (Hammerschmidt et al., 1996b; Mullins et al., SolnicaKrezel et al., 1996). Also, the reverse is true: mutations affecting epiboly do not lead to obvious defects in the DV pattern of the gastrula (Kane et al., 1996;

1 . Pattern Formation in Zebrafish

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Solnica-Krezel et al., 1996). Furthermore, epiboly is not affected significantly in embryos that were dorsalized or ventralized by various experimental treatments like injections of p-catenin, TARAM-A-D, BMP-2/4, and BMP-2/4 antagonists. The lack of a relationship between epibolic movements and DV patterning is not that surprising, considering that epiboly is initiated at the sphere stage just before the first asymmetric expression of dorsal-specific genes like gsc can be detected and that it occurs around the entire circumference of the embryo. However, there is a certain asymmetry to the rate of epibolic expansion of blastoderm along the DV axis, with the blastoderm spreading across the yolk slightly faster on the dorsal side of the embryo. As a consequence, the point at which the yolk plug is closed is not at the vegetal pole but slightly ventral to it (Kimmel et al., 1995). This asymmetry can be explained, though, by convergent extension movements that are concentrated on the dorsal side where they contribute to epibolic spreading of the blastoderm, creating the DV difference in the rate of epiboly (Kimmel et al., 1995). It will be important to investigate whether this asymmetry is decreased or eliminated in mutants in which convergent extension is impaired (see the following).

2. Involution/Ingression Movements Involution/ingression movements at or near the blastoderm margin lead to the formation of a mesendodermal hypoblast below the ectodermal epiblast in the teleost gastrula (Trinkaus, 1996; Warga and Kimmel, 1990). This movement occurs around the entire circumference of the embryo, although it might be initiated first and might be more intense on the dorsal side of the gastrula. None of the mutants identified so far exhibits a very obvious defect in hypoblast formation, but more subtle defects cannot be excluded at this point. However, strongly affected embryos injected with BMP-2/4 RNA exhibit a clear reduction of hypoblast at the tailbud stage that might be a consequence of impairment of involution/ingression movements (Neave et al., 1997; Nikaido et aZ., 1997).

3. Convergent Extension Convergent extension is an important morphogenetic movement of gastrulation in all vertebrate embryos that leads to the narrowing of an embryo in the mediolateral axis and its AP elongation (Keller et al., 1985). In teleosts, convergence of cells from ventral and lateral positions toward the dorsal side of the gastrula leads to thickening of the embryonic shield and formation of the embryonic body rudiment. Cells converging dorsally leave the ventral, and later the lateral, area, creating an evacuation zone that increases with the progress of convergent extension (Kimmel et al., 1995). Mediolateral cell intercalations that are thought to occur mostly in the dorsal region of the gastrula drive both further narrowing and extension of the embryonic axis and organ rudiments in the AP

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Lilianna Solnica-Krezel

direction. Not only the rudiment of the embryonic body but also the shape of the entire gastrula undergoes characteristic changes, most likely effected by these convergent extension movements. The embryo changes its shape from a sphere at the onset of morphogenetic movements to an oval (elongated in the animalvegetal axis) at the end of epiboly (Kimmel et al., 1995). Mutations defining at least 16 genes result in an abnormal shape of the gastrula that could be explained by abnormal convergent extension movements (Table 11). At the bud stage, kny, tri, slb, ogo, din, and oep mutant embryos exhibit a distinctly shorter embryonic axis, indicative of decreased AP extension. All of these mutants also are characterized by a spherical yolk cell at the tailbud stage, in contrast to the yolk cell of wild-type embryos, which is elongated in the animal-vegetal axis at this stage of development (Fig. 4). This is most likely a consequence of a decreased convergent extension movements. Indeed, the evacuation zone is smaller in kny and tri mutants than in the wild type at this stage of development (L. Solnica-Krezel, unpublished), supporting the notion that convergence extension movements are decreased in these embryos. Expression domains of several genes in the ectoderm and mesoderm are perturbed in the mutant embryos in a manner consistent with decreased convergence and extension; the expression domains are expanded mediolaterally and compressed along the AP axis of the embryo. Embryos with an abnormally elongated “squash” shape are observed in the swirl, somitabun (sbn), snailhouse (snh), piggytail (pgy), lost-a-$n (laf ), and minifin (mfn)mutants and for some alleles of chordino (Mullins et al., 1996; Solnica-Krezel et al., 1996), as well as embryos injected with RNAs encoding p-catenin, Noggin, and tBR. This phenotype appears to be opposite that observed Table I1 Mutations Affecting Convergent Extension of Embryonic Axis in Zebrafish

Phenotypic class Decreased convergence and extension; spherical gastrula shape

Locus name

Alleles recovered

knypek (kny)

m119

trilobite (tri)

m144; m209; m747; m778

spadetail (spt)

References Solnica-Krezel et al., 1996

Hammerschmidt et al., 1996a; SolnicaKrezel et al., 1996 b104; b333; Hammerschmidt et m423, rm41; tq5 al., 1996a; Kimmel rt al., 1989; Solnica-Krezel et al., 1996

Phenotype Ectopic accumulation of cells near the anus region at 1 dpf; rare partial cyclopia Frequent partial cyclopia Somites, blood, and pectoral fins missing

(continues)

25

1. Pattern Formation in Zebrafish Table I1 (Continued)

Phenotypic class

Alleles recovered

Locus name

References

volcano (vol) avalanche ( m a ) half baked (hub) lawine (law) weg (weg)

m712 tm94 dtv43 lts18 tx230

Kane et al., 1996; Solnica-Krezel er al., 1996

one-eyed pinhead (oep)

m134

Hammerschmidt et al., 1996a; SolnicaKrezel er al., 1996

m60

Solnica-Krezel et al.. 1996

b160; b19.5; m149, m550; tb244e; tc-41; ts260; tm84; tt250; m52; m70; m169; m282; m346; m586

Melby et al., 1997

Fisher et ul., 1997; Hammerschmidt et al., 1996b; SolnicaKrezel et al., 1996

tc300, ta72

Mullins et al., 1996

no rail

Decreased or increased extension, spherical or elongated gastrula

chordino (din)

Increased extensionelongated gastrula

swirl (swr)

somitabun (sbn) dtc24 snailhouse (snh) ty68a dty40, dti216, piggytail (PSY) tc22 7a. tm124a, ta206, tx223 tmll0b. mlOO lost-a-fin (laf)

minifin (mif)

tv96, tc263a, tt203a, tyl30a, tb241c, tf21 I a &'15a, tn217b

Mullins et al., 1996 Mullins er al., 1996 Mullins et al., 1996

Mullins et al., 1996; Solnica-Krezel et al., 1996 Mullins et al.. 1996

Phenotype Epiboly arrested, also convergent extension decreased; spherical shape of gastrula; the epiholy mutations most likely affect single locus (D. A. Kane, personal communication) Derivatives of prechordal plate missing; cyclopia and deficiencies in ventral aspects of CNS Decreased extension, spherical gastrula shape Decreased convergent extension

Multiplication of ventral fin fold correlated with decreased extension; tail truncations correlated with elongation of a gastrula Deficiencies in ventroposterior fates, premature tail eversion, tail and trunk deficiencies

Fig. 4 Morphology of selected gastrulation mutants exhibiting a convergent extension defect at the tailbud stage of development (9.5 hpf). (A-F) Lateral views and (G, H) dorsal views of live embryos. (A) Wild-type gastrula exhibits an oval shape: the head is positioned over the animal pole with polster reaching to the ventral side, and the tailbud is at the vegetal pole. In volcano mutant (B), epiboly and extension of the embryonic axis are defective; the gastrula also exhibits a spherical shape. The “ventralized’ din ( C ) and ogo (D) mutants as well as the kny (E) and tri (F) mutants exhibit a shortened embryonic axis and a spherical shape of the gastrula. In contrast, the swr mutant gastrula appears excessively elongated (H). Arrowheads in G and H indicate boundaries of the forming notochord, which appears broader in the swr mutant. [ ( C ) Reprinted from Development, Hammerschrnidt e f al. (1996b); (B, D, E, F) Reprinted from Development, Solnica-Krezel et al. (1996); (G, H) Reprinted from Development, Mullins et al. (1996). all with permission from the Company of Biologists Limited.]

27 for mutants with decreased convergent extension (Fig. 4). Surprisingly, the “squash” shape of swr embryos does not reflect a simple increase in convergent extension movements. Rather, the dorsal convergence of cells is decreased as judged by the thickened ventral anterior region of swr mutant embryos at 80% epiboly. Furthermore, the notochord of these embryos is broader, possibly a consequence of reduced convergence (Mullins et al., 1996). How can a reduction of convergence lead, on the one hand, to shorter and rounder embryos such as kny, tri, and slb, but on the other hand to the excessively elongated gastrula in the swirl-like mutants? Is it possible that the two classes of phenotypes arise due to different defects in the second type of cellular behavior thought to drive convergent extension, i.e., mediolateral intercalation. The rounder shape of the gastrula, the shorter but broader embryonic axis of the kny and tri class of mutants, might reflect a reduction in mediolateral intercalations concomitant to reduced dorsal convergence. In swirl-like mutants, cells would exhibit excessive mediolateral intercalation behavior, not only in the dorsal region of the gastrula but also around the circumference of the embryo. This would be exaggerated by the presence of cells on the ventral side of the embryo due to reduced convergence. Furthermore, the phenotype of din and ogo mutants might result from reduced rates of mediolateral intercalation without convergence being affected significantly (but see the following). Testing this hypothesis requires precise in vivo analysis of cell movements in these mutants by time-lapse videomicroscopy. Notably, very interesting parallels exist between the two types of convergent extension defects and patterning of the gastrula. din and ogo mutants from the “ventralized” class exhibit a rounder shape of the gastrula and shorter body axis, whereas the “dorsalized” class of mutants, like swr, is characterized by the elongated shape of the gastrula. Furthermore, embryos injected with RNA encoding Noggin or tBR also are characterized by the elongated shape of the gastrula. The dorsalization of the gastrula caused by these manipulations might be the underlying cause of convergent extension defects in such embryos. First, dorsal convergence is likely to be directed by a signal from the shield (Trinkaus et al., 1992). Furthermore, in Fundulus embryos the net rate at which cells move toward the dorsal side of the gastrula is inversely proportional to their distance from the shield. Hence, the signal for convergence might be graded, with a peak at the dorsal side (Trinkaus et al., 1992). It is also conceivable that cells might be actively moving away from the ventral side. Because in “dorsalized” embryos expression of dorsal-specific genes is expanded toward the ventral side, so might be the expression of the signal for dorsal convergence. In extreme cases, such a signal might be distributed uniformly around the circumference of the embryo, resulting in inhibition of convergence movements. Similarly, the ventral expansion of a signal for mediolateral intercalation behavior might result in ectopic mediolateral intercalations in the ventral part of the embryo, as discussed earlier. Indeed, in Xenopus embryos ectopic expression of Noggin can induce, either 1. Pattern Formation i n Zebrafish

28

Lilianna Solnica-Krezel

directly or indirectly, elongation of tissue explants (Smith et al., 1993). It has been demonstrated that narrowing and extension of the dorsal marginal zone explants is affected by mediolateral intercalations of cells in the explant (Shih and Keller, 1992; Wilson and Keller, 1991). In "ventralized" mutants the signals for mediolateral intercalation would be decreased, and this could explain both the shape of the gastrula and the decreased extension of the axis. As mentioned earlier, the convergence defect is not obvious in these mutants. The width of rudiments of neural plate and somite is decreased; however, this might reflect changes in the fates of cells and reduction of neuroectoderm and somitic mesoderm rather than changes in cell movements. The alteration of cell fates in the mutant gastrulae makes interpretation of morphogenetic defects difficult.

VI. Conclusions Cellular and molecular mechanisms involved in the establishment of the zebrafish embryonic body plan appear strikingly similar to those operating in amphibians or other vertebrates. The initial characterization of a collection of zebrafish mutations affecting early pattern formation provided key genetic evidence for the model of dorsoventral patterning of the gastrula via the antagonistic interactions between the ventral morphogen, BMP-2/4, and its dorsal inhibitors. It is likely that zebrafish mutants also will identify novel components of the early patterning system. Furthermore, they will be an important tool in the elucidation of the genetic hierarchies regulating this process as well as in the functional analysis of the genes involved.

Acknowledgments I thank Mary Mullins, Matthias Hammerschmidt, Nigel Holder, Roger Patient, and Derek Stemple for contributing images of mutant and experimental embryos. Furthermore, Mary Mullins, Matthias Hammerschmidt, Marnie Halpern, and Derek Stemple generously shared their results before publication. I also thank Chris Wright, Mary Mullins, Karuna Sampath, and members of my group for critical reading of the manuscript and discussions. Work in my laboratory has been supported by an NIH grant (ROI GM 55101) and the Basil O'Connor Starter Scholar Research Award (#5-FY97-0046) from the March of Dimes Birth Defects Foundation. NOTEADDED I N PROOF: one-eyed pinhead (oep) gene has been isolated by a positional cloning approach and shown to encode a novel EGF-related protein (Zhang, J., Talbot, W. S., and Schier, A. F. (1998). Cell 92, 241 -25 I). r n e r c e d e ~ ' ~and ~ ~ 'ogonnr6"mutations do not complement one another, indicating that they likely affect the same locus (Mamie Halpern and Matthias Hammerschmidt, personal communications).

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2 Molecular and Cellular Basis of Pattern Formation during Vertebrate Limb Development jennifer K. Ng,* Koji Tamura,* Dirk Biischer,* and Juan Carlos Izpisua-Belmonte Gene Expression Laboratory The Salk Institute La Jolla, California 92037

I. Introduction 11. The Proximal-Distal Axis

A. Outgrowth of a Limb B. Apical Ectodermal Ridge and FGFs C. Gene Expression in the AER and in the Progress Zone 111. The Anterior-Posterior Axis A. Positional Information and Nature of Polarizing Activity B. Hox Genes and Anterior-Posterior Patterning C. Specification of Anterior Region IV. The Dorsal-Ventral Axis A. Ectoderm and Mesoderm Transplantation Studies B. Dorsoventral Boundary and AER Formation C. Dorsal Positional Cues D. Ventral Positional Cues V. Conclusions References

The body plan is generated by cells and tissues that become arranged precisely in the embryo. This process, termed pattern formation, involves cell interactions in which a particular group of cells produce signals that specify new cell types or patterns of differentiation in responding cells. These patterning signals emanate from very discrete centers called “organizer centers,” such as the Hensen’s node or Spemann organizer, the midbrain-hindbrain junction, the notochord, or in the case of the limb, the zone of polarizing activity (ZPA) or the apical ectodermal ridge (AER). The developing vertebrate limb is an ideal model system for the study of pattern formation because, i n addition to surgical manipulations, molecular manipulations are now fCdSibk. In this review we summarize early experiments that established, by means of surgical manipulations, the different organizer centers of the vertebrate limb: the ectoderm covering the limb bud. the apical ectodermal ridge. the zone of polarizing activity, and the distal mesoderm (progress zone) underlying the AER. We then describe the domains of expression of various *Joint first authors Currenr Topics in Developmenml Sio/ogy, Vol. 41 Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved. 0070.2153/99 $25.00

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Ng et al. genes present during the development of the limb and discuss some of the functional approaches (overexpression and lack of function studies) undertaken to ascertain their role in limb outgrowth. The knowledge acquired in the last few years has had an enormous impact not only on our view of how limbs develop (perhaps now one of the most approachable vertebrate model systems) but also in a more general sense of how the embryo is organized in space and time. Copyright 0 1999 by Academic Press.

1. Introduction The vertebrate limb has served for a long time as a paradigm used to understand pattern formation. The relatively good accessibility of limb bud tissue in avian species has enabled researchers to address the question of how the threedimensional form of the limb is achieved. The vertebrate limb bud. composed of ectoderm- and mesoderm-derived cells, forms three axes: the anterior-posterior (AP), the dorsoventral (DV), and the proximal-distal (PD) axes (Fig. 1). The establishment of these axes has been shown to rely on many genes that are also involved in structuring other regions within the developing embryo, such as the nervous system and various organs. Early grafting experiments [reviewed by Tickle (199 l)] have indicated that ecto- and mesodermal tissues in limbs are not established independently but rather coordinately. This also holds true for the establishment of the three axes. These observations have been confirmed at the level of gene expression. Thus, the division of this chapter into three parts, corresponding to the proximal-distal, anterior-posterior, and dorsoventral axes, is rather arbitrary and is not meant to reflect the actual mechanism underlying limb bud development. Furthermore, we would like to emphasize how the patterning of the limb is dependent on interactions between various signaling pathways emanating from the different spatial regions of the limb bud. In this review, we will describe significant research in

Fig. 1 Schematic diagram of chick limb bud and its axes. Indicated in black is the apical ectodermal ridge (AER), fine spots are the progress zone, and course spots are the zone of polarizing activity (ZPA).

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limb development and models of axial patterning as well as provide insight into the future direction of vertebrate limb development.

II. The Proximal-Distal Axis A. Outgrowth of a Limb

1. Signals That Initiate Limb Outgrowth The actual limb-bud forming process starts long before a cell mass becomes visible and recognizable as a bud that protrudes from the body wall. Transplantation studies by Stephens et al. (1 989) showed that the entire region from future neck to future leg in stage 11-14 (Hamburger and Hamilton, 1951) embryos has the potential to form limbs. Microsurgical removal of the mesonephros adjacent to the presumptive limb region and foil barrier experiments resulted in reduced limb outgrowth. This suggested that signals originating from the lateral plate mesoderm play an inductive role in initiating limb bud development (Stephens and McNulty, 1981; Strecker and Stephens, 1983; Geduspan and Solursh, 1992) between stages 12 and 18 (Saunders and Reuss, 1974). Some limbless chick mutants have been described to lack limb structures and mesonephros (Waters and Bywaters, 1943; Zwilling, 1956), supporting a putative connection between kidney and limb formation. Another limbless mutation in chick described by Prahlad et al. (1979) showed no impairment of kidney development. Yet, because limb development in this mutant was reported to be normal up to stage 18 and differences in size were obvious from stage 22 onward (Carrington and Fallon, 1988), initial determination of intermediate mesoderm seemed to have occurred normally. Thus, analyses of chick mutants seem to support a putative correlation between development of mesonephros and limb initiation. On the contrary, Fernandez-Teran et al. (1997) showed that surgical termination of the development of the mesonephros at stages 9- 11 still causes limb bud outgrowth to occur normally, clearly indicating that the two developmental processes take place independently of each other. Two genes have been implicated to play a role in the limb-initiating process: fibroblast growth factors 8 and 10 (Fgf-8 and Fgf-lo). Fgf-8 was first proposed to function in the initiation of limb outgrowth because its spatio temporal expression is in the mesonephros at wing level (Crossley et al., 1996; Vogel et al., 1996). However, the experiment by Fernandez-Teran et al. (1997) mentioned earlier revealed that the mesonephric expression of Fgf-8 had been abolished, and therefore FGF-8 does not seem to initiate the outgrowth. Fgf-10, which is expressed solely in the mesoderm, has been shown to precede Fgf-8 expression in the intermediate mesoderm, but is dependent on Fgf-8 expression in the apical ectodermal ridge (AER) at later stages. This indicates the importance of Fgf-10 expression as a key element in limb initiation and outgrowth (Ohuchi et al.,

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1997). In the chick limbless mutant, formation of an AER is perturbed and ectodermal expression of Fgf-8, Bmp-2, and engrailed in the early AER was not detected (Noramly et al., 1996; Grieshammer et al., 1996; Ros et al., 1996). On the contrary, expression of Fgf-8 in the intermediate mesoderm of limbless mutants has been reported to occur normally (Ros et d., 1996). Because the mesoderm in limbless mutants is capable of inducing an AER when grafted to wildtype ectoderm (Carrington and Fallon, 1988),one would also expect Fgf-10 to be expressed normally. Additional studies of this mutant may further clarify the role of Fsfs in limb initiation.

2. Positioning of the Limb The position of limb outgrowth along the anterior-posterior main body axis was mapped by Chaube (1959) and shown to correspond to the position of Hensen’s node, an organizing center in chick that is comparable to the Spemann organizer in amphibians. Studies indicated a correlation between axial positioning and Hox gene expression. In particular, the expression domain of Hox-9 genes in the lateral plate mesoderm seems to have a striking relationship with the positioning of limb formation (Cohn et al., 1997). Another plausible candidate for regulating the positioning of limb outgrowth is retinoic acid (RA), which was shown to induce Hox gene expression (Lu et al., 1997). Nevertheless, very little is known about the genes and molecules defining the exact position of limb outgrowth. Studies have shown that Radical fringe is implicated in determining the position of the AER (Laufer et al., 1997; RodriguezEsteban et al., 1997a) and, hence, also could be involved in positioning limb outgrowth. The restricted expression patterns of the T-box genes 4 and 5 in the hindlimb and forelimb, respectively, suggest that these transcription factors are involved in limb identity (Ohuchi et al., 1998; Isaac et al., 1998; Gibson-Brown et al., 1998).

B. Apical Ectodermal Ridge and FGFs The first sign of the future chick limb bud can be detected at stage 16, when a condensation of the somatopleural mesoderm occurs opposite somites (15-20 (Hamburger and Hamilton, 1951). At stage 17, a cuboidal epithelium covers the presumptive wing bud, which at stage 18 differentiates into a pseudo-stratified columnar epithelium, the AER (Saunders, 1948; Todt and Fallon, 1984). The formation of the AER depends on signals from the underlying mesoderm, which was demonstrated by grafting stage 12-17 mesoderm into host flank. This mesoderm is capable of inducing an AER and limb bud outgrowth (Kieny, 1968; Saunders and Reuss, 1974). The outgrowth of limb buds is not initiated by increased proliferation but rather by reduced proliferation of the tissue flanking the future limb buds (Searls and Janners, 1971).It has been shown that the whole

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ectoderm overlying the somatopleural mesoderm will differentiate into the AER. Ectoderm overlying the paraxial intermediate and lateral somatopleural mesoderm gives rise to future dorsal and ventral limb bud ectoderm, respectively (Michaud et al., 1997). Removal of the AER leads to a truncation of the limb (Saunders, 1972), and because limb development proceeds from the proximal to the distal end, removal of the AER at later stages causes an absence of more distal structures. The discovery of the presence of FGFs in the AER was a significant step toward understanding limb outgrowth. So far, 15 FGFs have been described (Tickle and Eichele, 1994; Yamaguchi and Rossant, 1995; Martin, 1998), three of which, Fgf-2, Fgf-4, and Fgf-8 (Fig. 2), are expressed in the AER (Dono and Zeller, 1994; Savage et al., 1993; Niswander and Martin, 1992; Suzuki et al., 1992; Laufer et al., 1994; Niswander et al., 1994; Heikinheimo et al., 1994; Crossley and Martin, 1995; Mahmood et al., 1995; Vogel et al., 1996). FGFs are

Fig. 2 F@3 expression in the AER. At stage 20, Fgf’8 is expressed in the chick limb bud throughout the whole AER.

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Fig. 3 Skeletal staining of a 7-day-old embryo in which a FGF-2-soaked bead was implanted at stage 17. The bead was implanted on the right side only, close to the presumptive wing field. The skeletal structure of the ectopic limb is that of a wing. Implantation of a bead in the vicinity of the presumptive leg would have resulted in an ectopic leg.

thought to play a critical function in limb outgrowth. Removal of the AER and its replacement with a bead soaked in either FGF-2, -4, or -8 can substitute for an AER, thus resulting in normal limb outgrowth (Niswander et al., 1993; Fallon et al., 1994; Crossley et al., 1996; Vogel et al., 1996). Furthermore, FGFs are capable of inducing ectopic limbs (Fig. 3), as shown by beads soaked with either FGF-1, -2, -4, -8, or -10, which were then placed in the presumptive flanks of chick embryos (Cohn et al., 1995; Crossley et al., 1996; Vogel et al., 1996; Ohuchi et al., 1997). This suggests that FGFs may initiate limb outgrowth. The expression pattern of Fgf-4 and studies in the mouse mutant limb deformity ( I d ) as well as studies in chick have demonstrated that Fgf-4 is important for creating a positive feedback loop with Sonic hedgehog (Shh). Shh functions as a regulator of anterior-posterior axis formation (see Section 111; Niswander and Martin, 1992; Niswander et al., 1993; Laufer et al., 1994; Haramis et al., 1995), but is not required for the initial outgrowth of the limb bud (Chiang et al., 1996). The extensive studies on FGFs have demonstrated the importance that these signaling molecules have on AER induction and maintenance, but the role each particular FGF may play needs to be elucidated further.

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C. Gene Expression in the AER and in the Progress Zone

The AER is an important signaling center that keeps mesodermal cells in an undifferentiated and proliferative state, which subsequently leads to the outgrowth of the limb (Saunders, 1972; Globus and Vethamany-Globus, 1976). These mesodermal cells make up the so-called progress zone. Various genes have been reported to be expressed there and have been shown to interact with the overlying ectoderm or AER. Lhx2, a LIM-homeodomain-containing gene, has been cloned and implicated in the induction and maintenance of limb outgrowth. Its expression precedes AER formation and persists throughout limb outgrowth, but is then dependent on signals such as FGF-8 from the AER. Thereafter, its expression is confined to the most distal mesoderm. Suppression of Lhx2 leads to an arrest of outgrowth, suggesting a role in maintaining outgrowth (Rodriguez-Esteban et al., 1997b). Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor (TGF) superfamily, are thought to be involved in bone formation and repair [reviewed by Rosen and Thies (1992)l. Three members of the Bmp family (Bmp-2, -4, and -7) are expressed in the AER (Francis et al., 1994) and in the underlying mesoderm. Targeted disruption of Bmp-2 and -4 resulted in early embryonic lethality, and disruption of Bmp-7 caused several mutant phenotypes including polydactyly [reviewed by Hogan (1 996)]. Misexpression of Bmp-2 in chick limb buds led to ectopic activation of Fgf-4 in the AER (Duprez et al., 1996). In in vitro studies, FGF-4 was shown to promote limb growth, whereas BMP-2 but not BMP-4 suppresses limb growth (Niswander and Martin, 1994). FGF-4 also has been demonstrated to induce Msx- 1, a homeobox-containing gene that is expressed in the distal mesoderm (Fig. 4) and is dependent on AER interactions (Davidson et al., 1991; Kostakopoulou et al., 1996). Msx-1 has been shown to be induced ectopically by BMP-4 (Watanabe and Le Dourain, 1996), indicating another putative feedback loop between ectoderm- and mesodermderived factors.

Fig. 4 Expression domains of Msx- I and DIx-5 in stage 20-23 chick limb buds. Msx-1 staining is detectable in the AER and underlying mesoderm, whereas Dlx-5 is seen nearly exclusively in the AER.

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Another group of genes involved in proximal-distal limb patterning are members of the Distal-less (Dlx) family. Dlx-I, -2, and -5 are expressed in the AER (Fig. 5) and Dlx-5 and -6 in the perichondrial areas (Doll6 et al., 1992; Bulfone et al., 1993; Simeone et al., 1994; Ferrari et al., 1995). Zhang et al. (1997) have shown that MSX and DLX proteins can heterodimerize via their homeodomains, but can be blocked by DNA containing the corresponding homeodomain binding sites. Further, MSX-1 and -2 are reported to be transcriptional activators. Based on the overlapping expression domains of Msx and Dlx genes, one might expect MSX and DLX to function in a regulatory cascade necessary for limb pattern formation. Just like Fgfgenes, Wnt genes encode for secreted molecules. However, less is known about their function in limb patterning. Wnt-1, -5a, and -7a may be involved in limb development. Misexpression of Wnr-1 in transgenic mice caused augmented proliferation and reduced condensation of mesenchymal limb bud cells, resulting in distal truncation of skeletal elements, skeletal fusion, and interdigital webbing (Zakany and Duboule, 1993). Wnt-5a is expressed in the AER and mesenchyme in a gradient that is highest in the distal region and lowest in the proximal parts. This could indicate a function in proximal-distal structuring of limb buds (Dealy et al., 1993). Yet, in Id mutant mice, Wnt-5a expression is greatly reduced in limb buds even though proximodistal patterning is affected only slightly (Zeller et a!., 1989; Kuhlman and Niswander, 1997). Targeted disruption of Wnt-5a may provide more substantial insight into its role in limb patterning. Wnt-7a is expressed exclusively in the ectoderm and has been shown to participate in dorsoventral axis formation (see Section IV). However, as yet,

A

ZP A

G

B

RA

Fig. 5 Schematic drawing of induction of mirror image duplication. (A) Early studies showed that implantation of the ZPA or RA-soaked beads in the anterior region of a host limb bud results in a mirror image duplication of the digits. (B) Later studies indicated that the ZPA is mediated by Shh and that RA is solely activating Shh expression in the anterior region, thereby inducing mirror image duplication.

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no clear functional studies have implicated a role for it in the outgrowth of the vertebrate limb. Hox genes represent another group of genes that has been shown to play a key role in patterning. They have been implicated in patterning the AP main body axis as well as the limbs. They are unique in their genomic organization, Hox genes are located in clusters and their temporal expression follows the gene order within the cluster [Doll6 et al., 1989a; Izpisua-Belmonte et al., 1991a,b; reviewed by McGinnis and Krumlauf (1992)l. In the limbs, HoxA and HoxD genes are expressed in the progress zone. The expression pattern of HoxD genes suggests a role in early events of patterning; on the contrary, HoxA genes are expressed in a proximal-distal pattern (Doll6 et al., 1989a; Yokouchi et al., 1991; Izpis6a-Belmonte et al., 1992). The functional role of both complexes in limb development has been assessed by targeted disruption and misexpression studies (Morgan et al., 1992; Doll6 et al., 1993; Small and Potter, 1993; Duboule and Morata, 1994; Duboule, 1995; Davis and Capecchi, 1996; Yokouchi et al., 1995; Favier et al., 1995; Davis et al., 1995; Fromental-Ramain et al., 1996; Goff and Tabin, 1997). Overall, the main conclusion obtained from this extensive work is that Hox genes play an important role not only in the proliferation and patterning of the early limb bud but also in the regulation of later stages of limb development, mainly cartilage proliferation and differentiation. This sculpturing of the limb is one of the lesser known processes in the field. In addition to the Hox genes, other genes have been identified as participants in this process, like Indian hedgehog (Ihh) and the Brnps. Ihh is a member of the hedgehog family and has been shown to regulate cartilage differentiation. Misexpression of Ihh perturbed cartilage formation and caused the misexpression of many other genes involved in SHH signaling, including Hox genes (Vortkamp et al., 1996). Another aspect of sculpturing the limb is the controlled cell death (apoptosis) that occurs in the interdigital space of some vertebrate limbs. BMPs have been implicated in inducing this process, which was shown by implanting BMPsoaked beads as well as misexpressing dominant-negative BMP receptors in the distal limb bud region (Gafian et al., 1996; Yokouchi et al., 1996, Zou and Niswander, 1996; Macias et al., 1997). It is clear that our understanding of these processes is very limited, and future studies are needed to gain more insight into the regulatory events of limb sculpturing. Knowledge about the proximal-distal axis was derived from transplantation as well as from gene expression analysis. Most investigations have focused on gene expression in limb buds that already possess an AER and a progress zone. Some studies have looked more closely at the early initiating signals and, unlike previous studies, have shown that FGF-8 is not an initiating factor. Originally, it was believed to be essential for outgrowth, but studies by Fernandez-Teran et al. (1997) have shown that Fgf-8 expression in the mesonephros does not mediate limb outgrowth along the PD axis. It remains to be seen whether FGF-10 or a yet

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to be identified molecule can initiate outgrowth. One approach to determining this factor is surgical ablation in chickens. Another may lie in analysis of conditional knockout mice. But all of these approaches are based on the hypothesis that one factor alone can induce the initiation of outgrowth. Another possibility is that FGF-8 and FGF-10 function in concert. Both could be sufficient to initiate outgrowth, but have divergent functions at later stages of development. It also should be noted that little is known about the FGF receptors and their role in limb development [see De Moerlooze and Dickson (1997); Deng et al., 19971. The complexity of the Hox code makes it difficult to assess the role of each individual Hox gene. It is hoped that with the increasing numbers of knockout mice, we will be able to unravel the combinatorial effects of Hox gene expression. Most of the genes demonstrated to pattern the vertebrate limb possess a functional counterpart in Drosophila. Even though the initial outgrowth and complexity of Drosophila appendages vary significantly from vertebrate limbs, signaling pathways appear to be conserved throughout evolution. Thus, extrapolation of the results obtained in other model systems such as Drosophila will continue to provide us with more information that will advance our understanding of vertebrate limb outgrowth and patterning.

111. The Anterior-Posterior Axis The first visual anteroposterior difference in the limb skeletal patterns results in the branching or segmental bifurcation of two zeugopodal elements (the radiusulna or tibia-fibula) from the proximal single stylopodium (the humerus or femur). Additional branching events generate several autopod elements, including the carpal-tarsal and digits. The way in which these differences are established from an apparently homogeneous undifferentiated population of mesodermal cells is an issue that has fascinated numerous scientists over the past few decades.

A. Positional Information and Nature of Polarizing Activity

In 1968, Saunders and Gasseling (1968) identified a group of cells at the posterior margin of the chick limb bud that was able to induce supernumerary digits when implanted into the anterior margin of a host limb bud. These findings were soon extended to other vertebrates [Xenopus (Cameron and Fallon, 1977), snapping turtle (Fallon and Crosby, 1977) and mouse (Tickle et al., 1975)l. A seminal model was introduced by Wolpert (197 1) that provided a conceptual framework for these results. This model presumes that a morphogen produced in the zone of polarizing activity (ZPA) diffuses toward the anterior side of the limb in a graded fashion, so that cells at different positions along the anteroposterior axis are

47 exposed to different concentrations of morphogen. The different concentrations of the primary morphogenetic signal then are interpreted by particular cells as a specific “positional value,” which allows them to differentiate into precisely organized skeletal elements. In the beginning of the 198Os, retinoic acid was reported to be a putative morphogen based on its ability to mimic the effects of a ZPA graft (Tickle et al., 1982; Fig. 5A). Several reports supporting this idea were offered. They suggested that retinoic acid activity is concentration-dependent (Summerbell, 1983). Exogenously applied retinoic acid actually forms a concentration gradient along the anteroposterior axis (Tickle et al., 1985). The posterior region of the chick limb bud contains an elevated level of endogenous retinoic acid (Thaller and Eichele, 1987). However, other observations appear to contradict this hypothesis. The cells adjacent to the retinoic-acid soaked bead have polarizing activity when implanted into the anterior margin of another limb bud (Wanek et al., 1991; Noji et al., 1991; Tamura et al., 1993; Fig. 5B), suggesting that retinoic acid converts surrounding cells into ZPA cells. Moreover, Noji et al. (1991) demonstrated that retinoic acid applied anteriorly activates the expression of RAR-P, one of the retinoic acid receptors, in an intense and rapid manner. Because ZPA grafts are not able to confer polarizing activity to neighboring cells nor activate RAR-P, it was concluded that retinoic acid is unlikely to be the ZPA morphogen. The controversy still goes on, and Helms et al. (1996) have suggested that retinoic acid is required for the establishment of the ZPA. These apparently opposing views leave unresolved the issue of retinoic acid as a morphogen capable of organizing the anteroposterior axis of the vertebrate limb bud. In 1993, an important study describing novel aspects of this process was reported. This was the discovery of Sonic hedgehog in the developing chick limb bud (Riddle et al., 1993; Fig. 6A). Sonic hedgehog colocalizes with the ZPA of the developing chick limb bud. Grafting of Sonic-hedgehog-producing cells into the anterior margin of the chick limb bud produces mirror image duplications comparable to those produced by a ZPA graft (Riddle et al., 1993; LopezMartinez et al., 1995; Fig. 7). These findings implicate SHH as a key molecule in establishing the AP polarity of the limb bud and raise the issue of the relationship between these two signals and their modus operandi. On the other hand, retinoic acid applied anteriorly induces the ectopic expression of Shh (Riddle et al., 1993; see Fig. 5B) in the region where retinoic acid has been shown to induce an ectopic polarizing region (Tamura et al., 1993). On the other hand, Sonic hedgehog and retinoic acid cooperate in generating the ZPA. Ogura et al. (1996) have shown that a P19 embryonic carcinoma cell line transfected with a Shh expression vector is not sufficient to show high polarizing activity, but when cultured with retinoic acid or cotransfected with a constitutively active retinoic acid receptor, P19 Sonic hedgehog cells induce complete duplications. These results, taken together with the fact that in various limb mutants anteroposterior patterning of the limb bud can 2. Vertebrate Limb Development

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Fig. 6 Expression domains of Shh, pfc, and Bmp-2 in stage 20-24 chick limb buds. All three genes show a posterior restricted expression domain. The model proposes that SHH binds to its receptor PTC, thereby inducing prc as well as Bmp-2 expression.

occur in the absence of SHH activity (Noramly et al., 1996; Grieshammer et al., 1996; Ros et al., 1996; Rodriguez et al., 1996; Chiang et al., 1996), indicate that SHH, although essential, may not act alone in the ZPA signaling process. Various attempts have been made to try to elucidate the way SHH functions. Biochemical and genetic evidence suggests that the Patched gene is a mediator of SHH activity. Although Patched transcripts are upregulated in cells responding to SHH, there is not yet any direct evidence demonstrating that Patched regulation depends on SHH concentration. Furthermore, inmunochemistry studies suggest that SHH does not appear to diffuse from the cells in which it is synthesized (Marti et al., 1995; Lopez-Martinez et al., 1995), indicating that SHH may not act as a diffusible morphogen. Another possible mode of action could be by relay mechanism. Sonic hedgehog is able to induce Bmp-2 and Fgf-4 expression in the anterior mesoderm and ectoderm, respectively (Laufer et al., 1994; Niswander et al., 1994), and the expression of Bmp-2 and Fgf-4 overlaps that of Sonic hedge-

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Fig. 7 A duplicated skeletal pattern of a chick wing induced by the SHH-producing cells. The numbers indicate the digit number. Taken from Riddle et al. (1993). Copyright 1993 Cell Press.

hog in the mesoderm (Francis et al., 1994; Francis-West et al., 1995; Fig. 6C) and AER, respectively (Laufer et al., 1994; Niswander et al., 1994). However, because neither BMP-2 nor FGF-4 alone can respecify AP patterning (Francis et al., 1994), other factors are needed to assess whether SHH acts via long-range or local-acting mechanisms.

