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Current Topics in Developmental Biology Volume 44

Series Editors Roger A. Pedersen

and

Reproductive Genetics Division Department of Obstetrics, Gynecology, and Reproductive Sciences University of California, San Francisco, California 941 43

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

Editorial Board Peter Cruss 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

Yosh itaka 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 44 Edited by

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

Gerald l? Schatten Departments of Obstetrics-Gynecology and Cell and Developmental Biology Oregon Regional Primate Research Center Oregon Health Sciences University Beaverton, Oregon

Academic Press San Diego

London Boston New York

Sydney Tokyo

Toronto

Cover photo credit: Eggs labeled with wt-GFP or RSGFP. For more details, see Chapter 1 “Green Fluorescent Protein as a Vital Marker in Mammals” by Masahito Ikawa, Shuichi Yamada, Tomoko Nakanishi, and Masani Okabe.

This book is printed on acid-free paper.@ 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 US.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. 0070-21 53/99 $25.00

Academic Press a division of Harcouri Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:l/www.apnet.com

Academic Press 24-28 Oval Road, London NW 1 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Book Number: 0-12-153144-9

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Contents

ix

Contributors

Preface

xi

1 Green Fluorescent Protein (GFP) as a Vital Marker in Mammals Masahito Ikawa, Shuichi Yarnada, Tornoko Nakanishi, and Masaru Okabe

I. 11. 111. IV. V.

Introduction 1 Characteristics of GFP and Its Variants Expression of GFP in Mammals 4 Future Applications 14 14 Observation Methods References 16

2

2 Insights into Development and Genetics from Mouse Chimeras John D. West

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

Introduction 21 Mouse Chimeras as Experimental Organisms 22 Studies of Mouse Development with Chimeras 30 Genetic Studies with Chimeras 40 Studies of Developmental Genetics with Chimeras 46 Concluding Remarks 53 References 56

3 Molecular Regulation of Pronephric Development Thomas Carroll, john Wallingford, Dan Seufert, and Peter 0. Vize

I. Introduction

67

11. Structure and Function 69 111. Early Development 70

IV. Pronephric Patterning and Induction

71 V

Contents

vi V. Pronephric Tubules

74

VI. Pronephric Duct 83 VII. Glomus 88 VIII. Conclusions 90 References 92

4 Symmetry Breaking in the Zygotes of the Fucoid Algae: Controversies and Recent Progress Kenneth R. Robinson, Michele Wozniak, Rongsun Pu, and Mark Messerli

I. Introduction

101

11. Physiology of Fertilization

103 111. The Response to Light 105 IV. The Role of Calcium and Calmodulin in Photopolarization and Rhizoidal Growth 108 V. Cortical pH Gradients during Axis Formation and Rhizoidal Growth VI. Actin Microfilaments and Photopolarization 1 14 VII. Axis Fixation 115 VIII. The Signal Transduction Process for Photopolarization 1 17 IX. An Opsin-like Photoreceptor in Pelvetia? 118 X. A Speculative Model for Photopolarization 119 XI. Summary 121 References 122

I13

5 ReevaluatingConcepts of Apical Dominance and the Control of Axillary Bud Outgrowth Carolyn A. Napoli, Christine Anne Beveridge, and Kimberley Cathryn Snowden

I. IJ. 111. IV. V. VI.

Introduction and Overview 128 Plant Architecture and Meristem Potential 129 Apical Dominance I33 Use of Induced Mutations to Study Axillary Bud Outgrowth 135 Molecular and Genetic Approaches for Understanding Bud Outgrowth Conclusions and Perspectives 160 References 163

6 Control of Messenger RNA Stability during Development Aparecida Maria Fontes, lun-itsu /to, and Marcel0 Jacobs-Lorena

I. Introduction 171 11. Regulation of mRNA Stability in Plants

174

137

vii

Contents 111. Regulation of mRNA Stability in Caenorhabditis elegans

IV. V. VI. VII. VIII.

176

Regulation of mRNA Stability in Drosophila 178 Regulation of mRNA Stability in Xenopus 184 Regulation of mRNA Stability in Avians 186 Regulation of mRNA Stability in Mammals 189 Conclusions and Prospects 194 References 195

7 ECF Receptor Signaling in Drosophila Oogenesis Laura A. Nilson and Trudi Schupbach

I. 11. 111. 1V. V. VI.

Overview 203 Introduction 204 Spatial Regulation of Egfr Activation 212 Response of Follicle Cells to Egfr Activation 221 Determination of Embryonic DV Polarity by Local Egfr Activation Summary 236 References 237

Index 245 Contents of Previous Volumes

255

232

This Page Intentionally Left Blank

Contributors

Numbers in purenthrses indicate rhe pages on which rhr uuthors' conrriburions begin.