B. Hox Genes and Anterior-Posterior Patterning Once the initial cues are established, a different subset of genes is needed to interpret them and direct the growth and differentiation of the limb along the three axes. The vertebrate class 1 homeobox-containing (Hox) genes are good candidates for mediating these processes. In the developing chick embryo, there are at least 23 Hox genes expressed in the limb bud (Nelson et al., 1996). With regard to the anteroposterior axis, some members are believed to function in axis formation. Hoxb8 may play a role in localizing the ZPA and Sonic hedgehog expressions because ectopic expression of Hoxb8 induces an additional ZPA along the anterior margin of the limb bud (Charite et al., 1994). The expression domains of the 5' members, which belong to the HoxD cluster, are posteriorly and distally restricted in a nested fashion that is collinear with their position on the chromosome (with more 5' members being more highly restricted). For example, Hoxd9 and -10, located on the 3' side of the HoxD complex, are expressed anteriorly, whereas Hoxd13, located closer to the 5' end, is expressed in a more restricted manner along the posterodistal area (Doll6 et al., 1989a;

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Fig. 8 Expression domains of HoxD genes in a stage 21 chick limb bud. In situ hybridizations are of sectioned embryos. (A) Hoxdl3; (B) Hoxdll; (C) HoxdlO.

Izpisua-Belmonte et al., 1991b; Nohno et al., 1991; Fig. 8). Once this nested pattern of expression was discovered, several functional studies involving lossand gain-of-function approaches were undertaken to unravel the role of these genes during the outgrowth and differentiation of the vertebrate limb. ZPA grafts, retinoic acid, and Sonic hedgehog can induce the ectopic expression of HoxD genes anteriorly (Izpisua-Belmonte et al., 1991a, 1992; Nohno et al., 1991; Riddle et al., 1993; Nelson et al., 1996), prior to the appearance of any distal structure. These results strongly suggest that the anteroposterior asymmetry of the limb is established by the sequential activation of the HoxD complex in the proliferating mesoderm. Natural and engineered mutations of several of the HoxD genes have been quite useful in determining the role of these genes in the proliferation and differentiation of cartilage. Misexpression of Hoxdl 1 and -13 not only results in modifications in digit patterning but also alters the growth and condensation of the cartilage (Morgan e f al., 1992; Goff and Tabin, 1997). Several chick limb polydactylous mutations display ectopic Hox gene expression that correlates with the absence of AP patterning (Izpisua-Belmonte et al., 1992), as well as with the duplication of digit elements (Rodriguez et al., 1996). This also was observed in mouse polydactyly mutants like extra toes and Rim4, where HoxD genes have a broader domain of expression (Masuya ef al., 1995; Buscher et al., 1997). In addition, mutations in the alanine repeat region in the N-terminal part of the Hoxd13 human gene have been implicated in human synpolydactyly (Muragaki et al., 1996). Finally, analyses of the skeletal phenotypes in several of the gene knockout studies in mice clearly reveal that Hox genes are important components of the machinery regulating cartilage proliferation and differentiation during limb outgrowth (see Doll6 et al., 1993; Davis et al., 1995; Favier et al., 1995; Fromental-Ramain et al., 1996). One possibility concerning the way Hox genes may influence these processes

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is via controlling the adhesiveness of the cells in which they are expressed. When randomly selected cells from the limb bud are mixed together in vitro, cells derived from the same region aggregate together. However, cells from different stages and regions tend to segregate from each other (Ide et al., 1994). This has led to the suggestions that the surface properties of mesoderm cells in different regions of developing chick limb bud vary along the anteroposterior and proximodistal axes and that these differences are associated with cell adhesivity. Indeed, it was shown that Hoxal3 can induce changes in the adhesivity of the limb mesodermal cells (Yokouchi et al., 1995) and that some cell adhesion molecules could be putative targets for Hox genes (Jones et al., 1992).

C. Specification of Anterior Region

For a long time, it was believed that the information specifying the AP axis emanated exclusively from the posterior region of the growing limb bud. The cloning of genes expressed on the anterior side of the limb bud and the study of their expression in several mouse mutants (Masuya et al., 1997; Buscher et al., 1997) have led to the idea that an inhibitory signal produced in the anterior region of a limb bud might restrict or refine the expression of posterior genes (Anderson et al., 1994). When mesoderm cells from the anterior side of chick leg buds are dissociated, mixed, and placed in an ectodermal jacket, the reaggregate forms a nonpolarized digitlike structure. In fact, Hoxd9 and - 1 1, which are expressed anteriorly as well as posteriorly, are activated in the absence of Sonic hedgehog expression (Hardy et al., 1995). It was then proposed that there are two independent mechanisms that activate the HoxD complex: polarizing region-dependent and -independent pathways. The anterior region might have the capacity to regulate the AP patterning of the limb, and a “balance of power” might exist between the anterior and posterior sides of the limb bud. Although there is very little evidence supporting such a system, a suggestive analogy could be made with the processes regulating dorsoventral patterning during Xenopus embryogenesis. Mesodermal patterning along the dorsoventral axis of the frog embryo has been thought to be organized by the Spemann organizer (a group of cells located on the dorsal side). Yet this view has been challenged by the accumulation of evidence detailing the active role of BMPs in patterning the ventral mesoderm. It appears that correct DV patterning requires an interaction between signals emanating from both the ventral and dorsal sides of the embryo [reviewed by Thomsen (1997)l. The data indicated earlier regarding the expression patterns of several genes in the developing limb bud may support an analogous mechanism. This is especially true for such genes as the HoxC cluster, Gli, or Pax9, which all have an expression domain restricted to the anterior region of the limb bud (Nelson et al., 1996; Marigo et al., 1996; Neubuser et al., 1995).

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Although the discovery of Sonic hedgehog has provided us with considerable knowledge of how the limb is patterned, we are still a long way from a complete understanding of how cells along the anteroposterior axis interpret this information and differentiate accordingly. Is there really a morphogen that organizes the positional information along the anteroposterior axis of the vertebrate limb bud? This question, which was formulated several years ago, is still unknown. It is hoped that more studies about Sonic hedgehog signaling and other molecules involved in other cascades will lead us to a better understanding of how molecular and cellular cascades regulate positional information. Moreover, we hope that the accumulation of knowledge about the characteristics and functions of various molecules expressed anteriorly will facilitate our understanding of anteroposterior pattern formation.

IV. The Dorsal-Ventral Axis The vertebrate limb can be divided along the dorsoventral axis into two distinct regions of specialized morphology. For example, the chick leg is covered with small scales on the ventral face of the toes, yet large scales on the dorsal face of the tarsometatarsus and toes. The distal tips of the digits are ventrally convex due to the asymmetric distribution of tendons and muscles on the dorsal and ventral sides of the limbs. The feather pattern of the dorsal wing is denser than that of the ventral side. How do these structures develop from a morphologically homogeneous limb bud? At what point in development do these differences arise, and what are the molecular mechanisms underlying such a transformation? This section will describe research into dorsoventral patterning that has enabled us to propose hypotheses to answer such questions.

A. Ectoderm and Mesoderm Transplantation Studies Classic transplantationexperiments conducted by MacCabe and co-workers (1974) and Pautou and others (Pautou and Kieny, 1973; Pautou, 1977) suggested the importance of the ectoderm in dorsoventral patterning of the vertebrate limb. When ectodermal hulls of stage 19-22 chick leg buds were grafted in reverse dorsoventral orientation onto stage-matched hosts, the proximal leg structures corresponded to the original polarity of the mesoderm, but the distal skeletal structures and integumentary structures were reversed along the DV axis. The transplanted ectoderm controlled the development of ectodermal and mesodermal structures of the recombinant chick. When the experiment was conducted using later stage chicks, the effects on the underlying mesoderm ceased. These studies indicated that ectodermal control is stage-dependent and varies in degree along the proximodistal axis. Further work by Geduspan and MacCabe (1989) suggested that before the

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initial limb outgrowth ectodermal and mesenchymal cells communicate with each other to pattern the limb along its DV axis. At stage 14 or earlier, dorsoventral polarity is controlled primarily by the limb mesoderm. When the DV orientation of the stage 14 ectoderm is reversed onto stage 14 mesoderm and grafted to the flanks of stage 19-21 host embryos, the resulting limb structures arenot altered. However, when stage 16 ectoderm is reversed onto stage 16 mesoderm, the polarity corresponds to the ectoderm (Geduspan and MacCabe, 1989). From these studies, it appears that both the ectoderm and the mesoderm at particular stages of limb development can influence dorsoventral patterning. Evidence from Michaud and colleagues (1 997) suggests that signals from the somites and the lateral somatopleure induce polarity even before the appearance of a presumptive limb bud. Results from their quail chick chimeric transplantation studies showed that polarization of the wing results from a dorsalizing factor produced by the somites and a ventralizing factor produced by the lateral somatopleure. When a region of the somatopleure containing ectodermal and mesodermal tissue from a stage 13 quail is rotated 180" and grafted to the wing region of a stage 13 chick, the resulting wing bud appears morphologically normal. The expression pattern of dorsal and ventral markers in the limb bud is similar to the wild-type expression pattern. However, if the somatopleure and somites opposite the wing region are grafted in reverse dorsoventral orientation to the flank ectoderm of a chick, the resulting limb bud appears bidorsal, as determined by the ventral expression of dorsal markers. A possible explanation provided by Michaud and collaborators (1997) is that the somites might induce the mesoderm of the lateral somatopleure, which subsequently specifies dorsoventral identity to the overlying ectoderm. Another explanation they propose is that the somites and the lateral somatopleure confer DV polarity by acting directly on the future dorsal and ventral ectodermal cells. On the other hand, the ectoderm might already possess dorsoventral identity and, as it grows over the budding limb, it could specify polarity to the underlying mesoderm (Michaud et al., 1997). In conclusion, transplantation experiments have implicated the ectoderm and the mesoderm to be necessary for establishing DV patterning of the limb. However, it remains unclear how this is accomplished.

B. Dorsoventral Boundary and AER Formation

In the past decade, research on Drosophila has focused on the importance of dorsal and ventral compartments in the wing imaginal disc. The current view suggests that cells are grouped together into distinct compartments and that cellcell interactions at the boundaries of these compartments influence the patterning of the future wing. Outgrowth of the wing thus is regulated by the preestablished dorsoventral pattern of the imaginal disc. An analogous mechanism in vertebrate limb development has yet to be found. Evidence suggests that DV patterning and

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outgrowth along the proximodistal axis are not strictly linked, at least at the earliest stages of limb budding [reviewed by Zeller and Duboule (1997)l. Findings by Ros and collaborators (1996), Noramly and others (1996), and Greishammer et al., (1996) suggest that initiation of the limb bud can occur without the proper expression of dorsal and ventral markers. Limbless mutant chicks do not develop wings and legs, yet their limb buds are wild-type morphologically until stage 19, after which the limb buds regress, possibly because they do not form AERs. In situ hybridization studies have shown that the mutant limb buds express the dorsal markers Wnt-7a and Lmx-1 in the proper region as well as in the ventral ectoderm and mesoderm, respectively. Engrailed-1 (En-1), a ventral ectodermal marker, is not expressed in the limb bud, suggesting that the limb bud is bidorsal. These studies in the limbless chicks indicate that the proper dorsoventral patterning of the limb bud, as determined by the expression of dorsal and ventral markers, is not required for the initial outgrowth of the limb. Tanaka and co-workers (1997) have demonstrated that the implantation of the presumptive dorsal wing tissue of stage 10-11 or stage 16-17 chicks into the presumptive ventral wing field of the host induces an ectopic AER. Even when the reverse experiment was conducted (the presumptive ventral wing tissue was implanted into the presumptive dorsal wing field), an ectopic AER formed. Fgf-8, an AER marker, was detected in the AER of the ectopic limb. The ectopic limbs formed incomplete wing structures. To determine which cell layer contributes to the generation of the ectopic outgrowth, ectodermal only and mesenchyma1 only transplants were conducted. In a few cases, ectoderm-free wing tissue was able to induce ectopic AERs. However, ectodermal wing tissue was unable to do the same. These results imply that proper AER formation requires the formation of a precise DV border in the ectoderm and underlying mesenchyme (Tanaka et al., 1997). However, it has been demonstrated that the boundary of r-fig expression and nonexpression marks proper AER positioning, rather than a DV boundary (Rodriguez-Esteban et al., 1997a; Laufer et al., 1997). Ectopic expression of r-fig using a replication-competent retrovirus perturbs AER formation (Fig. 9), as noted by Fgf-8 expression in the ectopic limb buds. Misexpression of r-fig does not affect dorsoventral identity. These data and others [see Laufer et a1 (1997); Rodriguez-Esteban et al., (1997a)l demonstrate that proper DV patterning is not required for the initiation of limb outgrowth or the formation of the AER. However, without the proper dorsal and ventral positional cues, the vertebrate limb cannot develop normally.

C. Dorsal Positional Cues

Misexpression and loss-of-function studies have shown that WNT-7a acts as a dorsalizing signal along the DV axis (Parr and McMahon, 1995; Riddle et al.,

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Fig. 9 Ectopic expression of Fgf-8 in the ectopic AER induced by r-fig misexpression using a replication-competent retrovirus. The left limb bud was uninfected.

1995; Vogel et al., 1995). Wnt-7a is a member of the Wnt gene family, which has been shown to be important in the early development of vertebrates and flies. There are 12 or more closely related genes in vertebrates. It has been predicted that the Wnt gene products encode secreted proteins that act over short distances and appear to be bound by the extracellular matrix and cell surface molecules [reviewed by Nusse and Varmus (1992); Nusse, 19971. In E9.5 mouse, six Wnt genes (Wnt-3, Wnt-4, Wnt-Sa, Wnt-6, Wnt-7a, and Wnt-7b) are expressed in the developing limb ectoderm. However, only WNT-7a appears to be important for dorsoventral patterning (Parr and McMahon, 1995). Wnt-7a expression in the limb is similar in chick and mice. At stages 18-25, Wnt-7a transcripts are present throughout the dorsal ectoderm of the limb bud (Dealy et a/., 1993) (Fig. 10). In mice, Wnt-7a was shown to be essential for dorsal patterning of the limb mesoderm. Wnt-7a null mutant mice exhibit biventral limbs. A dorsal-to-ventral transformation of mesenchymal cell fates occurs in the footpads, skin, tendons, seamoid bones, and joints of the mutant limbs. However, the transformation is restricted to the dorsal mesenchyme. Expression of ventral markers such as En-1, Bmp-2, and Dlx-2 normally is restricted to the ventral ectoderm (Parr and McMahon, 1995). Experiments by Riddle and collaborators (1995) and Vogel el al. (1995) showed that dorsalization of the ventral mesenchyme results from misexpression of Wnt-7a. When replication-competent retroviruses encoding Wnt-7a are injected into the flank region of stage 10 chicks, the infected chicks at stage 37 exhibit gross malformations of the limbs. There are changes in the dorsoventral flexing of the limbs at the ankles and the hips, as well as a shortening of the distal

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Fig. 10 Expression domains of Wnt-7a, En-1, and r-fig in stage 20 chick embryos. In situ hybridizations are of sectioned embryos. Wnt-7a transcripts are restricted to the dorsal ectoderm. En-1 transcripts are restricted to the ventral ectodenn. r-fig transcripts are detected in the dorsal ectoderm and the AER. Dorsal = left; ventral = right.

skeletal structures. The ventral muscle pattern is perturbed, but the dorsal muscles appear normal. Ectopic expression of Wnt-7a in the ventral limb ectoderm induces Lmx-1 in the ventral distal mesoderm. Lmx-1 normally is restricted to the dorsal mesenchyme. Based on the surgical experiments mentioned earlier, these data suggest that Lm-1 could respond to Wnt-7a, thereby establishing dorsal patterning in the limb bud. Indeed, ventral misexpression of L m - 1 results in bidorsal limbs with severe morphological alterations (Riddle et al., 1995; Vogel et al., 1995). All of these results confirm, at the molecular level, previous indications obtained by surgical manipulations that the mesenchyme responds to DV signals derived from the ectoderm. How does Wnt-7a transduce its dorsalizing activity to ectodermal and mesodermal cells in the developing limb bud? Thus far, the signaling cascade has not been demonstrated in the vertebrate limb. In the vertebrate limb, the Wnt-3a signaling pathway is analogous to the Xenopus Wnt-1 pathway (Kengaku et al., 1998). As reviewed by Moon and colleagues (1997), Xenopus WNT-1 binds to receptors believed to homologues of members of the frizzled family. Dishevelled,

57 a cytoplasmic protein, when activated by a WNT signal, inhibits the activity of glycogen synthase kinase 3 (GSK-3). In the absence of WNT signaling, GSK-3 activity directly or indirectly leads to phosphorylation and subsequent degradation of p-catenin. In the presence of WNT signaling, GSK-3 is inhibited, thus increasing the cytosolic levels of p-catenin and promoting its interactions with LEF- 1, a high-mobility group box transcription factor. The complex then translocates to the nucleus, where it can activate target genes. If all of the Wnt gene products follow the same signal pathway, questions of redundancy and specificity of signal arise. At this point, the WNT-7a signal transduction pathway in vertebrate limbs remains unknown. 2. Vertebrate Limb Development

D. Ventral Positional Cues

If WNT-7a is a dorsalizing signal, is there a complementary ventral signal? The restricted expression pattern of Engruiled-I in the ventral ectoderm and the ventral half of the AER in developing limbs suggest a role in dorsoventral patterning. Wurst and collaborators ( 1994) used a gene-targeting strategy to replace the homeobox, thereby generating En- 1 knockout mice. These mice die within a day of birth; they display abnormally shaped forelimbs and sternum and are missing mid-hindbrain tissue (Wurst et ul., 1994). The malformations of the forepaw range from mild truncations of digits to fused digits to polydactyly. The mutant mice appear to have double dorsal paws. Dorsally restricted hair follicles in the wild-type are present on the ventral side of the digit surfaces of the mutants. Ventral tendons and bones in the paw were either absent or poorly developed. En- 1 appears to promote ventral epidermal differentiation and inhibit dorsal-type differentiation. A detailed analysis by Loomis and colleagues (1996) of the limb buds from E10.5 En-1 null mutant mice revealed that the AER of the mutant is thinner and extends more ventrally and proximally. The expression patterns of Fgf-8 and Bmp-2 also are expanded ventrally in the mutant limb buds. Wnt-7a expression was detected in the dorsal and ventral ectoderm, but not in the AER. One plausible explanation for the mutant phenotype is that competing molecular mechanisms in the dorsal or ventral regions of the limb bud lead to the induction of either dorsal or ventral cell types. Expression studies conducted by Logan and collaborators (1997) described a similar restricted expression pattern of En-1 in the ventral ectoderm and ventral half of the AER of the chick limb bud (Fig. 10). Misexpression studies also demonstrated that En- I represses dorsal-specific gene expression. The anatomical mutations to the virally infected chick limbs were similar to the morphological changes in En-1 null mutant mice. There were losses and fusions of skeletal elements. The feet of infected chicks often were hyperextended and unable to flex ventrally. Wnt-7a and Lmx-1 expression in the dorsal limb ectoderm was downregulated. The reduction or loss of L m - 1 expression might be caused

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indirectly by the loss of Wnt-7a expression or directly by a mechanism whereby En-1 might be interfering with the establishment or maintenance of L m - 1 (Logan et al., 1997). When En-1 was misexpressed on the dorsal side of the limb bud, one observed phenotype was dorsal outgrowths (Laufer et al., 1997; Rodriguez-Esteban et al., 1997a; see Fig. 11). En-1 appears to repress Wnt-7a, Lm-1, and r-fig. It has been suggested that En-1 expression, along with r-fig, is essential for the positioning and formation of the AER. Dorsoventral patterning of the vertebrate limb is controlled by the interactions between the ectoderm and the mesenchyme as the limb bud emerges from the trunk region of the embryo. Although early inductive signals have not been identified, the positioning of the AER appears to be mediated by the activity of the r-fig gene. Later signalling cues, such as Wnt-7a and En-1, that specify dorsal and ventral cell fates have been shown to be transduced from the ectoderm to the mesenchyme. In addition, the mesenchyme signals to the ectoderm to maintain dorsoventral polarity, as noted by the activity of Lm-1. Research into the dorsoventral patterning of the Drosophila wing imaginal disc has implicated Notch signaling as an important signal cascade. An analogous pathway in vertebrates might serve a similar role in establishing dorsoventral polarity in the limb. In order to gain a greater knowledge of limb development, we must understand the induction of DV patterning and determine whether it is mediated by local or long-range influences. Much has been learned about dorsoventral patterning in the vertebrate limb in the past decade, but much remains a mystery.

Fig. 11 Fgf-8 expression in the ectopic AER located in the additional dorsal outgrowth. The extra limb bud was generated by En- 1 misexpression.

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V. Conclusions This review has surveyed past and present research in limb development. We have described the role of the ectoderm and mesenchyme in the initial patterning of the three limb axes, as determined by transplantation experiments in the chick. We have discussed the possible mechanisms that establish and maintain the organizing centers: AER, ZPA, and the progress zone. In doing so, we have detailed the expression of known positional cues important for Patterning the limb bud. However, we have not described a complete model. It remains to be determined for each axis whether the creation of a specific cell type in threedimensional space is generated by competitive induction from opposing sides of the axis or whether there is a default cell type that can be repressed by an opposing signal. Thus far, there seem to be many signals yet to be discovered. For example, while SHH appears to be a posterior polarizing signal, one might expect a reciprocal organizing signal to emanate from the anterior of the limb bud. However, one has not been identified. Much remains unknown about the establishment and maintenance of patterning along each axis. However, there is some evidence suggesting that the axes are established cooperatively. In particular, it has been shown experimentally by manipulating Fgf-4 and Shh expression that proximodistal and anteroposterior patterning are mutually dependent (Yang and Niswander, 1995; Parr and McMahon, 1995). They interact to form a positive feedback loop in which FGF-4 in the AER regulates Shh expression in the posterior mesenchyme, and in response Shh expression is regulated by FGF-4 (Laufer et al., 1994; Niswander et al., 1994). It has also been shown by Yang and Niswander (1995) and Parr and McMahon (1 995) that WNT-7a can act as a dorsal ectodermal signal required for Shh expression and proper anterposterior patterning. As mentioned previously, WNT-7a serves as an essential dorsalizing signal important for dorsoventral patterning. It has been proposed that WNT-7a, SHH, and FGF-4 interact with each other to mediate positional information required for patterning the vertebrate limb. However, the direct interactions comprising this signaling network are not known. We would expect WNT-7a to interact with FGF-4 if these three signals functioned in a feedback loop. As yet, there is no evidence to prove so. An understanding of axial patterning along each individual axis will provide great insight; however, to fully understand vertebrate limb development, we must determine how proximodistal, anteroposterior, and dorsoventral signaling molecules act together to generate a limb. Within the past decade, our understanding of the vertebrate limb has increased significantly with the identification of genes required for its outgrowth and patterning. Much remains unknown and unimagined. However, empowered with surgical, molecular, and genetic techniques, we hope to unravel the complex mechanisms responsible for vertebrate limb development.

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Acknowledgments This work was funded by grants from the National Science Foundation and the G. Harold and Leila Y. Mathers Charitable Foundation (J.C.I.B.). D.B. is a recipient of a fellowship by the Deutsche Forschungsgemeinschaft. We thank Lorraine Hooks for her careful attention to details and expert editing of the manuscript.

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Nelson, C. E., Morgan, B. A,, Burke, A. C., Laufer, E., DiMambro, E., Murtaugh, L. C., Gonzales, E., Tessarollo, L., Parada, L. F., and Tabin, C. (1996). Analysis of hox gene expression in the chick limb bud. Developmenf 122, 1449-1466. Neubuser, A,, Koseki, H., and Balling, R. (1995). Characterization and developmental expression of Pax9, a paired-box-containing gene related to Pa.rI. Dev. Biol. 170, 701-716. Niswander, L., and Martin, G . R. (1992). FGF-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114, 755-768. Niswander, L., and Martin, G. R. (1994). FGF-4 and BMP-2 have opposite effects on limb growth. Nufure 361, 68-71. Niswander, L., Tickle, C., Vogel, A,, Booth, I., and Martin, G. R. (1993). FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75, 579-587. Niswander, L., Jeffrey, S., Martin, G. R., and Tickle, C. (1994). A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371, 609-612. Nohno, T., Noji, S., Koyama, E., Ohyama, K., and Myokai, F. (1991). Involvement of the Chox-4 chicken homeobox genes in determination of anteroposterior axial polarity during limb development. Cell 64, 1197-1205. Noji, S., Nohno, T., Koyama, E., Muto, K., Ohyama, K., Yoshinobu, A,, Tamura, K., Oshugi, K., Ide, H. J., Taniguchi, S., and Saito, T. (1991). Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud. Nature 350, 83-86. Noramly, S., Pisenti, J., Abbott, U., and Morgan, B. (1996). Gene expression in the limbless mutant: Polarized gene expression in the absence of Shh and an AER. Dev. Biol. 179, 339-346. Nusse, R. (1997). A versatile transcriptional effector of Wingless signaling, Cell 89, 321 -323. Nusse, R., and Varmus, H. E. (1992). Wnt genes. Cell 69, 1073-1087. Ogura, T., Alvarez, I. S., Vogel, A,, Rodriguez, C., Evans, R. M., and Izpisua-Belmonte, J. C. (1996). Evidence that Shh cooperates with a retinoic acid inducible cofactor to establish ZPAlike activity. Development 122, 537-542. Ohuchi, H., Nakagawa, T., Yamamoto, A,, Araga, A,, Ohata, T., Ishimaru, Y., Yoshioka, H., Kuwana, T., Nohono, T., Yamasaki, M., Itoh, N., and Noji, S. (1 997). The mesenchymal factor, FGF-10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF-I, an apical ectodermal factor. Development 124, 2235-2244. Ohuchi, H., Takeuchi, J., Yoshioka, H., Ishimaru, Y., Ogura, K., and Noji, S. (1998). Correlation of wing-leg identity in ectopic FGF-induced chimeric limbs with the differential expression of chick Tbx5 and Tbx4. Developmenr 125, 5 1-60, Pam, B. A., and McMahon. A. P. (1995). Dorsalizing signal Wnt-7a required for normal polarity of DV and AP axes of mouse limb. Nature 374, 350-353. Pautou. M. P. (1977). Establissement de I’axe dorsoventral dans the pied de I’embryon de poulet. J. Embryol. Exp. Morphol. 42, 177-194. Pautou. M. P., and Kieny, M. (1973). Interaction ectomesodermique dam I’estlissement de la polarite dorsoventrale du pied de I’embryon de poulet. J . Embryol. Exp. Morphol. l l , 765-789. Prahlad, R. B., S k d h G . , Jones, D. B., and Briles, W. S. (1979). Limbless: A new genetic variant mutant in the chick. J. Exp. Zool. 209, 427-434. Riddle, R. D., Randy, L. J., Laufer, E., and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401-1416. Riddle, R. D., Ensini, M., Nelson, C., Tsuchida, T., Jessell, T., and Tabin, C. (1995). Induction of the LIM homeobox gene Lmx-1 by Wnf-7a establishes dorsoventral pattern in the vertebrate limb. Cell 83, 631 -640. Rodriguez, C., Kos, R., Macias, D., Abbott, K., and Izpisua-Belmonte, J. C. (1996). Shh, HoxD, Bmp-2, and Fgf-4 gene expression during development of the polydactylous talpid2, diplopodial, and diplopodia4 mutant chick limb buds. Dev. Genet. 19, 26-32. Rodriguez-Esteban, C., Schwabe, J. W. R., De La Peiia, J., Foys, B.. Eshelman, B., and IzpisbaBehnonte, 5. C. (1997a). Radical fringe positions the AER at the dorsoventral boundary of the vertebrate limb and induces elaboration of the proximodistal axis. Nature 386, 360-366.

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3 Wise, Winsome, or Weird? Mechanisms of Sperm Storage in Female Animals Deborah M. Neubaum and Mariana F. Wolfner* Section of Genetics and Development Cornell University Ithaca, New York 14853

I. Introduction A. Sperm Storage in Overview B. Sperm Transfer C. Why Transfer So Many Sperm? 11. Mechanisms of Sperm Storage A. Male and Female Anatomy Serves to Channel Sperm into Some Areas but Restrict Them from Other Areas B. Muscular Contractions Can Pump Sperm into Storage C. Male Seminal Fluids Can Facilitate Movement of Sperm to Storage D. Female Genital Tract Secretions Help Release Sperm from Spermatophores and Can Push or Draw Sperm into Storage E. Sperm Can Travel into Storage via Their Own Motility F. “Helper” Sperm or “Helper” Filaments Might Carry Sperm into Storage G. Chemotaxis-Are Sperm Lured into Storage? 111. The Fate of Unstored Sperm and Secretions I v. Sperm inside the Storage Organs A. The Fate of Previously Stored Sperm B. Which Sperm Will Be Used First? C. The Release of Stored Sperm Molecules Important for Sperm Storage V. A. Biochemical and Genetic Approaches B. Sperm Storage in D.rnelanogaster Requires Acp36DE The Adaptive Significance of Sperm Storage VI. VII. Conclusions References

Female sperm storage is an integral part of the reproductive pattern of many species. In the female, sperm become sequestered in specialized storage organs or reservoirs, where they may remain for several days, weeks, months, or years before being used to fertilize eggs. Several different but interrelated mechanisms are used by animals to target the sperm to the portion of the female genital tract adapted for sperm storage. Both males and females influence this process. This review describes themes among the mechanisms and molecules neces-

*Corresponding author. Currenr Topics m Develr,prnmral Urology, Val. 41 Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved. 0070-2153/99 $25.00

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Neubaum and Wolfner sary for sperm to become efficiently stored in females and the roles that the female storage organs play in the nourishment, protection, and release of stored sperm. Copyright 0 1999 by Academic Press.

1. Introduction In several animal species, females store sperm received during mating in specialized organs or storage reservoirs. In some cases, stored sperm are used quickly; in others, sperm are preserved for weeks, months, or years before being used to fertilize eggs. Often, the female’s ability to store sperm is an integral part of the species’ reproductive strategy and can provide important advantages. The structures and mechanisms required for sperm storage are likely to be under intense evolutionary selection, due in part to male-female conflicts of interest (Partridge et al., 1987; Chapman et al., 1995; Rice, 1996; Eberhard, 1996) and in part to sperm competition, which arises when females mate multiple times (Parker, 1970; Birkhead and Maller, 1992). To fully appreciate the impact of sperm storage on animal biology, it is important to understand how animals deliver sperm to storage locations, how they maintain sperm viability, and how they regulate the release of stored sperm. The mechanisms of sperm storage are diverse. This review highlights some recurring themes and recounts some examples seen in insects, birds, mammals, and other animals. In large part, our current understanding of the mechanisms of sperm storage is based on detailed observation of anatomy and mating behavior. We know relatively little about the molecules provided by the male or the female that are essential for the process. This is beginning to change and recent advances will be described.

A. Sperm Storage in Overview

The variety of animals that store sperm is large and spans several taxa (Table I). Sperm may remain stored for weeks or months, a period equivalent to or longer than the breeding season of many animals, and may last over winter. In some cases, sperm can last several seasons. The Eastern box turtle Terrupene carolina, for example, stores sperm for up to 4 years (Ewing, 1943) and the Javan wart snake Acrochordus javanicus for up to 7 years (Magnusson, 1979). However, sperm storage is not always prolonged. For example, most mammals store sperm for less than 48 hr. Caution must be exercised when comparing reports of storage duration among different animals, because reported values in some studies represent the mean storage time, whereas other studies report the maximum duration observed. Moreover, the duration of storage sometimes is measured as the length of time motile sperm can be found in the female tract and other times as the length of time females can produce fertile offspring without remating. Some instances of the latter may not be due to long sperm storage but to partheno-

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3. Mechanisms of Female Sperm Storage Table I Sperm Storage in Female Animals<' Taxa

Duration stored

Insects Grasshopper Water strider Stick insect Drosophilu

26-1 13 days 30 days 77 days 14 days

Reptiles Turtles-tortoises Snakes Lizards Crocodiles

90-1460 days (4 years) 90-2555 days (7 years) 30-365 days (1 year) 7 days

Birds Chicken Turkey Finch Canary

21-30 days 56- 1 I7 days 8-16 days 68 days

Mammals Marsupials Bats Mouse Human

0.5-16 days 16 days-6 months 0.6 days 5 days

Fish Shark

Up to several years

"Adapted from Birkhead and Moller (1993).

genesis, which has been documented in some species of lizards and snakes (Schuett et al., 1997; Dubach and Sajewicz, 1997; Darevsky et al., 1985). Despite these cautionary remarks, however, it is clear that sperm can survive for long periods of time in the female genital tract in many species. Sperm storage and survival in females is explained in part by the presence of specialized storage organs or regions in the female tract. Sperm generally congregate at these sites within a few hours after a mating. In some cases, the storage organshegions are thought to provide nourishment or protection for the sperm, allowing them to remain viable for longer periods (Tingari and Lake, 1973; Smith and Yanagimachi, 1990; Krishna, 1997; Uhl, 1994a). Storage organs vary in shape and number depending on the animal. For example, insects often have 1 3 saclike structures attached to the female tract by a thin duct or long coiled tubules that attach directly to the tract (Davey, 1958). The 300-20,000 sperm storage tubules (SSTs) of birds are long, thin, blind-ended tubules in the epithelial lining of the female genital tract, usually near the junc-

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tion of the uterus and vagina (Fig. 1; Zavaleta and Ogasawara, 1987; Briskie and Montgomerie, 1993; Briskie et al., 1997). Similarly, snakes and lizards have furrows or tubular receptacles in the vagina or infundibulum (Fox, 1956, 1963; Conner and Crews, 1980; Halpert et al., 1982). Some species do not have discrete organs for sperm storage, but collect sperm at particular areas (“storage reservoirs;” Morton and Glover, 1974b; Austin, 1975; Gomendio and Roldan, 1993b). In some of these cases, storage can simply be the prolongation of normal events that occur in a mating, for example, in penaenoid shrimp, which simply retain the sperm they received in the lumen of their oviduct [reviewed in Bauer, (1991)l. In other cases, sperm may move to specialized regions (though not discrete storage organs) for storage. Mammalian sperm collect initially at the cervix or at the uterotubal junction (UTJ; Fig. l), but their primary site of storage (the “functional storage reservoir”) is the caudal isthmus, a specialized region of the fallopian tube (Suarez et al., 1991; Bedford and Breed, 1994; Smith and Yanagimachi, 1990; Hunter, 1995). These regions (cervix, UTJ, and isthmus) may contain crypts and secretions (Smith and Yanagimachi, 1990; Bedford et al., 1997), which may prolong the viability of sperm that accumulate therein (Smith and Yanagimachi, 1990).

ov

Fert.

Ut

BIRDS

EUTHERIAN MAMMALS

INSECT (0.melanogasfer)

Fig. 1 Diagram of the reproductive tract of female birds, eutherian mammals, and a representative insect, showing the sites of sperm storage. Abbreviations: SSTs, sperm storage tubules; SSR, sperm storage reservoir; SR, seminal receptacle; Sp, spermatheca; Ejac., site where sperm are ejaculated; Fert., site of fertilization; Cap., site where sperm undergo capacitation; ov, ovaries; ovid, oviduct; isthm, isthmus; UTJ, uterotubal junction; ut, uterus, cerv, cervix; vag, vagina. Birds and mammals redrawn with permission from Gomendio and Roldan (1993b). Reprinted from Trends Ecol. Evol. 8, M. Gomendio and E. R. S. Roldan, Mechanisms of sperm competition: Linking physiology and behavioral ecology, pp. 95- 100. Copyright 1993, with permission from Elsevier Science. Drosophilu melunogusrer redrawn with reference to Miller (1 950).

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The percentage of stored sperm ultimately used to fertilize eggs is high in some species (insects and nematodes), but low in others (birds and mammals). The hermaphrodite Caenorhabditis elegans uses sperm with nearly 100% efficiency; indeed, the limiting factor in hermaphrodite self-progeny production is sperm rather than eggs (Ward and Carrell, 1979). The fruit fly Drosophila melanogaster stores 25% of the sperm it receives and uses 50% or more of its stored sperm for fertilization (Lefevre and Jonsson, 1962; Gilbert et al., 1981). In contrast, birds and mammals use only a small fraction of the transferred sperm. The chicken Gallus domesticus transfers 100 million sperm per copulation, stores less than 1 million in the SSTs, and only a few thousand sperm reach the site of fertilization (Brillard and Antoine, 1990; Birkhead and Mprller, 1993). In mammals, both the cervix/UTJ and the caudal isthmus act as major selective barriers to sperm transport into the upper regions of the reproductive tract (Shalgi and Phillips, 1988; Hunter, 1995; Longo, 1997). Thus, although the male hamster ejaculates 108-109 sperm per mating, only 0.001% of them reach the site of fertilization, and few of those actually fertilize an ovum (Shalgi and Phillips, 1988; Drobnis and Overstreet, 1992; Longo, 1997). Although it may appear wasteful for species to make, transfer, and store sperm that will never be used, several authors have suggested reasons why this could be of adaptive significance (see the following discussion).

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B. Sperm Transfer

Because sperm are transferred in a variety of ways, a discussion of the mechanisms of female sperm storage begins with consideration of the mode of sperm transfer. If sperm are not deposited in the storage organs directly, then mechanisms must exist to move them to their final storage site (Mann, 1984). Species that transfer motile sperm need mechanisms to channel the sperm toward the site of storage. Species that transfer sperm encased in packets (see the following discussion) require ways to release the sperm inside the female and move them the remaining distance to storage. Vertebrates and some invertebrates transfer sperm as a suspension of individual cells bathed in seminal fluids (Cohen and Tyler, 1980; Morton and Glover, 1974a; Uhl, 1994a,b). Several insects, crustaceans, and mollusks transfer “spermatophores,” packets in which sperm are enfolded in male reproductive tract secretions that have gelled, hardened, or coagulated around the sperm [see Mann (1 984) for a review]. Spermatophores range from simple coatings to elaborate multilayered structures with appendages [reviewed in Mann (1984); see also Bauer (1991) for Crustacea]. Delivery of spermatophores or free sperm directly into the storage organs is relatively rare, but does occur among some insect species. The insect order Odonata (dragonflies and damselflies), for example, have male genitalia specialized to reach directly into the storage organs, or

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“spermathecae.” Alternatively, sperm may be deposited in a chamber called the bursa copulatrix (or “bursa”) immediately adjacent to the storage organs (SivaJothy, 1987a). In contrast, sperm of other insects, mammals, or birds are deposited in the vagina or uterus (or cloaca) and must be transported the length of the female tract. As will be discussed later, the anatomy, secretions, and copulatory behaviors of both male and female play critical roles in this second stage of sperm transfer, moving sperm from the site of delivery to the site(s) of storage. Occasionally, sperm transfer can follow unorthodox routes. Two female giant squid (Architeuthis)were found to have spermatophores stored under the skin of their ventral arms (Norman and Lu, 1997). Sperm may have been delivered to these females through small wounds in the skin, apparently made by males. It is unclear how (or whether) sperm travel from the embedded spermatophores to sites where they fertilize eggs (Norman and Lu, 1997). This method of delivery has certain similarities to the “hypodermic or hemocoelic insemination” methods of the insect superfamily Cimicoidea [Davis, 1965; for reviews, see Davey (1965) and Hinton (1964)l. The male bed bug Cimex Zectularius uses a sharp stylus to penetrate the body wall integument of the female abdomen and deposits sperm into a pouch called the ectospermalege (Hinton, 1964). Sperm traverse the wall of this organ and enter the female’s circulatory system. Eventually, the sperm find their way to two pockets off the lateral oviducts, which function as temporary storage (Davey, 1965). Some insects that use hemocoelic insemination have no ectospermalege, and the male point of entry is different at each insemination and leaves a scar. In other species, sperm deposited in each ectospermalege never enter the hemocoele at all, but travel via a tissue bridge to a specialized pouch near the ovaries (Davis, 1956). Thus, the sites of sperm delivery and sperm storage may occur in wholly different parts of the animal, and mechanisms have evolved to transport the sperm through the body to storage.

C. Why Transfer So Many Sperm?

Males of many types of animals transfer numbers of sperm far in excess of what can be stored or used for fertilization. Why should males make and transfer sperm that will not be used? First, the transfer of large numbers of sperm may increase the efficiency of storage. For example, in birds, large inseminations correlate with increased fecundity (Brillard and Antoine, 1990; Wishart, 1987). Second, a large portion of the transferred sperm die or get lost. In mammals, for example, the majority of transferred sperm leak out of the female tract or are lost from the infundibulum into the peritoneal cavity. Moreover, the uterus is thought to be a hostile environment for sperm, killing many of them (Austin, 1975; Hunter, 1995). Also, due to their adhesive properties, mammalian sperm become dispersed by nonspecific interactions with the wall of the female tract or

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agglutination with other sperm (Hunter, 1995; Drobnis and Overstreet, 1992). Others may degenerate or fail to complete capacitation before ovulation occurs in the female (Hunter, 1995; Diesel, 1989, 1991; Halpert et al., 1982). Third, large transfers of sperm may represent a form of “paternity insurance.” Eady (1995) showed that decreasing the number of sperm transferred in the beetle Cullosobruchus maculatus did not decrease the number of offspring produced by a female, but it did affect the proportion of offspring sired by that male if the female mated more than once. Thus, transfer of a large number may provide a hedge against sperm competition. In this light, it is interesting that several animals can adjust the number of sperm transferred or the frequency of copulation to the likelihood that their mate has copulated recently with another male (Birkhead and Fletcher, 1994; Ginsberg and Rubenstein, 1990). For example, the Mediterranean fruit fly Cerutitis cupitata inseminates more sperm when a rival is present during the matings (Gage, 1991). Thus, males that transfer large numbers of sperm may ensure that plenty of healthy sperm are available to be stored and improve the chances of their sperm in competition with those of other mates.

II. Mechanisms of Sperm Storage For effective sperm storage to occur, females need to (1) sequester sperm within the sperm storage organs or reservoirs (a limited area) and (2) accomplish storage within a few hours after the end of mating (a limited time). The mechanisms used to accomplish these two objectives are likely to be almost as diverse as the animal kingdom itself. The collective evidence suggests that both males and females contribute to the mechanisms of storage. Although the present understanding of the processes of sperm storage is limited, the following themes recur and are likely to facilitate storage in some animals. Although listed individually here, these mechanisms for storing sperm often are used in combination.

A. Male and Female Anatomy Serves to Channel Sperm into Some Areas but Restrict Them from Other Areas

The profound influence of morphology on sperm storage can be highlighted by two examples, the specialized genitalia of insect males and the shape of the vertebrate female genital tract. One way to facilitate sperm storage is simply to deposit sperm as near as possible to the storage sites. Many insect species have specialized genitalia for strategic placement of sperm in or near the female storage sites. For example, Bonhag and Wick (1953) describe an extremely long intromittent organ used by the male milkweed bug Oncopeltus fusciutus to deposit sperm directly in the single spermatheca. The organ is coiled up in the male until secretions fill it

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during mating, causing it to uncoil and elongate until its tip reaches, and can be inserted into, the spermatheca. Similarly, the rove beetle (Aleocharu curtula uses a long intromittent organ to deliver a spermatophore to the spermathecal duct (Gack and Peschke, 1994). As the spermatophore swells into the spermatheca, the female’s spermathecal muscle contracts, causing two large internal spermathecal teeth to “bite” the spermatophore open and release the sperm (Gack and Peschke, 1994). In vertebrate species, the shape of the female genital tract itself is sufficient to impose limitations on the location of spermatozoa deposited within it. Morphological constriction of the female genital tract at the cervix or UTJ likely accounts for the incipient collection of sperm in these areas and represents a major selective barrier that only a fraction of the sperm can cross (Suarez and Osman, 1987; Suarez et al., 1990). The caudal isthmus itself also is morphologically constricted; its narrow lumen generally is occluded by the contracted state of the surrounding smooth muscle layers (Hunter, 1995). In addition to anatomical restrictions, the mucosal secretions that accumulate at the isthmus (Jansen, 1978; Suarez et al., 1990), the attachment of sperm to the mucosal surface (Suarez and Osman, 1987; Smith et al., 1987; Smith and Yanagimachi, 1990; Suarez et al., 1991), and a slightly decreased temperature (Hunter and Nichol, 1986; Smith and Yanagimachi, 1990) all contribute further to the retention or survival of sperm at this location (Smith and Yanagimachi, 1990). Moreover, the caudal isthmus contains shallow crypts (- one-third the length of a spermatozoan) that enhance the survival of the sperm they house (Smith and Yanagimachi, 1990; Bedford et al., 1997). Thus, females use morphology as a means to control sperm storage, but do so in combination with other controls such as temperature gradients, mucosal secretions, and muscular contractions.

6. Muscular Contractions Can Pump Sperm into Storage

A second widely used means of manipulating the location of sperm in females is muscular contractions of the female genital tract. Both contractions of the storage organs themselves (insects) and contractions of the uterus and oviduct (insects and mammals) are thought to play a major role in moving sperm to storage. Insects provide several examples of this: Eady (1994) describes a muscle connecting the distal end and main body of a comma-shaped spermatheca in the beetle C. maculatus. During mating, sperm and fluid begin to enter the spermatheca, causing it to swell and the spermathecal muscle to stretch. When distended, the spermathecal muscle gives a large contraction, pushing out previously stored sperm, and then relaxes as the fresh sperm enter (Eady, 1994). Peristaltic contractions of the spermathecal duct, in combination with a one-way valve, are thought to facilitate sperm storage in the honeybee Apis mellifera (Sander, 1985). In the bug Rhodnius, the opaque secretions produced by one accessory gland lobe are

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proposed to act through the nervous system to cause contractions of the female genital tract (Davey, 1958). If contractions are prevented by removing the opaque secretion or by paralyzing the female muscle by oxygen deprivation, sperm do not travel to the spermatheca. Consistent with the importance of muscular contraction in bringing sperm into storage in Rhodnius, killing sperm does not affect their transfer from bursa to spermatheca (Davey, 1965). Muscular contractions in the female also are important in mammalian sperm storage, and coordinated smooth muscle contractions appear to be one of the primary means of sperm transport through the genital tract (Drobnis and Overstreet, 1992; Hunter, 1988). Contractions may be stimulated by coitus or by molecules present in the seminal fluid (Hunter, 1995).

C. Male Seminal Fluids Can Facilitate Movement of Sperm to Storage

Male seminal fluid components can facilitate the storage of sperm by several means. They may (1) stimulate sperm motility, (2) bind to sperm, (3) interact with the female genital tract, (4) coagulate within females to force sperm into (or restrict them from) certain regions, or (5) stimulate changes in osmotic pressure that push sperm out of the spermatophore. 1. Motility The seminal fluid of flies, mosquitoes, butterflies, moths, and beetles is thought to contain substances that activate sperm motility (Davey, 1965; Hinton, 1964; Davis, 1965). Davis (1965) found that seminal fluid activated sperm motility in the bed bug (C. lectularius and showed that activation of sperm was required for their migration from the site of insemination into the female abdomen through the hemolymph to the temporary storage site (Davis, 1965).

2. Sperm Association In mammals, birds, and insects, products of the male seminal fluid may associate with sperm (Moore, 1980, 1981; Millette, 1977; Esponda and Bedford, 1985; Morris et al., 1987; Neubaum, 1997; Neubaum, and Wolfner, in preparation). Although the specific functions of individual seminal fluid molecules bound to sperm are not known, various important phenomena have been attributed to them. For example, the passage of vertebrate sperm through the epididymis, where seminal fluid products bind to the sperm, has been shown to be critical for the subsequent ability of sperm to fertilize (Moore, 1981). Modification of the surface of spermatozoa that occur in the epididymis affects the fertility of sperm and may alter their propensity for agglutination with other sperm or modify their threshold of motility (Hammerstedt et al., 1982; Moore, 1981).

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3. Stimulation of Female Genital Tract Contractions

The seminal fluid of mammals contains hormones, metabolites, and enzymes, which may stimulate the contractions of the female tract needed for sperm transport (Drobnis and Overstreet, 1992). For example, prostaglandins in the seminal fluid of mammals and insects can stimulate the contraction of reproductive tract smooth muscle in vitro (Freund, 1973; Drobnis and Overstreet, 1992; Hunter, 1988). Similarly, the opaque secretions of the accessory gland appear to stimulate female genital tract contractions in Rhodnius (Davey, 1958, 1965). 4. Coagulation

The localization of seminal fluid constituents in mated females can play an important role in sperm storage in some organisms. In an intriguing hypothesis, Bairati (1968) suggests that the coagulating mating plug secretions of D. melanogaster form a scaffolding for sperm to move along on their way to the anterior uterus. The mating plug secretions of D. melanogaster also are proposed to help confine the sperm to the anterior uterus, near the sperm storage organs (Bertram et al., 1996). Moreover, sperm may be restricted from entry into the oviduct by the presence of a barrier in the oviduct, of which the male accessory gland proteins may be a part (Bertram er al., 1996; see the following discussion). In this light, it is interesting that a D. melanogaster seminal fluid protein, Acp76A, is a member of a class of protease inhibitors that includes proteins involved in the coagulation of blood proteins during clotting (Wolfner et al., 1997). Thus, male seminal fluid molecules may help confine sperm to a limited region in the anterior uterus, from which they will enter the storage organs or may participate in forming a matrix along which sperm may move to storage. 5. Osmosis

An example of the importance of osmosis in pushing sperm into storage is seen in the cricket Gryllus domesticus. Male crickets produce a highly specialized spermatophore that they place in the female with the spermatophore neck inserted into the female sperm storage organ (Khalifa, 1949). Once inside the female, two pressure bodies in the center of the spermatophore take up fluid from other parts of the spermatophore(Fig. 2). The resulting expansion to the pressure bodies squeezes sperm out of the spermatophore and into the spermatheca (Khalifa, 1949). D. Female Genital Tract Secretions Help Release Sperm from Spermatophores and Can Push or Draw Sperm into Storage

Female secretions can be used to facilitate movement of sperm in a desired direction. The forces at play here include digestion, turgidity, suction, and viscosity.

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Fig. 2 Diagram of the spermatophore of Gryllus domesricus. The spermatophore is composed of several layers: a thin outer membrane, a layer of “evacuating fluid,” and a firm internal layer. Sperm are present in a sperm sac in the internal part of the spermatophore. In the female, the evacuating fluid moves into the pressure bodies, causing them to swell and push out the sperm. The process takes about 20 min, during which time the handle keeps the spermatophore positioned in the female’s ovipositor. Redrawn with permission from Davey (1965).

1. Digestion-Turgidity In species that transfer spermatophore, female secretions have been proposed to contain proteolytic enzymes that help dissolve the spermatophore and release the sperm (Hinton, 1964). Alternatively, female secretions may coagulate or accumulate within the female genital tract and press upon the spermatophore, forcing sperm out. In such cases, female secretions can act as a type of pressure body similar to the situation described earlier for the spermatophore of male crickets (Hinton, 1964).

2. Suction The importance of suction (in combination with osmosis) for sperm storage can be seen in the midge, Culicodes melleus. As the spermatophore in the bursa swells and sperm are pushed out, the female simultaneously absorbs fluid from inside the spermatheca, causing a suction that draws sperm up the duct and into the storage organ (Linley and Simmons, 1981; Linley, 1981). Linley and Simmons (1981) suggest that fluid absorption is a common means of sperm transport in the midge and other insects whose spermathecal ducts are noncontractile and

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devoid of muscles. In these cases, sperm are drawn by suction through narrow ducts. Not only are the ducts noncontractile but their narrowness may be important for increasing the suction pressure (Linley and Simmons, 1981; Linley, 1981).

3. Viscosity The viscous nature of some fluids within the female genital tract can assist sperm storage in several species in two ways: it may serve (1) to align sperm, encouraging them to swim in a particular direction, and (2) at the storage reservoir itself, to impede sperm and prevent their inappropriate dispersal (Austin, 1975). In cows, sheep, and humans, the cervix secretes a mucus that flows toward the vagina. Strands of molecules within this mucus appear to orient sperm and may help direct them toward the uterine cavity (Settlage et al., 1973; Austin, 1975).

E. Sperm Can Travel into Storage via Their Own Motility

The motility of sperm can be an important means of transport through the female genital tract. It is generally accepted that mammalian sperm play an active role in their movement through the female [reviewed in Drobnis and Overstreet (1992); Hunter, 1988, 1995; Longo, 19971. Motility appears to be critical for sperm to cross the cervix or UTJ (Drobnis and Overstreet, 1992). Smith et al. (1988) and Olds-Clarke (1988) noted that vigorous motility was required for rat sperm to traverse the UTJ in vitro: dead or abnormal sperm failed to cross the UTJ barrier or crossed it poorly (Gaddum-Rosse, 1981; Blandau, 1969; Mortimer, 1983). In conjunction with uterine contractions, motility appears to play a role in the transport of sperm to the upper reproductive tract (Hunter, 1988). Some researchers suggest that the function of motility during sperm transport may be less to move sperm in a certain direction than to keep them in suspension and prevent their sticking to the walls of the uterus (Austin, 1975; Hunter, 1988). Once stored in the isthmus, sperm are relatively quiescent until motility again becomes important for their transport to the ampulla for fertilization (Hunter, 1988, 1995). Whereas motility of mammalian sperm clearly is important for their storage, the role of sperm motility in sperm storage by insects is less clear. Vigorous thrashing movements are exhibited by insect sperm in vitro, but it is not clear whether these motions reflect their motility in vivo. Moreover, Linley and Simmons (1981) suggest that the diameter of insect spermathecal ducts may be too narrow in some species to permit the amplitudes of sperm movement that would be optimal for propulsion (Linley and Simmons, 1981). Given this restriction and the probability that only a few sperm may enter the storage duct simultaneously, Linley and Simmons (1981) conclude that sperm motility is insufficient to account for the rate of sperm storage observed in the lower Diptera, e.g., midges,

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mosquitoes, and flies (Linley and Simmons, 1981; Linley, 1981). Although it is possible that the spherical twisting movements observed in extremely long Drosophila sperm (Pitnick et al., 1995) could circumvent this problem, it remains to be shown.

F. “Helper” Sperm or “Helper” Filaments Might Carry Sperm into Storage

Males of some insect, worm, and mollusk species make and transfer more than one type of sperm to females during mating (Snook, 1997; Bressac and Hauschteck-Jungen, 1996; Beatty and Sidhu, 1970; Braidotti and Ferraguti, 1982; Healy and Jamieson, 1981). Males of other insect species transfer large filamentous structures along with sperm (Bairati, 1968). Because filaments and anucleic morphs of sperm do not appear to participate in fertilization, but in some cases enter the storage organs, some authors have suggested that they may aid the transfer or storage of normal sperm (Friedlander and Gitay, 1972; Sander, 1985; Riemann, 1970). Friedlander and Gitay (1972) and Iriki (1942) suggest that the smaller and more motile anucleic sperm of the silkworm Bombyx mori may serve a mechanical function in helping the larger and less motile typical sperm move into the storage site. A similar mechanical function for aiding storage has been proposed for the filamentous microtubule-like structures observed in the D. melanogaster male accessory glands (Bairati, 1968). These filaments appear to be transferred to the female, but cannot enter the storage organs if sperm are not present (Hihara, 1981). Although mechanical aids to transfer and storage are an intriguing idea, whether they are actually used in the manner proposed is debated (Baker and Bellis, 1988, 1989; Harcourt, 1989, 1991).

C . Chemotaxis-Are Sperm lured into Storage?

The ability of sperm to sense, turn, and follow a chemical gradient has been demonstrated in the sperm of several marine creatures that fertilize externally, including cnidarians, tunicates, chitons, and echinoderms [reviewed in Lop0 (1983)l. Whether chemotaxis also operates in internally fertilizing animals as a mechanism to attract sperm to specific sites remains controversial. Human sperm can respond in vitro to a chemoattractant present in ova, vestments, or follicular fluid (Villanueva-Diaz et al., 1991, 1992, 1995; Ralt et al., 1991, 1994; Cook et al., 1994; Cohen-Dayag et al., 1994, 1995), although whether this also occurs in vivo is debated [e.g., see Makler et al. (1992)l. Progesterone, calcitonin, acetylcholine, and calcium levels have been proposed as possible chemoattractants for sperm (Villanueva-Diaz et al., 1995; Sliwa, 1994, 1995; Cook et al., 1994). Most of the proposed examples of chemotactic responses of sperm relate to an

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interaction between sperm and egg. Chemotaxis as a mechanism to attract sperm into areas of storage is less documented, but has been proposed as a possible mechanism to attract the sperm of nematode (C. elegans) males to the spermathecae (Ward and Carrell, 1979). In addition, Weidner (1934) observed that insect sperm (Rhodnius) readily entered a capillary tube connected to the storage organ, but did not enter a similar unconnected tube [cited in Davey (1965)l. This suggested that the storage organ contained substances that diffused through the capillary tube and attracted sperm into the tube. Although true chemotaxis is still a subject of debate, sperm of all major animal groups are thought to respond to chemokinetic factors, defined as substances that stimulate motility [reviewed in Longo (1997); Ward, 19931. Chemokinetic factors do not necessarily stipulate the direction of motility (Longo, 1997), but may keep sperm active and prevent their agglutination or adhesion to the female tract.

111. The Fate of Unstored Sperm and Secretions In most species, unstored sperm are unlikely to remain viable for long inside the female (Briskie et al., 1997; Austin, 1975). A majority of mammalian sperm deposited in the female leak out or are flushed out within a few hours of coitus by mucus or urine or are phagocytosed by leukocytes (Austin, 1975; Hunter, 1995). Most sperm that remain are likely to die; even at the storage reservoir the best survival rate is only 51-69% (for sperm embedded in crypts at the isthmus; Smith and Yanagimachi, 1990). In birds and insects, eggs push out any spermatozoa remaining in the lumen of the genital tract by a few hours after mating (Gomendio and Roldan, 1993b; Miller 1950). After their release of sperm to storage, the spermatophores of insects often are absorbed or eaten by the females (Boucher and Huignard, 1987; Mann, 1984). Spermatophores often are large (40% of body weight in some crickets; Mann, 1984) and can be a nutritive source to females in some cases (Boucher and Huignard, 1987).

IV. Sperm inside the Storage Organs The arrangement of sperm inside the storage organs generally is highly ordered, although its details differ among organisms. A tight, even arrangement might help conserve the energy of sperm by keeping them in a relatively immobile state and possibly facilitates a smooth release of sperm from storage [see Van Krey et al. (1981) for birds and Fox (1956) for snakes]. Ordered sperm storage occurs in lizards (Fox, 1963; Conner and Crews, 1980), snakes (Halpert et al., 1982; Fox, 1956), and chickens (Bobr et al., 1964; Tingari and Lake, 1973). In all of these cases, stored sperm are arranged in a parallel fashion, sometimes in bundles, with their heads pointed toward the distal end of the sperm storage tubules. Stored

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sperm of birds and some mammals agglutinate in a "head-to-head" fashion (Tingari and Lake, 1973; Smith and Yanagimachi, 1990). An important question is whether females actively participate in maintaining the viability of sperm inside the organs. In many cases, the sperm storage organdregions do not merely hold the sperm together, but can serve nutritive, protective, or anchoring functions as well. In chickens, bats, boars, hamsters, and other mammals, a close association between sperm and the walls of the storage organs (or crypts) is observed (Smith and Yanagimachi, 1990; Suarez et al., 1991; Gomendio and Roldan, 1993a; Tingari and Lake, 1973). Nonciliated cells deep in the sperm storage tubules are rich in lipids, P-glycogen, mitochondria, and secretary granules (Tingari and Lake, 1973). suggesting that these cells produce secretions that bathe the sperm. Cells in the female storage organ may provide nutrients or metabolites to sperm to maintain viability or encourage quiescence during long storage. In addition to forming tight associations with sperm, the female genital tract can support sperm in more indirect ways. For example, close association with the wall of the storage organs and the thick mucous secretions of some animals could serve to anchor the sperm or protect them from the sometimes hostile environment of the female genital tract (Smith and Yanagimachi, 1990, 1991; Suarez er al., 1990, 1991; Gomendio and Roldan, 1993b). The ability of sperm to become anchored inside the storage organs also is important for animals such as the nematode, in which eggs pass through the storage organs directly and are fertilized there. The amoeboid sperm of C. eleguns use their pseudopod tails to burrow into the spermathecal wall, which may guard against their being swept out as eggs pass through the chamber (Ward and Carrell, 1979). Intimate association of the sperm with the wall of the storage organ is not observed in lizards and birds (Fox, 1963; Conner and Crews, 1980; Tingari and Lake, 1973), although the female could still actively support them in other ways. In any case, the sequestration of sperm into enclosed areas probably serves a protective function for sperm that remain viable in the female genital tract for weeks or longer.

A. The Fate of Previously Stored Sperm

When females remate before their sperm stores are depleted, incoming sperm can disrupt the storage of previous sperm in several ways (Table 11). If the storage organ is elastic, the female may simply take up the additional sperm (Uhl, 1994a). When this happens, incoming sperm can mix with the sperm already in storage, so that both continue to be used in proportion to their representation. Alternatively, instead of mixing, stratified layers of sperm may form, so that the sperm of one male are used more frequently (Eady, 1995). In some cases, stratification of sperm can be extreme. For example, before transferring his packet of spermatophores, the male ghost spider crab Znachus phalangium trans-

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Table I1 The Fate of Previously Stored Sperm upon Remating Animal Mediterranean fruit fly Cerafitis capitata Fruit fly Drosophila melanogaster Beetle Callosobruchus maculatus Damselfly Lestes vigilax Fly Dryomyza anilis

Tree cricket Truljulia hibinonis Giant water bug Abedus herberri Ghost spider crab lnachus phalangium Dunnock (bird) Prunella modularis

Fate of previous sperm

References

Mixes with new

Saul and McCombs, 1993

Displaced and restoredmixed? Mixed in storage organs? Pushed out by spermathecal contraction Scraped out by horns on male penis Extruded by female when males “taps” external genitalia Flushed out by a large ejaculate volume Diluted by frequently repeated copulation Completely sealed off by a layer of seminal plasma Squirted out when male pecks cloaca

Lefevre and Jonsson, 1962 Newport and Gromko, 1984 Eady, 1994; Villavaso, 1975 Waage, 1979 Otronen, 1990; 1997

Ono et al., 1989 Smith, 1979 Diesel, 1989; 1990 Davies, 1983

fers seminal plasma that pushes the previous male’s sperm to a distal part of the storage organ. This seminal plasma forms a hardened layer around the previously stored sperm packets, so that they are completely sealed off and cannot be used (Fig. 3; Diesel, 1989, 1990, 1991). If the storage organs are nearly full or will not stretch to accommodate more sperm, some of the previous sperm must be displaced. The process of sperm displacement is best understood in the Odonata, an order of insects with specialized genitalia that reach into the storage organs to scrape or flush out previously stored sperm (Waage, 1979, 1986; Miller and Miller, 1981; Ono et al., 1989; Siva-Jothy, 1987b; Yokoi, 1990). These insects are extraordinarily effective at removing previous sperm; several species can remove 85-100% of the stored sperm (SivaJothy, 1987a,b; Waage, 1979). The exact events that occur when new sperm meet previous sperm are less clear in other animals, and whether sperm displacement occurs at all in mammals is debated (Hunter et al., 1993). B. Which Sperm Will Be Used First?

Females that mate more than once usually can store sperm from more than one male in the storage organs. Even if a female has mated only once, however, the

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Fig. 3 Seminal receptacle of the ghost spider crab Inachusphalungium. The sperm packet (containing many small spermatophores) and seminal fluid of the first male to mate (“I”) are pushed into the distal part of the storage organ and sealed off completely by the seminal plasma of the subsequent male to mate (‘TI”).A sphincter-like muscle, the vellum, monitors the release of sperm to the fertilization chamber. Redrawn from Diesel ( I 990), with permission from Cambridge University Press.

sperm stored in certain regions are likely to be used before others. The determination of which sperm are used first requires consideration of the distribution of sperm among the organs, the extent of female control over release, the mechanisms of sperm release (see the following discussion), and the morphology of the storage organs. Species with more than one sperm storage organ may preferentially use sperm from one of the organs first. In D. melanogaster, sperm from the seminal receptacle are used before those of the spermathecae (Fowler, 1973); in Drosophila subobscuru, the reverse is true (Bressac and Hauschteck-Jungen, 1996). Likewise, female Dryomyza unilis flies preferentially use sperm stored in the singlet spermathecae over those in the doublet spermathecae (two organs with a common duct) or those in the bursa (Otronen, 1997). Some authors suggest that if females could actively allocate sperm of different males to different storage organs and draw upon the sperm of some organs preferentially, this would give them a measure of control over the paternity of their offspring (Ward, 1993; Otronen, 1997). In other cases, the prioritized use of sperm from one type of organ probably reflects the mechanics of sperm release rather than active control by the female. Sperm may, for example, be less securely anchored in one type of organ and therefore be released more quickly. A mechanism for sperm precedence that does not require any active participation by the female was suggested by Walker (1980) and Austad (1984). These authors proposed that the morphology of the female sperm storage organ could

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predict sperm precedence in some animals. For example, anatomical considerations suggest that sperm entering a storage organ with a “cul-de-sac” shape (one aperture) might be used on a last in-first out basis. Conversely, sperm that enter “conduit” organs, which have separate entry and exit points, will be positioned for use on a first in-first out basis (Austad, 1984; Walker, 1980; Watson, 1991). Thus, the cul-de-sadconduit proposal relies more on the number of entry and exit points rather than whether the organ is saclike or tubular in nature. Although the shape of the storage organ may suggest a basis for predicting sperm precedence in many organisms, the arrangement of sperm inside the organ also is important in predicting which sperm will be used first. This is illustrated by the filling of the flask-shaped storage organs of the rabbit flea Spilopsyllus cuniculi, which have a single opening (Rothschild, 1991). Incoming sperm are aligned side by side along evenly spaced ridges beginning on the proximal side of the organ. This circular arrangement leaves a cavity in the middle, through which subsequent sperm pass and become stored more distally. Thus, the alignment of sperm as they enter the storage organ makes the first sperm that enter the most likely to be the first used, even though the spermatheca has a “cul-de-sac” shape (Rothschild, 1991).

C. The Release of Stored Sperm

For sperm storage to operate effectively, it is important not only to acquire and maintain sperm adequately in the storage organs but to release them when they are needed. The mechanisms by which sperm are released from storage will determine in part whether sperm are used efficiently or lavishly by the female. Two opposing types of mechanisms might govern sperm release. First, sperm might be released from storage in a gradual but sustained manner, independent of any active female control. Second, sperm might remain stored until an action from the female, such as muscular contraction of the spermatheca, squeezed a few sperm out. Support for both types of mechanisms has been proposed, though active release seems to be more generally observed. 1. Passive, Gradual Release Upon noting the orderly array of sperm stored in hen SSTs, Van Krey et al. (1981) proposed that the agglutination among the sperm heads might break down slowly over time, resulting in their slow release. This type of mechanism would result in a slow, steady release of sperm and may help remove sperm from storage that are aged and cannot agglutinate properly (Senger and Saacke, 1976). However, a slow continuous release of sperm usually is not observed in most animals.

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2. Active, Regulated Release

In several organisms, eggs passing through the oviduct may exert mechanical pressure on the sperm storage organs, causing a small number of sperm to be squeezed out [see Fowler (1973) for Drosophilu, Grigg (1957) for chickens, and Fox (1956) for snakes]. In addition, more direct mechanisms for femalecontrolled release also exist, largely involving muscular and endocrine controls. The spermathecal muscles of the boll weevil Anthonornus grandis (Villavaso, 1975) and the rabbit flea S. cuniculi (Rothschild, 1991) play important roles in sperm release. Alternatively, sperm release may be controlled by means of a sphincter. The storage organ of the female ghost spider crab I. phulungiurn is connected to the fertilization chamber by means of a ringlike diaphragm muscular membrane called a “velum,” which opens to release sperm and may regulate their release (Fig. 3; Diesel, 1989). Muscular contractions or relaxation could be coordinated with endocrine or other signals to release sperm when eggs are available to be fertilized. A coordination of sperm release and ovulation is observed in several mammals and may be primarily under endocrine control (Hunter, 1995), although others have suggested that the release of follicular fluid or “products of ovulation” into the ampulla may play a role in sperm release (Hunter, 1995). In mammals, the coordination of sperm release with ovulation may be important for two reasons: (1) Sperm still have the distance of the isthmus and ampulla (upper oviduct) to travel before they reach the egg (Hunter, 1995). Insect species, however, generally fertilize eggs in or immediately outside of the storage organs (Sander, 1985). (2) Mammalian sperm may need to complete the final stages of capacitation before fertilization is possible. These processes require minutes to hours to complete. However, once capacitated, sperm have a limited life span. The female endocrine system serves to coordinate sperm release and ovulation, ensuring that capacitated sperm are ready and available to fertilize newly ovulated eggs. The contributions of males may have some influence on the sperm release process. Null mutations in two Drosophilu male seminal fluid genes, Acp36DE and esteruse-6, affect the rate of sperm loss from the storage organs (Neubaum, 1997; Neubaum and Wolfner, in preparation; Gilbert et al., 1981). Because sperm release is an event that continues long after mating, the influence of males on sperm release is more likely to be indirect, via their influence on sperm storage or other target tissues in the female, for example.

V. Molecules Important for Sperm Storage Relatively little is known about the molecules that are required for sperm to be efficiently stored and used. Important molecules could include ones that (1) activate, direct, or assist sperm transport; (2) assist sperm to be stored in orderly

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arrangements; (3) nourish sperm while in storage or stabilize the pH of the storage environment; (4) help retain sperm at the storage locations or facilitate their sustained release; or ( 5 ) protect sperm and/or the female against fungal or bacterial infection.

A. Biochemical and Genetic Approaches

Many, if not all, of the mechanisms for storage discussed earlier rely in some measure on molecules produced by the male or female for their operation. Because almost all organisms whose sperm storage has been studied are not currently amenable to genetic analysis, primarily biochemical methods have been used to identify male seminal fluid or female secretions that might play a role in sperm storage. Drosophila, however, has provided a molecular genetic means to identify and test molecules proposed to function in sperm storage and will be discussed later. As discussed earlier, seminal fluid contains molecules that participate in sperm storage, as well as ones that mediate other reproductive functions [e.g., Wolfner (1997) for Drosophila]. Progress is being made toward biochemical identification of the seminal fluid components. In an electrophoretic characterization of the cellar spider seminal fluid, Uhl described 19 proteins of 10-130 kDa, including 6 glycoproteins and 5 lipoproteins (Uhl, 1996). Proteins, lipids, simple sugars, amino acids, fatty acids, and carbohydrates, including glycoproteins and polysaccharides, are common constituents of seminal fluid in insects (Leopold, 1976; Taber, 1977; Wolfner, 1997) and mammals (Vishwanath and Shannon, 1997; Shivaji et a f . , 1990; Moore, 1981). The function of individual seminal fluid molecules is known in some cases, particularly those for which a biological assay can be devised, e.g., egg-laying stimulation, depressed receptivity to mating, or antibacterial properties in Drosophila (Chen et af., 1988; Samakovlis et al., 1991; Ohashi et al., 1991; Herndon and Wolfner, 1995; Baumann, 1974), but few specific components have been identified that influence sperm storage. Some of the molecules produced by the female that are important for sperm storage have been identified. Prostaglandins and peptide hormones, present in the seminal fluid or ovarian follicular fluid (Hunter and Poyser, 1985), may interact with receptors (Ayad et al., 1990) in the ovarian epithelium to stimulate muscle contraction [see Hunter (1995) for a review]. The genetic approach has been a powerful tool for the study of Drosophila seminal fluid proteins [reviewed in Wolfner (1997); Chen, 19961. Seminal fluid proteins, which are transferred to females during mating, mediate the storage, displacement, and release of stored sperm (Neubaum and Wolfner, in preparation; Gilbert et al., 1981; Clark et al., 1995; Lefevre and Jonsson, 1962; U. Tram and M. F. W., unpublished results). Genetic analysis of a seminal fluid protein, Acp36DE, of D. mefanogaster demonstrated that this protein is required for

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sperm storage in the fruit fly (Neubaum, 1997; Neubaum and Wolfner, in preparation).

B. Sperm Storage in D. rnelanogaster Requires Acp36DE

Acp36DE is a large, novel glycoprotein made in the male accessory gland and transferred to the female during mating in the seminal fluid (Wolfner et al. 1997; Bertram et al., 1996). In the mated female, Acp36DE localizes specifically to the posterior oviduct, just above the openings of the sperm storage organs, and associates with the mass of sperm present in the uterus (Bertram et al., 1996). Acp36DE also enters the storage organs themselves. The localization of Acp36DE suggested that the protein might play a role in sperm storage (Bertram et al., 1996). In the course of mating, sperm and seminal fluids collect in the anterior uterus. Sperm entry into the storage organs begins immediately but requires 6 or more hours for completion (Gilbert et al., 1981; Gilbert, 1981). Normal females mated to males lacking Acp36DE received sperm in the anterior uterus as usual, but stored only 15% as many sperm in their storage organs as females mated to normal males (Neubaum, 1997; Neubaum and Wolfner, in preparation). Females mated to males lacking Acp36DE produced only 10% as many progeny as controls (Neubaum, 1997; Neubaum and Wolfner, in preparation). Thus, although sperm from Acp36DE-deficient males appear normal and are capable of fertilization, they are not stored efficiently. How does Acp36DE cause efficient sperm storage? A barrier forms in the female genital tract at the site of binding of Acp36DE (Bertram et al., 1996). Bertram et al. proposed that this barrier prevented sperm from progression into the anterior oviduct and hence channeled them into the region from which they could enter the storage organs. Acp36DE is not essential for the formation of the barrier, but the intensity of Acp36DE localization at the site of the barrier nevertheless correlates with the efficiency of sperm storage (Neubaum, 1997; Neubaum and Wolfner, in preparation). Sperm storage in D. melanogaster is proposed to occur in two steps (Neubaum, 1997; Neubaum and Wolfner, in preparation). The first step, mediated by the barrier, would restrict the sperm to a limited region near the storage organs. This would then permit the second step, in which sperm are actively moved into the storage organs. From its location in the oviduct, Acp36DE could participate in the second step by stimulating contractions of the spermathecal ducts and proximal seminal receptacle that could aid in the uptake of sperm. Alternatively, Acp36DE might stimulate the motility of sperm so that they might swim up the ducts, although this model has the caveat that the extremely narrow entry ducts of the D. melanogaster storage organs may impose limitations on sperm movement, as discussed earlier (Linley, 1981; Linley and Simmons, 1981). Acp36DE is tightly associated with sperm in the mated female and can bind

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sperm in vitro in the absence of other accessory gland proteins. The interaction of Acp36DE with sperm may be important for their orderly arrangement inside the storage organs, or it could affect their rate of release from storage.

VI. The Adaptive Significance of Sperm Storage There are several reasons why sperm storage in females may be advantageous. One important advantage is the potential for increased fecundity. With a supply of sperm at hand, animals can fertilize a large number of eggs in rapid succession (Cook, 1970; Lbpez-Lebn et al., 1994). The storage of sperm enables insects and other species to produce hundreds of offspring from a single mating (Gilbert, 1981; L6pez-Le6n et al., 1994). In birds, Brillard and Antoine (1990) demonstrated a positive correlation between the number of stored sperm, the number of sperm at the site of fertilization, and the probability of fertility. The mollusk Crepidula fornicara stores enough sperm in its seminal receptacle to fertilize the 50,000-100,000 eggs it lays over the period of 1 year or more (Fretter and Graham, 1994). These examples demonstrate the high level of fecundity that can be achieved through the use of female sperm storage. Secondly, female sperm storage forms the basis of the reproductive habits of several species, because it enables mating and ovulation to occur at widely separated intervals (Hatch, 1983; Briskie and Montgomerie, 1993). A female redsided garter snake Tharnnophis sirtalis parietalis mates and stores sperm, but delays ovulation for weeks after mating in the spring and for months after mating in the fall (Halpert et al., 1982). The female petrel Fulrnarus glacialis (a seabird) departs for weeks after mating. The male prepares the nest in her absence and may depart without re-encountering the female. The female lays an egg almost immediately upon her return, whether or not the male is present (Hatch, 1983). In this case, the storage of sperm in females makes possible a sequence of eventsmating, extended foraging, nesting-that may be difficult to achieve by other means. Thus, female sperm storage can allow species to occupy niches they otherwise could not. Similarly, sperm storage may significantly improve the survival of the desert lizard species Anolis carolinensis, which may encounter mates infrequently in submarginal habitats or insular colonies (Fox, 1963; Conner and Crews, 1980). Sperm storage can extend the breeding season of species. Although the testes of lizards atrophy in the fall, females can continue to lay fertilized eggs at this time using sperm stored previously [for a review, see Fox (1963)l. When sperm are stored, matings can be minimized. Mating requires time for courtship and copulation; it may disrupt feeding and increase exposure to predation, disease, and parasite transmission (Thornhill and Alcock, 1983; Crews, 1973; Smith, 1984; Hunter et al., 1993). In D. rnelanogaster, seminal fluid components received during mating shorten the life span of females (Chapman et

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al., 1995), as does the exposure to males (Partridge et al., 1987). Thus, sperm storage allows females to minimize the costs associated with mating. In conjunction with other ejaculate products, the presence of stored sperm in insects can trigger behaviors in the female that contribute to the efficient production of progeny. In Drosophila (Hihara, 1981; Manning, 1962, 1967; Scott, 1987; Kalb et al., 1993) and many other insects [reviewed in Gillott and Friedel ( 1 977); Ringo, 19961, stored sperm can cause the female to maintain a high egg-laying rate and low receptivity to remating. The coordination of the supply of stored sperm with egg laying and remating can allow females to minimize the number of unfertilized eggs laid and the risks associated with frequent mating. Finally, an important consequence of sperm storage is sperm competition (Parker, 1970). Sperm competition, as originally defined by Parker, is the competition between sperm from different males to fertilize the eggs of a single female (Parker, 1970). Potentially, sperm competition can result in the healthiest sperm being used for fertilization. In a study of Swedish adders, who store sperm for months, Madsen et ul. (1992) demonstrated that multiply mated females had a decreased proportion of stillborn offspring relative to singly mated females. This study suggests competition can cull inferior sperm before fertilization (Madsen et ul., 1992). An extension of this argument would suggest the theoretical possibility that sex ratio or other traits of offspring could be selected for or against by differential survival of sperm in storage, even among sperm from a single mating. A broader definition of the term sperm competition incorporates both behavioral and physiological adaptations in males or females [for a review, see Birkhead and Hunter (1990); Garcia-Bellido, 1964; Edwards, 19931. Because the competition among sperm for storage relates to their potential for successful fertilization, sperm competition may be under sexual selection (Anderrson, 1994; Eberhard, 1985). Thus, at some level the mechanisms of sperm storage and the mechanisms of sperm competition become inseparable.