Christine Anne Beveridge (l27), Department of Botany, The University of Queensland, Brisbane, Queensland 4072, Australia Thomas Carroll (67), Center for Developmental Biology, Department of Zoology, University of Texas, Austin, Texas 78712 Aparecida Maria Fontes (171), Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 Masahito Ikawa (l), Genome Information Research Center, Osaka University, Suita, Osaka 565-087 1, Japan Jun-itsu Ito (171), Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44 106 Marcel0 Jacobs-Lorena ( 1 7 l), Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 Mark Messerli ( 10 I), Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Tomoko Nakanishi (I), Genome Information Research Center, Osaka University, Suita, Osaka 565-087 1, Japan Carolyn A. Napoli ( 127), Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 Laura A. Nilson (203), Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 Masaru Okabe ( I ) , Genome Information Research Center, Osaka University, Suita, Osaka 565-087 1, Japan Rongsun Pu ( l O I ) , Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Kenneth R. Robinson (101), Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Trudi Schiipbach (203), Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

ix

X

Contributors

Dan Seufert (67), Center for Developmental Biology, Department of Zoology, University of Texas, Austin, Texas 787 12 Kimberley Cathryn Snowden ( 127), School of Biological Sciences, University of Auckland, Auckland, New Zealand Peter D. Vize (67), Center for Developmental Biology, Department of Zoology, University of Texas, Austin, Texas 787 12 John Wallingford (67), Center for Developmental Biology, Department of Zoology, University of Texas, Austin, Texas 78712 John D. West (21), Department of Obstetrics and Gynaecology, University of Edinburgh, Centre for Reproductive Biology, Edinburgh EH3 9EW, United Kingdom Michele Wozniak (101), Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Shuichi Yamada (l), Program for Promotion of Basic Research Activities for Innovative Biosciences; and Genome Information Research Center, Osaka University, Suita, Osaka 565-087 1, Japan

Preface

This volume continues the custom of Current Topics in Developmental Biology in addressing developmental mechanisms in a variety of experimental systems. In an effort to ensure that green organisms are well represented in this eclectic series, we are pleased that three chapters consider developmental biology in these groups. Chapter 4, by Kenneth R. Robinson, Michele Wozniak, Rongsun Pu,and Mark Messerli from Purdue University, presents symmetry breaking in the zygotes of the fucoid algae: controversies and recent progress. In Chapter 5, by Carolyn A. Napoli from the University of Arizona, Christine Anne Beveridge from the University of Queensland, and Kimberly Cathryn Snowden from the University of Auckland, the authors reevaluate concepts of apical dominance and the control of axillary bud outgrowth. The volume’s first chapter considers a new green organism, namely mice created as GFP transgenics: Masahito Ikawa, Shuichi Yamada, Tomoko Nakanishi, and Masaru Okabe from Osaka University present green fluorescent protein (GFP) as a vital marker in mammals. Insights into development and genetics from mouse chimeras by John D. West of the University of Edinburgh is our second chapter. Thomas Carroll, John Wallingford, Dan Seufert, and Peter Vize from the University of Texas consider the molecular regulation of pronephric development in Chapter 3. This volume concludes with a consideration of the control of messenger RNA stability during development in Chapter 6, by Aparecida Maria Fontes, Jun-itsu Ito, and Marcelo Jacobs-Lorena from Case Western Reserve University. In Chapter 7, Laura A. Nilson and Trudi Schiipbach from Princeton University discuss EGF receptor signaling in Drosophila oogenesis. Together with the other volumes in this series, this volume provides a comprehensive survey of major issues at the forefront of modern developmental biology and transgenic strategies. These chapters should be valuable to researchers in the fields of plant and animal 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 biology and molecular controls of differentiation. This volume in particular will be essential reading for anyone interested in gene regulation of development, transgenic approaches, organogenesis, chimeras, signaling molecules, plant development, and embryonic axis formation. 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 xi

xii

Preface

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 topics and authors, and Liana Hartanto and Michelle Emme for their 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 Green Fluorescent Protein (GFP) as a Vital Marker in Mammals Masahito Ikawa, Shuichi Yamada,’,* Tomoko Nakanishi,2 and Masaru Okabe,2,* I Program for Promotion of Basic Research Activities for Innovative Biosciences Genome Information Research Center Osaka University Suita, Osaka 565-087 1, Japan I. Introduction 11. Characteristics of GFP and Its Variants 111. Expression of GFP in Mammals A. In Vitro

B. In Vivo IV. Future Applications V. Observation Methods A. Fixation B. Fluorescence Microscopic Analysis C. Fluorometry D. Analysis by Flow Cytometer E. Immunostaining and Western Blotting References

1. Introduction Reporter genes have long been used for analysis of biological events (Bronstein et ul., 1994; Cui et al., 1994). These transgenes often, but not always, encode enzymes, e.g., P-galactosyltransferase (lacZ), chloramphenicol acetyltransferase (CAT), and firefly luciferase (luc). In such cases, the addition of a substrate is required to detect gene expression. Therefore, application of the enzymes in living cells as a reporter gene demands noninvasive loading of substrates. Recently, several substrates have been developed that penetrate the plasma membrane and are processed in the cytoplasm for use in living cells (Reddy et al., 1992). However, it is still necessary to load the substrate with these markers. Since Chalfie et al. reported that the green fluorescent protein (GFP)derived from the jellyfish Aequoreu victoria can serve as a useful marker of gene expression without substrate loading or any other pretreatment (Chalfie et al., 1994), GFP has opened the door for the use of intact cells and organisms as experimental systems (see Cubitt et al., 1995; Prasher, 1995; Misteli and Spector, 1997). Here we present an overview *Author to whom correspondence should be addressed. Currenr Topics in Developmental Biol0g.x Vol. 44 Copyright 8 1999 by Academic Press. All rights of reproduction in any form reserved. 0070-2153/99 $25.00

1

2

Masahito Ikawa et al.

of the application of GFP and its variants as vital markers in mammals both in vitro and in vivo.