VII. Conclusions Storage of sperm by mated females is an important component of animal reproduction. The study of mechanisms that govern sperm storage is useful not only for our improved understanding of cell transport, targeting, and communication but to clarify the forces that shape sexual selection and ecology. The tools of light and electron microscopy have been invaluable for establishing the existence of female sperm storage and for describing the organs and the arrangement of sperm within them. The identification of the specific molecules that mediate sperm storage will improve our understanding of the ways that males and females contribute to the sequestration of sperm in defined areas and may clarify how sperm can be kept viable and then released in a predictable fashion for use in fertilization.

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Acknowledgments We are grateful to U. Tram, K. Kemphues, A. Blackler, T. Fox, and V. Vogt for helpful suggestions, information, and comments on the manuscript. Our studies of the role of seminal fluid proteins in sperm storage have been supported by sequential grants from the NSF to M. F. W. NIH Training Grant T32-GM07617 supported D. M. N. during part of the time she investigated sperm storage.

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Morris, S. A., Howarth, B., Jr., Crim, J. W., Rodriguez de Cordoba, S., Esponda, P., and Bedford, J. M. (1987). Specificity of sperm-binding Wolffian duct proteins in the rooster and their persistence on spermatozoa in female host glands. J . Exp. Zool. 242, 189-198. Mortimer, D. (1983). Sperm transport in the human female reproductive tract. In “Oxford Reviews in Reproductive Biology,” pp. 30-61. Oxford University Press, Oxford, UK. Morton, D. B., and Glover, T. D. (1974a). Sperm transport in the female rabbit: The effect of inseminate volume and sperm density. J . Reprod. Fertil. 38, 139- 146. Morton, D. B., and Glover, T. D. (1974b). Sperm transport in the female rabbit: The r61e of the cervix. J. Reprod. Fertil. 38, 131-138. Neubaum, D. M. (1 997). A Drosophila seminal fluid protein, Acp36DE. is required for sperm storage in mated females. Ph.D. Thesis, Cornell University, Ithaca, NY. Newport, M. A . , and Gromko, M. H. (1984). The effect of experimental design on female receptivity to remating and its impact on reproductive success in Drosophila melanogaster. Evolution 38, 1261-1272. Norman, M. D., and Lu, C. C. (1997). Sex in giant squid. Nature 329, 683-684. Ohashi, Y. Y., Hamo-Fukushima, K., and Fuyama, Y. (1991). Purification and characterization of an ovulation stimulating substance from the male accessory glands of Drosophila suzukii. In.sect Biochem. 21, 413-419. Olds-Clarke, P. ( 1988). Genetic analysis of sperm function in fertilization. Gamete Res. 20,241-264. Ono, T., Siva-Jothy, M. T., and Kato, A. (1989). Removal and subsequent ingestion of rivals’ semen during copulation in a tree cricket. Physiol. Ent. 14, 195-202. Otronen, M. (1990). Mating behavior and sperm competition in the fly, Dryomyza anilis. Behav. Ecol. Sociohiol. 26, 349-356. Otronen, M. (1997). Sperm numbers, their storage and usage in the fly Dryomyza anilis. Proc. R. Soc. London B 264,777-782. Parker, G. A. (1970). Sperm competition and its evolutionary consequences in the insects. B i d . Rev. 45, 525-567. Partridge, L., Green, A,, and Fowler, K. (1987). Effects of egg-production and of exposure to males on female survival in Drosophila melanogaster. J. Insect Physiol. 10, 745-749. Pitnick, S., Markow, T. A,, and Spicer, G. S. (1995). Delayed male maturity is a cost of producing large sperm in Drosophila. Proc. Natl. Acad. Sci. USA 92, 10614-10618. Ralt, D., Goldenberg, M., Fetterlof, P., Thompson, D., Dor, J., Mashiach, S., Garkers, D. L., and Eisenbach, M. (1991). Sperm attraction to a follicular fluid factor(s) correlates with human egg fertilizability. Proc. Natl. Acd. Sci. USA 88, 2841-2844. Ralt, D., Manor, M., Cohen-Dayag, A,, Tur-Kaspa, I., Ben-Shlomo, I., Makler, A., Yuli, I., Dor, J., Blumberg, S . , et al. (1994). Chemotaxis of human spermatozoa to follicular factors. B i d . Reprod. 50, 774-785. Rice, W. (1996). Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature 381, 232-234. Riemann, J. G. (1970). Metamorphosis of sperm of the cabbage looper, Trichoplusia ni, during passage from the testes to the female spermatheca. In “Comparative Spermatology, Proceedings of the International Symposium, Rome and Siena, 1-5 July 1969” (B. Baccetti, ed.), pp. 321-332. Academic Press, New York. Ringo, J. (1996). Sexual receptivity in insects. Annu. Rev. Entomol. 41, 473-494. Rothschild, M. L. (1991). Arrangement of sperm within the spermatheca of fleas, with remarks on sperm displacement. B i d . J . Linnean Soc. 43, 313-323. Samakovlis, C., Kylsten, P., Kimbrell, D. A,, Engstrom, A,, and Hultmark, D. (1991). The andropin gene and its product, a male-specific antibacterial peptide in Drosophila melanogaster. EMBO J. 10, 163-169. Sander, K. (1985). Fertilization and egg cell activation in insects. In “Biology of Fertilization” (C. B. Metz, ed.), pp. 409-430. Academic Press, Inc., Orlando, FL.

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4 Developmental C enet ics of Caenorhabdifis elegans Sex Determination Patricia E. Kuwabara MRC Laboratory of Molecular Biology Cambridge CB2 2QH, United Kingdom

I. Introduction A. Overview B. Sexual Dimorphism in the Nematode C . elegans 11. The Role of the X:A Ratio A. Primary Sex Determination B. Genetic Basis of Dosage Compensation C. Molecular Analysis of Dosage Compensation 111. Genetic Analysis of Sex Determination A. Identification of Genes Controlling Sex Determination B. Building a Regulatory Pathway by Genetic Epistasis C. The Coordinated Control of Sex Determination and Dosage Compensation IV. Molecular Analysis of Sex Determination A. Transcriptional Regulation B. Cell-to-Cell Signaling and Cell Nonautonomy in Sex Determination C. Signal Transduction Controls the Activity of TRA-I D. Control of Sexual Fate Involves Protein-Protein Interactions E. Identifying Sexual Partners V. TRA- I Targets and the Conservation of Sex-Determining Mechanisms VI. How to Count Chromosomes: The X:A Ratio Revisited VII. Analysis of Germ-Line Sex Determination VIII. The Hermaphrodite Sperm-Oocyte Decision A. Anatomy of the Somatic Gonad B. Germ-Line-Specific Controls Regulate the Sperm-Oocyte Decision IX. Phylogenetic Comparisons and the Evolution of Sex-Determining Genes X. Unresolved Questions A. Why So Many Genes? B. Dosage Compensation in the Germ Line? C. Parallel Pathways and Feedback Regulation XI. Future Perspectives References

The nematode Caenorhabditis elegans has two naturally occumng sexes: a self-fertile XX hermaphrodite that first produces sperm, then oocytes, and an XO male. The primary determinant of sex is the X:A ratio, the number of X chromosomes to sets of autosomes. The X:A ratio regulates not only sex determination, but also dosage compensation. In the intervening years since the identification of the X:A ratio, most of the key regulatory genes that Currenr Topics i n Dwelupmenml Biology, k11. 4 / Copynghl 0 1999 hy Academic Press. All rights of reproduction in any form reserved. 0070-2153/99 $25.00

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respond to the X:A ratio have been genetically identified and ordered into regulatory hierarchies. Advances have also been made in identifying the X chromosome numerator elements of the X:A ratio. This review highlights the genetic, molecular, and biochemical approaches that have led to an understanding of how these genes interact to control sex determination and dosage compensation. The review also discusses the differences between the control of sexual cell fate in the soma and germ line of C. elegans and addresses the role of germ-line-specific regulation in controlling the sperm-oocyte decision in the hermaphrodite germ line. Finally, strategies that take advantage of the availability of the entire C. elegans genome sequence, which is expected to be completed in 1998, are discussed for identifying hitherto unidentified genes that may play a role in the control of sexual cell fate. Copyright 0 1999 by Academic Press.

1. Introduction A. Overview

Caenorhabditis elegans sex determination and dosage compensation are among the best understood developmental pathways due to the insights provided by many laboratories. The intent of this chapter is to highlight the genetic, molecular, and biochemical approaches that have led to the identification of regulatory genes and to an understanding of how they interact to control sex determination and dosage compensation in C. elegans. It will become apparent that the lessons learned about the mechanisms underlying sex determination and dosage compensation also are relevant to understanding many basic cell biological mechanisms, ranging from chromatin architecture and transcriptional control to signal transduction and translational regulation. The chapter opens by addressing the nature of sexual dimorphism in the nematode C . elegans and the primary determinant of sex-the X:A ratio. This is followed by a discussion of dosage compensation and sex determination, processes that are each regulated by the X:A ratio. Early events in dosage compensation and sex determination are regulated coordinately by a set of early acting genes, but this coordinated control resolves into separate pathways with independent sets of genes responsible for executing each process (Tables I and 11). Until the complexities associated with the coordinated control of sex determination and dosage compensation were appreciated, progress in analyzing the X:A ratio was delayed. Therefore, the nature of the X:A ratio and the strategies for identifying counting elements are revisited after a discussion of this coordinated control. The review then turns to the topic of germ-line sex determination and addresses the nature of germ-line-specific regulatory mechanisms that allow the self-fertile hermaphrodite to produce sperm first and then switch to producing oocytes. Finally, the evolution of sex-determining genes and the impact of the complete C. elegans genome sequence on future studies are discussed.

4. Sex Determination in C. elegans Table I

101

List of Abbreviations and Genetic Nomenclature Copulation defective Deficiency Duplication Dumpy enhanced gain-of-function Fern binding ,factor Feminization Feminization of the germ line Feminizing site on X chromosome Gain-of-function Green fluorescent protein Germ-line proliferation abnormal Hennaphroditization P-Galactosidase Lethal and feminizing 13-12 and glp phenotype Loss-of-function Male abnormal Masculinization of the germ line Mixed character RNA-mediated interference (antisense) Sex and dosage compensation Transformer XO lethal

B. Sexual Dimorphism in the Nematode C. elegans

The small nematode C. elegans has two naturally occurring sexes: male and selffertile hermaphrodite (Fig. 1). The hermaphrodite has a female soma, but her germ line first produces a limited number of sperm before making oocytes. Otherwise, she can be considered the female of the species. One consequence of the production of both sperm and oocytes in an essentially female animal is that the mechanisms controlling somatic and germ-line sex determination are not identical, although many of the same global genes are involved. The specification of germ cells to become sperm requires the repression of female-promoting genes and the activation of male-promoting genes. Subsequently, the male-promoting genes must be repressed to allow the switch to oogenesis. In C. elegans, the decision to follow the hermaphrodite or male fate gives rise to differences in body patterning, behavior, and biochemistry. The lineage of each cell in the hermaphrodite (959 somatic nuclei) and in the male (1031 somatic nuclei) is known in its entirety (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979; Sulston et al., 1980, 1983; Hodgkin, 1988). Most of the differences in sexual phenotype arise postembryonically; however, the first sign of sexual

Table I1 Mutant Phenotypes and Molecular Identities of Genes Required for Sex Determination and Dosage Compensation in C. elegans ~~~

Gene class”

fox- I xul-1 sdc-1‘ S~C-2 S~C-3” DPY Tra Null

dpy-21 d~y-26~ dpy-27’ d~y-28~ dpy-30

XX phenotype

XO phenotype

Sex and Dosage Compensation Hermaphrodite Male Hermaphrodite Inviable; feminized Dumpy; masculinized Male Dumpy; masculinized Male

Dumpy hermaphrodite Nondumpy; masculinized Dumpy hermaphrodite

~~

Molecular identity RNA binding protein Novel Zinc finger motif Novel Zinc finger motifs ATP binding

Male Male Male

Dosage Compensation‘ Weak dumpy Inviable, dumpy escapers Inviable, dumpy escapers Inviable; dumpy escapers Inviable; dumpy Viable, scrawny

Unknown Novel SMC family Unknown Novel

Global Sex Determination

her-I

(If) (gfl

Secreted protein (ligand) Hermaphrodite Masculinized soma and germ line

rra-2

(If) (gf) (W (eg)

~ a - 3 ~ fem-16 fem-26 fem-36

(0 (gf)

Pseudomale Female Female Hermaphrodite Pseudomale Female Female Female Female soma; sperm

tra-1

(0

fog-] fog-2 f w 3 mug-I to mug-6 lafl/+c f b f l :fbf-2 (RNAi)d

Mating male; sperm, some oocytes Female

Hermaphrodite Male Membrane protein (receptor) Male Male; yolk and oocytes Male Hermaphrodite Male Calpain cysteine protease Female Ankyrin repeats SerRhr phosphatase Female Novel Female Male Zinc finger motifs Male Female

Germ-Line Sex Determination Female Male; oocytes Female Male Female Male; oocytes Female soma; sperm Male Female and hermaphrodite Male Female soma; sperm Male

Unknown Novel Unknown Unknown Unknown Pumilio repeats

“Unless otherwise stated, phenotypes are loss-of-function. For individual references, refer to the text. 6These genes show maternal effects, which for simplicity have not been discussed in the text. ‘iafl/+ shows impenerrant dominant feminization in XX animals and recessive lethality (Goodwin ef al., 1997). dloss-of-function phenotypes are deduced from RNAi (Zhang et al., 1997). eXX phenotypes are all hermaphrodite; XO phenotypes are all male.

103

4. Sex Determination in C. elegans pharynx

/

sperm in spermatheca

vulva

embryos in uterus

intestine single lobed gonad

sperm

Fig. 1 Schematic illustration of the C. elegcms hermaphrodite (top) and male (bottom)

dimorphism appears during embryogenesis and is marked by two sets of programmed cell deaths. These deaths remove four CEM (male-specific cephalic neurons) in XX hermaphrodites and two HSN (hermaphrodite-specific neurons required for egg-laying) in XO males. Over 30% of somatic nuclei in the adult hermaphrodite and 40% in the adult male are sexually dimorphic; the most obvious differences are manifested in tissues involved in reproduction and mating (Fig. 1). The XX hermaphrodite has a bilobed ovotestis that reflexes to meet centrally at the vulva (opening for egg-laying on the ventral surface), whereas the male has a single-lobed gonad that produces only sperm. The hermaphrodite also has neurons and muscles that are involved in egg-laying. The tail of the hermaphrodite tapers to a simple spike, whereas the male has an elaborate tail with specialized features and sensory structures that are necessary for mating and copulation. Males also display a characteristic mating behavior, and it has been possible to identify genetic mutants that disrupt this behavior. These include many mab (for male abnormal) mutants (Shen and Hodgkin, 1988), which have abnormal male tail morphology, and many cod (for copulation defective) mutants (Liu and Sternberg, 1995; Emmons and Sternberg, 1997), which have no obvious physical defects in male tail morphology, yet fail to display normal mating behavior. At the biochemical level, yolk proteins (vitellogenins) are expressed exclusively by the intestine of XX hermaphrodites (Kimble and Sharrock, 1983). Ectopic expression of yolk proteins by the intestine of XO males has proven to be a sensitive reporter for inappropriate XO feminization. Males, although not essential for the propagation of wild-type C. elegans, are

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Patricia E. Kuwabara

found at a frequency of 0.2% of the natural population. C. elegans XO males do not have a Y chromosome and arise through meiotic non-disjunction. Although wild-type XX hermaphrodites are self-fertile, they can be mated with XO males to produce cross-progeny. Sperm provided by XO males have a competitive advantage over hermaphrodite sperm, so that after mating, only cross-progeny are produced in a Mendelian sex ratio of 1: 1 (Ward and Carrel, 1979). Certain mutant strains with defects in meiotic segregation also produce a higher frequency of males than found in wild-type C. elegans (e.g., him mutants, for high incidence of males) (Hodgkin et ul., 1979).

II. The Role of the X:A Ratio A. Primary Sex Determination

It was shown by Nigon (1949) that wild-type diploid XX animals are hermaphrodites and XO animals are males. What is the primary determinant of sex in C.elegans? Is it the absolute number of X chromosomes? To address this question, Madl and Herman (1979) varied chromosomal ploidy to determine how it affected sex determination. Normally, diploid XO animals (X:A ratio, 1X:2A = 0.5) are male and diploid XX animals (2X:2A = 1) are hermaphrodite. Their results indicated that the primary determinant of sex is the X:A ratio (number of X chromosomes to the number of sets of autosomes) and not the absolute number of X chromosomes. By way of example, XX tetraploids (2X:4A = 0.5) develop as males, not hermaphrodites, which would have been the case if the absolute number of X chromosomes determined sex. Further examination of different X:A ratios revealed that animals with an X:A ratio of 0.67 (2X:3A) developed as males and those with an X:A ratio of 0.75 (3X:4A) developed as hermaphrodites. Animals with an intermediate X:A ratio, 0.67 < X:A < 0.75, developed as intersexes.

B. Genetic Basis of Dosage Compensation

One consequence of the dimorphism in X chromosome number between the sexes is that X-linked expression must be equalized through dosage compensation. Several dosage compensation schemes have been documented in other organisms. In mammals, dosage compensation is achieved through X inactivation [for reviews, see Grant and Chapman (1988) and Borsani and Ballabio (1993)l. X inactivation does not appear to be the basis for C. elegans dosage compensation, because XX animals heterozygous for an X-linked recessive marker invariably are wild type in phenotype. Instead, measurements of the steady-state mRNA levels of several X-linked C. elegans genes indicate that

4. Sex Determination in C. elegans

10.5

dosage compensation is achieved by downregulating, by one-half, the expression of each X chromosome in an XX hermaphrodite so that it is equivalent to the output from the single X in XO males (Meyer and Casson, 1986). Variations in ploidy that lead to severe overexpression (4X:2A) (Hodgkin et al., 1979) or underexpression (IX:3A) (Hodgkin, 1987b) of the X chromosome result in lethality or severe sickness. These excessive imbalances in X chromosome dosage probably cannot be redressed by the dosage compensation machinery. Similarly, mutations in genes controlling dosage compensation might also be expected to produce sex-specific lethality in diploids. A set of five autosomal genes (dpy-21,-26,-27,-28,and -30)were identified that implement X chromosome compensation (Tables I and 11) (Hodgkin, 1983b; Meneely and Wood, 1984; Plenefisch et d., 1989; Hsu and Meyer, 1994). It is generally observed that mutations in genes controlling dosage compensation are lethal to XX animals and have little or no effect on XO animals. Strong loss of function or null mutations in either dpy-26,-27,or -28 lead to XX inviability and rare dumpy (Dpy, short and fat) escapers, which give rise to the dpy gene name; in contrast, mutations in these same genes have essentially no effect on XO males with a single X chromosome (Hodgkin, 1983b; Plenefisch er al., 1989). dpy-21 mutants show less severe, but similar defects; XX dpy-21 hermaphrodites are Dpy and XO males essentially are wild type (Hodgkin, 1983b). dpy-30 alleles are temperaturesensitive and their phenotype is somewhat different from other dosage compensation dpy mutants (Hsu and Meyer, 1994). XX dpy-30 mutants are almost always inviable, but at permissive temperatures rare Dpy escapers are observed; XO dpy-30 animals are viable males, but scrawny, indicating that dpy-30 also plays a role in XO development. Mutations in the dosage compensation dpy genes have no direct effect on sexual phenotype, although the implementation of dosage compensation is determined by the X:A ratio. Therefore, the dosage compensation process is distinct from the pathway controlling sex determination (Fig. 2). It has been observed, however, that mutations in the dosage compensation dpy genes can indirectly influence sex determination. 2X:3A (X:A = 0.67) animals that would normally develop as males can be shifted toward the hermaphrodite mode of sex determination by mutation in the dosage compensation dpy-21 or dpy-27 gene (Hodgkin, 1 9 8 7 ~ )This . observation has been interpreted to indicate that there may be a feedback mechanism operating between the sex determination and dosage compensation pathways. Additional evidence for feedback comes from studies of the sdc-3 gene (see Section 1II.C; DeLong et al., 1993). Mutations in the dosage compensation dpy genes have been shown to elevate X-linked expression in XX animals by using phenotypic assays (Meneely and Wood, 1984; Wood et al., 1985; DeLong et al., 1987) and by measuring X-linked expression (Meyer and Casson, 1986), consistent with the proposed wild-type role of these genes in downregulating X-linked expression. Because of the similar mutant phenotype shared by most dosage compensation dpy genes, they have

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Patricia E. Kuwabara

Coordinated control of sex determination and dosage compensation sdc-1

X:A xol-1 X:A ratio fox-1

Dosage compensation

f

dPY-21, d ~ y - 2 6dpy-27, , dpy-28, dpy-30 (X:A = 1 .O, ON; X:A = 0.5, off)

4sdc-2

?

Sex determination

S~C-3

\

her-1

--Ifern-2 4tra-1

4

-'''-3

fem-3

Transcnptional Cell-to-cell Signal transduction regulation signaling

(2X:2A) low (IX:2A) HIGH

HIGH

low

low HIGH

HIGH low

low HIGH

brnab-3, ?

Transcriptional regulation

HIGH low

Female Male

Fig. 2 The regulatory pathway of C. elegans somatic sex determination. The genes regulating sex determination (and dosage compensation) are ordered by genetic epistasis in a pathway whereby each gene negatively regulates the activity of its nearest downstream neighbor (see Tables I and 11). The activity of each gene is determined in response to the X:A ratio, as indicated. Thefox-1 gene encodes a potential X chromosome counting element; a question mark is placed below fox-I to indicate that other numerator elements exist. The xol-1 and sdc genes at the beginning of the pathway coordinately regulate sex determination and dosage compensation; however, the pathway bifurcates into separate branches controlling only dosage compensation or only sex determination. In the pathway controlling sex determination, the sdc and tru genes promote XX female development and xol-1, her-I, and the fern genes promote XO male development. The tra-1 gene ultimately controls the decision to develop a male or female soma. Potential transcriptional feedback is illustrated, because it has been observed that TRA-1 may positively regulate tra-2 expression (Okkema and Kimble, 1991).The mab-3 gene is shown as a potential downstream target of rru-1 activity, but other targets must exist as indicated by the question marks. The genes that regulate dosage compensation share similar mutant phenotypes and hence could not be ordered into a genetic pathway.

not been ordered by genetic means, but it is postulated that the dpy gene products function together to implement dosage compensation. Not all X-linked genes are dosage-compensated in XX animals; the levels of an unknown X-linked mRNA, uxt-2, are 2-fold higher in XX hermaphrodites than in XO males (Meyer and Casson, 1986). It is possible that the X:A numerator elements also will escape dosage compensation (see Section VI). C. Molecular Analysis of Dosage Compensation

Substantial progress has been achieved toward understanding the molecular and biochemical basis of dosage compensation in C . elegans. The molecular analysis of dpy-27 has provided the first clues that dosage compensation functions to lower X-linked chromosome expression by specifically altering the architecture of the X chromosome. By using antibodies directed against the DPY-27 protein,

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it was shown that, prior to the 30-cell stage, DPY-27 protein is present in the nuclei of both XX and XO animals; however, at the 30-cell stage, DPY-27 associates with each X chromosome in XX hermaphrodites, but fails to localize to the single X in XO males (Chuang el al., 1994). Thus, the onset of dosage Compensation is associated with the 30-cell stage. The dpy-27 gene encodes a homologue of the SMC family of proteins (Structural Maintenance of Chromosomes), which play essential roles in chromosome condensation and segregation in yeast and frogs (Strunnikov et al., 1993, 1995; Hirano and Mitchison, 1994). In XO xol-Z mutants (see Section III.C), DPY-27 is inappropriately associated with the single X chromosome; therefore, XOL-1 normally prevents DPY-27 from associating with the single X chromosome in XO animals (Chuang et af., 1994). The dosage compensation gene, dpy-26, was cloned and found to encode a novel acidic protein (Lieb et a f . , 1996). Similar to DPY-27, antibodies directed against DPY-26 protein detect DPY-26 in the nuclei of both XX and XO animals; at the 30-cell stage, DPY-26 is associated with each X chromosome in XX, but not XO animals (Lieb et al., 1996). DPY-26 also co-localizes with DPY-27 to the X chromosome at each cell cycle; however, the staining patterns of DPY-26 and DPY-27 are not identical. Unlike DPY-27, DPY-26 protein is associated with all condensed chromosomes as they undergo mitosis prior to the 30-cell stage. However, by the 60-cell stage, DPY-26 staining is limited to the mitotic X chromosome in XX animals. Genetic analyses indicate that, in addition to its role in dosage compensation, dpy-26 plays a role in meiotic segregation; dpy-26 mutants display a higher level of X chromosome non-disjunction as detected by the increase in XO males (Hodgkin, 1983b; Plenefisch et al., 1989). Examination of the XX hermaphrodite germ line using anti-DPY-26 antibodies shows that DPY-26 co-localizes with germ cell chromosomes as they condense and enter pachytene (Lieb et al., 1996). In contrast, DPY-27 is not detected in the hermaphrodite germ line. It has been further shown that mutations that abolish DPY-26 staining in embryos (see the following discussion) do not affect the localization of DPY-26 protein in the hermaphrodite germ line. Therefore, DPY-26 has an independent function associated with meiotic chromosome segregation in the hermaphrodite germ line that is separable from its role in dosage compensation. To examine how the dosage compensation dpy genes and their upstream regulators interact to regulate the assembly of a complex on the X chromosome, the effects of mutations in these genes on the immunochemical staining patterns of DPY-26 and DPY-27 were examined. As described in Section IILC, the sdc genes are responsible for activating dosage compensation in XX animals, and xof-I is responsible for repressing the sdc genes in XO animals (Fig. 2). It was found that sdc-2, sdc-3, and dpy-30 are necessary for localizing DPY-27 to the X chromosome in XX animals. DPY-27 fails to localize to the X chromosome in XX animals mutant for sdc-2, sdc-3, or dpy-30, and the staining pattern instead

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resembles that of XO animals (Chuang et al., 1996). Similarly, DPY-26 also fails to localize to the X chromosome in sdc-2, sdc-3, dpy-27, and dpy-30 mutants (Lieb et al., 1996). The dpy-26 and dpy-28 genes affect the synthesis or stability of DPY-27. Mutations in these genes do not significantly alter dpy-27 mFWA steady-state levels, but they completely abolish DPY-27 staining (Chuang et al., 1996). Mutations in dpy-27 and dpy-28 also diminish or abolish DPY-26 staining, but do not affect dpy-26 mRNA steady-state levels (Lieb et al., 1996). It was also reported that null mutations in sdc-1 or dpy-21 have no effect on the staining patterns of either DPY-27 (Chuang et al., 1996) or DPY-26 (Lieb et al., 1996), indicating that these genes do not play a direct role in recruiting DPY-27 or DPY-26 to the X chromosome. At the biochemical level, co-immunoprecipitation experiments indicate that DPY-26 and DPY-27 form a complex and that this interaction may be important for protein stability (Chuang et al., 1996). Additional studies suggest that DPY-28 may also complex with DPY-26 and DPY-27 (Chuang et ul., 1996). It remains to be determined how XOL-1 regulation of the sdc genes or their products prevents a dosage compensation complex from forming on the single X of XO males. It is likely that xol-1 directly regulates either sdc-2 or sdc-3 and not sdc-I, because mutations in sdc-1 do not effect the recruitment of DPY-26 or DPY-27 to the X chromosome in XX animals (Fig. 2).

111. Genetic Analysis of Sex Determination A. Identification of Genes Controlling Sex Determination As demonstrated in Section II.B, perturbations in dosage compensation often lead to sex-specific lethality; however, sexual transformations in either direction generally are not lethal. Thus, early genetic screens led to the recovery of many sex reversal mutants that have phenotypes opposite to that specified by the X:A ratio (Tables I and 11).These mutants were shown to carry mutations in genes that globally regulate both somatic and germ-line sex determination. Genetic selection and suppressor screens subsequently have made it possible to identify novel genes that display tissue specificity and rare alleles of known sex-determining genes, which have shed light on tissue- and sex-specific regulatory mechanisms (Tables I and 11). The three tru-I, -2, and -3 genes were among the first identified loss-offunction mutations in genes that regulate sex determination (Hodgkin and Brenner, 1977). Mutation in any tra gene (tra-1,2,3) transforms XX animals into males; however, the phenotypes of these transformed XX males are not identical (Table 11). XX tra-l(lf) males are capable of siring cross-progeny, whereas XX tra-2(lf)or tra-3(lf)males are not. XX tra-2 and tra-3 males show defective tail morphology and an absence of mating behavior; hence, these males also are

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referred to as pseudomales. In contrast to their effect on XX sexual phenotype and behavior, mutations in trc1-2 and tru-3 do not affect the normal development of XO males; however, tru-1 XO males have variably abnormal gonads, indicating that tru-1 plays a role in the normal development of the XO gonad (Hodgkin, 1987a; Schedl et ul., 1989). XX tra-2/+ animals are partially masculinized because they have an Egl (egg-laying defective) phenotype resulting from missing or abnormal HSNs (hermaphrodite-specific neurons) (Trent et al., 1983). The haplo-insufficiency associated with tru-2 is one indication that sexual cell fate decisions are sensitive to perturbations in the dose or activity of the sexdetermining genes. Taken together, the normal wild-type role of the tru genes, as inferred from their loss-of-function phenotypes, is to promote female development in the XX hermaphrodite. Genes that promote XO male development also were identified through genetic screens by searching for mutations that feminize XO animals (Table 11). her-lflf)mutations transform XO animals into fertile hermaphrodites, but have no effect on XX animals (Hodgkin, 1980; Trent et al., 1988). Null mutations in the fern-I, -2, and -3 genes transform both XO and XX into spermless hermaphrodites, that is, females (Klass et ul., 1976; Nelson etnl., 1978; Doniach and Hodgkin, 1984; Kimble et al., 1984; Hodgkin, 1986). Thefern genes are essential for both XX hermaphrodite and XO male spermatogenesis. Dominant gain-offunction feminizing mutations in the tra-1 gene also were recovered (Hodgkin, 1983a. 1987a); both XX and XO tru-l(gf)mutants develop as females, which can be mated to produce cross-progeny. Temperature-sensitive alleles were identified for many of the sex-determining genes and dosage compensation dpy genes, which have proven to be valuable tools for identifying extragenic suppressors. Temperature shift studies using temperature-sensitive alleles of sex-determining genes have revealed that the sexual fate of individual tissues is determined at different times in development. It has also been shown that many of the sex-determining genes are required continuously throughout development to maintain a sexual cell fate decision. For example, her-/ activity is required continuously in XO males to ensure the maintenance of the male state; when XO her-l(ts) males raised continuously at permissive temperatures are shifted as adults to restrictive temperatures, they produce oocytes and their intestine begins synthesizing yolk proteins (Schedin et a/., 1994). The genes described so far are global regulators that control sexual cell fate decisions in both somatic and germ-line tissues, but have no apparent role in dosage compensation. The control of germ-line sex determination involves additional tissue-specific genes and tissue-specific controls that regulate the activity of global sex-determining genes. Therefore, the following discussion pertains only to the control of somatic sex determination. A discussion of germ-line sex determination is deferred to Section VII.

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B. Building a Regulatory Pathway by Genetic Epistasis

Epistatic interactions among the different sex-determining genes have been analyzed extensively (Hodgkin and Brenner, 1977; Nelson et ul., 1978; Hodgkin, 1980, 1984, 1986; Doniach and Hodgkin, 1984). For example, XX animals carrying a single mutation in either tru-2 or fern-1 develop as males or females, respectively; XX animals doubly mutant for tra-2 and fern-1 develop as females, not males. Therefore, in a control hierarchy fern-1 acts later than tru-2. After testing all possible double-mutant and some triple-mutant combinations, the sexdetermining genes were ordered in a hierarchical regulatory pathway based on negative regulation (Fig. 2) (Hodgkin, 1987~).In this pathway, sex determination is controlled by a series of on-off or high-low switches, whereby each gene functions to negatively regulate the activity of its nearest downstream neighbor. The tru-1 gene is epistatic to all sex-determining genes; tru-1 is placed downstream of tru-2 and tru-3 because XX tru-2 (or tru-3); tru-I(&) double mutants develop as females, not males. Therefore, tru-1 is the master regulator of somatic sex determination; female somatic development ensues when tru-l is active and male development when tru-1 is inactive.

C. The Coordinated Control of Sex Determination and Dosage Compensation

The genes discussed so far function to promote only dosage compensation or only sex determination. However, additional genes have been identified that coordinate both pathways (Tables I and 11). The first of these genes to be identified was sdc-1 (for sex and dosage compensation) (Villeneuve and Meyer, 1987). Mutations in sdc-1 have no effect on XO males, but they produce dumpy and variably masculinized XX hermaphrodites. Thus, sdc-1 disrupts both dosage compensation and sex determination. Two lines of evidence support the view that sdc-1 mutations disrupt dosage compensation: first, X-linked genes are overexpressed in XX sdc-1 mutants; second, the Dpy phenotype is associated with XX animals, regardless of their sexual phenotype. It was further shown that loss-offunction mutations in her-I, which transform XO animals to hermaphrodites, suppress the masculinization of XX sdc-1 mutants, but not their inability to activate dosage compensation. This indicates that sdc-1 independently regulates dosage compensation and sex determination. On the basis of genetic epistasis arguments, sdc-1 has been placed upstream of both the dosage compensation and sex determination pathway (Fig. 2) (Villeneuve and Meyer, 1990). sdc-1 does not function alone to coordinately regulate dosage compensation and sex determination. .sdc-2 and sdc-3 mutants also exhibit XX sexual transformations and defects in dosage compensation, while having no effect on XO males (DeLong ei ul., 1993; Nusbaum and Meyer, 1989). Of the sdc genes, null mutations in sdc-2

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exhibit the most severe phenotype; XX sdc-2 mutants are inviable, but rare escapers are Dpy and sexually transformed (Nusbaum and Meyer, 1989). sdc-3 is unusual in that, in addition to null mutations that produce Dpy XX hermaphrodites, certain sdc-3 alleles affect only dosage compensation or only sex determination (Table 11). Thus, sdc-3 appears to have independently mutable regions that discriminate between its effects on sex determination and dosage compensation (DeLong et al., 1993; Klein and Meyer, 1993). The sdc genes are not the earliest genes in the regulatory hierarchy that respond to the X:A ratio; this position presently is occupied by xol-1 (for XO lethality) (Fig. 2 ) (Miller et al., 1988). xol-1 null mutations have no effect on XX animals, but they are lethal and feminizing to XO animals. Ectopic expression of xol-I also is lethal to XX animals because the male mode of dosage compensation is activated inappropriately (Rhind et al., 1995). The lethality of xol-1 mutants is suppressed by mutations in any of the dosage compensation dpy genes, whereas the sex determination defects are suppressed by mutations in any of the .sdc genes. Taken together, genetic epistasis suggests that xol-1 functions upstream of the sdc genes and that xol-I is a direct target of the X:A ratio (Fig. 2). Therefore, xol-1 is the earliest acting regulatory switch gene affecting both sexual cell fate and dosage compensation. xol-1 appears to be transcriptionally regulated because steady-state RNA levels are higher in XO than in XX embryos (Rhind et a/., 1995). In addition, xol-I also may be regulated post-transcriptionally, because the xol-I locus expresses multiple transcripts, only one of which is sufficient to rescue all aspects of the xol-1 mutant phenotype (Rhind et al., 1995). A heat-shock-driven xol-1::lacZ reporter that rescues xol-1 mutants was used to show that xol-1 functions early in development and that its activity is required only during the gastrula stage to promote male development.

IV. Molecular Analysis of Sex Determination The molecular cloning of the major regulatory genes controlling sex determination provided insights into, but not an immediate understanding of, the mechanisms underlying the genetic pathway of sex determination. Several of the sexdetermining genes were shown to have sequence motifs found in other proteins with characterized function; however, many of the sex-determining genes encoded novel proteins. Among those proteins with known sequence motifs were sdc-I (Nonet and Meyer, 1991), sdc-3 (Klein and Meyer, 1993), and tra-1 (Zarkower and Hodgkin, 1992; Hodgkin, 1993); each of these genes encodes proteins with zinc finger motifs, which are associated with transcriptional regulation. From this information, it was surmised that transcriptional regulation plays a key role in the initiation and execution of sexual cell fate decisions (Fig. 2). However, transcriptional regulation is not the only mechanism controlling sexual cell fate; as discussed in Sections 1V.B and IV.C, cell-to-cell signaling

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and signal transduction also play central roles in regulating the final activity state of tra-1.