II. Characteristics of GFP and Its Variants In Aequorea, GFP is responsible for the green bioluminescence from the margin of its bells with Ca2+-mediatedactivation (Fig. I ,+ see color plate). Aequorea bioluminescence is first activated when Ca2+binds to aequorin following emission of blue fluorescence. Blue fluorescence and subsequent energy transfer from activated aequorin cause GFP to emit green fluorescence. Therefore, unlike enzymes, GFP needs no substrate to emit fluorescence. Rather, it requires only energy to excite the fluorophore (Fig. 2a). GFP is a single peptide of 238 amino acids (Prasher et al., 1992). Using deletion analysis, Li et al. defined the minimal domain in GFP required for fluorescence to amino acids 7-229 (Li et al., 1997). To become fluorescent, GFP needs to form its fluorophore by post-translational autocyclization of Ser65, Tyr66, and Gly67 following oxidation (Fig. 2b). Finally, the chromophore locates in the center of a barrel-like basket composed of I 1 P-sheets (Fig. 2c) (Ormo et al., 1996). GFP becomes fluorescent even when expressed in the heterologous species, probably because the conformational changes require no substrate(s) or cofactor(s). After the formation of fluorophore, GFP is quite stable and remains fluorescent up to 65°C pH 11, 1% SDS or 6 M guanidinium chloride (Ward and Bokman, 1982; Cubitt et al., 1995); Prasher, 1995). Since the first report of expression of GFP in C. elegans, this novel and simple reporter has attracted much interest for its potential as an in vivo marker of gene expression and cell lineage (Fig. 3). Moreover, GFP can serve as a unique tag to monitor protein localization since it remains fluorescent even after the formation of fusion protein. Both the N- and C-termini have been successfully fused to a wide range of cellular proteins without affecting the function of the host protein (Cubitt et al., 1995). However, one must be cognizant of the effects of the GFP tag (which may be added to the fusion protein) such as increased solubility and tendency toward steric hindrance. The emission spectrum of wild-type GFP (maximum at 508 nm) is proportional to that of the well-known dye fluorescein isothiocyanate (FITC; maximum at 5 15 nm), but its excitation spectrum (maximum at 395 nm) differs considerably from that of FITC (maximum at 493.5 nm). After the initial report of GFP as a marker protein, considerable improvements in both spectrum and intensity were accomplished through modification to the GFP (Heim et al., 1994; Delagrave et al., 1995; Crameri et al., 1996; Heim and Tsien, 1996). Many of the GFP variants contain mutations within the chromophore region itself. Replacement of Ser65 by Thr (S65T) shifts the excitation spectra to a slightly longer wavelength (maximum 'Figure lb, c: Reprinted from Trends in Generics 11, D. C. Prasher, Using GFP to see the light. pp. 320-323. Copyright 1995, with permission from Elsevier Science.

I . GFP as a Vital Marker in Mammals

3

(a) aequorin (apoaequorin + coelenterazine + 0,)

Ca-apoaequori&lenterazine*

blue light nm)

(5,=469

GFP

’ GFP’T GFP

green fluorescence (5,1509 nm)

H

OH

H

OH

Fig. 2 (a) Bioluminescent pathway in Aequurea victoria. The luciferin involved in the pathway is coelenterazine. The photoprotein aequorin responds directly to Ca2+by oxidizing the bound coelenterazine. The excited-state luciferin (marked with an asterisk) releases blue light in the absence of GFP. Stimulation by activated luciferin or blue light causes GFP to emit green fluorescence. (b) Proposed biosynthetic mechanism for green fluorescent protein chromophore, in which cyclization precedes oxidation. (c) Crystal structure of GFP. The light-emitting fluorophore is located in the center of a barrel-like basket. [(a) Reprinted from Trends in Generics 11, D. C. Prasher, Using GFP to see the light. pp. 320-323. Copyright 1995, with permission from Elsevier Science; (b) Reprinted from Trends in Biuchernical Science 20, A. B. Cubitt et al., Understanding, improving and using green fluorescent proteins, pp. 448-455.Copyright 1995, with permission from Elsevier Science; (c) Ormo etal., 1996.

Masahito Ikawa et al.

4

Fig. 3 (a) GFP as a reporter gene. (b) The insertion of an IRES (internal ribosomal entry site) sequence enables bicistronic expression of GFP and target protein as a separated structure. (c) GFP can be used as a tag to indicate the localization. (d) The addition of a signal peptide leads the GFP to localize into intracellular organelles.

at 489 nm) and results in an optimal observation opportunity with FITC filter sets or for fluorescence-activated cell sorting (FACS) analysis (Heim et al., 1995). To date, various GFPs emitting blue (BFP) or yellow (YFP)have been made commercially. Without mutation, GFP can emit red fluorescence with green excitation under the condition of low oxygen (Elowitz e l al., 1997). This implies that a fluorescent protein emitting red fluorescencemight also be available in the near future. It has been reported that the mutations affect not only spectra but also other characteristics of GFP such as magnitude and solubility (Cormack et al., 1996; Siemering et al., 1996). Several publications have already demonstrated the utility of these GFP variants and their combinations in creating new applications. The characteristicsof GFP and its variants are listed in Table I.

111. Expression of GFP in Mammals A. In Vitro 1. Turning Cells “Green”

The expression of GFP cDNA in cultured mammalian cells turns these cells fluorescent green (Fig. 4, see color plate). GFP has a significant advantage over other commonly used reporters. Since GFP emits green fluorescence without any substrates or cofactors, one can monitor the presence of GFP by illuminating living cells. The significance is that the observation of GFP expression can be done noninvasively, without fixation or permeabilizing cells. In the earliest studies on GFP, it was reported that the formation of the wt-GFP chromophore is time-consuming (about 4 h at 22°C (Heim et al., 1994)). making it difficult to determine the exact point at which gene expression begins. Although culturing at a low temperature (30-33°C) facilitates chromophore formation

Table I GFP and Its Variants

variant

Max. Max excitation emission (nm) (nm)

Mutations

wt-GFP

-

395

509

Cycle3 RSGFP S65T GFPmut1 EGFP

F99S. M153T. V163A F64M. S65G, Q69L S65T F64L. S65T F64L, S65T

395 490 489 488 488

509 505 51 1 507 507

P4-3

Y66H. Y145P S65G, V68L, S72A. T203Y

380 513

440

1OC

_ _ _ ~

527

EM (cm-I M-I)

21,000 (7,150) nd nd 39,200 250,000 250,000 37,000 36,500

Characteristics

Ref.

Green emission

Chalfie et al., 1994

X 18 brighter Excited at 488 nm Excited at 488 nm X35 brighter Codon optimized for human cells Blue emission Greenish-yellow emission

Crameri et al., 1996 Delagrave et nl., 1995 Heim et al.. 1995 Cormack et al., 1996 B a n g et nl.. 1996 Heim et al.. 1994

Ormo et al., 1996 ~

Note: The EM has been measured at optimal excitation. The EM for 488-nm excitation of wt-GFP is presented in parentheses. Many of the GFP variants are available from companies (Clontech, hnp://www.clontech.com/;PharMingen, hnp://www.pharmingen.com/;Life Technologies, htrp://ww.lifetech.com/; Quantum Biotechnologies,hnp://ww.qbi.com/).

6

Masahito Ikawa et al.

(Ogawa et al., 1995), this presents obvious problems for the culture of mammalian cells. Recently, however, several GFP variants were demonstrated to have overcome these disadvantages. The S65T mutation forms the chromophore about four times more rapidly than wild-type GFP (Heim et al., 1995). Some mutations were reported to improve the thermosensitivity of GFP (Siemering er af., 1996; Kimata et al., 1997). The double-amino-acid substitutions (P64L and S65T) in the EGFP (GFPmutl) make it about 35-fold brighter than wt-GFP due to an increase in its extinction coefficient (EM) (Cormack et al., 1996). Moreover, EGFP contains more than 190 silent base changes that correspond to human codon-usage preferences in gaining expression in mammalian cells. However, it is still difficult to determine when the gene shuts down since GFP is quite stable after the chromophore formation. Some of the applications of GFPs in mammalian cells are summarized in Table 11.

2. Attaching a “Green” Tag to Protein The advent of GFP has made possible an entirely new way of looking at intracellular protein traffic. GFP and its variants retain fluorescence even after the formation of fusions to many proteins as well as maintain the normal biological activity of the fused proteins. By taking advantage of GFP, Ogawa er al. reported the translocation of a fusion protein between GFP and human glucocorticoid receptor from cytoplasm to a nucleus in a single living cell in real time (Ogawa et al., 1995). GFP fusions can provide enhanced sensitivity and resolution in comparison to standard techniques using antibodies (Wang and Hazelrigg, 1994). In addition, GFP utilization made it possible to examine protein kinetics in v i v a Time-lapse analysis of protein transport through secretary pathways has been reported by several groups (Kaether and Gerdes, 1995; Presley et al., 1997; Scales et al., 1997; Wacker et af., 1997), with some of them provided as an mpeg demonstration on the internet. It is also possible to tag GFP with a signal peptide. GFPs having a signal peptide finely label organelles in the cell. Visualization of the nucleus and mitochondria in a cell was possible by adding the nuclear localization signal or mitochondria targeting signal to GFP or BFP, respectively (Rizzuto et al., 1996). Using this approach, Sawin reported an efficient method for identifying nuclear localization signals in yeast by screening in living yeast (Sawin and Nurse, 1996).

3. As an Intracellular Biosensor By combining GFP and other proteins, we can create new molecules, which can visualize the change at the molecular level. Translocation of GFP-glucocorticoid receptor reflects hormone-mediated signal transduction (Ogawa et al., 1995). Although G protein-coupled receptors (GPCR) represent the single most important drug targets for medical therapy, a method either to identify the function of new GPCRs or to associate them with cognate ligands was lacking in a growing num-

Fig. 1 (a) Green fluorescent protein is responsible for the green color of the bioluminescence emitted by the jellyfish Aequorea vicroria. A photograph of the living jellyfish under normal (reflected) lighting. GFP is normally sequestered in photogenic masses around the bell margin and when they glow, the bell margin region emits light, in the form of a broken ring. This photo was kindly provided by Dr. Claudia E. Mills, University of Washington. (b, c) Aequorea bioluminescence originates from light organs called photocytes, which are located in clusters beside the tentacle bulbs near the marginal nerve ring at the margin of the bell. (b) is a light micrograph of one tentacle bulb and (c) is a fluorescent photograph of the same preparation, showing the localized GFP in siru. [(b,c) Prasher, 1995.1

Fig. 4 GFP turns cells "green." (a) Double labeling of nucleus and mitochondria with GFP and BFP, respectively. (M)Eggs labeled with wt-GFP or RSGFP were distinguishable by the emitted light. Eggs expressing wt-GFP (c) emit green when excited with V-filter sets, whereas eggs expressing RSGFP(d) are fluorescent with B-filter sets. [(a) Rizzuto, era/., 1996.)