A. Transcriptional Regulation

What is the transcriptional target of sdc-1 and sdc-3? One potential target is her-1. At the beginning of the pathway controlling only sex determination, her-1 is the earliest acting switch gene (Fig. 2) (Hodgkin, 1980; Trent et al., 1988). her-1 also is active in XO males and is inactive in XX hermaphrodites. This difference in her-1 activity arises through transcriptional regulation; her-1 mRNA steady-state levels are high in XO males and low or absent in XX hermaphrodites (Trent et al., 1991). The steady-state levels of her-1 mRNAs respond to the X:A ratio and are not affected by mutations in downstream sexdetermining genes. For example, although XX tru-1 mutants develop as phenotypic males, the levels of her-1 mRNAs in XX tra-1 males are comparable to those found in wild-type XX hermaphrodites but not XO males (Trent et al., 1991). The sdc genes are good candidates for mediating the transcriptional repression of her-1. Consistent with this notion, her-1 mRNA steady-state levels are 20-fold higher than those of wild type in animals carrying an sdc mutation (Trent et al., 1991; DeLong e f al.,1993). As stated earlier, sdc-1 and sdc-3 each encode proteins with zinc finger motifs; however, the zinc fingers in SDC-3 have been shown to be necessary for dosage compensation, but not sex determination (Klein and Meyer, 1993). Presently, the mechanism by which SDC-3 and the other SDC proteins repress her-1 is unknown.

B. Cell-to-Cell Signaling and Cell Nonautonomy in Sex Determination

A more difficult challenge has been to understand how the presence or absence of her-1 is detected and subsequently transduced through tra-2, tra-3, and the fern genes to control tru-1 activity. Part of the understanding came from observations indicating that sex determination contains a cell nonautonomous component. Mosaic analysis indicated that her-1 (Hunter and Wood, 1992) and sdc-1 (Villeneuve and Meyer, 1990) behave cell nonautonomously. Molecular analysis subsequently has revealed that her-1 encodes a predicted secreted protein called HER-I (Perry et al., 1993). Because sdc-1 regulates the expression of her-1 mRNAs, this might also explain why sdc-1 functions cell nonautonomously despite encoding a zinc finger protein that might be expected to function cell autonomously (Villeneuve and Meyer, 1990; Nonet and Meyer, 199 1). Another piece of the puzzle was provided when it was shown that one of the tra-2 transcripts encodes a large predicted membrane-spanning protein named TRA-2A (Kuwabara et al., 1992), which might act as a receptor for the HER-1 ligand.

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C. Signal Transduction Controls the Activity of TRA-1

A simple model was proposed that took into account the genetic relationships of the genes controlling sex determination, the cell nonautonomy of sexual cell fate decisions, and the molecular identities of the sex-determining genes (Kuwabara et al., 1992; Kuwabara and Kimble, 1992). The key aspects of this model involved cell-to-cell signaling mediated by HER- 1 and TRA-2A and signal transduction mediated through TRA-2A and the intracellular FEM proteins to TRA- I . As shown in Fig. 3, TRA-2A is depicted as being present in both sexes, because

Fig. 3 Molecular model for the control of somatic sex determination in C. elegans [adapted from Kuwabara and Kimble (1992)l. This model is based on the molecular identities o f the sex-determining genes, their genetic interactions, and the knowledge that there is cell nonautonomy in sexual fate decisions. In XX hermaphrodites, TRA-2A is depicted as a membrane protein that functions constitutively to inhibit the activity of the FEM proteins. There is evidence that the carboxy-terminal tail (intracellular)of' TRA-2A interacts with FEM-3; therefore, it is hypothesized that TRA-2A may bind and sequester FEM-3 (Kuwahara and Kimble, 1995; A. M. Spence and P. E. Kuwabara, unpublished). FEM-3 also has been shown to interact with FEM-2 as shown (Chin-San and Spence, 1996); however, it is not known whether this interaction occurs in both sexes. The inhibition of the FEM proteins allows TRA-I to function in the nucleus as a transcriptional regulator that promotes female somatic development. In XO males, there is evidence indicating that HER- I functions cell nonautonomously to repress the activity of TRA-2A (Hunter and Wood, 1992; Perry et al.. 1993; Kuwahara, 1996h). This repression is predicted to occur only in XO animals, because her-1 is transcriptionally regulated; her-] mRNAs are detected in XO, but not XX animals (Trent e f al., 1991). Because TRA-2A is repressed in XO animals, this allows the FEM proteins to inhibit TRA-I, There is no evidence indicating that any of the FEM proteins bind TRA- 1, but because FEM-2 encodes a Ser/Thr type 2C pbosphatase and TRA- 1 has a potential GSK3 kinase site (Pilgrim er a/., 1995; de Bono et a/., 1995; Chin-San and Spence, 1996), it is speculated that these proteins may interact. The proposed interaction between TRA-1 and the FEM proteins may be transient and result in a post-translational modification of TRA-I that interferes with its localization to the nucleus or its ability to promote female development. (Reprinted from Trends Gener. 8, P. E. Kuwahara and J. E. Kimble, Molecular genetics of sex determination in C. elegans, pp. 164-168. Copyright 1992, with permission from Elsevier Science.)

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tra-2 mRNAs are present in both sexes, albeit at levels 10-fold lower in XO males (Okkema and Kimble, 1991). In XO males, HER-1 is present and is postulated to bind to and inactivate TRA-2A (Trent et al., 1991; Perry er al., 1993). In turn, the FEM proteins (Rosenquist and Kimble, 1988; Spence et al., 1990; Pilgrim er al., 1995) are active and repress TRA-1, possibly through protein-protein interactions or post-translational modification. In XX hermaphrodites, HER-1 is absent and TRA-2A is predicted to function constitutively, promoting female development in the hermaphrodite. TRA-2A is postulated to bind to and inactivate one or more of the FEM proteins, possibly through sequestration, allowing TRA- 1 to promote female development.

D. Control of Sexual Fate Involves Protein-Protein Interactions

Many aspects of this model have now received support and clarification through additional molecular and biochemical studies (Fig. 3). It has been demonstrated that, although the tra-2 gene expresses multiple transcripts (Okkema and Kimble, 199l), the 4.7-kb tra-2 mRNA encoding TRA-2A provides the primary feminizing activity of the tru-2 locus (Kuwabara and Kimble, 1995). When TRA-2A expression is driven by the heat-shock promoter, it rescues the soma of XX tra-2 transgenic animals, which otherwise would develop as males. Furthermore, heat-shock-driven TRA-2A completely transforms XO transgenic animals into fertile hermaphrodites. Therefore, if TRA-2A levels are sufficiently high in an XO animal, HER-I repression can be overcome to allow hermaphrodite development. This switch in XO fate supports the notion that HER-1 repression may be required to prevent even low levels of TRA-2A from inappropriately feminizing XO animals. It is speculated that if TRA-2A is active in an XO animal, it may activate a transcriptional feedback that increases tra-2 mRNA steady-state levels and leads to XO feminization (see Section X.C). To further support the hypothesis that HER-1 binds and represses TRA-2A, a novel class of dominant fra-2(eg) feminizing mutants were identified in a genetic selection for XO feminizing mutations (Hodgkin and Albertson, 1995). The tru-2(eg) mutants mimic the phenotype of her-1 null mutants; XO tru-2(eg) mutants are transformed to fertile hermaphrodites, whereas XX tru-2(eg)mutants remain hermaphrodites (Kuwabara, 1996a). The tra-2(eg) mutant phenotype suggests that TRA-2A(EG) is specifically insensitive to HER- 1 regulation, because it retains feminizing activity and is sensitive to other forms of regulation (Kuwabara, 1996a). To test this hypothesis, an XX tra-2(eg);her-l(gf)double mutant was created; XX her-1(gf) mutants are masculinized because her-I mRNA steady-state levels are elevated inappropriately (Trent et al., 1991; Peny et al., 1994). It was found that the tra-2(eg)mutation suppresses her-l(gf) XX masculinization, because XX tru-2(eg);her-l(gf) mutants are no longer masculinized (Kuwabara, 1996a). Molecular analysis of the 10 tru-2(eg) mutants

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revealed that they all carried an identical base change that produced an R177K change in a predicted extracellular region unique to TRA-2A called the EG domain; none of the other tra-2 transcripts encoded proteins carrying the EG domain (Okkema and Kimble, 1991; P. E. Kuwabara, P. G. Okkema, and J. Kimble, unpublished). Therefore, it has been postulated that the tra-2(eg) mutants define a potential HER- 1 binding site (Kuwabara, 1996a).

E. Identifying Sexual Partners

How does TRA-2A repress the activity of the FEM proteins to promote female development in XX animals? The novelty of the TRA-2A (Kuwabara et al., 1992), FEM-1 (Spence et al., 1990), and FEM-3 (Ahringer et al., 1992) sequences meant that there was little available information to help predict how TRA-2A would interact with one or more of the FEM proteins. The first clue that a potential TRA-2A-FEM interaction site resided in a hydrophilic carboxyterminal region of TRA-2A was provided from a heat-shock-activated transgene that expressed only this region of TRA-2A (Kuwabara and Kimble, 1995). It was observed that this transgene induced expression of the hermaphrodite-specific yolk proteins and led to the partial feminization of the male tail of tra-2 null and XO animals. Subsequently, a direct interaction between the carboxy-terminal region of TRA-2A and FEM-3 has been detected using a yeast 2-hybrid interaction screen (A. M. Spence and P. E. Kuwabara, unpublished). The 2-hybrid system also was used to demonstrate the FEM-3 and FEM-2 interact directly (Chin-Sang and Spence, 1996). Therefore, FEM-3 can partner either TRA-2A or FEM-2, but it is not known whether the three proteins can form a complex or whether interactions among these proteins are sex-specific (Fig. 3). Ironically, the ankyrin motifs in the FEM-1 sequence suggest that FEM-1 also is involved in a protein-protein interaction (Spence et al., 1990), yet the molecular role of FEM-1 in the sex-determining process has remained elusive. It is not known how the FEM proteins inactivate TRA- 1, although a mutation in any one fern gene is sufficient to prevent inactivation of tra-1 in an XO animal. Given the requirement for all three FEM proteins to inactivate TRA-1, it is postulated that the FEiM proteins may interact as a complex. There are hints that phosphorylation plays a regulatory role in controlling TRA-1 activity and that FEM-2 may be involved in this regulation. The cloning of fern-2 revealed that it encoded a type 2C serinelthreonine phosphatase with in vitro phosphatase activity (Pilgrim et al., 1995; Chin-Sang and Spence, 1996). The identity of FEM-2 as a phosphatase suggests that post-translational modifications probably play an important role in modulating protein-protein interactions involved in controlling sex determination. The analysis of tra-I(&) mutants also supports the notion that phosphorylation-dephosphorylation regulates protein-protein interactions between sex-determining proteins (Fig. 3). The tra-l(&) mutations dominantly

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feminize both XX and XO animals (Table 11) (Hodgkin, 1983a, 1987a). Molecular analysis of 26 tru-l(gf) mutants defined a region that may participate in an inhibitory protein-protein interaction; this region contains a potential glycogen synthase kinase-3 phosphorylation site (de Bono et ul., 1995). A kinase with a specific role in sex determination has yet to be identified, although a glycogen synthase kinase-3 homologue has been identified by the C. eleguns sequencing consortium. Given that tru-1 is the terminal gene in the pathway and functions to promote female somatic development, the presumption is that TRA- 1 activates female somatic gene expression and/or represses male somatic gene expression (see Section V). The tru-3 gene is the last of the global sex-determining genes remaining to be positioned in the molecular model for sex determination (Hodgkin and Brenner, 1977; Hodgkin, 1986). Genetically, tru-3 is postulated to function as a coactivator of tru-2, because strong tru-2(gf) feminizing mutations can partially overcome the requirement for wild-type tru-3 activity (Fig. 2) (Hodgkin and Brenner, 1977; Hodgkin, 1986; Doniach, 1986). The TRA-3 protein is a member of the calpain family of neutral cysteine proteases (Barnes and Hodgkin, 1996). Experiments support the notion that TRA-3 has proteolytic activity; site-directed change of an invariant cysteine found in the TRA-3 catalytic domain to a serine abolishes the ability of a tru-3 transgene to rescue tru-3 null mutants (S. Sokol and P.Kuwabara, unpublished). TRA-3 might promote female development either by cleaving and activating a protein involved in female development or by repressing a protein involved in male development.

V. TRA-1 Targets and the Conservation of Sex- Determining Mechanisms What are the downstream transcriptional targets of TRA- 1 that execute sexual cell fate decisions? Genetically, tru-I promotes female development either by activating the expression of female-specific genes, such as yolk proteins (vitellogenins), or by repressing the expression of male-specific genes (Fig. 2). The mub-3 gene (for male abnormal) has been the most promising candidate for a downstream target for TRA- 1 because of its specific role in promoting male development (Shen and Hodgkin, 1988). Mutations in mub-3 lead to inappropriate expression of yolk proteins by the intestine of XO males and to defects in the differentiation of sense organs in the male tail. Surprisingly, mab-3 encodes a protein with sequence similarity to Drosophilu doublesex (dsx), a DNA binding protein that plays a key role in the regulatory pathway of Drosophilu sex determination (Burtis and Baker, 1989; Raymond et ul., 1998). MAB-3 contains two copies of the DNA binding motif that is found in DSX; this motif has been named DM. Not only do MAB-3 and DSX share structural similarities, but they also appear to have conserved function (Raymond er ul., 1998). The dsx gene pro-

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duces two sex-specific alternatively spliced transcripts that encode proteins DSXF and DSXM with alternative carboxy termini. DSXM controls sex-specific neuroblast formation and yolk protein gene expression, similar to the role of MAB-3 in worms. Transgenic expression of DSXM, but not DSXF, partially restored the formation of male tail sensory organs in XO mab-3 mutants. The conservation of two proteins involved in controlling sexual cell fate across phyla has been hitherto unprecedented. Additional downstream targets of TRA- 1 may possibly be identified by taking advantage of the C. elegans sequence, which will be > 95% complete in 1998 (Waterston and Sulston, 1995). The TRA-I protein has zinc finger motifs that preferentially bind a nonamer sequence (TGGGT/AGGTC), similar to that identified for the human GLI protein; TRA-I is a homologue of GLI and GLI3 (Ruppert et al., 1990; Zarkower and Hodgkin, 1992, 1993). Therefore, TRA-1 binding targets would be predicted to carry one or more copies of the TRA-1 binding domain in their promoters. Although a number of candidate genes with such properties have been identified by the C. elegans genome sequencing consortium, appropriate tests have not yet been performed to examine whether these genes are regulated by TRA- 1.

VI. How to Count Chromosomes: The X:A Ratio Revisited The demonstration that the X:A ratio is the primary determinant of sex was one of the earliest contributions to our understanding of C. elegans sex determination. However, the identification of “counting” elements has been more refractory to analysis. In partial explanation, duplications or deficiencies harboring numerator elements may not have been recovered because many of the pre-existing X chromosome duplications and deletions were isolated on the basis of their viability in XX and XO animals. Chromosomal duplications that increase the number of numerator elements would be predicted to cause XO-specific lethality, and simirly, deletions that lower the dose of numerator elements might cause XX-specific lethality. A greater understanding of the coordinated control of sex determination and dosage compensation has led to the development of strategies that allow the recovery of deletions and duplications that might otherwise be detrimental because they result in the implementation of an inappropriate mode of dosage compensation (Akerib and Meyer, 1994; Hodgkin et al., 1994; Hodgkin and Albertson, 1995). For example, duplication of X chromosome numerator elements that are feminizing and lethal to XO animals can be suppressed by mutations in an sdc or dosage compensation dpy gene or by constitutive expression of x o l - 1 (Fig. 2). Conversely, deletions that remove numerator elements, and would be predicted to be lethal and masculinizing to XX animals, can be suppressed by an x o l - 1 mutation, which locks animals into the XX mode of dosage compensation and sex determination (Akerib and Meyer, 1994).

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Using the strategies described previously, the left end of the X chromosome was found to harbor potential numerator elements; duplications of this region had a detrimental effect on XO viability, which could be suppressed by a mutation in a dosage compensation dpy gene (Akerib and Meyer, 1994; Hodgkin et al., 1994; Hodgkin and Albertson, 1995). It was further found that the region of the X chromosome harboring these numerator elements could be subdivided into three regions, each containing numerator activity (Akerib and Meyer, 1994). Duplications covering two or more X chromosome subregions revealed that the combination of numerator elements increased XO lethality synergistically. On the other hand, removal of a single dose of all of the left-hand X chromosome numerator elements produced only a weak Sdc phenotype (slightly masculinized and dumpy), indicating that XX animals may better tolerate increases in X-linked expression than XO animals can tolerate decreases (Akerib and Meyer, 1994). Taken together, these studies indicated that numerator elements are likely to be discrete elements and that there are probably only a limited number of these elements. Molecular identification of one numerator element was aided by comparing two duplications, eDp26 and mnDp73, which differ in XO lethality because mnDp73 carries only the two weak regions at the left end of the X, whereas eDp26 carries an additional strong region. The region of the X chromosome unique to eDp26, and not mnDp73, was delineated by fluorescent in situ hybridization (Hodgkin et al., 1994). A small number of overlapping cosmids in this region, when introduced as multicopy transgenes, were found to mimic the XOspecific lethality and feminization produced by eDp26, and a likely candidate gene with potential numerator activity was identified, fox-1 (feminizing on X ) . the fox-I region encodes a putative RNA binding protein, suggesting that it may function as a transcriptional or post-transcriptional regulator of xol-I (Hodgkin et al., 1994). Subsequent genetic screens to identify X-linked suppressors of the XO lethality associated with duplications of the left region of the X chromosome identified two point mutations in thefox-l gene (Nicoll et al., 1997). Therefore, fox-I mutations function in a dose-dependent manner to suppress the effect of X chromosome duplications that are lethal to XO animals; however, XX animals deleted for thefox-l gene are fully viable, indicating thatfix-l is not an essential gene and the other signals participate in the counting process (Nicoll et al., 1997; M. Skipper and J. Hodgkin, personal communication). To explore the notion that fox-l directly controls the regulatory switch gene xol-I, the expression of an integrated lacZ reporter driven by the xol-1 promoter, Pxol-l::lacZ, was examined in fox-I mutants (Nicoll et al., 1997). Integrated copies of the Pxol-1::lacZ reporter transgene behave appropriately by sex; high levels of reporter activity are detected in XO animals and low levels in XX animals (Mind et al., 1995). It was found that FOX-1 does not directly regulate the transcription of xol-1, because the Pxol-1::lacZ reporter remains repressed in

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XX fox-1 mutants. However, when transgenic animals carrying a bifunctional reporter composed of xol-I genomic sequences fused in frame to GFP (green fluorescent protein) were exposed to high levels of transgenic FOX- 1, both the reporter and the rescuing activity of the construct were repressed. This result suggests that FOX- 1 regulates xol-1 post-transcriptionally (Nicoll et al., 1997). The previous report that the xol-1 locus expresses multiple transcripts raises the possibility that FOX-1 regulates the alternative splicing of xol-1 (Rhind et al., 1995). Therefore, X chromosome numerator elements appear to function by regulating xol- I activity at both the transcriptional and post-transcriptional levels. At the present time, denominator or autosomal counting elements have not been identified. One might surmise that if the denominator were a single gene, then screens for XO feminizing mutants would have already identified such an element. However, if the denominator is polygenic or is not a chromosomal entity per se (e.g., the nuclear volume occupied by all chromosomes), then simple genetic screens may not easily identify such elements. Future screens for polygenic autosomal elements might take advantage of using animals poised at an ambiguous X:A sex ratio ( e g , 2X:3A) to identify denominator elements that are not sufficiently strong, by themselves, to shift the X:A ratio of diploids toward the hermaphrodite fate.

VII. Analysis of Germ-Line Sex Determination The global sex-determining genes (her-I, tra-1,2,3, and fern-1,2,3) affect not only somatic sex but also germ-line sex (Table 11). However, there are several important differences between the pathways controlling sex determination in the soma and in the germ line (Fig. 4). First, although tra-1 is involved in germ-line sex determination, it is not the terminal regulator of cell fate in the germ line (Hodgkin, 1987a; Schedl et al., 1989). Doubly mutantfern;tra-1 animals have a male soma, but they produce oocytes, indicating that tra-1is not directly required for specifying female fate in the germ line. Second, additional rnog andfog genes have been identified as tissue-specific regulators of germ cell fate (Tables I and 11).These genes regulate the specification of oocytes and sperm, respectively, but play no apparent role in somatic sex determination Third, the fern genes, fog-Z, and fog-3 are the terminal regulators of sex in the germ line (Barton and Kimble, 1990; Ellis and Kimble, 1995). These genes are absolutely required for spermatogenesis to occur in XX and XO animals, and their absence results in the specification of all germ cells as oocytes (Fig. 4). Finally, several of the global regulators of sexual cell fate have germ-line-specific control regions that are involved in restricting their activities during the hermaphrodite sperm-oocyte decision.

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Fig. 4 Model for the control of germ-line sex determination. Sexual cell fate decisions in the germ line are controlled by global sex-determiding genes and by tissue-specific regulators, such as the nzog and fog genes (Table 11). Unlike somatic sex determination, tru-1is not the terminal regulator of germ cell sexual fate. Although fru- / is involved in germ-line sex determination, its specific role is unclear (Hodgkin, 1987a; Schedl etul., 1989). The three fern genes,fog-1, and fog-3 are the terminal regulators of germ cell fate and are essential for specifying spermatogenesis in XO males and XX hermaphrodites (Barton and Kimble, 1990; Ellis and Kimble, 1995). The absence of any one of these genes results in all germ cells developing as oocytes. In XO males, her-1 is active and thus represses the activity of tra-2 so that the fern genes are active and only sperm are produced (left). However, in an XX hermaphrodite, first sperm and then oocytes are produced (right).The tru-2 and fern-3 genes are the key regulators controlling the sperm-oocyte decision and their activity state (off-on) during each decision is indicated in the following. The analysis oftra-2 germ-line feminizing mutations suggests that there are two controls that repress rru-2 activity to allow the onset of hermaphrodite spermatogenesis: one control involves translational regulation of tru-2, which is mediated through the 3’ UTR and may involve / u f l (Goodwin e f ul., 1993). and the second may involve a protein-protein interaction between TRA-2A and FOG-2 (Schedl and Kimble, 1988; P.E. Kuwabara, P. G. Okkema, and J . Kimble, unpublished). Repression of fern-3 appears to be the primary control that allows the hermaphrodite germ line to switch from spermatogenesis to oogenesis (Barton et ul., 1987).Thejbfgenes are likely to encode proteins that bind Lo the,fern-3 3’ UTR and possibly function as translational repressors of ,fern-3 activity in the germ line (Ahringer and Kimble, 1991; Zhang et ul., 1997). The rnog genes may encode proteins that also help to regulate the switch (Graham and Kimble, 1993; Graham et ul., 1993).

VIII. The Hermaphrodite Sperm-Oocyte Decision A. Anatomy of the Somatic Gonad

The hermaphrodite has a bilobed tubular somatic gonad that meets centrally at the vulva (proximal opening for egg-laying). A pool of mitotically proliferating germ cells are maintained at the distal end of the gonad; a somatic cell named the distal tip cell (DTC) plays a key role in maintaining this pool of proliferating germ cells (Fig. 1). Laser ablation of the DTC results in all germ cells entering the meiotic pathway; this phenotype is mimicked by mutations in the glp-1 gene (for germ-line proliferation) (Austin and Kimble, 1987). Molecular analyses reveal that the maintenance of mitotically proliferating germ cells is dependent

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on cell-to-cell signaling mediated by the GLP- 1 receptor in the germ line and the LAG-2 ligand, which is expressed by the DTC (Henderson et a!., 1994). GLP- 1 is a transmembrane protein related to the C. elegans LIN-12 and Drosophila Notch receptors. LAG-2 is a membrane protein with sequence similarity to Drosophilu delta, which encodes a ligand for Notch. In each arm of the hermaphrodite gonad, the first 160 germ cells to enter meiosis become sperm and the remaining germ cells develop as oocytes [for a review, see Schedl (1 995)]. Therefore, the total number of self-progeny produced by a hermaphrodite is limited by the total number of sperm produced. Hermaphrodite spermatogenesis requires the activity of the male-promotingfern and fog-I and fog-3 genes in an essentially female animal. Subsequently, activity from these five genes must be negatively regulated to allow the germ line to switch and produce oocytes (Barton and Kimble, 1990; Ellis and Kimble, 1995). How is this exquisite regulation of germ cell fate specification achieved? The first clues came from the identification of mutations in global sex-determining genes that affect only the determination of germ cell fate and not somatic cell fate. XX tra-2(gf) and tra-2(mx) mutants produce only oocytes, but they can be crossfertilized; these mutants appear to be defective in allowing the onset of spermatogenesis (Table 11) Doniach, 1986; Schedl and Kimble, 1988). mfem-3(gf) mutants are sterile because they produce sperm, but not oocytes; these mutants appear to be defective in switching from spermatogenesis to oogenesis (Barton et al., 1987). Neither thefern-S(gf) or tra-2(mx) mutant shows somatic sex determination phenotypes; however, strong tra-2(gf) alleles partially feminize XO males (Doniach, 1986; Schedl and Kimble, 1988).

B. Germ-Line-Specific Controls Regulate the Sperm-Oocyte Decision 1. Germ-Line Regulation of tru-2 The molecular analysis of mutant alleles that specifically affect the regulation of tra-2 and fern-3 activity in the hermaphrodite germ line indicated that posttranscriptional regulation mediated through 3' untranslated regions (UTRs) and protein-protein interactions are important in controlling the hermaphrodite sperm-oocyte decision. The two classes of tru-2 germ-line-feminizing mutants identify two distinct controls that regulate the onset of hermaphrodite spermatogenesis (Table 11). The six tru-2(gf) mutations all disrupt at least one copy of a 28-bp direct repeat element (DRE) found in the tra-2 3' UTR (Goodwin ef af., 1993). The strongest tru-2(gf) mutation, e2020, is a 108-bp deletion that removes both copies of the DRE; tru-2(e2020gf) also feminizes the intestine (yolk, expression) and germ line (sperm and oocytes) of XO males. Northern blot analysis indicates that the steady-state level of tru-2 mRNAs expressed in tra-2(gf) mutants is not significantly elevated compared to wild-type animals. Two lines of evidence suggest that the tra-2(gf) mutants identify a binding site for a factor that

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mediates translational regulation of tru-2 mRNA through its 3' UTR. First, polysome gradient analysis indicates that tru-2 mRNAs isolated from tru-2(gf) mutants are associated with larger polysomes than wild-type tru-2 mRNAs, and second, the activity of a lac2 reporter carrying the wild-type tru-2 3' UTR is repressed in the hermaphrodite germ line, whereas the same reporter carrying the tru-2(gf) 3' UTR is not. A candidate for the DRE binding factor is encoded by the luf-I (for lethal and feminizing) gene, which decreases expression of the lucZ::tru-2 3' UTR reporter (described earlier) in the germ line (Goodwin er ul., 1997). The molecular identity of luf-1 is as yet unknown. The tru-2(mx) mutants feminize the germ line of XX hermaphrodites; however, unlike strong tru-2(gf) mutants, none of the tru-2(mx) mutants feminize XO males (Doniach, 1986; Schedl and Kimble, 1988). The tru-2(m) mutations are point mutations that result in nonconservative amino acid changes in a 22-amino acid region, named the MX domain (P. E. Kuwabara, P. G. Okkema, and J. Kimble, unpublished). The MX domain is likely to identify a domain involved in a regulatory protein-protein interaction. Genetically, her-1 and fog-2 are candidate repressors of tru-2 activity (Tables I and 11) (Hodgkin, 1980; Trent et d., 1988; Schedl and Kimble, 1988). However, XX her-I mutants develop as hermaphrodites, not females, and therefore do not affect the sperm-oocyte decision. In contrast, fog-2 mutations mimic the phenotype of tru-2(mx) mutations; fog-2 mutations transform XX animals into females, but have no effect on XO sex determination (Schedl and Kimble, 1988). Therefore, fog-2 may encode a candidate MX binding factor. fog-2 has been cloned and shown to encode a novel protein (R. Clifford and T. Schedl, personal communication). Although both controls of tru-2 germ-line activity affect the onset of hermaphrodite spermatogenesis, it has not been established that translational control mediated by the 3' UTR normally plays a direct role in allowing the onset of hermaphrodite spermatogenesis (Fig. 4). It could be argued that, because the tru-2(gf) mutants also affect XO males, translational control of tru-2 plays a more general role in maintaining TRA-2A protein at low levels. Otherwise, if TRA-2A expression is too high, its activity cannot be post-translationally regulated. 2. Germ-Line Regulation of f e r n 3 fern-3(gf) mutants produce excess sperm and do not undergo the switch to oogenesis (Fig. 4) (Barton et ul., 1987). Seventeen fern-j(gf) mutations alter and one deletes a 5-nt sequence from the fern-3 3' UTR, named the PME (point mutation element) (Ahringer and Kimble, 1991). This sequence is likely to identify a binding site for a negative regulator of fern-3 germ-line activity. To support this notion, it was shown that transgenic expression of the fern-3 3' UTR appears to titrate a trans-acting factor and masculinize the XX germ line (Ahringer and Kimble, 1991). To identify a protein that specifically interacts with thefern-3 PME, an elegant series of experiments was performed using a modified yeast 3-hybrid system to detect RNA binding proteins (SenGupta et ul., 1996;

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Zhang et al., 1997). A single cDNA-activation domain clone was found that interacted specifically with the wild-type fern-3 PME, but not a mutated PME from afern-3(q96gf) mutant. When this cDNA clone was used as a hybridization probe, two distinct genes,fbf-1 and,fbf-2, were identified (fern-3 binding factor), encoding proteins that are 9 I % identical in amino acid sequence. Although lossof-function mutations forfbf-l andpf-2 have not yet been identified, RNAi (Guo and Kemphues, 1995) was used to demonstrate that Jbf-l(RNAi) orJbf-2(RNAi) mimics the .fern-3(gf) mutant phenotype, which would be expected of a fern-3 germ-line repressor (Zhang et al., 1997). Therefore, FBF-I and FBF-2 are excellent candidates for the fern-3 germ-line repressor. It was further shown that FBF-I and FBF-2 each contains eight repeats that are similar to those found in Drosophila Pumilio (Barker et al., 1992: Macdonald, 1992), a protein that binds to nanos-response elements in the 3’ UTR of hunchback (hb) mRNA and causes translational repression and instability of hb mRNA. Taken together, these observations suggest that FBF may be responsible for regulating the hermaphrodite sperm-oocyte switch by mediating translational repression of fern-3 through its 3’ UTR. Six mog genes also have been identified that display a phenotype similar to that offern-jfgf) mutants and may be involved in regulating fern-3 germ-line repression (Graham and Kimble, 1993; Graham et al., 1993). The rnog genes may also play a more general developmental role because the progeny of animals carrying a feminizing mutation in combination with a rnog mutation dies as embryos. What controls the timing of the sperm-oocyte switch? The relative invariance in wild-type hermaphrodite brood size, 330 +- 34, which is also an indicator of total sperm number, (Hodgkin et al., 1979), suggests that there is likely to be a timing or counting mechanism that determines when the switch occurs. Although the answer is not yet known, it may be found that FBF is responsible for regulating the switch (Zhang etal., 1997) and that factors regulating the availability or activity of FBF are responsible for controlling the timing of the switch.

IX. Phylogenetic Comparisons and the Evolution of Sex-Determining Genes It has been generally observed that mechanisms controlling sexual fate are not conserved across phyla. Therefore, it is surprising to find that the C. elegans rnab-3 and Drosophilu dsx sex-determining genes appear to be conserved in both structure and function (Raymond et al., 1998). It will be of interest to determine whether additional sex-determining regulatory genes can be identified that show a similar conservation across phyla or whether this is an example of convergent evolution whereby transcriptional regulation by DM domain proteins is used to control sexual fate in different phyla. Otherwise, phylogenetic comparisons of the sequences between C. elegans and its closely related hermaphroditic relative Caenorhabditis briggsae indicate that the sex-determining genes are the most rapidly evolving of all

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genes examined so far in the genus Caenorhabditis (de Bono and Hodgkin, 1996; Kuwabara, 1996b). The TRA-I and TRA-2A proteins share less than 49% identity with their C. briggsae homologues. Because both C. elegans and C. briggsae are hermaphroditic, identification of FEM-3 and TRA-2A homologues from closely related male-female species (e.g., Caenorhabditis remanei) will provide valuable comparative information about the evolution of regulatory mechanisms, especially in regard to the control of germ-line sex determination. A C. briggsae Ce-HER- 1 homologue has not yet been identified, but the Brugia Genome Project has identified a homologue from the filarial parasite Brugia malayi through an EST sequencing project. Thus, for the purposes of phylogenetic comparison, it also will be of interest to determine whether other homologues of C. elegans sex-determining genes will be identified in B. malayi. The C. elegans sex determination pathway makes use of control mechanisms common to many organisms, such as transcriptional and translational regulation. However, unlike the RASIMAP kinase cascade, which has been strongly conserved across phyla, the same is not true of the signal transduction pathway controlling sexual cell fate in C. elegans. Is this signal transduction pathway unique to the control of sex determination in C. elegans? The simple answer is that it may be too early to know. Vertebrate homologues have been identified for TRA-I (Kinzler et al., 1988; Ruppert et al., 1990), TRA-3 (Dear et al., 1997), FEM-1 (J. Hodgkin, personal communication), and FEM-2 (Wenk et al., 1992), but their functions have not been fully characterized. In time, vertebrate homologues may also be identified for HER-1, TRA-2A, and FEM-3. Several similarities have been noted between the genes and the mechanisms controlling C. elegans sex determination and Hedgehog (Hh)-Patched (Ptc) positional signaling. TRA-2A shares marginal sequence similarity and a hydropathy profile similar to that of Ptc (Nakano et al., 1989; Hooper and Scott, 1989; Kuwabara et al., 1992). Both are membrane proteins with multiple membranespanning domains. In addition, TRA-2A and Ptc each are repressed through ligand binding, although there is no obvious sequence similarity between HER- 1 and Hh (Perry et al., 1993; Mohler and Vani, 1992). tra-2 mRNA levels also are positively regulated by TRA-I (Okkema and Kimble, 1991), and ptc mRNA levels similarly are regulated by the Drosophila Cubitus interruptus protein (Orenic et al., 1990; Alexandre et al., 1996), a homologue of TRA-1 and vertebrate GLI. Because of these similarities, it is speculated that the C. elegans sexdetermining pathway originally may be derived from the Hh/Ptc pathway.

X. Unresolved Questions A. Why So Many Genes? One question that is often raised in regard to the C. elegans sex determination pathway is the following: Why are there so many genes, especially when somatic

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sexual fate ultimately is determined simply by tru-l? It probably will be found that the number of C. eleguns sex-determining genes is not unusual, but instead is a measure of how extensively the pathway has been analyzed at the genetic and molecular levels. One of the advantages to having a gene cascade regulating sexual cell fate is that this allows multiple levels of control to be imposed at different points in the pathway, with the consequence that sexual cell fate decisions can be either refined, restricted, or amplified. This point can be illustrated by the control of the sperm-oocyte decision in the hermaphrodite germ line. The imposition of tissue-specific controls regulating the germ-line, but not the somatic, activities of tru-2 and fern-3 is responsible for allowing the onset of hermaphrodite spermatogenesis and the switch to oogenesis, respectively. Additional levels of control also can be achieved through feedback regulation, which can amplify or help to maintain a commitment to a specific cell fate.

B. Dosage Compensation in the Germ line?

It is not known whether there is a need for dosage compensation in the germ line. Antibody detection of DPY-27 and DPY-26 proteins indicates that the dosage compensation complex does not appear to assemble until the 30-cell stage of embryogenesis (Chuang et ul., 1994). However, it has also been observed that mutations in the dosage compensation dpy-21, -26, or -28 genes greatly ameliorate the fertility of XO her-1 and XO tru-2(eg) hermaphrodites (Hodgkin, 1983b; Kuwabara, I996a). Dosage compensation would not be expected to be active in XO animals; therefore, mutations in the dosage compensation genes should not affect XO development or fertility. It remains to be determined whether there is a feedback mechanism controlling dosage compensation, which is detected when the XO germ line is feminized (Kuwabara, 1996a).

C. Parallel Pathways and Feedback Regulation

The genes controlling C. elegans sex determination have been ordered in a linear pathway on the basis of epistatic interactions; however, this linear organization does not preclude the existence of parallel regulatory pathways or feedback regulation. A linear pathway does not explain why null mutations in tru-2 or tru-3, unlike mutations in tru-1, fail to transform XX animals into complete males. This observation indicates that there is residual tru-1 activity in XX tru-2 or tru-3 mutants; genetic arguments suggest that the element controlling this tru-l activity resides upstream of her-1. It has also been shown that a xol-1 mutation in combination with a tru-2 or tru-3 mutation has the apparently paradoxical effect of transforming (masculinizing) XX animals into complete mating males, whereas the same xol-1 mutation is lethal and feminizing to XO animals.

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A potential feedback loop involving the transcriptional regulation of tra-2 mRNAs by TRA-1 also has been documented. It was shown that, in wild-type animals, XX hermaphrodites have 10-fold higher tru-2 mRNA steady-state levels than XO males (Okkema and Kimble, 1991). This difference in tra-2 mRNA levels appears to be a consequence of phenotypic sex and not chromosomal sex dictated by the X:A ratio. For example, XO fern-3 females have levels of tra-2 mRNA similar to those found in XX hermaphrodites and not XO males; tra-1 activity appears to positively regulate tra-2 mRNA steady-state levels. The consequence of this potential feedback may be to ensure the maintenance of a sexual cell fate decision. Additional feedback also appears to be operating at an early point in the pathway that was revealed by various interactions between dosage compensation and sex determination, as described in Section 1I.B. Exploration of these effects probably will require a more detailed understanding of the coordinate regulators of sex and dosage compensation, in particular, the SDC-2 protein, which may be a key player in these events.

XI. Future Perspectives The potential for identifying novel genes involved in the control of sex determination using standard genetic screens has probably been exhausted. However, as more information is gained about the mechanisms regulating sex determination, genetic selection strategies are being designed to obtain genes that might not have been identified previously because of their pleiotropic or deleterious effects on development (e.g., numerator elements) (Akerib and Meyer, 1994; Hodgkin and Albertson, 1995). In addition, the development of new techniques, such as the yeast 3-hybrid system, promises to help identify components responsible for mediating post-transcriptional and translational control (SenGupta et al., 1996; Zhang et al., 1997). In 1998, the sequence of the entire C. elegans genome will be completed (Waterston and Sulston, 1995). The availability of the entire genome sequence offers unique opportunities for identifying new components of the sex determination pathway (Kuwabara, 1997). In C. elegans, the potential loss-of-function mutant phenotype associated with a genomic sequence of unknown activity can be deduced quickly by antisense injection (RNAi). RNAi should prove to be particularly valuable in identifying genes with redundant activities, which might not be easily obtained as single mutants from genetic screens (e.g., fbf-1 and fbf-2; Zhang et al., 1997). In addition, potential TRA-1 targets, which have a TRA-I consensus binding sequence, can be analyzed by RNAi to determine whether they show sex-determining phenotypes. The genome sequence also can be used to identify genes with sex-specific expression. For example, the Bargmann lab fused the promoters of predicted serpentine receptor genes to a GFP

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reporter and observed that some constructs were expressed only in subsets of neurons in a sex-specific manner (Troemel et al., 1995). These screens have the potential for identifying genes downstream of tru-1 that execute cell fate decisions affecting the sex-specific behavior and cell biology of worms. Finally, microchips carrying arrays of C. elegans genomic sequence will provide powerful tools for identifying genes that are expressed differentially between the two sexes (Lashkari et ul., 1997). The sensitivity of this technique will make it possible to detect even subtle differences in gene expression.