Fig. 5 (a) Structure of pCX-GFPs. The SalI-BamHI fragment. containing CMV-IE enhancer, chicken p-actin promoter, GFP cDNA, and rabbit P-globin poly-A signal. was used to make transgenic mice. (b. c) Approximately half of the fertilized eggs derived from wild female and hemizygous males were tluorescent green, starting from the 4-cell stage (the photo shows the blastocyst-stage eggs). (d) Implanted embryos and placentas (the photo shows the day 12 embryo and placenta; note that the wild-type embryo and the placenta were not visible at all under this condition). (e-f) Newborn mice were also green under excitation light. [(a) Ikewa e r a / . , 1995b; (bf) Okabe er a/.. 1997.1

..

Fig. 6 Green sperm. (a) There is an exocytotic vesicle, called the acrosome, located on the head of the sperm. The acrosome reaction with release of the contents packed in the acrosome is a Acrosin promoter driven GFP, which is prerequisite physiological change before fertilization. (k) tagged by acrosin signal peptide, exists only in spermatogenic cells and is localized to the sperm acrosome.

Fig. 7 Production of chimeric mice between wild-type and green mice embryo. (a-b) Eight-cell stage embryos were aggregated in the depression made by a darning needle in the microdrops. (c-f) Contributions of green cells were easily identified following culture ( c 4 morulae; e-f, blastocyst). (g-j) Chimeric mice carrying green cells were obtained. (All photos were taken under a stereotyped fluorescence microscope.)

1 . GFP as a Vital Marker in Mammals

7

ber of orphan receptors. By observing the translocation of a fusion protein between GFP and 0-arrestin as a biosensor, it was possible to recognize the activation of pharmacologically distinct GPCRs (Barak et al., 1997). Tsien’s group proposed applying a phenomenon called FRET (fluorescence resonance energy transfer) as a new way to utilize GFP in biological systems. This is based on the shift of emission wavelength of closely associated GFPs of a different wavelength. Using FRET, they showed that GFP could serve as a fluorescent indicator for Ca2+concentration by making a fusion protein named “chameleon” (Miyawaki et al., 1997). Chameleon consists of tandem fusion of a BFP, calmodulin, calmodulin-binding peptide M13, and an EGFP. Binding of Ca2+to M 13 causes calmodulin to wrap around the M 13 domain, increasing the FRET between the flanking GFPs. Although chameleon was designed to sense Ca2+concentration, one can imagine numerous extensions of this novel work by arranging a combination of proteins that might give rise to FRET. B. In Vivo 1. GreenMice

To determine if GFP can be used as a vital marker in vivo, we made transgenic mice expressing GFPs (Ikawa et al., 1995a; Okabe et af., 1997) (Fig. 5 , see color plate). First, we tested polypeptide chain elongation factor la (EF) promoter, for it is known to have a ubiquitous expression (Mizushima and Shigekazu, 1990).However, in EF-wt-GFP transgenic mice, very weak expression in the liver was observed (unpublished data). When we chose a CAG promoter (combination of 0-actin promoter and hCMV enhancer) reported by Miyazaki (Fig. 5a) (Niwa et d., 1991), a strong expression was observed in some tissues, especially in muscle and pancreas. Finally, we reported mice expressing wt-GFP, the first application of GFP in mammals in vivo (Ikawa et al., 1995a). However, to our surprise, when EGFP instead of wt-GFP was expressed by a CAG promoter, almost all tissues fluoresced bright green with EGFP, whereas a few were fluorescent in the former transgenic mice (Okabe et al., 1997) (Fig. 5b). The difference was not simply due to greater sensitivity of EGFP, allowing detection in tissues with lower expression. For example, whereas blood vessels were always negative in transgenic mice expressing wtGFP (n > 20), this tissue was one of the brightest parts of the body in all of the transgenic animals expressing EGFP (more than 50 lines were observed). Since the difference between EGFP structure and others is only a few amino acid substitutions, codon optimization might be responsible for the ubiquitous expression. In the “green mice” (expressing GWs by CAG promoter), green fluorescence can be initially detectable in the preimplantation stage and remains throughout pregnancy (Figs. 5c and 5d). The characteristic green fluorescence was easily detected upon the birth of transgenic mice under a hand-held UV lamp (Fig. 5a). However, all transgenic mice lines looked normal and healthy, despite a significant amount of EGFP expression throughout the body, including brain, nerve cells,

m

Table II Application of GFPs in Cultured Mammalian Cells Cell A. Marker for gene expression CHO HeLa and human primary fibroblast T-cell line (CEM) NEI3T3 NlH3T3 and ES PEER B. Localization of fusion proteins HeLa CV 1 HeLa COS-7, HeLa, and LM(TK-)

cos

Hela and U20S PtK, NM3T3, BHK, and COS-7 HeLa NIH3T3 and RBL

Fusion

Characteristics

Ref.