Acknowledgments I thank Bob Clifford, Jonathan Hodgkin, Magdalena Skipper, and Tim Schedl for sharing unpublished data and Jonathan Hodgkin for helpful comments on the manuscript. I am also grateful to Jonathan Hodgkin, Judith Kimble, and Tim Schedl for their continued support over the years.

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5 Petal and Stamen Development Vivian F. Irish Department of Molecular, Cellular and Developmental Biology Yale University New Haven, Connecticut 06520

1. Introduction 11. Petal and Stamen Ontogeny Ill. Genes Controlling the Specification of Petal and Stamen Identities IV. Differentiation of Petals A. Petal Shape B. Petal Epidermal Cell Differentiation V. Differentiation of Stamens VI. Coordination of Gene Expression and Tissue Differentiation VII. Summary References

1. Introduction Flowers are composed of four types of floral organs: sepals, petals, stamens, and carpels (Fig. 1). These organs each have different roles in assuring the reproductive success of the plant. Sepals enclose the developing flower bud and are thought to play a protective role. Sepals generally are green and photosynthetic and often resemble foliage leaves. Petals often are the most conspicuous organs of the flower. In many species, the large, brightly colored petals are thought to have evolved to maximize visual and olfactory signals to attract pollinators. The male reproductive structures, the stamens, are composed of an elongated filament that bears the anther, in which the pollen grains containing the male gametes develop. Carpels, the female reproductive structures, can arise separately or can be fused to form a gynoecium, which contains the ovules. It is clear that each floral organ type is morphologically distinct. What is the rationale, then, for considering the development of petals and stamens together? The floral organs, while serving different functions, have all been hypothesized to be derived from modifications of a lateral leaflike organ (Weberling, 1989). Goethe, in 1790, was one of the first to articulate this hypothesis, proposing that plants are composed of a series of similar organs that undergo a progressive metamorphosis (Goethe, 1790). Goethe theorized that this metamorphosis was currvnr lopic., m I)evel~~pme,zrd €li<,log.", VOl. 41 Copyright D 1999 by Academic Prcss. All rights of reproductinn in any form reserved. 0070-2153/99 $25.00

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gynoecium ovule

I

Fig. 1 The angiosperm flower. Cutaway diagram showing the position of the floral organs. The stamen is the male reproductive organ and is composed of an elongated filament topped by the anther in which the pollen grains develop. The gynoecium, or female reproductive structure, contains the developing ovules. The reproductive organs are surrounded by the petals and sepals.

brought about by the action of a sap that became refined as it progressed up the plant, culminating in the successive development of the different types of floral organs. More recent phylogenetic analyses have refined this view and suggested that while carpels and stamens appear to have each evolved prior to the origin of the angiosperms, petals and sepals may have been derived more recently, and in particular, petals are thought to have been derived from other floral organs (Cronquist, 1988; Takhtajan, 1991; Drinnan et al., 1994; Endress, 1994). In the majority of angiosperm groups, including all of the higher eudicots and monocots, petals are thought to be derived from stamens, a condition termed andropetaly. Among the lower eudicot and magnolid dicot families, there are species that contain andropetals, as well as species in which petals appear to have been derived from sepals or other leaflike organs, termed bracteopetaly. The predominance of andropetaly in the angiosperms suggests that, in the majority of angiosperm species, the developmental programs controlling the specification of petal and stamen identities are similar and evolutionarily linked. Almost all of the experimental work on flower development that has been carried out has involved higher eudicot species, including Arabidopsis, Antirrhinum (snapdragon), Nicotiana (tobacco), and Petunia. The emerging genetic and molecular evidence indicates that there are several genes that are uniquely required for the development of both petals and stamens in these species, supporting the idea that a common molecular mechanism is involved in the specification of these two organ types. Nonetheless, the petal and stamen primordia differentiate into anatomically and functionally very different structures. In this chapter, I will discuss the similar strategies by which petal and stamen identities

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are established in the higher eudicots, as well as some of the genes involved in specifying terminally differentiated cell types characteristic of each organ. The differentiation of particular cell types in the flower also depends on intercellular interactions. Because the ultimate fate of a plant cell depends on its final position and not its lineage, it is clear that cells must be signaling to each other to coordinate their development and differentiation (Huala and Sussex, 1993; Irish, 1993). In addition, cell fates can be respecified until their final division, indicating that intercellular interactions must be occurring throughout development. Investigations of flower development, in particular, have been instrumental in beginning to elucidate some of the underlying molecular mechanisms by which plant cells signal to each other. Several lines of evidence indicate that such intercellular interactions are critical for petal and stamen development and will be reviewed herein.

11. Petal and Stamen Ontogeny Petals and stamens arise from cells derived from the florally determined shoot apical meristem. The floral meristem of dicots and some monocots is organized into three cell layers, which generate the tissues of the flower (Fig. 2 ) . The Ll, or

A

i""".;((I:: primordium

side view

top view

C

Fig. 2 Meristem signaling possibilities. (A) The three-layered organization of the meristem is apparent in longitudinal section (left). Cells in the LI and L2 layers divide to maintain their layered organization, whereas cells in the L3 layer can divide in any plane. At the right is a top view of the meristem, showing a whorl of four sepal primordia developing on the flanks of the meristem. (B-D) Dark arrows show possible inductive interactions between whorls of developing primordia (B), between developing organs within a whorl (C), and between cell layers (D).

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outer, layer of meristematic cells gives rise to the epidermis, whereas the underlying L2 and L3 cells contribute to the mesophyll and vascular tissues of the flower (Satina and Blakeslee, 1941; Steeves and Sussex, 1989). Cells in all three cell layers of the flanks of the floral meristem change their patterns of growth and division to form the floral organ primordia. In general, the floral organs are initiated sequentially, with sepals arising first, followed by the almost simultaneous appearance of petal and stamen primordia, and finally the carpel primordia become apparent (Sattler, 1973; Steeves and Sussex, 1989). Histological and lineage analyses carried out in Arubidopsis have given us a good picture of the ontogeny of petals and stamens. Arubidopsis, like other crucifers, has four petals and six stamens. The four petal and the four medial stamen primordia arise nearly simultaneously, and the two lateral stamen primordia become apparent slightly later in development (Hill and Lord, 1989; Smyth et al., 1990). The cell division patterns that take place during the development of these organ types are distinct from the outset (Crone and Lord, 1994). The petal primordia initially enlarge primarily by expansion and division of cells in the L2 layer, whereas divisions in both the L2 and L3 layers are associated with the more rapid early growth in the stamen primordia (Crone and Lord, 1994). These patterns of divisions can be different in other species, although in general the patterns of cell divisions in the petal and stamen primordia are distinct from each other (Satina and Blakeslee, 1941; Dengler, 1972; Hicks, 1973). Although all three cell layers contribute to the development of the petals and stamens, the contribution of each cell layer can be somewhat variable (Satina and Blakeslee, 1941; Tilney-Bassett, 1986; Dawe and Freeling, 1990; Goldberg et al., 1993; Bossinger and Smyth, 1996; Bouhidel and Irish, 1996; Furner, 1996). Marked cell lineages have been used to show that each cell layer gives rise to different extents of an organ (Bossinger and Smyth, 1996; Bouhidel and Irish, 1996 Furner, 1996). Furthermore, a cell in one layer can invade another layer and differentiate according to its new position, and such invasion events appear to occur stochastically (Klekowski, 1988; Furner, 1996). These observations raise several questions about how floral organ primordia are specified. Because subsets of meristematic cells proliferate in specific regions on the flanks of the meristem, some mechanism must be acting to define regions of primordial outgrowth. Furthermore, these regions are bounded by cells that apparently do not proliferate to the same extent. Within a given species, the number of floral organ primordia is almost always constant, indicating that the processes defining organ primordia must be tightly regulated. Despite this orderly pattern of growth, the occurrence of cell layer invasion events indicates that floral primordial cells are not committed to a particular developmental fate. In turn, this suggests that intercellular interactions must be taking place to coordinate the cell division and growth events required for appropriate organ development. Several types of such spatially regulated signaling events have been proposed to explain the regular pattern of organ primordium initiation, as well as how the

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development of cells within a floral organ primordium is coordinated (Fig. 2). These include signals between whorls of organ primordia, signaling between organ primordia within a whorl, and signaling between the cell layers of a developing organ. In addition to spatial control of signaling, it appears that such inductive interactions may be regulated temporally as well. The results of experiments in which meristems have been manipulated surgically have been interpreted to suggest that signals between whorls are necessary for the development of the floral organ primordia (Wardlaw, 1957; McHughen, 1980). Researchers have suggested that inductive signals from one whorl are required to promote the formation of the next whorl of organ types. For instance the incipient sepal primordia were postulated to induce the formation of the second whorl petal primordia (Wardlaw, 1957). These models, however, did not clearly distinguish between the specification of an organ primordium in a distinct position and the specification of organ identity. It is clear from the existence of floral homeotic mutants, which can have organs of any identity in any position, that such inductive interactions are not required to specify particular organ identities. These types of interwhorl inductive signals may, however, operate to specify the position of the next whorl of organs (Wardlaw, 1957; Green, 1988). Furthermore, these models do not attempt to explain how individual organs arise in specific locations on the flanks of the meristem, nor have any corresponding signaling molecules been identified. Genetic ablation studies indicate, however, that if such interwhorl signals exist, they can only be operating at very early times in floral development (Day et al., 1995). Signaling between organs within a whorl may account for how the spacing of organ primordia in a particular whorl occurs (Day et al., 1995). Evidence supporting such intrawhorl signaling comes from experiments in which developing tobacco floral meristems were bisected. Depending on the time of surgery, halfmeristems could regenerate some or all of the missing organs, implying that the pre-existing organ primordia on the unoperated side of the half-apex were sufficient to induce the development of new organs on the operated side (Hicks and Sussex, 1971). Again, the nature of such an interwhorl signal has not yet been established. Both the interwhorl and intrdwhorl signals postulated to specify the inception of organ primordia in particular locations could be similar or identical to those signals that have been proposed to operate in the positioning of leaf primordia during vegetative development. Phase-specific (e.g., vegetative or floral) cues must be overlaid upon such a positional signaling mechanism in order to specify leaf or floral organ primordia. Within a floral organ, it does appear that intercellular signals operate to coordinate development. Periclinal chimeras, in which one cell layer is genotypically different from the other cell layers, have been used to demonstrate that interlayer signaling does occur within developing floral organ primordia. Camellia periclinal chimeras in which the L1 was derived from a species containing stamens and carpels, whereas the L2 and L3 were derived from a sterile line lacking stamens

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and carpels, develop normal stamens and carpels (Stewart et al., 1972). In tomato, meristem size depends, to a large extent, on the genotype of the L3 layer (Szymkowiak and Sussex, 1992). Specific genes also have been shown to play a role in such interlayer signaling. For instance, periclinal chimeras in which the cell layers differ only by the function of a single gene have been used to show that the product of the Antirrhinum FLO gene acts non-cell-autonomously, and its presence in just the L l layer of the flower can almost completely rescue the phenotype of otherwise genetically $0 mutant floral meristem cells (Hantke et al., 1995). These results indicate that there are likely to be inductive interactions between all layers of cells in the floral meristem, although the mechanisms by which such cues are disseminated are far from clear. It also is not clear whether there is a temporal window during which such interlayer interactions have to occur. What all of these studies point out is that the development of the floral organs appears to rely on a complex interplay between different cells. Inductive interactions may occur to specify the correct positioning of organ primordia, whereas within an organ intercellular interactions must be acting to coordinate the development of different tissue types. Ongoing molecular and genetic analyses are beginning to identify many of the genes required for various aspects of floral development. It seems clear that at least a subset of these genes will be involved in producing or responding to those signaling molecules that are required for organogenesis. The next sections will review the molecular and genetic analyses that have been carried out to identify genes involved in elaborating petal and stamen organ identities. In a few cases, the products of several of these genes have been implicated in the cell-cell signaling events required to coordinate the division and differentiation of cells in a developing organ primordium.

111. Genes Controlling the Specification of Petal

and Stamen Identities A number of mutations have been identified that cause homeotic transformations of the floral organs. In Arabidopsis, the APETALA3 (AP3) and PISTLLATA (PI) genes are required to specify petals and stamens. Mutations in either of these genes result in a homeotic conversion of petals into sepal-like structures, and stamens are converted into carpels (Bowman et al., 1989; Hill and Lord, 1989). The homologous genes in Antirrhinum, DEFICIENS (DEF) and GLOBOSA (GLO), not only are similar to their counterparts at the sequence level, but also are required to specify petal and stamen identities in this species (Sommer et al., 1990;Trobner et al., 1993).Homologues of AP3 and PI also have been identified in a number of other angiosperms, and at least within the higher eudicots, it appears that the functions of these genes in specifying petal and stamen identities are conserved (Irish and Kramer, 1998).

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On the basis of the phenotypes produced by several floral homeotic mutations, a combinatorial model (the ABC model) has been proposed to explain how floral organ identities are established (Bowman et al., 1991; Coen and Meyerowitz, 1991; Meyerowitz et al., 1991). Briefly, combinations of homeotic gene functions in different regions of the developing flower are thought to act to specify particular organ identities. These functions are proposed to correspond to the actions of particular sets of genes. In Arabidopsis, the two A group genes, APETALAI (API) and APETALA2 (AP2), are both required for sepal development. AP3 and PI, the B group genes, in combination with the A group genes act to specify petal identity, whereas the B group genes, in combination with the action of the C group gene, specify stamens. Lastly, the C group gene, AGAMOUS (AG), is required in the fourth whorl to specify carpel identity. In addition, the A and C functions are proposed to negatively regulate each other. The phenotypes produced by mutations in these floral homeotic genes, or in doubly mutant combinations, are largely consistent with the ABC model (Weigel and Meyerowitz, 1994). The AP3, PI, AG, and AP1 genes have been cloned, and they share homology with a family of yeast and animal genes that encode DNA binding transcription factors (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994). The 56-amino acid region of homology has been termed the MADS box domain and corresponds to the DNA binding domains of the yeast and mammalian gene products (Sommer et al., 1990). MADS box domain containing transcription factors binds to a consensus DNA sequence, CC(A/T),GG, which is called the CArG box (Norman et al., 1988; Passmore et al., 1988; Wynne and Treisman, 1992). Binding sites for several of the plant MADS box containing gene products have been defined in vitro, and these fit the known CArG box consensus motif (Schwarz-Sommer et al., 1992; Trobner et al., 1992; Huang et al., 1993, 1995, 1996; Shiraishi et al., 1993). These observations suggest that the plant MADS box gene products may act as transcriptional regulators. Among the plant MADS box containing genes, a second region of homology has been identified and called the K box, which may be required for protein-protein interactions (Ma et al., 1991; Pnueli et al., 1991; Krizek and Meyerowitz, 1996b). The AP2 gene also has been postulated to encode a transcription factor, because it contains two copies of a motif that is similar to the DNA binding domains of ethylene response element binding proteins (Jofuku et al., 1994; Weigel, 1995). In Arabidopsis, then, AP3 and PI are uniquely required for specifying petal and stamen identities and presumably act as transcription factors to regulate the expression of other petal- and stamen-specific genes. Although neither the AP3 nor PI protein can bind to DNA independently, AP3-PI heterodimers have been demonstrated to bind to CArG box sequences in vitro (Riechmann et al., 1996b; Hill et al., 1998; Tilly et al., 1998). Similarly, the products of the Antirrhinum

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DEF and GLO genes also bind to DNA as a heterodimer (Schwarz-Sommer et al., 1992). Several other lines of evidence suggest that AP3 and PI must interact with each other to mediate their proposed functions. AP3 and PI both need to present together in the same cell for AP3 and PI fusion proteins to be localized to the nucleus (McGonigle et al., 1996). Epitope-tagged AP3 protein can co-immunoprecipitate PI, and conversely, epitope-tagged PI can co-immunoprecipitate AP3 (Goto and Meyerowitz, 1994; Riechmann et al., 1996a). Finally DEF and GLO can interact in the yeast two-hybrid system, further supporting the contention that these classes of proteins bind to each other to effect their function (Davies et al., 1996b). The transcriptional expression patterns of the B group genes generally are consistent with their roles in specifying petal and stamen identity. AP3 transcripts are seen early in floral development in the meristematic domain that is thought to give rise to the petals and stamens (Jack et al., 1992). AP3 expression persists in the developing petal and stamen primordia until the flowers mature, with transcript levels declining at the time of fertilization (Jack et al., 1992). PI transcripts are also detectable early in floral development, but are initially found in the meristematic cells that will give rise to petal, stamen, and carpel primordia (Goto and Meyerowitz, 1994) As the organ primordia emerge, this pattern of expression becomes refined, with PI transcripts becoming limited to the developing petals and stamens (Goto and Meyerowitz, 1994). That the AP3 and PI expression patterns become coincident appears to reflect the fact that the AP3 and PI gene products are required for both auto- and crossregulatory interactions. A functional AP3 gene product is required to maintain both AP3 and PI transcription and similarly, PI is required to maintain AP3 and PI transcription (Jack et al., 1992; Goto and Meyerowitz, 1994). The coordinate expression of AP3 and PI together appears to be sufficient to confer petal or stamen identify in the flower, because ectopic expression of both AP3 and PI in the first whorl is sufficient to confer petal identity (Krizek and Meyerowitz, 1996a; McGonigle et al., 1996). Ectopic expression of AP3 in the fourth whorl, where PI is already expressed, results in the development of stamens (Jack et al., 1994). In the case of AP3, this maintenance of expression presumably is mediated in part by the binding of an AP3-PI heterodimeric transcription factor to at least three sequences in the AP3 promoter (Hill et al., 1998). In some other higher eudicot species, the expression patterns of the AP3 and PI homologues are reversed. For instance, in Antirrhinum, DEF, the orthologue of AP3, is expressed initially in the petal, stamen, and carpel primordia, whereas expression of the PI orthologue GLO is restricted to just the developing petals and stamens (Schwarz-Sommer et al., 1992; Trobner et al., 1992). A similar switch also has been seen in the expression of B group genes in the Solanaceae (Pnueli et al., 1991; Garcia-Maroto et al., 1993; Hansen et al., 1993; Davies et al., 1996a). Despite this switch in expression patterns, the functions of these genes appear to be similar to those of their Arabidopsis counterparts. This pre-

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sumably reflects the fact that the overlap in B group gene expression patterns, and consequently the expression domain that can be maintained by auto- and cross-regulatory mechanisms, is the critical factor in establishing petal and stamen identities. The restriction of AP3 gene expression to just floral tissues may also depend on epigenetic mechanisms. Mutations in the Arabidopsis CURLY LEAF (CLF) gene result in ectopic AP3 expression in the leaves (Goodrich et al., 1997). CLF encodes a product with similarity to the Drosophila Polycomb group of proteins, which have been proposed to regulate gene expression through modulating chromatin structure (Goodrich et al., 1997). Alterations in methylation status also have been implicated in AP3 regulation, because plants in which genornic cytosine methylation levels have been reduced display ectopic AP3 expression (Finnegan et al., 1996). In addition to maintaining B group gene expression within a developing organ primordium, some mechanism must ensure that the boundaries of B group gene expression are in register with whorl boundaries. It appears that these boundary conditions are established early in floral development. Lineage analyses in Antirrhinum have shown that a lineage restriction arises between whorls at the same stage that B group gene expression commences (Vincent et al., 1995). This observation suggests that either cells are dividing more slowly or they have altered planes of division at the whorl boundary, thus restricting the proliferation of cells into the adjacent domain. Although it is possible that the B group genes function to establish these boundaries, it seems unlikely because B group gene expression is just commencing at the time at which these lineage restrictions are being laid down. An alternative possibility is that another gene operates to set the whorl boundary, and this same gene also functions to delimit the boundary of B group gene expression. The product of the Antirrhinum FIMBRIATA (FIM) gene may be playing this role; FIM is expressed at whorl boundaries and is required for B group gene expression (Simon et al., 1994). The product of the FZM gene has been implicated in the control of cell division, because it can complex with proteins that have homology to yeast gene products required for protein degradation and cell cycle progression (Ingram et al., 1997). The Arabidopsis homologue of FIM, UNUSUAL FLORAL ORGANS (UFO) also has been postulated to regulate the domain of B group gene expression in a similar fashion (Ingram et al., 1995; Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995). At least part of the repertoire of B group gene function may include the regulation of cell division within the B group domain. It is clear that some aspects of cell proliferation within an organ can be controlled by B group gene function, as overexpression of PMADSl, the AP3 homologue in Petunia, stimulates extra lateral cell divisions (van der Krol et al., 1993). Furthermore, SUPERMAN (SUP), which encodes a zinc finger domain containing protein, appears to control the rates or planes of cell division at the third-fourth whorl boundary, as mutations in SUP result in excessive proliferation of third whorl cells (Sakai et

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al., 1995).AP3 gene function is required to activate SUP expression, and, in turn, SUP activity itself appears to be required to restrict AP3 expression to the third whorl (Bowman et al., 1992; Sakai et al., 1995). This feedback control mechanism to regulate the boundary of the third whorl may rely in part on intercellular interactions, because the patterns of SUP and AP3 expression are not entirely coincident. Coordination of the development of the different cell layers in the petals in stamens appears to depend on intercellular signaling mediated by B group gene activity. Periclinal chimeras have been used to assess whether expression of a particular floral homeotic gene in a single cell layer of the developing floral bud is sufficient to confer a wild-type phenotype. Expression of the Antirrhinum DEF and GLO B group genes in just the inner L2 and L3 layers is sufficient to induce the formation of petaloid epidermal cells in the L1 layer of the second whorl (Perbal et al., 1996). The fact that the DEF and GLO proteins can be found in the L1 layers of these chimeric plants suggests that these floral homeotic proteins are being translocated between cell layers via the plasmodesmata (Perbal et al., 1996). Despite the apparent ability of floral homeotic proteins to move between cell layers, there is no evidence for lateral intralayer signaling. Revertant wildtype DEF or GLO sectors in an otherwise mutant L1 layer are sharply bounded, indicating that intralayer movement either does not occur or is limited (Carpenter and Coen, 1990; Perbal et al., 1996). The directional cell-to-cell communication between layers may reflect the distribution of plasmodesmata in the developing flower bud. Within the floral meristem, cells are connected within a cell layer by primary plasmodesmata; however, cells in different layers must form secondary plasmodesmata to allow the interlayer passage of molecules (Lucas, 1995). This difference in plasmodesmatal origin allows for the possibility of regulated trafficking in only certain directions within the developing floral meristem. The continued expression of the floral homeotic genes in particular organs presumably serves to direct the expression of an array of target genes. Few such candidate downstream target genes have been identified, however. There are a number of problems associated with identifying such target genes. First, the floral homeotic genes are expressed throughout much of floral development and have been shown to have a functional role late in floral development as well (Bowman et al., 1989; Carpenter and Coen, 1990; Trobner et a1.,1992; Zachgo et al., 1995). Because it is likely that the repertoire of target genes is changing over time, this implies that the specificity of floral homeotic gene function must also be modulated during development. A second difficulty lies in the fact that the DNA binding specificities of AP1, AG, and AP3-PI dimers are very similar in vitro, making the identification of targets using in vitro binding approaches unreliable (Riechmann et al.,l996b; Riechmann and Meyerowitz, 1997b). In addition, expression of chimeric API, AP3, PI, or AG cDNAs containing heterologous MADS box domains does not affect their homeotic activity, indicating that DNA binding specificity alone is not a good predictor of in vivo function (Krizek and

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Meyerowitz, 1996b; Riechmann and Meyerowitz, 1997a). The specificity of the floral homeotic gene products for particular downstream target sequences may depend in part on as yet unidentified cofactors that may modulate DNA binding activity in vivo. Precedent for this exists in yeast and mammalian systems, in which MADS domain containing gene products have modified DNA binding specificities depending on the presence of accessory factors (Treisman, 1994; Shore and Sharrocks, 1995). In fact, most of the putative downstream targets of the floral homeotic genes have been identified by other criteria and only later shown to be possible targets of the floral homeotic genes. However, a novel approach to identify genes specifically upregulated in response to homeotic gene action in the absence of protein synthesis has led to the identification of one putative direct target of the A P 3 and PI floral homeotic genes (see the following discussion) (Sablowski and Meyerowitz, 1998). Although a number of petal- and stamen-specific differentiation-associated genes have been described, much work needs to be done to demonstrate how these genes are regulated by the floral homeotic genes.

IV. Differentiation of Petals Petals are relatively simple laminar structures, with a sheet of epidermal cells covering an internal layer of mesophyll (Weberling, 1989). Petals develop by divisions in all three meristematic cell layers, and the formation of the lamina depends on division in submarginal initial cells (Martin and Gerats, 1993). Cell divisions cease in the developing petal at a relatively early stage, and the final phase of petal growth depends entirely on cell expansion (Martin and Gerats, 1993). The terminally differentiated petal mesophyll cells usually are uniform in size and generally rounded, whereas the petal epidermis lacks stomata and trichomes and is composed of regular cone-shaped epidermal cells. Generally, the epidermal cells are pigmented, and in some species there can be very elaborate patterns of pigmentation across each petal. For instance, many species have petals in which the base is differently pigmented from the tip of the petal. Because these coloration patterns are not clonal and pigment biosynthesis occurs after cell division has ceased, these patterns must reflect some underlying mechanism that functions to coordinate expression across a region of petal cells (Coen et al., 1986). Despite the simple laminar structure, petal shape and size can vary dramatically in different species. Many of the elaborate petal shapes appear to have evolved to maximize pollinator attraction. Individual petals can be shaped differently within a flower, resulting in bilaterally symmetric flowers as in the pea family. In many species, one or more petals are modified from a spur, which can be a very pronounced saclike structure. For instance, in some orchids, one petal forms a spur, which gives these flowers their characteristic appearance and serves

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to guide insects, bats, or other pollinators to the pollen (Heywood, 1993). Some flowers have fused petals that form a tube, such as tobacco, whereas other species have what are termed “window” flowers. Ceropegia species have elaborate window flowers in which the petals are fused at the tips, forming lateral openings, or windows, through which insect pollinators can reach the center of the flower (Weberling, 1989). The fact that individual petals within a flower can have different shapes or intricate color patterns indicates that an underlying patterning mechanism is operating to specify regional differences in those few cell types that make up the petal. A number of genes have been identified that are expressed predominantly or solely in the petals, and several mutations affecting different aspects of petal development also have been characterized. The analyses of the corresponding genes, as well as investigations into differential gene regulation, have provided inroads into elucidating how regional differences in petal development are established.

A. Petal Shape

Many flowers are zygomorphic, or bilaterally symmetric, and this departure from radial symmetry most often is exemplified in radical changes in the form and size of the petals. For instance, in Antirrhinum, the two dorsal petals shaped differently and have larger lobes than the lateral and ventral petals, contributing to the elaborate shape of these flowers. In addition to differences in shape between petals, the individual petals themselves can have an asymmetric shape. Flower symmetry has been shown to have a functional role as well, in that it can be a factor in the discrimination of pollen sources by insects (Giurfa et al., 1996). The genetic control of zygomorphy has been investigated recently in Antirrhinum, although interest in understanding the genetic basis of zygomorphy dates back to Darwin (1868). It appears that the asymmetry across the flower, as well as the asymmetric shape of individual petals in zygomorphic Antirrhinum flowers, is controlled by the same underlying genetic mechanism. The Antirrhinum cycloidea (cyc) mutation causes a partial transformation of dorsal petals into more lateral, and lateral petals into smaller, more ventral-like form (Luo et al., 1996). Each partially transformed petal itself also shows a graded ventralized effect. Plants doubly mutant for cyc and another mutation called dichotoma (dich) show a much more complete transformation of all petals to a ventral-like state, resulting in a radially symmetric flower (Luo et al., 1996). Not only are these genes required to establish a dorsal-ventral axis of symmetry across the flower, but it is also clear that these genes participate in the asymmetric development of the individual petals along the dorsoventral axis. Although the symmetry-inducing mutations have the most dramatic effect on petal form, they also affect more subtle aspects of zygomorphy in the other organs of the flower.

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The CYC gene is expressed early in floral development in the dorsal part of the flower primordium and then later in the two dorsal petal primordia and the dorsally placed stamenode (Luo et al., 1996). DICH, like CYC, is expressed in the dorsal part of flower, suggesting that together, these gene products are responsible for establishing dorsal cell fates (Luo et d., 1996). These genes also appear to negatively regulate the DIVARICATA gene, which is responsible for establishing ventral identity in the Antirrhinum flower (Almeida et al., 1997). The patterns of DICH and CYC expression, however, do not explain the phenotypic effects of the corresponding mutations on lateral petal development. It is possible that DICH and CYC actually are expressed in the developing lateral petals but below the level of detection, or these genes may act in a nonautonomous fashion to regulate lateral petal identity and form (Luo et al., 1996). One possibility is that these genes are involved in establishing a morphogenetic dorsal-ventral gradient that specifies positional information across the flower and that this information is interpreted independently by each cell in each petal primordium. Surprisingly, the maize gene teosinte-branched (fb) shows remarkable sequence homology to CYC and DICH (Doebley et al., 1997). Although these genes show no sequence similarities to other known genes, making it difficult to propose specific biochemical functions for their products, it is possible that these genes have somewhat similar functional roles. The maize tb gene is required for the growth of axillary organs, including the lodicules and stamens in the flower (Doebley et a]., 1997). It is possible that these genes generally are required for regulating differential cell growth, a role that has been proposed previously for the action of CYC (Luo et al., 1996).

B. Petal Epidermal Cell Differentiation Perhaps the most overt example of a regionalized expression pattern in the petal is the often highly elaborate pattern of pigmentation that occurs in many flowers. These patterns of pigmentation do not follow clonal boundaries, and so some type of intercellular signaling mechanism must also be operating at this level to specify these pigmented domains. One of the major classes of pigments is the anthocyanins, which are found predominantly in the epidermis, and concomitant with this distribution, many of the genes required for anthocyanin biosynthesis are specifically transcriptionally upregulated in petal epidermal cells (Dooner et al., 1991). The transcriptional activation of many of these genes in petal epidermal cells occurs during the last cell expansion phase of petal development (Jackson et al., 1992). Many of the anthocyanin biosynthetic genes appear to be coordinately regulated at the transcriptional level (Martin and Gerats, 1993; Quattrocchio et al., 1993). A number of investigations into the control of anthocyanin biosynthesis have been instrumental in beginning to define those gene products that may be essential for the control of region-specific pigmentation patterns.

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Chalcone synthase, one of the anthocyanin biosynthetic enzymes, normally is expressed in the petal epidermal cells during the expansion phase (Martin and Gerats, 1993). Mutations in the Antirrhinurn DELILA (DEL) gene result in chalcone synthase expression in mesophyll cells as well, indicating that the distinction between epidermal and mesophyll expression is being made even at postdivision stages of development (Jackson et al., 1992). DEL may function as a transcriptional regulator, as it encodes a product with similarity to myc-type basic helix-loop-helix proteins (Goodrich et al., 1992). The DEL gene also has other regulatory effects on anthocyanin biosynthesis, as mutations in DEL abolish the expression of several anthocyanin biosynthetic genes in the basal part of the petals (Martin et al., 1991). One possibility for DEL function is that it acts as a global regulator of anthocyanin biosynthesis in all regions of the petal, and its specific functions in activating the expression of some anthocyanin biosynthetic genes and repressing others in a tissue-specific manner are conferred by interactions with different cofactors. At least some of these cofactors may correspond to myb-related transcriptional regulators, which have been described in Antirrhinurn and also are required for anthocyanin pigmentation and activation of petalspecific promoters (Sablowski et al., 1994, 1995). Interactions between these types of proteins have been documented in maize, lending credence to the idea that direct interactions between myc-type basic helix-loop-helix proteins and myb domain proteins are required for the differential transcriptional regulation of anthocyanin biosynthesis genes (Goff et al., 1992; Mol et al., 1996). However, spatially regulated expression or function of such putative myb-related cofactors has not yet been established, leaving open the question of whether these factors play a role in the regional expression of chalcone synthase and other anthocyanin biosynthetic genes. A similar type of regulation has been implicated in the regional activation of anthocyanin biosynthetic genes in Petunia. The Petunia an1 and an2 genes are required for the appropriate transcriptional activation of a subset of anthocyanin biosynthetic genes, and they encode a basic helix-loop-helix protein and an myb-related gene product, respectively (de Vetten et al., 1997). Although little is known about how these putative transcription factors act to specify the regional activation of anthocyanin biosynthetic genes in this system, at least one component in this signaling pathway has been identified. The Petunia an11 gene encodes a cytoplasmically localized WD repeat containing protein that appears to act upstream of the an2 gene (de Vetten et al., 1997). The an11 gene product has been postulated to regulate the activity of an2 and other transcription factors involved in anthocyanin biosynthesis by modulating post-translational modifications required for transcriptional function (de Vetten et al., 1997). It is also possible that the coordinated upregulation of anthocyanin biosynthetic genes is brought about in part by the action of the floral organ identity genes or other MADS box containing genes. CArG box sequences have been found in the promoter regions of a number of anthocyanin biosynthetic genes in Antir-

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rhinum, including the NIVEA gene, which encodes chalcone synthase (Martin et al., 1991; Martin and Gerats, 1993). In addition, several petal-specific genes encoding putative zinc finger containing transcription factors have been identified, which also contain CArG boxes in their promoters (Takasuji et al., 1994). These observations suggest at least part of the genetic hierarchy required for regional activation of anthocyanin biosynthetic genes may include a transcriptional regulatory cascade and may be regulated directly by the floral homeotic genes in concert with regional specific cofactors. These observations suggest that differential transcription factor activity may play a role in the spatial regulation of anthocyanin biosynthesis. Nonetheless, the mechanistic basis by which the anthocyanin biosynthetic genes are activated in spatially restricted domains within the petal is still unclear. Furthermore, because regional differences in pigmentation patterns are nonclonal, global or local cellcell interactions must be operating in the petal to specify these specific domains of expression. Based on the very dramatic region-specific patterns of pigmentation that arise in cosuppressed transgenic plants, a model has been proposed that posits a morphogenetic gradient of transcription factor activity that could serve to specify positional information across the developing petal (Jorgensen et al., 1996). Cosuppression occurs when an introduced transgene results in the silencing of both the endogenous gene as well as the introduced copies (Napoli et al., 1990). The loss of chalcone synthase expression and concommitant development of unpigmented white petal cells in Petunia flowers due to the introduction of a chalcone synthase A (chsA) gene, is a well-characterized example of cosuppression (Jorgensen, 1995). Cosuppression is not unique to chalcone synthase expression but has been observed for a number of other loci, including other anthocyanin biosynthetic genes (van der Krol et al., 1990; Meyer and Heidmann, 1994). However, it does appear that some types of genes may be more prone to silencing, as the chalcone synthase gene NIVEA from Antirrhinum also is susceptible to cosuppression-like silencing effects (Bollman et al., 1991). Flowers that display a chalcone synthase cosuppressed phenotype have patches of normal, intensely pigmented epidermal cells adjacent to completely unpigmented cells (Jorgensen, 1995). Although these patterns of pigmentation are nonclonal, such patterns are heritable to the next generation (Napoli et al., 1990).These phenomena lead to the question of how these epigenetic states are generated and maintained reproducibly. The all-or-none difference in pigmentation from cell to cell suggests that the cosuppressed state may sensitize the response of the cosuppressed gene to subtle region-specific differences. The basis for this regional specificity of cosuppression could be due to the regulation of transgene expression by a gradient of transcription factors (Jorgensen et al., 1996). Because there does appear to be a correlation between a particular transgene construct, the number of inserted copies, and the site of insertion(s) with particular types of patterns, it seems possible that position effects on transgene

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expression could play a role in setting up the pattern of pigmentation (Jorgensen et al., 1996; Meyer and Saedler, 1996). chsA cosuppression appears to be due to regulation at the post-transcriptional level (Van Blokland et al., 1994; Metzlaff et al., 1997), and such post-transcriptional effects could act to amplify regionspecific differences in transcriptional activity. Thus, a combination of nonclonal variation in transgene expression with post-transcriptional regulation of silencing together would form a metastable state resulting in a particular pattern of petal coloration (Jorgensen et al., 1996). One other aspect of petal epidermal cell differentiation that has been examined at the molecular genetic level is the development of epidermal cell shape. Petal epidermal cells are very regular and conical, and this shape is thought to maximize petal color by enhancing light absorption by pigments (Kay et al., 1981). In Antirrhinum, a mutation in the MZXTA gene causes the epidermal cells to develop as flattened rather than cone-shaped cells, resulting in paler colored petals (Noda et al., 1994). The MIXTA gene encodes a putative myb domain related transcription factor, and its function is required to generate the specialized, thickened cell walls that give the petal epidermal cells their conical shape (Noda et al., 1994). In turn, this conical shape enhances the optical properties of the epidermal cells, in part by focusing light specifically in the pigmented parts of the epidermal cells (Gorton and Vogelmann, 1996). It has been proposed that MZXTA represents a relatively late-acting target gene activated by DEF and GLO (Noda et al., 1994); however, there is, as of yet, no direct evidence supporting this suggestion.

V. Differentiation of Stamens Stamens are more complex structures morphologically than petals, with a number of distinct tissue types, many of which are associated with the formation of pollen. Stamens are composed of an elongated filament topped by an anther, which contains the tissues involved in sporogenesis. The anther is composed of a layer of epidermal tissue surrounding four microsporangia (Fig. 3). The micro-

,vascular bundle

pollen tapetum Fig. 3 Anther cross section. The four microsporangia contain the developing pollen. The cells of the tapetum surround the pollen sac and secrete sporopollenin, a polymer of carotenoids, which coats the pollen grains. At maturity, the anther splits along the stomium, releasing the pollen.