GFP does not confer a growth disadvantage Monitoring BAC-gene transfer

Gubin et al., 1997 Baker and Cotten, 1997

CRMl cre recombinase -

Method for monitoring HIV infectivity titers Cell cycle dependent expression of CRM 1 Marker for cre expression IRES-mediated bicistronic expression with c-myc 6106-143

GeNaiX el d.,1997 Kudo et al.. 1997 Gagneten et al., 1997 Mosser et al., 1997

NLS and mitochondria targeting signal Peroxisomal targeting signal1 NLS Calreticulin Lamin B receptor CENP-B MAP4 NAGT I Elastase

Double labeling of mitochondria and nucleus with BFP and GFP Visualization of peroxisome Comparison of NLSs Localization of calreticulin to nucleus Localization of lamin B to nuclear membrane Localization of CENP-B to centromers Localization of MAP4 to microtubules" Localization of NAGT to Golgi Visualization of nuclear envelope and ER calcium stores

Rivuto et nl., 1996

-

Wiemer et al., 1997 Chatterjee et al.. 1997 Roderick er al., 1997 Ellenberg et al., 1997 Shelby et al.. 1996 Olson er al., 1995 Shima eta!.. 1997 Subramanian and Meyer, 1997

C. Translocation of GFP fusion proteins HeLa and Vero Chromogranin B

Visualization of protein transport in the secretory pathway

Kaether and Gerdes, 1995: Wacker et a/., 1997 Presley et al.. 1997; Scales et al., 1997 Tarasova et a[., 1997

COS and Vero

VSVG

Visualization of ER-to-Golgi transporth

CHO, NIH3T3. HeLa, and cos 1 COS-7 HEK293 and 32D.3 CHO

Cholecystokininreceptor type A OTC Raf- 1 GLUT4 PML protooncogene and glucocorticoid receptor

Visualization of G protein-coupled receptor trafficking Visualization of mitochondria1protein import Targeting of Raf-1 to mitochondria by Bcl-2 Insulin-stimulatedtranslocation of GLUT4 Hormone-stimulated translocation of glucocorticoid receptor and thermosensitivity of GFP

Yano et a/.. 1997 Wang eta/.. 1996 Dobson et al., 1996 Ogawa et al., 1995

HEK293 and COS

p-arrestin 2

Bar& et al., 1997

HeLa

Calmodulin, M13, and BFP

Biosensor for detecting G protein-coupled receptor activation Fluorescent indicators for Ca2 based on FRET system

cos-1 D. Other

+

Miyawaki et al., 1997

Note: BAC, bacterial artificial chromosome: PEER, human acute lymphoblastic leukemia; IRES, internal ribosomal entry site; NLS, nuclear localization signal; MAP, microtubule-associated protein; NAGT, N-acetylglucosaminyltransferase;OTC, ornithine transcarbamylase;VSVG, viral glycoprotein ts045; GLUT, glucose transporter; FRET, fluorescence resonance energy transfer. a http://www. rochester.edu/College/BIO/olmtedhp.hrml. http://diir.nichd. nih.gov/CBMB/pbl labob.hrml.

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Masahito Ikawa et al.

immune cells, and hormonal glands. Histochemical analysis also showed no abnormalities in any of the tissues examined. These data demonstrate that GFP is not toxic for most cells, from early development to maturity (Okabe et al., 1997). According to Chiocchetti, GFP has a higher sensitivity when compared to lacZ, even when the amount of proteins is comparable under the same promoters (Chiocchetti et al., 1997). 2. GreenEggs

The discernment of transgenic pups from nontransgenic siblings is an important step in producing homozygous transgenic mice and establishing transgenic lines. The main methods used in detection of transgenes are polymerase chain reaction (PCR) analysis or southern blotting following extraction of DNA from the tail of 3- to 4-week-old pups (Gendron and Gridley, 1993). However, both techniques require skill and consume time if applied to a large number of transgenic animals. A quick separation of transgenic mice after birth has been reported to be possible by coinjecting a marker transgene (Overbeek er al., 1991; Bonnerot and Nicolas, 1993). For example, mice lacking tyrosinase become albino and the injection of a tyrosinase minigene into albino eggs will render the coat color of the transgenic mice agouti or black (Overbeek et al., 1991). However, this method is only applicable to strains lacking a tyrosinase gene (e.g., FVB or CD-1). Since the newborns from all the green mice were distinguishable by their green fluorescence as already mentioned, the pCX-GFPs should be useful as a transgenic marker when coinjected with a desired transgene. In our case, when a GFP gene was injected with a target gene (mole ratio 1 : l), more than 80% of the transgenic founder mice carried both genes, whereas the remaining 20% carried one (unpublished data). It is also possible to separate transgene-bearing embryos by blastocyst biopsy and subsequent PCR analysis (Sheardown et al., 1992). However, biopsy procedures include embryo holding, partial dissection of the zona pellucida, removal of a single blastomere from the embryo, and transfer of the blastomere into the PCR tube. Moreover, the PCR procedures include DNA extraction, amplification of the objective gene, and electrophoresis. Since much skill and time are required, it is difficult to handle large numbers of embryos at one time (Han et al., 1993).Moreover, PCR amplification from a small number of cells tends to cause false positive or false negative signals. Therefore, noninvasive, easy, and reproducible detection methods are desirable and the development of such techniques has been much anticipated. Using GFP,it was easy to separate transgenic eggs before implantation when the transgenic eggs were fluorescent green (Fig. 5b) (Ikawa er al., 1995b). Morulae obtained from a wild female mated with a hemizygous transgenic male were separated into “green” or “nongreen” groups under a fluorescent microscope. All the morulae survived the selection procedure and formed blastocysts within the

1. GFP as a Vital Marker in Mammals

11

following 18 h. The individual embryos were subjected to PCR analysis to check the transgene. A total of 49 embryos were separated and subjected to PCR analysis and accurate separation was confirmed. When separated embryos were transferred into the uterus of pseudopregnant females (day 2.5), all the mice born from green eggs proved to be carrying the GFP transgene whereas the nongreen eggs did not carry the transgene at all. For propagation of large transgenic animals such as bovine or porcine, detection of the transgene at the preimplantation stage would be desirable considering the long gestation period and limited number of offspring. The real usage of the separation of the transgenic embryo would emerge during in vitro fertilization using one transgenic male for many nontransgenic females, since numerous gametes could be obtained from the male but not from the female. Therefore, a more successful detection method of paternally inherited transgene is required. GFP-expressing constructs are also effective in producing transgenic animals. To date, the low efficiency of transgenic animal production by microinjection has been a serious problem, especially in large mammals. Using an EF promoter combined with CMV enhancer sequences, Takada et al. have shown that the eggs injected at the pronuclear stage become fluorescent until the blastocyst stage and transgenic eggs can be selected in a considerably high ratio before implantation (Takada et al., 1997). These data imply that an efficient production and propagation of transgenic livestock could be achieved by GFP coinjection. Even after maturation, transgenic GFP can be introduced to animals by direct injection of DNA, direct injection of DNA particles, or virus-mediated gene transfer. For example, living olfactory neurons were labeled in vivo and the physiological function of an odorant receptor was analyzed by bicistronic expression of GFP and odorant receptor (Zhao et al., 1998). The insertion of an IRES sequence between the genes for a target protein and GFP enables simultaneous expression of them as separate proteins in the same cell (Fig. 3b). It is therefore possible to determine if a given cell is transfected without affecting the function of the target protein or damaging cells. Recently, embryo sexing by PCR analysis from a part of the embryo has been performed in a variety of species such as human, bovine, and mouse (Handyside et al., 1990; Cui et al., 1993). By producing a sex chromosome-linked GFP transgenic mouse, it was possible to separate male and female embryos before implantation (unpublished data). These X- and Y-bearing embryos are good models for identifying the developmental differences between male and female during embryogenesis (e.g., sex-related growth difference, HY-antigen), which have been the subject of much debate for many years.

3. Green Sperm and Others Green mice produced green eggs but not green sperm as few cytosol are left on sperm after spermiogenesis. Certain, cell- or organ-specific expression of GFP is

12

Masahito Ikawa er al.

possible if we use an appropriate promoter to drive GFP in transgenic mice and a signal peptide leads GFP to the target organelle. EGFP driven by acrosin promoter results in male germ cell specific expression. Moreover, fusion protein in which the amino terminus of EGFP has been fused at the carboxyl terminus of the acrosin signal peptide has been localized into the sperm acrosome. This reflects the endogenous phenomenon that acrosin, a trypsin-like protease, is expressed only in male germ cells and localized to sperm acrosome (Fig. 6, see color plate). Thus, prepared green sperm is very useful for studying fertilization mechanisms. Although motile upon leaving the male reproductive tract, mammalian sperm are not fertile at this stage. They require a period to complete the maturation initiated in the epididymis. The final maturation phase is called capacitation. A subsequent exocytotic event known as the acrosome reaction (Fig. 6), which occurs in the female reproductive tract, has been extensively studied in mice. Since the size of the acrosome in mice is very small, it is difficult to observe the occurrence of the acrosome reaction morphologically. To detect the change in the acrosome, antibodies, fluorescent dye, and agglutinins have been utilized. However, all of these probes need time to react with sperm and could affect sperm function. The green sperm we made enabled us to monitor swimming in a dark field and the release of acrosomal contents during the acrosome reaction, a prerequisite for fertilization (in preparation). Green mice have green organs, but it was difficult to point out the shape of cells since most cells were green. By expressing GFPs under the astrocyte-specificglial fibrillary acidic protein (GFAP) promoter, visualization of astrocytes throughout the central nervous system was demonstrated (Zhuo et ul., 1997). Such transgenic mice were applicable to visualizing dynamic changes in astrocyte morphology by various physiological stimuli, since fluorescence microscopic analysis can be carried out on living preparations. In such mice, Zhuo er a/. compared GFP and lacZ gene driven by the same promoter and showed that GFP expression matched endogenous GFAP expression better than IacZ. The difference suggests either that GFP is more sensitive than lacZ or that other features of the lacZ sequences somehow interfere with gene expression as previously reported. The visualization of developmental stage specific gene expression has also been demonstrated. GFP was expressed under the transcriptional control of yeast UAS and targeted to specific cell types during development by crossing a variety of GAL4-expressing enhancer trap lines. This was done in Drosophilu, but this enhancer trap system is also applicable as a means to identify the enhancers regulating dynamic change in gene expression during mammalian development. Table 111 summarizes the applications of GFPs in mammalian cells in vivo. 4. Chimeras

Green mice are a useful source of green-tagged cells or organs for transplantation and eggs for making chimeric mice to monitor their lineage (Figs. 7a-j, see color

Table III Application of GFPs in Mammalian Cells in Vivo Species

Vector

A. Transgenic mice Mouse Nonviral Mouse

Nonviral

Mouse Mouse Mouse

Nonviral Nonviral Nonviral

Promoter

Rat

Adenoviral and lentiviral Adenoviral

Visualization of live astrocyte in transgenic mice Separation of transgenic-bearingeggs Selective production of transgenic mice

Zhuo et al.. 1997 Ikawa et al., 199% Takada et al., 1997

Ubiquitous Hippocampus Transplanted ANIP973

GFP labeling of blastomere and green ES cells Labeling of neural cells Visualization of metastasis in vivo