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sporangia are composed of several cell layers: an outer endothecial cell layer, a middle layer, and the inner tapetum, surrounding the sporogenous tissue, which ultimately will give rise to the pollen. The form of the stamen can vary dramatically between species and reflects the method of pollination (D’Arcy and Keating, 1996). Those species that are wind-pollinated tend to have long, slender filaments so that the anthers are well exposed to the elements, whereas species that are pollinated by animals have stamens with diverse forms, which are thought to have evolved in reponse to the specific pollination strategies. During stamen development, the anther primordia generally become apparent first, followed by the developing filaments (Sattler, 1973). Later in development, the growth of the filaments is quite rapid and occurs predominantly through cell elongation (Greyson, 1994). In the developing anther primordium, the precursors of the microsporangial tissue increase their rates of cell division, and the cells themselves can also enlarge (Bhandari, 1984; Greyson, 1994). The microsporangial cell layers appear to arise from different lineages of cells, depending on the species (Bhandari, 1984; Goldberg et al., 1993). Within the sporogenous tissue, internal cells enlarge to become the microsporocytes, which undergo meiosis to produce tetrads of microspores that develop into the pollen grains. Despite the importance of regulating male fertility in hybrid seed production and controlling crop plant fertility, comparatively little work has been done to characterize the genes required for stamen development. Several genes involved in stamen development have been identified either mutationally or by searching for transcripts that are abundant in stamen tissues. Many genes that are highly expressed during stamen development have been isolated on the basis of differential or subtractive hybridization methods (Gasser, 1991; Scott et al., 1991). The predominant class of genes isolated in such screens is expressed in the tapetum, and many encode putative secreted proteins (Scott et al., 1991). This is consistent with the role of the tapetum in providing nutrients and cell wall components to the developing microspores. Functions for some of these gene products have been inferred on the basis of homology with other sequences. These include a lipid transfer protein and a stearoyl-acyl carrier protein desaturase, both thought to be involved in membrane biogenesis, as well as a number of glycine-rich proteins that may be required for pollen wall synthesis (Koltunow et al., 1990; Chen et al., 1994; Slocombe et al., 1994; Matsunaga et al., 1996). The high level of recovery of tapetum-expressed genes in such screening procedures appears to reflect the transcriptionally very active state of the tapetum, as tapetal cells themselves do not make up a large portion of the stamen (Koltunow et al., 1990; Scott et al., 1991). Differential hybridization methods have been used to identify genes that expressed in wild-type flowers, but not in mutants that lack stamens. For instance, several genes have been obtained from Antirrhinurn that are not expressed in DEF mutants, which lack stamens (Sommer et al., 1990). Several of the genes obtained in this screen, including FILl, FIL2, and TAPI, are expressed in stamen

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tissue and could represent target genes that are upregulated by the DEF gene product (Nacken et al., 1991; Steinmayr et al., 1994). The FZLl and FZL2 genes are expressed predominantly in the filaments whereas TAPl is expressed in the tapetum, and all three genes contain putative CArG box elements in their promoters. The differential expression patterns of FILl, FZL2, and TAPl imply that other factors, potentially other MADS domain containing transcription factors, must be acting to regulate these genes. Both TAPl and FZLl encode putative secreted proteins with N-terminal hydrophobic domains (Nacken et al., 1991). FIL2 encodes a putative extracellular matrix protein with leucine-rich repeats (Steinmayr et al., 1994). These observations suggest that not only are these genes potential targets of DEF, but they may also be involved in mediating intercellular signaling events required for stamen development. A potentially powerful differential screening approach has been used to identify a gene involved in stamen development that is activated directly by the B group floral homeotic genes. By inducing AP3 expression in Arabidopsis plants that have been treated with cyclohexamide and that constitutively express PZ, genes that are upregulated directly in response to B group gene action could be identified (Sablowski and Meyerowitz, 1998). One gene identified in this screen, called NAP, appears to have a role predominantly in later aspects of stamen development, as loss of NAP function results in plants that have shorter stamens due to defects in filament cell expansion (Sablowski and Meyerowitz, 1998). AP3-PI heterodimeric proteins have been shown to bind in vitro to a CArG box contained in the first intron of the NAP gene (Sablowski and Meyerowitz, 1998). Because the NAP expression pattern only partially overlaps with that of the B group genes, and low levels of expression are seen even in the absence of functional AP3-PI proteins, it is clear that other gene products also are involved in NAP transcriptional activation (Sablowski and Meyerowitz, 1998). Mutational studies have been carried out in an attempt to identify those genes that are required for different aspects of stamen development. It has been estimated that there are approximately 25,000 genes expressed in a tobacco anther (Kamalay and Goldberg, 1984). Many such genes also are expressed in other tissues, making the identification of the corresponding mutations and the definition of their role in stamen development more problematic. Most mutational analyses have focused on identifying and characterizing male sterile mutations. Male sterile mutations have been loosely grouped into three classes: structural, functional, and sporogenous (Kaul, 1988). Structural mutants lack anthers or are otherwise so impaired morphologically that they rarely produce pollen. Functional mutants can produce pollen, but have abnormal morphologies such that the pollen is not released effectively. Sporogenous mutants are morphologically normal, but do not produce functional pollen. Most mutants that have been wellcharacterized are sporogenous male sterile mutants and, thus, are likely to be defective in terminal differentiation processes (Chaudhury, 1993; McCormick, 1993; Preuss et al., 1993; Gorman and McCormick, 1997). Comparatively little

151 work has been carried out on functional or structural mutants, although such lesions have been identified in several systems (Chaudhury, 1993; Dawson et al., 1993; Chaudhury et al., 1994; Gorman and McCormick, 1997). Several structural mutants resulting in male sterility appear to be due to defects in hormone responses. In particular, gibberellin (GA) appears to play a critical role in stamen differentiation in a number of species. For instance, the stamenless-2 mutation of tomato, which results in short, thin stamens, can be rescued by exogenously added GA (Sawhney and Bhadula, 1988). A number of dwarf mutations have been described in maize, which cause the proliferation of stamens in the normally pistillate ear (Dillaporta and Calderon-Urrea, 1994). Several of these dwarf mutants have been shown to be defective in GA biosynthesis (Phinney and Spray, 1982). The tasselseed2 gene, which is required for the production of the staminate tassel in maize, has been shown to encode a putative steroid dehydrogenase and may be involved in the regulation of the GA-mediated balance between male and female flower development (DeLong et al., 1993; Dellaporta and Calderon-Urrea, 1994). Other plant hormones, in particular auxins, cytokinins, and brassinosteroids, have also been shown to have an effect on the development of the reproductive organs in some species (Dellaporta and Calderon-Urrea, 1993; Clouse er al., 1996; Szekeres et al., 1996). Therefore, it appears that the regulation of the relative levels of hormones in particular tissues may have a critical role in stamen differentiation. Male sterility also can be caused by a mitochondrially inherited trait, known as cytoplasmic male sterility, which has been documented in many plant species (Hanson et al., 1989). Cytoplasmic male sterility results in defects in anther function and concomitant defects in pollen development. The mitochondrial genes that are affected in cytoplasmic male sterility have been identified in several species. In maize, one form of cytoplasmic male sterility appears to result from a rearranged mitochondrial sequence, which encodes a chimeric polypeptide that spans the inner mitochondrial membrane (Levings, 1993). In Petunia, another chimeric mitochondrial gene produces a polypeptide of unknown function, which is associated with cytoplasmic male sterility in that species (Levings, 1993). Mitochondrially induced male sterility in tobacco also is associated with defects in petal development, suggesting that a common metabolic component required for petal and stamen development is perturbed in these lines (Kofer et al., 1993). Although the mechanism by which altered mitochondrial function can cause cytoplasmic male sterility is unclear, at least one possibility is that any defect in mitochondrial function in the tapetal cells would adversely affect the ability of this tissue to deal with the heightened demands of pollen development (Warmke and Lee, 1978; Levings, 1993). This is consistent with the high numbers of mitochondria that are normally observed in the tapetum (Wannke and Lee, 1978). Despite the wealth of genes involved in stamen development that have been identified by these approaches, little work has been done to establish the mecha5. Petal and Stamen Development

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nisms by which these genes function to produce the complex tissues of the stamen. A few studies have been undertaken to examine whether there are inductive interactions within the tissues of the anther during late stages of development. Stamen-specific gene promoters have been used to drive cytotoxic gene expression in the tapetum to examine the effects of the consequent cell ablation on the development of the pollen. The ablation of tapetal cells at different stages in development has markedly different effects on fertility. Tapetal cell ablation early, at the tetrad stage of pollen development, results in a lack of functional pollen and concomitant sterility (Mariani et al., 1990). On the other hand, tapetal cell ablation at the later stage of uninucleate microspore development has little effect on fertility (Mariani et al., 1990; Roberts et al., 1994). During terminal differentiation of the anther, the pollen is released by a developmentally programmed splitting of the anther along a line of specialized stomium cells (Fig. 3) (Weberling, 1989). Ablation of the stomium cells has shown that functional stomium cells are required for dehiscence and release of pollen (Beds and Goldberg, 1997). These observations indicate that temporally and spatially coordinated interactions between the anther cells and the developing pollen grains are critical for insuring male fertility.

VI. Coordination of Gene Expression and

Tissue Differentiation Despite the fact that the activation of certain genes associated with differentiation processes in the petals and stamens can be detected, it is clear that cells in a developing organ primordium are not committed to a particular fate. In fact, it appears that cells continuously are reassessing their state relative to their surroundings as well as in response to the environment. One of the clearest examples of how cells can be respecified as to their fate is the phenomenon of floral reversion. Many species can undergo either natural or experimentally induced floral reversion in which flower primordia that are already initiated differentiate as vegetative structures in response to changes in day length (Battey and Lyndon, 1990). Impatiens, which is a short day plant, can be induced to revert to vegetative growth with long day treatments. The newly formed leaves that arise are arranged in a typical floral phyllotatic pattern, indicating that morphogenesis but not the inception of organ primordia has been altered (Battey and Lyndon, 1984). Organs that are initiated at the time of reversion can display a partial reversion, with the tip determined before, and the base determined after, the switch to noninductive conditions (Krishnamoorthy and Nanda, 1968; Battey and Lyndon, 1988). In second whorl organs, for instance, that have undergone partial reversion, cells at the boundary can display chimeric features in that individual cells can have characteristics of both petal and leaf (Battey and Lyndon, 1988). Similarly, partial restoration of the AP3

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homologous PMADSI function in Petunia can result in chimeric petal-leaf cells (van der Krol et al., 1993). Therefore, it appears that cells are not committed to a particular fate even up the their last division, and their differentiated fate depends on a combination of cues perceived at a particular time point in development. The specification of terminally differentiated cell fates also depends on the continued expression of the floral homeotic genes. Several of the floral homeotic genes required for petal and stamen development appear to be required late in development to specify a particular cell fate. Temperature shift experiments indicate that the Arubidopsis AP3 gene product is required until the petals and stamens begin to differentiate (Bowman et ul., 1989). Activation of floral homeotic gene expression late in development can also trigger a change in differentiated cell fate. For instance, an unstable DEF mutation induced by the Tam transposable element in Antirrhinum can revert by excision of the element, restoring DEF function (Carpenter and Coen, 1990). Such excision events can occur so late in development as to result in patches of petal epidermis as small as four cells (Carpenter and Coen, 1990). Nonetheless, the mosaic patch of petal tissue has the appropriate dorsal and epidermal characteristics or petal cells that would normally arise in that location (Carpenter and Coen, 1990). This indicates that even at one or two divisions prior to terminal differentiation, cells retain the ability to alter their fate in response to changes in gene expression.

VII. Summary Analyses of petal and stamen development are beginning to illuminate the molecular genetic processes that are required to elaborate these organ types. Floral homeotic genes are required to specify certain organ identities, and these functions also are required throughout organogenesis. These genes, either directly or indirectly, presumably control a wide array of tissue- and cell-type-specific differentiation processes. At least part of this repertoire seems to include the regulation of cell proliferation, coupling the specification of organ identity with changes in growth dynamics in different regions of the developing flower. Furthermore, cells have an enormous amount of developmental plasticity, which means that they have to be able to integrate multiple sources of information as they terminally differentiate. Some of the identified inputs include the position of the cell in the developing organ, the status of gene expression and epigenetic information, and environmental signals. How this information is disseminated between cells is largely unknown. Not only do individual cells need to respond to this information, but fields of cells must coordinate their differentiation to form a functionally complex structure. The challenge that is before us is to understand how this plasticity of response is regulated to give a reproducible and speciesspecific pattern of differentiated tissues.

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Acknowledgments I thank Iain Dawson and members of my laboratory for their insightful comments on the manuscript. Work on flowering in my laboratory is supported by grants from the National Science Foundation and the United States Department of Agriculture.

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6 Gonadotropin-Induced Resumption of Oocyte Meiosis and Meiosis-Activating Sterols Clam Yding Andersen, Mogens Baltsen, and Anne Crete Byskov* Laboratory of Reproductive Biology The Juliane Marie Centre for Children, Women, and Reproduction University Hospital of Copenhagen DK-2100 Copenhagen, Denmark

I. Introduction 11. Gonadotropin-Induced Resumption of Meiosis

A. Effect of the Preovulatory Surge of Gonadotropins on Maturation of the Follicle and Its Oocyte B. Localization of Gonadotropin Receptors in the Follicle and Heterogeneity of the Granulosa Cell Population C. Oocyte Maturation and Ovulation Triggered by Gonadotropins and Gonadotropin. Releasing Hormones 111. Possible Signal Transduction Pathways Involved in Resumption of Meiosis A. Oocyte-Cumulus-Granulosa Cell Interaction B. Cyclic Adenosine 5’-Monophosphate (CAMP),Phosphodiesterases (PDE), and Protein Kinase A (PKA) C. Nuclear Purines D. Calcium and IP, Pathway E. MAS IV. Hypothesis of a Role for MAS in Gonadotropin-Induced Resumption of Meiosis V. Possible Implications for Fertility References

1. Introduction The female germ cell line follows a unique life cycle that begins in fetal life and ends much later at ovulation time. Only a small number of oocytes complete their life cycle and mature, become fertilized, and create new offspring. After an initial wave of mitosis, usually seen during fetal life, the number of female germ cells reaches a maximum. After that the population invariably *Author to whom correspondence should be addressed. Current Topics in Developmental Biology, Vol. 41 Copyright 6 1999 by Academic Press. All rights of reproduction in any form reserved M)70-2153/99 $25 W

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declines throughout life, and around menopause the ovaries are exhausted and deprived of germ cells. In humans, the fetal ovaries contain a maximum of 6-7 million germ cells at midgestation. By birth this number is reduced to approximately 1-2 million and at the beginning of fertile life 0.5-1 million oocytes remain. During fertile life only 400-500 oocytes will undergo full maturation and ovulate. The vast majority of germ cells degenerate before full maturation without completing meiosis (Baker, 1972). In most mammalian species, the initiation of meiosis in female germ cells commences early in life after the gonads have undergone sexual differentiation. At this stage, oogonia embark on the prophase of the first meiotic division, pass through the leptotene, zygotene, pachytene, and diplotene stages, and are finally arrested in the late stage of the first meiotic prophase, the dictyate stage. Pairing and exchange of genetic material between the sister chromatids takes place during the meiotic prophase. Immediately after each oocyte enters the dictyate stage, it becomes surrounded by a single layer of somatic cells, the granulosa cells, lined with a basal membrane. This closed germ cell compartment is the primordial follicle. The follicle is the functional unit of the ovary and provides a unique environment for development and maturation of the oocyte, including regulation of oocyte resumption of meiosis. Once primordial follicles start to grow they can either reach full maturation and ovulate or become atretic. The time span between the commencement of growth and ovulation of a fully mature follicle differs among species. In the mouse it takes around 5 weeks (Pedersen, 1970), whereas human follicles take at least 14 weeks to reach ovulation (Gougeon, 1996). During this growth period the oocyte enlarges, e.g., in the rat from approximately 15 to 80 pm in diameter (Hirshfield, 1991), and the number of granulosa cells increases from approximately 10 to many thousands (Pedersen, 1970; Gougeon, 1996). When a few layers of granulosa cells have developed, the spindle-formed theca cells differentiate around the basal membrane. In later stages of follicular growth, an antrum containing follicular fluid forms between the granulosa cells. During follicular growth the original stem granulosa cells multiply and differentiate into functional and morphological heterogeneous subpopulations. At least two distinct populations, namely, the pseudostratified peripheral granulosa cell layer, i.e., the mural granulosa cells lining the follicular wall, and the cells that surround the oocyte, i.e., the cumulus cells, are distinguishable. All of these cells are interconnected with gap junctions, which facilitate intercommunication and transportation of hormones and other low-molecular-weight components from the theca layer and blood vessels surrounding the follicular basal membrane. Many studies have documented the close interaction between granulosa cells and oocytes during follicular development and maturation (Eppig et al., 1997). The paracrine interaction between oocyte and granulosa cells perhaps is one of the most crucial mechanisms involved in oocyte maturation.

165 The oocyte of the growing follicle is blocked meiotically in the so-called germinal vesicle stage (GV), in which the nuclear membrane and the nucleolus are visible through the microscope. The resumption of meiosis in the oocyte of that particular follicle only takes place when the preovulatory follicle matures by the preovulatory surge of gonadotropins. This is visualized by the breakdown of the germinal vesicle and is referred to as germinal vesicle breakdown (GVBD). The gonadotropins induce changes in the follicular environment, which insure that the meiotic process is resumed and proceeds. The completion of the first meiotic division results in a secondary oocyte with a haploid number of chromosomes and the extrusion of a first polar body. However, the meiotic process will not be completed as long as the oocyte resides in the follicle. Instead it will proceed to the metaphase of the second meiotic division (i.e., metaphase 11) and stop for the second time. Metaphase I1 is the state in which the oocyte is found most often when it is released from the follicle by ovulation. The oocyte will remain in metaphase I1 until it becomes fertilized. Under normal circumstances this takes place in the fallopian tubes, outside the ovaries. The intruding sperm cell induces completion of the meiotic process of the oocyte, and the second polar body is extruded. Therefore, it is a rare event that an oocyte completes meiosis. This chapter focuses on the regulation of meiosis within the follicular compartment. How is meiosis prevented from resuming in an untimely manner, and how do gonadotropins induce the resumption of meiosis? In this context, we will describe the possible role of a newly described group of paracrine hormones, the meiosisactivating sterols, MAS (Byskov et al., 1995), in relation to gonadotropin-induced resumption of meiosis. MAS may also play a role in stimulating the oocyte to complete meiosis after fertilization, which, however, will not be covered by this review. By incorporating the effects of these MAS hormones, we propose a novel hypothesis for gonadotropin-induced resumption of meiosis. Finally, we will discuss how these newly discovered hormones may be relevant to the development of future contraceptives as well as the treatment of infertility. 6. Gonadotropin-Induced Meiosis

II. Gonadotropin-Induced Resumption of Meiosis The preovulatory surge of gonadotropins induces two specific biological responses in viva (i) resumption of meiosis in oocytes of large preovulatory follicles and (ii) initiation of the ovulatory process itself, resulting in the degradation of the follicle wall and the expulsion of the cumulus-oocyte complex from the follicle. The tight synchronization of these two events insures the release of mature oocyte(s), which can be fertilized and undergo further embryonic development (Espey and Lipner, 1994). In the following we will mainly focus on the resumption of oocyte meiosis.

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A. Effect of the Preovulatory Surge of Gonadotropins on Maturation of the Follicle and Its Oocyte

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are released from the anterior pituitary upon stimulation by gonadotropin-releasinghormone (GnRH) from the hypothalamus. It has long been known that both LH and FSH can cause ovulation after systemic injection (Greep et al., 1942). Later it was shown that in the rabbit 10 times more FSH than LH is needed for ovulation induction after systemic as well as intrafollicular injection of the hormones (Jones and Nalbandov, 1972). In women the actual preovulatory peak of FSH seems to occur 1-2 hr later than the LH peak (Yussman et al., 1970, Hillier, 1994), with the peak level being around 10-20 IUAiter). The peak level of LH is about 50-100 IU/liter, which is 10-50 times higher than that seen in the follicular phase (Ross et al., 1970). It takes several hours before the preovulatory rise of gonadotropins can be measured in the follicle. Although low-molecular-weight substances have been shown to enter the follicle from circulation quite rapidly (i.e., within minutes) (Yding Andersen, 1997), certain proteins like LH enter the follicle more slowly (McNatty et al., 1981). Two hours after the induction of the LH surge the plasma concentration of LH in ewes had increased 200-fold compared to the initial concentration, whereas the level of LH within the preovulatory follicle was still unchanged. Only after 5-6 hr was the intrafollicularconcentration of LH comparable to that in plasma (McNatty et al., 1981). Similar conditions seem to apply for the entry of FSH into the follicle (Dieleman et al., 1983). Consequently, it is likely that the follicular compartment experiences a temporal effect of LH earlier than FSH. Furthermore, the mural granulosa cells are prone to be affected earlier than the more centrally situated cumulus cells. The midcycle surge of gonadotropins initiates the degradation of the gap junctional complexes of the granulosa cells (Section 1II.A). The temporal pattern for the entry of LH and FSH, therefore, supports earlier studies suggesting that gap junction degradation takes place in the mural granulosa cells before it occurs in the cumulus-oocyte complex. In fact, the temporal pattern for the entry of LH and FSH into a follicle resembles that of gap junctional degradation and makes it conceivable that these events are linked. Because LH receptors within the follicle are found exclusively on the mural granulosa cells, it is conceivable that gonadotropin-mediated effects may appear first and most pronounced in the mural granulosa cell layer. The prominent influence on mural granulosa cells by LH is supported further by experiments that demonstrate that mural granulosa cells from large human preovulatory follicles in culture produced a 30-fold higher level of CAMP (see section 1II.B) when stimulated with LH than with similar concentrations of FSH (Yong et al., 1994). The precise functions of LH and FSH in oocyte maturation and ovulation are not fully understood, but it is well-documented that LH-human chorion go-

167 nadotrophin (HCG) have an indirect effect on the resumption of meiosis (Tsafriri et al., 1972). The confusion surrounding the effects of gonadotropins on oocyte maturation can be illustrated in the following way: (i) an LH surge without a concomitant rise in FSH induces GVBD in vivo; (ii) pure LH induces GVBD in cultured intact follicles; (iii) in contrast, LH does not induce GVBD in cultured, isolated cumulus-oocyte complexes or in naked oocytes (Eppig and Downs, 1988; Byskov et al., 1997). Previously, it was assumed that only LH, and not FSH, was responsible for initiating the ovulatory process. Indeed, there is much evidence to show that LH is able to evoke both ovulation and resumption of meiosis without the presence of FSH. However, a large bolus of FSH, without LH activity, also is able to induce the resumption of meiosis and ovulation, as shown in the hamster (Greenwald and Papkoff, 1980), rat (Galway et al., 1990), and monkey (Schenken et al., 1984). Actually, FSH can induce both follicular growth and ovulation in the same animal, as shown in hypophysectomized rats treated with recombinant FSH (Galway et al., 1990). Therefore, each of the gonadotropins may substitute for one another, and stimulation of ovulation seems to depend more on the total amount of gonadotropins rather than the actual hormone itself. In any event, under normal conditions, the gonadotropins seem to act together to regulate the two key events, resumption of meiosis and expulsion of the oocyte from the follicle. 6. Gonadotropin-Induced Meiosis

B. Localization of Gonadotropin Receptors in the Follicle and Heterogeneity of the Cranulosa Cell Population

Information on the distribution of FSH and LH receptors (FSH-R and LH-R) in the gonads originally was obtained from binding studies using radioactively labeled ligands (Amsterdam et al., 1975). The distribution of gonadotropin receptors subsequently was confirmed through receptor-directed antibodies (Takao et al., 1997) and in situ hybridization (Zheng et al., 1996) with labeled riboprobes or reverse transcriptase-polymerase chain reaction (RT-PCR) on mRNA extracted from the tissue. A parallelism between FSH-R mRNA levels and receptor expression seems to be evident (Dunkel et al., 1994). However, the functional significance of receptor activation-desensitization (Lindner et al., 1977), which may be accomplished by receptor internalization, G-protein uncoupling, or expression of variant forms of receptors, has not yet been shown. The oocyte itself does not seem to express receptors for LH and FSH (Dekel, 1988). In the female, FSH-R is found almost exclusively on the granulosa cells (GoreLangton and Armstrong, 1994). One study detected the expression of FSH-R mRNA in theca cells, human ovarian surface epithelium, and the fallopian tubes (Zheng et al., 1996). FSH-R is already expressed on the granulosa cells of the primary (early growing) follicles and throughout the rest of follicular development in humans (Zheng et al., 1996), sheep (Tisdall et al., 1995), cattle (Xu et al.,

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1995), and rabbit (Roy et al., 1987), and the granulosa cell compartment undoubtedly represents the major target for FSH. In contrast to FSH-R, LH-R is not expressed in smaller follicles. In large antral follicles the expression of LH-R differs among the mural granulosa cells. Those that are close to the basal membrane exhibit the highest immunohistochemical signal, and less is seen in cells lining the follicular fluid. The cumulus cells and the oocyte itself seem to lack expression of LH-R (Peng et al., 1991; Eppig et al., 1997). In fact, the expression pattern of LH-R seems to be one of the clearest markers for distinguishing between different types of granulosa cells, although other activity gradients also are evident, e.g., steroidogenesis (Zoller and Weisz, 1979; Vanderhyden and Tonary, 1995). The LH-R, in addition to the granulos cells, also is present in the theca cells at all stages of follicular development, in cells of the corpus luteum, and in ovarian interstitial cells without much apparent variation among species (Takao et al., 1997; Bukovsky et al., 1995; Chegini et al., 1991). Substantial expression of both LH-R and FSH-R of the mural granulosa cells from large follicles occurs upon gonadotropin stimulation, reflecting the different responses in the granulosa cell populations (Zeleznik et al., 1974; Richards et al., 1976; Segaloff et al., 1990). The role of the oocyte in regulating these differences by suppressing the granulosa cell expression of LH-R mFWA has been shown (Eppig et al., 1997). GnRH receptors and GnRH-R mRNA also are expressed in the ovary (Whitelaw et al., 1995; Goubau et al., 1992), although the physiological significance has not been defined as yet.

C. Oocyte Maturation and Ovulation Triggered by Gonadotropins and Gonadotropin-Releasing Hormones

Several studies have evaluated whether oocyte maturation and ovulation exhibit different sensitivities to the midcycle surge of gonadotropins. Actually, there is some evidence suggesting that the resumption of meiosis in follicle-enclosed oocytes is induced at a considerably lower threshold level than the actual ovulatory process itself, both in vivo and in vitro (Bomsel-Helmreich et al., 1989; Peluso, 1990; Mattheij et al., 1993, 1994; Dekel et al., 1995). Peluso tested the effect of subovulatory doses of gonadotropins on pregnant mare serum gonadotrophin (PMSG)-stimulated rats by evaluating oocyte maturation, progesterone secretion, and follicular rupture (Peluso, 1990). The PMSG-treated ovaries were placed in a perfusion culture for 21 hr and exposed to 5, 30, and 85% of the ovulatory gonadotropin surge. He found that the threshold level for maximal induction of the resumption of meiosis and progesterone production was reached when the ovaries were exposed to 5% of the gonadotropin surge, whereas the induction of ovulation itself was only partly induced by 85%. Similar results have been obtained by Dekel et al., who showed that, in PMSG-treated rats, 1.4 IU of HCG was sufficient to induce a maximal response in GVBD without affecting ovulation, which in turn required at least 4 IU (Dekel et al., 1995).

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Mattheij and co-workers showed that, in rats, subovulatory doses of both LH and FSH were able to induce an asynchronous resumption of meiosis in follicleenclosed oocytes and ovulation in vivo (Mattheij et al., 1993, 1994). The follicleenclosed oocytes were triggered to resume meiosis before the midcycle surge of gonadotropins. GnRH also may play a role in regulating oocyte maturation. Although circulating levels of GnRH are extremely low (Clayton and Catt, 1981), local ovarian production seems to take place (Oikawa et al., 1991; Goubau et al., 1992), and GnRH agonists stimulate the resumption of meiosis in intact follicles (Hillensjo and LeMaire, 1980). The presence of GnRH receptors and GnRH receptor mRNA in the ovaries suggests that GnRH, via its receptor and in a paracrine way, may participate in controling meiotic maturation (Whitelaw et al., 1995). However, more research is needed to elucidate the precise role of GnRH in follicular maturation. Taken together, the two basic events occurring during the midcycle surge of gonadotropins, the resumption of meiosis and the ovulatory process itself, most likely are activated at different levels of gonadotropins. Resumption of meiosis requires a much lower level than the ovulatory process itself. Furthermore and equally important, each of the two gonadotropins, LH and FSH, can substitute for one another in these two events, in vivo.

111. Possible Signal Transduction Pathways Involved in Resumption of Meiosis A. Oocyte-Cumulus-Cranulosa Cell Interaction

From the early stages of follicular growth, the oocyte and its cumulus cells are closely connected through gap junctional complexes, and the cumulus cells likewise are connected to the mural granulosa cells (Anderson and Albertini, 1976; Gilula et al., 1978). Thus, the oocyte is able to communicate with the most remote granulosa cells at the basal lamina through this network of interconnections. The gap junctions appear to play the most important role in follicular growth and maturation. They have been postulated to transfer substances from and to the oocyte that affect the resumption of meiosis both negatively and positively. In fact, studies of cultured follicles showed that more than 85% of the metabolites derived from the culture medium enter the granulosa cells first and subsequently the oocyte via gap junctions (Heller et al., 1981). The observation that fully grown oocytes undergo spontaneous meiotic resumption when removed from the follicular compartment supported the idea that follicular cells produce a meiosis-preventing substance or an oocyte maturation inhibitor (Tsafriri and Channing, 1975). Apparently, follicular fluid is not essential for maintaining meiotic arrest, because contact between the cumulus-oocyte complex and the follicle wall is sufficient to sustain meiotic arrest (Liebfried and

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First, 1980; Motlik et al., 1986; Sirard and Bilodeau, 1990). The theca layer also seems to play a role in preventing oocyte maturation (Kotsuji et al., 1994; Richard and Sirard, 1996). A meiosis-preventing substance from the theca may diffuse into the mural granulosa layers and be transmitted to the oocyte via the network of the gap junctions. During natural gonadotropin-induced ovulation at midcycle, the number of gap junctions between the granulosa cells decreases in parallel with the meiotic resumption of the oocyte (Eppig, 1982; Larsen etal., 1986, 1987). The degradation, or rather internalization, of the gap junctions starts in the periphery of the follicle and moves toward the more centrally situated cells. The gap junctions between the cumulus cells and the oocyte disappear later on (Dekel and Beers, 1978; Larsen et al., 1986; Dekel, 1988). This disconnection is believed to abolish the transfer of the meiosis-preventing substance-originating in the mural granulosa cells-to the cumulus cells and to the oocyte. FSH enhances the reduction of gap junctions between cumulus cells in cumulus-oocyte complexes from rats in vitro (Wert and Larsen, 1989). The importance of LH is indicated by the finding that expression of connexin-43, a protein present in ovarian gap junctions, is negatively correlated with LH exposure (Granot and Dekel, 1994). On the other hand, it has been shown that the oocyte and cumulus cells nevertheless remain metabolically coupled via gap junctions during meiotic resumption of the oocyte (Moore et al., 1981; Eppig, 1982; Larsen et al., 1986; Fagbohun and Downs, 1991). FSH and epidermal growth actor (EGF) induce GVBD in cultured mouse oocytes. It has been suggested that both components trigger the production of a positive meiosis-activating signal within the cumulus cells and that the transfer of this signal to the oocyte requires the presence of gap junctions (Downs et al., 1988). It also has been shown that FSH triggers cultured, intact cumulus-oocyte complexes, but not cumulus cells deprived of the oocyte, to produce a diffusible meiosis-activating component (Byskov et al., 1997) (Fig. 1). This component is able to induce the resumption of meiosis in cumulus-deprived mouse oocytes. FSH seems to induce an oocyte signal in the intact cumulus-oocyte complex, which triggers the cumulus cells to produce a meiosis-activating substance. Gap junctions seem to be crucial for the regulation of meiosis in the preovulatory follicle. A meiosis-preventing substance may be transferred to the oocyte from the mural granulosa cells before the preovulatory surge of gonadotropins. After the surge, the transfer of this signal is interrupted by internalization of the gap junctions in the mural granulosa cell layers. The rise in FSH levels, on the other hand, induces positive signal transduction between the oocyte and cumulus cells, which results in the resumption of meiosis.

B. Cyclic Adenosine 5'-Monophosphate (CAMP), Phosphodiesterases (PDE), and Protein Kinase A (PKA)

FSH and LH exert their function by activating membrane receptors of target cells. The receptors activate the membrane-bound adenylate cyclase (AC), lead-

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LANOSTEROL

FF-MAS HO

T-MAS CVP51

ZVMOSTEROL

0 '

C14-reductase

HO

CHOLESTEROL

4-demethylase HO

Fig. 1 Conversion of lanosterol to cholesterol. FF-MAS (4,4-dimethyl-5a-cholest-8,14,24triene-3P-01) and T-MAS (4,4-dimethyl-5u-cholest-8,24-diene-3~-ol) are intermediates of cholesterol biosynthesis. The conversion of lanosterol to FF-MAS depends on the activity of cytochrome P450 enzyme I4a-demethylase (CYF51) (Aoyama and Yoshida, 1986). which has been cloned (Stromstedt et al., 1996). FF-MAS is converted to T-MAS by C14-reductase (Aoyama and Yoshida, 1986). FF-MAS (isolated from human follicular fluid) and T-MAS (isolated from bull testes) can trigger the resumption of meiosis in mature mouse oocytes (Byskov er al., 1997).

ing to the production of cyclic adenosine 5'-monophosphate (CAMP).cAMP acts as the intracellular messenger for gonadotropin stimulation, and the production of cAMP leads to the phosphorylation of cellular proteins and the induction of specific cellular events. cAMP is degraded by the family of phosphodiesterase enzymes (PDE), which produces inactive second messenger products.

1. cAMP When oocytes are removed from the follicular compartment and cultured with membrane-permeable cAMP analogues (e.g., dibutyryl-CAMP), they remain in the GV stage, e.g., in the mouse (Cho et al., 1974) and humans (Tornell and Hillensjo, 1993). Activation of AC with forskolin or inhibition of PDE with the nonselective 3-isobutyl-1-methylxanthine (IBMX) also prevents GVBD by maintaining elevated levels of cAMP within the oocyte (Downs and HunzickerDunn, 1995). However, the activation of the cumulus-oocyte complex with FSH causes GVBD concomitantly with an increase in the amount of cAMP in the cumulus-oocyte complex (Eppig and Downs, 1988), but with a decrease in cAMP levels in the oocyte itself (Schultz, 1988). There is no obvious decrease in the concentrations of cAMP in oocytes of other mammals, such as the hamster (Racowsky, 1985; Hubbard, 1986), sheep (Moor and Heslop, 1981), and pig (Racowsky, 1985), indicating that other mechanisms may participate in the regulation of meiosis. Natural gonadotropin-induced ovulation (Tsafriri et al., 1972) or induced oo-

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cyte maturation with forskolin in vivo (Yoshimura et al., 1992) leads to major increases in cAMP in ovarian follicles simultaneously with the resumption of oocyte meiosis. These apparently opposing effects of cAMP on oocyte maturation may be explained by compartmentalization and differential response to gonadotropins by the mural granulosa cells, cumulus cells, and the oocyte (Eppig et al., 1997). Maturation of oocytes in vitro can be induced by transient elevations of cAMP using analogues of CAMP, such as dibutyryl-CAMP (Tsafriri et al., 1972) and 8-bromo-CAMP(Hillensjo et al., 1978), by inhibiting PDE with IBMX (Dekel et al., 1988; Cho et al., 1974), or by stimulating AC with a brief exposure to forskolin (Dekel, 1986; Downs et al., 1988; Yoshimura et al., 1992), which causes the production of a meiosis-activating substance (Guoliang et al., 1994; Downs and Hunzicker-Dunn, 1995). However, these studies also show that permanently elevated levels of cAMP caused by prolonged exposure to these agents maintain oocytes in meiotic arrest. It has been suggested that the magnitude of a peak change (i.e., the relative change) in cAMP concentration may be the more important trigger for the resumption of meiosis (Yoshimura et al., 1992) and that the absolute level of cAMP in the oocyte may be of less importance (Downs et al., 1988). Other signal transduction pathways also may be involved in oocyte maturation. A study of oocytes from juvenile gonadotropin unprimed mice showed that the resumption of meiosis can be triggered by exposure to lithium ions (Bagger et al., 1993). This ion interferes not only with the cAMP pathway but also with the phosphatidylinositol cycle (Halsher and Sherman, 1980) (see Section 1II.D). 2. Differential Regulation of cAMP Levels within the Follicle: PDE and PKA Tsafriri et al. have demonstrated that the oocyte mainly expressed PDE type 3, whereas the granulosa cells predominantly expressed type 4 (Tsafriri et al., 1996). Furthermore, LH-induced maturation of follicle-enclosed oocytes was blocked by inhibitors of type 3 but not by inhibitors of type 4 PDE. Treatment with type 4, but not type 3, PDE inhibitors induces the resumption of meiosis, possibly by increasing the level of cAMP in granulosa cells. Based on these results, they suggest that the selective regulation and expression of PDE may be involved in the regulation of cAMP levels in the oocyte-cumulus complex and the rest of the follicle cells, thereby regulating maturation of the follicle-enclosed oocyte. cAMP affects the meiotic process through the activation of the CAMPdependent protein kinase A (PKA). Activation of PKA results in phosphorylation of proteins in serine and threonine residues (Francis and Corbin, 1994). Corbin et al. have described two major isoenzymes of PKA, types I and I1 (Corbin et al., 1978). It was shown that the mouse oocyte exclusively contains type I PKA, while both type I and type I1 are present within the cumulus cells (Downs and

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Hunzicker-Dunn, 1995). The differential expression of the two isoenzymes within the oocyte and the cumulus cells may dictate the net result of the cAMP response. It was suggested that type I PKA within the oocyte mediates meiotic arrest, whereas type I1 PKA within the cumulus cells is responsible for inducing meiotic resumption. Data also suggest that inhibitory factors from theca cells can regulate PKA in the cumulus cells (Richard and Sirard, 1996). In addition, the two PKA isoenzymes exhibit different affinities for CAMP,and the enzymes are differentially activated by cAMP analogues (Beebe and Corbin, 1986; Downs and Hunzicker-Dunn, 1995). It appears that CAMP has a higher affinity for type I PKA than for type I1 PKA (Hunzicker-Dunn et af., 1985). Consequently, lower levels of cAMP may preferentially activate type I PKA, keeping the oocyte in meiotic arrest, while higher levels of CAMPas experienced during the midcycle surge of gonadotropins may activate type Il PKA in the cumulus cells, leading to the synthesis of substances that promote meiotic resumption (Downs and Hunzicker-Dunn, 1995). The pivotal role of CAMP as a second messenger in the signal transduction pathway of gonadotropins is well-established. Furthermore, several lines of evidence indicate that cAMP also plays an important role in maintaining meiotic arrest.

C. Nuclear Purines

Certain purines, particularly hypoxanthine and adenosine, are present in follicular fluid (FF) from mouse and pig follicles, and when added to a culture medium in concentrations similar to those in follicles, oocyte resumption of meiosis is blocked in the mouse (Downs et af., 1985; Eppig et af.,1985), rat (Miller and Behrman, 1986), cow (Sirard and First, 1988), monkey (Warikoo and Bavister, 1989), and rabbit (Thibault et al., 1987). Hypoxanthine is an inhibitor of cAMP PDE. As such, it is likely to prevent the hydrolysis of oocyte CAMPand thereby maintain elevated levels of CAMPin the oocyte. Meanwhile, the concentration of hypoxanthine decreases only slightly in mouse FF in response to the midcycle surge of gonadotropins (Eppig et al., 1985), suggesting that a stimulatory component may override the meiosis inhibitory effect of hypoxanthine after the gonadotropin surge (Downs et al.,1988; Byskov et al ., 1997). Purine nucleotides such as hypoxanthine may be important in other control mechanisms. They appear to be required for the signal transduction pathway of FSH (Downs, 1997). If the purine nucleotide-generating pathways are blocked, FSH’s capacity to induce the resumption of meiosis is reduced significantly, and de novo synthesis of purines seems to be required for the FSH-induced oocyte resumption of meiosis (Downs, 1997). However, these findings contradict results from previous studies, such as those by Shim et al. (1992), which showed that microinjection of small amounts of hypoxanthine into the oocyte is effective in

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preventing GVBD. The precise physiological significance of hypoxanthine in the control of the meiotic process therefore is still unclear, and more research is needed to clarify the role of follicular purines. Two other purines, adenosine and guanosine, also maintain meiotic arrest. Adenosine stimulates the adenylate cyclase and at the same time acts as a substrate for the generation of CAMP. Guanosine and guanosine derivatives like guanosine monophosphate also have proved to be of importance for maintaning meiotic arrest (Downs, 1995). D. Calcium and IP, Pathway

The inability to demonstrate a cAMP decrease in the oocyte in connection with the resumption of meiosis in some mammalian species (see previous discussion) has paved the way for other suggestions regarding intracellular regulation of meiosis in the oocyte. It has been proposed that the actual GVBD-inducing signal from the cumulus cells may be either free calcium (Ca2+) or a calcium-releasing factor such as inositol 1,4,5-triphosphate (IP,) (Homa et al., 1993; Eppig, 1993; Homa, 1995). This hypothesis is based on the findings that chelation of the intracellular Ca2+ inhibits GVBD. This effect has been observed in some species, such as the cow (Homa, 1991) and pig (Kaufman and Homa, 1993), whereas oocytes from mice were unaffected (Carroll and Swann, 1992). Other studies have shown that the resumption of meiosis can be manipulated by affecting the intracellular concentration of free Ca2+: (i) In oocytes from hamster and mouse, Ca2+ ionophores partly overcome CAMP-maintained meiotic arrest; (ii) Ca2+ channel blockers may enforce the effect by the Ca2+ ionophores (Powers and Paleos, 1982; Racowsky, 1986); and (iii) microinjection of IP, into cow oocytes induces GVBD probably by increasing intracellular concentrations of free Ca2+ (Homa et al., 1991). The hypothesis suggesting that Ca2+/IP3 plays a role in the resumption of meiosis is supported by the finding that LH, in addition to activating AC, also enhances IP, production (Davis et al., 1986; Alila et al., 1989). This suggests that LH triggers the release of Ca2+ in the granulosa cells, which, via gap junctions, could be transferred to the oocyte and act as the positive GVBDinducing signal overcoming cAMP inhibition. Alternatively, IP, produced in the granulosa cells may enter the oocyte via gap junctions and then cause a rise in free Ca2+ in the oocyte, triggering the resumption of meiosis (Homa et al., 1993). However, it remains to be proved whether the changes in intracellular Ca2+ levels follow the ovulatory process and resumption of meiosis and whether the concentration of Ca2+ actively plays a role in the control of meiotic resumption. E. MA5

It is now well-established that, in addition to reducing an uphold signal for meiotic progression, the preovulatory surge of gonadotropins also provides a

175 positive signal that induces the resumption of meiosis. Identification and characterization of a family of meiosis-activating sterols (MAS), which trigger the resumption of meiosis in cumulus-enclosedand naked oocytes in vitro, show that they may be candidates for such a signal (Byskov et al., 1995). However, it cannot be excluded that the resumption of meiosis is controlled by several different substances. MAS was purified from human follicular fluid and identified as 4,4dimehtyl-5a-cholest-8,14,24-triene-3~-ol (FF-MAS), and another closely related MAS from bull testis was identified as 4,4-dimethyl-5a-cholest-8,24-diene-3~-01 (T-MAS). In addition, two synthesized substances closely resembling FF-MAS and T-MAS also induced GVBD and polar body formation in hypoxanthinearrested oocytes, in vitro. FF-MAS and T-MAS are intermediates in the conversion of lanosterol to cholesterol and therefore are naturally occurring substances in all tissues with de novo biosynthesis of cholesterol (Byskov et al., 1995) (Fig. 1). Lanosterol itself is inactive with respect to stimulation of meiotic resumption. De novo synthesis of cholesterol takes place in the granulosa cell compartment, as shown in the human (Ryan er al., 1968; Endresen, 1990) and pig (Spicer, 1990). Lanosterol 14a-demethylase (CYP5l), the enzyme that converts lanosterol to FF-MAS, has been cloned (Stromstedt et al., 1996). It also has been shown that gonadotropins stimulate the expression of CYP5 1, suggesting a mechanism for the effect of FSH on the production of MAS (Yoshida et al., 1996). It has been shown that only the cumulus cells and not the mural granulosa cells produce a meiosis-activating signal after stimulation with FSH. Furthermore, a recombinant LH preparation without any concomitant FSH activity was unable to induce meiotic resumption in intact cumulus-oocyte complexes or the production of a positive signal from the mural granulosa cells. The results indicated that intact cumulus-oocyte connections are a prerequisite for the ability of FSH to induce the synthesis of this signal. However, transfer of the cumulus-derived signal (MAS?) to the oocyte was not dependent on the presence of intact gap junctions (Byskov et al., 1997). We therefore suggest that a key event in the regulation of gonadotropininduced resumption of meiosis involves the production of MAS, which by transfer to the oocyte stimulates the resumption of meiosis. However, the precise intracellular events in the oocyte after MAS stimulation are not clear, but may involve the CAMPand IP, intracellular signal transduction pathways. 6. Gonadotropin-Induced Meiosis

IV. Hypothesis of a Role for MAS in Conadotropin-Induced Resumption of Meiosis There are two hypotheses regarding the mechanism by which gonadotropins induce the resumption of meiosis. The first hypothesis involves the action of a meiosis-preventingmechanism or substance within the follicle that is lowered or

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eliminated by the surge of gonadotropins. This idea initially was based on the finding that removal of the fully grown oocyte from its follicle will cause meiosis to resume spontaneously (Pincus and Enzmann, 1935; Edwards, 1965; Biggers, 1972). The results demonstrated that (i) hormonal induction of ovulation is not necessary for the oocyte to develop competence to resume meiosis and (ii) it is likely that the factors that maintain the oocyte in the GV stage originate in the follicle itself. The first hypothesis predicts that the midcycle surge of gonadotropins, especially LH, terminates the signal that prevents the oocyte from resuming meiosis. Much research has been directed toward revealing the nature of this upholding signal. Within the oocyte it may be cAMP itself or compounds or a process controlled by cAMP (Section 1II.B) (Eppig, 1993). The second hypothesis focuses on the production of a positive signal within the follicular compartment, which actively induces the resumption of meiosis. Gonadotropins appear to promote the generation of such a positive signal produced in the cumulus cells and transferred to the oocyte, thus inducing the resumption of meiosis (Section 11). The two hypotheses for the gonadotropin-inducedresumption of meiosis do not necessarily exclude each other. We have tried to combine them into one new hypothesis by incorporating the effects of MAS on oocyte meiotic resumption. A number of observations suggest that the midcycle surge of LH exerts a major role in governing the functional and physical dismantling of the mural granulosa cells from the oocyte-cumulus complex. This also involves decreasing the flow of meiosis-arresting or -upholding substances (i.e., CAMP?)from the mural granulosa cell layer to the oocyte via the cumulus cells. Cumulus cells remain metabolically coupled to the oocyte for a longer period than to the mural granulosa cells. Our studies have shown that FSH stimulation of isolated intact oocyte-cumulus complexes results in the transfer of heat-stable substances (MAS?) to the oocyte as well as to the surrounding medium, which subsequently can overcome hypoxanthine-prevented meiosis and reinitiate meiosis. A functional metabolic coupling between the cumulus cells and the oocyte is, however, necessary for FSH to trigger the cumulus cells to produce this signal. Therefore, oocyte and cumulus cells must interact before the cumulus cells can provide the oocyte a positive signal (Fig. 2). In contrast, FSH stimulation of mural granulosa Fig. 2 Hypothesis of the role of FSH and MAS in oocyte resumption of meiosis based on studies of mouse oocytes (Byskov ef al., 1997). The drawings show sections through a cumulus cell with (panel 1) or without (panels 2-4) connection to the oocyte. Panels 3 and 4 indicate that FSH has no effect on GVBD when added to a naked oocyte, no matter whether cumulus cells are close by. Panel 2 indicates that MAS can trigger GVBD in naked oocytes. Panel 1 summarizes the hypothesis of the action between FSH and the oocyte during GVBD initiation: FSH activates receptors on the cumulus cells. Simultaneously or shortly after, the cumulus cells receive a signal from the oocyte, which is crucial for the cumulus cells to produce MAS. MAS produced by the oocyte-connected cumulus cells is transferred to the oocyte (and to the surroundings, e.g., the follicular fluid) where the resumption of meiosis is triggered. Whether MAS acts through a receptor or by other means is, so far, not known.

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cells or cumulus cells alone seems to be unable to produce a meiosis-activating substance (Byskov et al., 1997). We propose that gonadotropin-inducedresumption of meiosis in vivo is a twostep regulated process. During the midcycle surge of gonadotropins, temporally determined sequences of events take place that collectively result in the resumption of meiosis. The initial step is likely to be an action on the mural granulosa cells predominantly exerted by LH, which, via its receptors, stimulates the synthesis of high levels of the upholding signal (CAMP?).These high levels result in a degradation of the gap junctions beginning among the mural granulosa cells, which terminates the transfer of meiosis-arresting substances (which ironically also seems to be CAMP)to the oocyte. LH may also release the meiosis-arresting brake. When the flow of meiosis-arresting substances from the mural granulosa cells decreases, the cumulus cells and the oocyte remain metabolically coupled for a while. At this stage LH and FSH may reach the cumulus cells, which are affected only by FSH. In the intact oocyte-cumulus complex FSH will trigger a signal from the oocyte to the cumulus cells, which induces the synthesis of the positive meiosis-activating signal [MAS(?)]. MAS(?) is transferred directly to the oocyte either via the still functioning gap junctions and/or by secretion to the follicular fluid to act as a paracrine meiosis-activating hormone. If LH releases the brake for reinitiating meiosis, FSH may be said to press the acclerator and produce a trigger substance. The elimination of the brake and the production of a meiotic trigger will insure that a resumption of meiosis will actually take place in the right follicle with a sufficient number of gonadotropin receptors. We propose that the gonadotropin-inducedresumption of meiosis in vivo is an intrafollicular two-cell, two-gonadotropin-regulated process. LH primarily acts on the mural granulosa cells to terminate the synthesis of a meiosis-arresting signal, and FSH primarily acts on the cumulus cells to induce the synthesis of MAS, inducing the resumption of meiosis. Our hypothesis combines the two current hypotheses and may explain some of the observations seen in a number of studies. One of the previous experimental obstacles to combining the two hypotheses has been difficulty explaining how an exogenous bolus of LH is able to induce ovulation, including the resumption of meiosis and expulsion of the oocyte from the follicle. However, to our knowledge the vast majority of animal studies have used exogenous gonadotropins to stimulate follicular growth, thereby introducing an experimental bias because the endogenous levels of FSH invariably increase as a result of the positive feedback action. Therefore, although the exogenous stimulus to ovulate is an injection of HCG without FSH activity, endogenous levels of FSH, which during the midcycle surge are much lower than LH, may still be sufficient to allow for induction of a positive signal. During the course of exogenous gonadotropin stimulation, a temporal relation should allow the intrafollicular concentrations of FSH to equilibrate with circulation. Consequently, most of these studies may actually have looked at a combined effect of

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LH and FSH. A similar argument may apply to humans, in which HCG commonly is used to induce ovulation. HCG usually is most effective when given along with a treatment that tends to increase the endogenous levels of FSH. However, HCG alone also may be effective, and most likely there are other control mechanisms involved that may work separately or in conjunction with the gonadotropins. More work certainly is needed to substantiate the hypothesis outlined here.

V. Possible Implications for Fertility The different levels of sensitivity of LH-HCG and FSH toward the two key events in ovulation, resumption of oocyte meiosis and the ovulatory process itself, have been used to explain poor reproductive performance in women who have higher levels of LH than expected during the follicular phase. In these women, who either may be undergoing ovarian stimulation (Howels et al., 1986) or may be in their normal menstrual cycle (Regan et al., 1990), reduced fertility is related to a premature resumption of meiosis. When the ovulatory process itself is induced, aged oocytes with a reduced potential for pre-embryo formation and conception may be released (Shoham et al., 1993). Although women with increased levels of LH indisputably show reduced fertility,there is no evidencethat the underlying mechanisms are causally related to premature resumption of meiosis. Actually, Dekel et al. (1999, using doses of LHHCG that were shown to induce premature resumption of meiosis in a rat model, were unable to show any negative effect on the subsequent pre-embryo development. Again, abnormalFSH levels also have been shown to have negative effects on fertility (Mattheij et al., 1993). In rats, increased FSH levels, without concomitant LH increases, were shown to induce premature resumption of meiosis correlated with a reduction in fertility from 91 to 30%. Because FSH has not been ascribed adverse effects on folliculardevelopment,it was suggestedthat the reduced fertility was causally related to the premature resumption of meiosis. A similar conclusion was reached in a study of normal gonadotropin pituitary downregulated women (Yding Andersen, 1997). It was found that whereas levels of LH were barely detectable, levels.of FSH during ovarian stimulation reached values above those found during the normal midcycle surge of gonadotropins. Levels of FSH on day 8 of ovarian stimulation showed a significant negative correlation with the subsequent ability of the retrieved oocytes to undergo fertilization and pre-embryo development, whereas levels of LH throughout the stimulation period showed no such correlation. Thus, the preovulatory follicles develop different sensitivities for gonadotropin stimulation toward ovulation time and the resumption of meiosis. We propose that it may be possible to develop ways to control the meiotic process and affect fertility without affecting ovulation and steroidogenesis. It

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may be possible-by using MAS, MAS-related compounds, or compounds inducing the synthesis of MAS (e.g., FSH)-to manipulate the meiotic process of oocytes from preovulatory follicles before ovulation is induced. At ovulation, “old” oocytes with a low probability for fertilization will be released. The premature induction of meiosis may not interfere significantly with ovulation or steroidogenesis. Several laboratories are now attempting to develop a new anticonceptional method in which the theory is to manipulate the meiotic process of the follicleenclosed oocyte in a way that inhibits proper fertilization after a naturally occurring ovulation. In contrast to the present anticonceptional methods, which mainly focus on making the environment for the developing embryo hostile, the new concept based on MAS focuses on affecting one or a few cells, namely, the preovulatory oocyte.

Acknowledgments We thank Susan Peters for correcting the language. This study was supported by the Danish Medical Research Council, Grant Nos. 9400824 and 9502022.

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Index

A Acp36DE, female sperm storage role, 87-88 Anterior-posterior patterning Danio rerio pattern formation, 20-22 vertebrate limb formation, 46-52 Hox gene role, 45-46, 49-51 polarizing activity characteristics, 46-49 positional information, 46-49 region specification, 51-52 Antirrhinum, flower development, 134-135, 138 AP3 gene, flower development role, 138-143, 153 Apical ectodermal ridge, vertebrate limb formation dorsal-ventral axis, 53-54, 58-59 proximal-distal axis differentiation, 40-42 gene expression, 43-46 Arabidopsis, flower development, 134- 136, 138

B @-Catenin dorsal blastula organizer establishment role, 4-7 gastrula organizer induction, 7-9 Blastomeres, dorsoventral polarity establishment, see Pattern formation Blastula organizer, see Dorsal blastula organizer Bone morphogenetic proteins anterior-posterior patterning, 20-22 dorsal-ventral patterning, 14-20 mesoderm-ectoderm dorsalization. 12- 14 C

Caenorhabditis elegans, sex determination, 99-127 dimorphism, 101- 104

future research directions, 126-127 gene evolution, 123-124 genetic analysis, 108-1 11 coordinated control, 110-1 11 gene identification, 108-1 10 regulatory pathway, 110 germ-line analysis, 119-120 hermaphrodite sperm-oocyte decision, 120123 fern-3 gene regulation, 122-123 somatic gonad anatomy, 120-121 tra-2 gene regulation, 121-122 mechanism conservation, 116- 1 17 molecular analysis, 111-1 16 cell nonautonomy, 112 cell-to-cell signaling, 112 protein-protein interactions, 114- 115 sexual fate regulation, 114-1 15 sexual partner identification, 115-1 16 signal transduction role, 113-1 14 TRA-1 activity regulation, 113-1 14 transcriptional regulation, 1 12 overview, 99-100 phylogenetic comparisons, 123-124 TRA- 1 activity regulation, 113-1 14 targets, 116-117 unresolved questions, 124-126 feedback regulation, 125-126 gene numbers, 124-125 germ line dosage compensation, 125 parallel pathways, 125-126 X:A ratio role chromosome count, 117-1 19 dosage compensation, 104-108 primary sex determination, 104 Calcium ion, gonadotropin-induced meiosis resumption role, 174 Cell-to-cell signaling, sex determination regulation, 112 Chemotaxis, sperm storage, 79-80 Convergent extension, Danio rerio pattern

187

I aa Convergent extension (conr. ) formation, gastrulation movement coordination, 23-28 Cyclic adenosine 5’-monophosphate, gonadotropin-induced meiosis resumption role, 170-172

D Danio rerio, pattern formation, 1-28 dorsal blastula organizer establishment, 4-7 gastrula organizer induction, 7-9 dorsal gastrula organizer affector mutations, 15-16 anterior-posterior patterning, 20-22 bone morphogenetic protein role, 12-20 dorsal-ventral patterning, 12-20 dorsoventral neural patterning, 20-22 ectoderm dorsalization, 12-14 embryonic shield equivalence, 9- 10 induction, 7-9 mesodem dorsalization, 12-14 molecular genetic characteristics, 10-22 dorsoventral polarity establishment, 2-4 gastrulation movement coordination, 22-28 convergent extension, 23-28 epiboly, 22-23 involution-ingression movements, 23 overview, I , 28 DEF gene, flower development role, 138-143 Dimorphism, Caenorhabdiris elegans, 101-104 Dorsal blastula organizer, Danio rerio pattern formation role establishment, 4-7 gastrula organizer induction, 7-9 Dorsal gastrula organizer, Danio rerio pattern formation role affector mutations, 15-16 anterior-posterior patterning, 20-22 dorsal-ventral patterning, 12-20 dorsoventral neural patterning, 20-22 ectoderm dorsalization, 12- 14 embryonic shield equivalence, 9-10 induction, 7-9 mesoderm dorsalization, 12- 14 molecular genetic characteristics, 10-22 Dorsal-ventral patterning Danio rerio pattern formation, 2-4 vertebrate limb formation, 52-58 apical ectodermal ridge formation, 53-54

Index dorsal positional cues, 54-57 dorsoventral boundary, 53-54 ectoderm transplantation studies, 52-53 mesoderm transplantation studies, 52-53 ventral positional cues, 57-58 Dosage compensation, Caenorhabditis elegans sex determination coordinated control, 110- 111 germ line, 125 X:A ratio role genetic basis, 104- 106 molecular analysis, 106-108 dpy gene, sex determination regulation, 109111 Drosophila melanogaster; sperm storage, 70, 76, 85-88

E Ectoderm dorsalization, Danio rerio, dorsal gastrula organizer role, 12-14 vertebrate limb dorsoventral patterning studies, 52-53 vertebrate limb formation, apical ectodermal ridge dorsal-ventral axis, 53-54, 58-59 proximal-distal axis, 40-46 Embryogenesis, pattern formation, see Pattern formation Embryonic shield, dorsal gastrula organizer equivalence, 9-10 Epiboly, Danio rerio pattern formation, gastrulation movement coordination, 2223

F .fern genes, sex determination regulation

epistatic interactions, 110 germ-line analysis, 119-122 identification, 109- 110 sexual partner identification, 115-1 16 signal transduction controls, 113-1 14 Fibroblast growth factor, vertebrate limb formation role, 39-42 Flower development, see Plant development Follicle-stimulating hormone, oocyte meiosis resumption induction follicle maturation role, 166-167 germinal vesicle breakdown induction, 170

Index granulosa cell population heterogeneity, 167- I68 meiosis-activating sterol role, 174-178 membrane receptor activation, 170

189 I Inositol triphosphate, gonadotropin-induced meiosis resumption role, 174

L G Gastrula organizer, .see Dorsal gastrula organizer Germ-line analysis, Cuenorhuhditis elegans sex determination, 119-120, 125 GLO gene, flower development role, 138-143 Gonadotropins, oocyte meiosis resumption induction, 163-179 fertility implications, 178-179 follicle maturation, 166- 167 gonadotropin receptor localization, 167- 168 granulosa cell population heterogeneity, 167-168 meiosis-activating sterol role, 174-178 oocyte maturation, 168- 169 overview, 163-165 ovulation induction, 168-169 preovulatory surge effects, 166-167 signal transduction pathways, 169- 175 calcium pathway, 174 cyclic adenosine 5’-monophosphate, 170172 inositol 1.4.5-triphosphate pathway, 174 meiosis-activating sterols, 174- 175 nuclear purines, 173-174 oocyte-cumulus-granulosa cell interactions, 169- I70 phosphodiesterases, 172-173 protein kinase A, 172-173 Granulosa cells, gonadotropin-induced meiosis resumption role oocyte-cumulus cell interactions, 169I70 population heterogeneity, 167-1 68

H her-1 gene, sex determination regulation, 112114, 119-122 Hermaphrodites, Carnurhabditis eleguns sex determination, sperm-oocyte decision, 120-123 Hux genes, vertebrate limb formation role, pattern formation, 45-46, 49-51

Limb development, vertebrate pattern formation, 37-59 anterior-posterior axis, 46-52 Hox gene role, 45-46, 49-5 1 polarizing activity characteristics, 46-49 positional information, 46-49 region specification, 5 1-52 dorsal-ventral axis, 52-58 apical ectodermal ridge formation, 53-54 dorsal positional cues, 54-57 dorsoventral boundary, 53-54 ectoderm transplantation studies, 52-53 mesoderm transplantation studies, 52-53 ventral positional cues, 57-58 overview, 37-39, 59 proximal-distal axis, 39-46 apical ectodermal ridge role differentiation, 40-42 gene expression, 43-46 fibroblast growth factor role, 39-42 limb outgrowth, 39-40 Luteinizing hormone, oocyte meiosis resumption induction follicle maturation role, 166- 167 germinal vesicle breakdown induction, 170 granulosa cell population heterogeneity, 167-168 meiosis-activating sterol role, 174- 178

M Meiosis-activating sterols, oocyte meiosis resumption role, 174-178 Meiosis, gonadotropin-induced resumption in oocytes, 163-179 fertility implications, 178-179 follicle maturation, 166-167 gonadotropin receptor localization, 167- 168 granulosa cell population heterogeneity, 167-168 meiosis-activating sterol role, 174- I78 oocyte maturation, 168-169 overview, 163-165 ovulation induction, 168-169

190 Meiosis ( conr. ) preovulatory surge effects, 166-167 signal transduction pathways, 169-175 calcium pathway, 174 cyclic adenosine 5’-monophosphate, 170172 inositol 1,4,5-triphosphate pathway, 174 meiosis-activating sterols, 174- 175 nuclear purines, 173-174 oocyte-cumulus-granulosa cell interactions, 169-170 phosphodiesterases, 172- 173 protein kinase A, 172-173 Mesoderm dorsalization, Danio rerio dorsal gastrula organizer role, 12- 14 vertebrate limb dorsoventral patterning studies, 52-53

N Nicotiana, flower development, 134-135 Nieuwkoop center, Danio rerio pattern formation role establishment, 4-7 gastrula organizer induction, 7-9

0 Oocytes Caenorhabditis elegans sex determination, hermaphrodite sperm-oocyte decision, 120- 123 fern-3 gene regulation, 122- 123 somatic gonad anatomy, 120-121 tra-2 gene regulation, 121-122 gonadotropin-induced meiosis resumption, 163-179 fertility implications, 178- 179 follicle maturation, 166-167 gonadotropin receptor localization, 167168 granulosa cell population heterogeneity, 167- 168 meiosis-activating sterol role, 174- 178 oocyte maturation, 168-169 overview, 163-165 ovulation induction, 168-169 preovulatory surge effects, 166-167 signal transduction pathways, 169-175 calcium pathway, 174

Index cyclic adenosine 5’-monophosphate, 170-172 inositol 1,4,5-triphosphate pathway, 174 meiosis-activating sterols, 174-175 nuclear purines, 173-174 oocyte-cumulus-granulosa cell interactions, 169- 170 phosphodiesterases, 172- 173 protein kinase A, 172-173

P Pattern formation Danio rerio, 1-28 dorsal blastula organizer establishment, 4-7 gastrula organizer induction, 7-9 dorsal gastrula organizer affector mutations, 15-16 anterior-posterior patterning, 20-22 bone morphogenetic protein role, 1220 dorsal-ventral patterning, 12-20 dorsoventral neural patterning, 20-22 ectoderm dorsalization, 12-14 embryonic shield equivalence, 9- 10 induction, 7-9 mesoderm dorsalization, 12-14 molecular genetic characteristics, 10-22 dorsoventral polarity establishment, 2-4 gastrulation movement coordination, 2228 convergent extension, 23-28 epiboly, 22-23 involution-ingression movements, 23 overview, 1, 28 vertebrate limb formation, 37-59 anterior-posterior axis, 46-52 anterior region specification, 51-52 Hox gene role, 45-46, 49-51 polarizing activity characteristics, 46-49 positional information, 46-49 dorsal-ventral axis, 52-58 apical ectodermal ridge formation, 5354 dorsal positional cues, 54-57 dorsoventral boundary, 53-54 ectoderm transplantation studies, 52-53 mesoderm transplantation studies, 5253 ventral positional cues, 57-58

Index overview, 37-39, 59 proximal-distal axis, 39-46 apical ectodermal ridge differentiation, 40-42 fibroblast growth factor role, 39-42 gene expression, 43-46 limb outgrowth, 39-40 Petal development differentiation, 143- 148 epidermal cells, 145-148 gene coordination, 152-153 shape, 144-145 genetic controls gene expression, 152-153 identity specification, 138- 143 tissue differentiation, 152-153 ontogeny, 135-138 overview, 133-135, 153 Petunia, flower development, 134- 135 Phosphodiesterases, gonadotropin-induced meiosis resumption role, 172-173 PI gene, flower development role, 138-143 Plant development overview, 133-135, 153 petals differentiation, 143-148 epidermal cells, 145-148 gene coordination, 152-153 shape, 144-145 genetic controls gene expression, 152-1 53 identity specification, 138-143 tissue differentiation, 152- 153 ontogeny, 135-138 stamen differentiation, 148- 152 genetic controls gene expression, 152-153 identity specification, 138-143 tissue differentiation, 152-153 ontogeny, 135-138 Progress zone, vertebrate limb formation, gene expression, 43-46 Protein kinase A, gonadotropin-induced meiosis resumption role, 172-173 Proximal-distal patterning, vertebrate limb formation, 39-46 apical ectodermal ridge role differentiation, 40-42 gene expression, 43-46 fibroblast growth factor role, 39-42

191 limb outgrowth, 39-40 Purines, gonadotropin-induced meiosis resumption role, 173- 174

S sdc genes, sex determination regulation, dosage

compensation coordination, 110- 111 Sex determination, Caenorhabditis elegans, 99-127 dimorphism, 101-104 future research directions, 126-127 gene evolution, 123-124 genetic analysis, 108-1 11 coordinated control, 110- 111 gene identification, 108-1 10 regulatory pathway, 110 germ-line analysis, 119-120 hermaphrodite sperm-oocyte decision, 120123 fern-3 gene regulation, 122- 123 somatic gonad anatomy, 120- 121 tra-2 gene regulation, 121-122 mechanism conservation, 116-1 17 molecular analysis, 111- 1 16 cell nonautonomy, 112 cell-to-cell signaling, 112 protein-protein interactions, 114-1 15 sexual fate regulation, 114-1 15 sexual partner identification, 115-1 16 signal transduction role, 113-1 14 TRA-1activity regulation, 113-1 14 transcriptional regulation, 1 12 overview, 99-100 phylogenetic comparisons, 123- 124 TRA-1 activity regulation, 113-1 14 targets, 116- 117 unresolved questions, 124-126 feedback regulation, 125- 126 gene numbers, 124-125 germ line dosage compensation, 125 parallel pathways, 125-126 X:A ratio role chromosome count, 117-1 19 dosage compensation, 104-108 primary sex determination, 104 Signal transduction oocyte meiosis resumption induction, 169175 calcium pathway, 174

192 Signal transduction (conf.) cyclic adenosine 5’-monophosphate, 170172 inositol 1,4,5-triphosphate pathway, 174 meiosis-activating sterols, 174- 175 nuclear purines, 173-174 oocyte-cumulus-granulosa cell interactions, 169-170 phosphodiesterases, 172- 173 protein kinase A, 172- 173 sex determination regulation, 113-1 14 Sonic hedgehog gene, vertebrate limb formation study, 47-50, 52 Sperm Caenorhabditis elegans sex determination, sperm-oocyte decision, 120-123 female sperm storage, 67-89 adaptive significance, 88-89 overview, 67-73, 89 storage mechanisms, 80-85 previous stored sperm fate, 81-82 sperm fate, 80, 84-85 sperm preference, 82-84 storage molecules, 85-88 Acp36DE, 87-88 biochemical studies, 86-87 genetic studies, 86-87 transfer mechanisms, 7 1-80 chemotaxis, 79-80 female secretions, 76-78 helper sperm, 79 morphology role, 73-74 muscular contractions, 74-75 seminal fluid, 75-76 sperm motility, 78-79 transfer numbers, 72-73 Stamen development differentiation, 148- 152 genetic controls gene expression, 152- 153 identity specification, 138-143 tissue differentiation, 152- I53

Index ontogeny. 135-138 overview, 133-135, 153

T fra genes, sex determination regulation conservation of mechanisms, 1 16- 1 17 germ-line analysis, 119-122 identification, 108-1 10 protein-protein interactions, 114- 1 15 sexual partner identification, 115-1 16 signal transduction controls, 112-1 14 Transcription, sex determination regulation, 112 Transforming growth factor-@,dorsal gastrula organizer establishment role, 7-9 TRA- 1 protein, Caenorhahditis elegans sex determination activity regulation, 113-1 14 targets, 116-117

w Wnt genes, vertebrate limb formation, 44-45, 54-55,59

n X:A ratio role, Caenorhabditis elegans sex determination chromosome count, 117-1 19 dosage compensation, 104-108 overview, 100 primary sex determination, 104

2

Zebrafish, see Danio rerio Zone of polarizing activity, vertebrate limb formation, 46-47, 55-59

Contents of Previous Volumes

Cellular and Molecular Procedures in Developmental Biology Guest edited by Flora de Pablo, Alberto Ferrus, and Claudio D. Stern 1 The Avian Embryo as a Model in Developmental Studies Elisabeth Dupin, Catherine Ziller, and Nicole M. Le Douarin

2 Inhibition of Gene Expression by Antisense Oligonucleotides in Chick Embryos in Vitro and in Vivo Aixa V Morales and Flora de Pablo

3 Lineage Analysis Using Retroviral Vectors Constance L. Cepko, Elizabeth Rydec Christopher Austin, Jeffrey Golden, and Shawn fields-Berry

4 Use of Dominant Negative Constructs t o Modulate Gene Expression Giorgio Lagna and Ali Hernmati-Brivanlou

5 The Use of Embryonic Stem Cells for the Genetic Manipulation of the Mouse Miguel Torres

6 Organoculture of Otic Vesicle and Ganglion Juan J. Garrido, Thomas Schirnrnang, Juan Represa, and Fernando Giraldez

7 Organoculture of the Chick Embryonic Neuroretina Enrique J. de la Rosa, Begona Diaz, and Flora de Pablo

8 Embryonic Explant and Slice Preparations for Studies of Cell Migration and Axon Guidance Catherine E. Krull and Paul M. Kulesa

9 Culture of Avian Sympathetic Neurons Alexander v. Holst and Herrnann Roher

193

194

Contents of Previous Volumes

10 Analysis of Gene Expression i n Cultured Primary Neurons Ming-Ji Fann and Paul

H. Patterson

11 Selective Aggregation Assays for Embryonic Brain Cell and Cell Lines Shinichi Nakagawa, Hiroaki Matsunarni, and Masatoshi Takeichi

12 Flow Cytometric Analysis of Whole Organs and Embryos Jose Serna, Belen Pirnentel, and Enrique J. de la Rosa

13 Detection of Multiple Gene Products Simultaneously by in Situ Hybridization and lmmunohistochemistry in Whole Mounts of Avian Embryos Claudio D. Stern

14 Differential Cloning from Single Cell cDNA libraries Catherine Dulac

15 Methods in Drosophila Cell Cycle Biology Fabian Feiguin, Salud Llarnazares, and Cayetano Gonzalez

16 Single CNS Neurons in Culture Juan Lerrna, Miguel Morales, and Maria de 10s Angeles Vicente

17 Patch-Clamp Recordings from Drosophila Presynaptic Terminals Manuel Martinez-Padron and Alberto Ferrijs

Meiosis and Cametogenesis Guest edited by Mary Ann Handel 1 Recombination in the Mammalian Germ Line Douglas L. Pittrnan and John C. Schimenti

2 Meiotic Recombination Hotspots: Shaping the Genome and Insights into Hypervariable Minisatellite DNA Change Wayne F1 Wahls

3 Pairing Sites and the Role of Chromosome Pairing i n Meiosis and Spermatogenesis in Male Drosophh Bruce D. McKee

Contents of Previous Volumes

195

4 Functions of DNA Repair Genes during Meiosis W. Jason Cummings and Miriam E. Zolan

5 Gene Expression during Mammalian Meiosis E. M. Eddy and Deborah A. O'Brien

6 Caught in the Act: Deducing Meiotic Function from Protein lmmunolocalization Jerry Ashley and Annemieke Plug

7 Chromosome Cores and Chromatin at Meiotic Prophase Peter B. Moens, Ronald E. Pearlman, Walther Traut, and Henry H. Q. Heng

8 Chromosome Segregation during Meiosis: Building an Unambivalent Bivalent Daniel I? Moore and Terry 1. Orr-Weaver 9 Regulation and Execution of Meiosis in Drosophila Males Jean Maines and Steven Wasserman

10 Sexual Dimorphism in the Regulation of Mammalian Meiosis Mary Ann Handel and John J. Eppig 11 Genetic Control of Mammalian Female Meiosis Patricia A. Hunt and Renee LeMaire-Adkins

12 Nondisjunction in the Human Male Terry J. Hassold

1 Paternal Effects in Drosophila: Implications for Mechanisms of Early Development Karen R. Fitch, Glenn

K. Yasuda, Kelly N. Owens, and Barbara T. Wakimoto

2 Drosophila Myogenesis and Insights into the Role of nautilus Susan M. Abrnayr and Cheryl A. Keller

3 Hydrozoa Metamorphosis and Pattern Formation Stefan Berking

4 Primate Embryonic Stem Cells James A. Thomson and Vivienne S. Marshall

196

Contents of Previous Volumes

5 Sex Determination in Plants Charles Ainsworth, ]ohn Parker, and Vicky Buchanan- Wollaston

6 Somitogenesis Achim Gossler and Martin Hrab6 de Angelis

1 The Murine Allantois Karen M. Downs

2 Axial Relationships between Egg and Embryo in the Mouse R. L. Gardner

3 Maternal Control of Pattern Formation i n Early Caenorhabdifis eregans Embryos Bruce Bowerman

4 Eye Development in Drosophila: Formation of the Eye Field and Control of Differentiation Jessica E. Treisman and Ulrike Heberlein

5 The Development of Voltage-Gated Ion Channels and I t s Relation to Activity-Dependent Developmental Events William 1. Moody

6 Molecular Regulation of Neuronal Apoptosis Santosh R. D’Mello

7 A Novel Protein for Ca2+ Signaling at Fertilization 1.Parrington, F. A. Lai, and K. Swam 8 The Development of the Kidney Jonathan Bard

1 Homeobox Genes in Cardiovascular Development Kristin D. Patterson, Ondine Cleaver, Wendy b! Gerber, Matthew W. Grow, Craig S. Newman, and Paul A. Krieg

2 Social Insect Polymorphism: Hormonal Regulation of Plasticity in Development and Reproduction in the Honeybee Klaus Hartfelder and Wolf Engels

Contents of Previous Volumes

197

3 Getting Organized: New Insights into the Organizer of Higher Vertebrates ]odi L. Smith and Gary C. Schoenwolf

4 Retinoids and Related Signals in Early Development of the Vertebrate

Central Nervous System A. 1. Durston, 1. van der Wees, W. W. M . Pijnappel, and S. F. Godsave

5 Neural Crest Development: The Interplay between Morphogenesis and Cell Differentiation Carol A. Erickson and Mark V Reedy

6 Homeoboxes in Sea Anemones and Other Nonbilaterian Animals: Implications for the Evolution of the Hox Cluster and the Zootype lohn R. Finnerty

7 The Conflict Theory of Genomic Imprinting: H o w Much Can Be Explained? Yoh lwasa

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