Zernicka Goetz et al., 1997 Moriyoshi et al., 1996 Chishima etal., 1997

Hematopoietic cells

Tracking of gene modified hematopoietic cells in vivo

Persons et al., 1997

Transplanted 208F cells Olfactory neurons

Doxycycline-dependent expression Functional expression of odorant receptor IRES-mediated bicistronic expression with odorant receptor Efficient gene transfer into photoreceptor

Watsuji et al., 1997 Zhao et al., 1998

Hemopexin and PI integrin GFAP CAG EF

Liver and embryo

CMV and Rhodopsin NSE and PDGF B-chain

Ref

Ikawa er al., 1995a; Okabe etal., 1997 Chiocchetti er a/., 1997

Ubiquitous

C. Marker for gene expression Mouse Retroviral rtTA system Rat Adenoviral CAG

Characteristics Ubiquitous GFP expression in transgenic mice as “green mice” GFP worked better than lacZ

CAG

B. Trac.Ag of label& :ells in vivo Mouse Nonviral cdc2 Rat Adenovirus CAG Mouse Nonviral Adenovirus major late promotor Mouse Retroviral MSCV LTR

Rat

Tissue

Photoreceptor and retinal cells Spinal cord

Efficient gene transfer into spinal cord neurons

Flannery et al., 1997; Miyoshi et al.. 1997 Peel et al., 1997

Note: CAG, human cytomegarovirus enhancer and chicken p action promoter; GFAP, astrocyte-specific glial fibrillary acidic protein; EF, polypeptide chain elongation factor la; ANIP973, human lung adenocarcinota cell; MSCV, murine stem cell virus; rtTA, reverse tetracycline-regulated;NSE,neuron-specific enolase; PDGF, plateletderived growth factor.

14

Masahito Ikawa et al.

plate). The behavior of mutant ES cells can be followed with simple microscopic observation of chimeric embryos or mice. Thus, chimeric animals could be used to monitor the phenotypes of embryo lethal mutant ES cells. If we prepare Xlinked GFP mice, chimeric mice between ES cells and green (female) eggs result in a 100% of germ-line cell from ES cells if it was transmittable. A combination of GFP variants allows labeling of cells of multiple origin in different colors (Fig. 4b). As one advantage, GFP-tagged cells were easily traced in intact organisms on a real-time basis. For instance, blastomeres labeled by injecting mRNA encoding GFP were used to analyze their progeny during preimplantation development (Zernicka Goetz et al., 1997). By labeling tumorigenic cells with GFP, micrometastases of lung cancer cells distant from the primary tumor in recipient mice were visualized (Chishima et al., 1997).

IV. Future Applications Compared to lacZ, the use of GFP in thin sections is limited. Moreover, CAT and luciferase enzyme assays are more sensitive than detecting direct fluorescence from GFP in a quantitative assay. However, it would be incorrect to compare the characteristics of GFP with those of other properties. The most striking and novel nature of GFP is that we can observe the expression in real time in a noninvasive manner. In this context, GFP possess an exclusive advantage. Many experiments made possible solely through the use of GFP have been reported and still more are expected to be published. The field of developmental biology will also benefit from the usage of GFP-tagged cells in experiments. By using GFP variants, several groups (including ours) succeeded in expressing them and detecting fluorescence in mouse embryonic stem (ES) cells (Gagneten et al., 1997; Zernicka Goetz et al., 1997; unpublished data). Green-ES cells were easily traced during chimerization and embryogenesis. These results suggest GFP can be a reporter for knock-in, gene trap, or negative selection in ES-cell-mediated transgenesis. As a source for the “green eggs” or “green stem cells,’’ we are in the process of submitting our “green mice” with a background of C57BL/6 to the Jackson Laboratories Induced Mutant Resource (Bar Harbor, ME).

V. Observation Methods A. Fixation

Since GFP is very soluble, care has to be taken regarding fixation. Even after acetone, ethanol, or methanol treatment, GFP remains fluorescent but dissolves and diffuses rapidly into the aqueous phase when phosphate-buffered saline (PBS)

15 or other aqueous solutions are added to the section for observation. We could use 4% paraformaldehyde in PBS to fix the samples without a significant loss in fluorescence. In the fixing of organs, perfused fixation with 4% paraformaldehyde solution is preferable (Akagi er al., 1997). Formalin fixation and paraffin embedding are not applicable because the background fluorescence hinders the GFP fluorescence. 1. GFP as a Vital Marker in Mammals

B. Fluorescence Microscopic Analysis

Although GFP can be excited with an FITC filter set because of its minor absorption peak at 475 nm, emission is about an order of magnitude lower compared to fluorescein. Therefore, proper excitation is necessary for fluorescence microscopy. We use filter sets (BP 405 nm for excitation and LP 455 nm for emission). For red-shifted variants such as S65T and EGFP, common FITC filter sets (BP 495 nm for emission and LP 5 15 nm for emission) are suitable. To suppress the background fluorescence when observing the culturing cells, we recommend using culture medium devoid of phenol red to lower the background. As an alternative, one can change the culture medium to PBS during observation. Some cell types or organs (liver, kidney, etc.) have an autofluorescence likely due to flavin coenzymes or mitochondrially bound NADH (Aubin, 1979). Paran-embedded section also emits strong background fluorescence. The selection of filter sets reduces the autofluorescence but improvement is limited. Furthermore, it must be noted that some nail polishes also emit fluorescence (Chalfie er al., 1994; Wang and Hazelrigg, 1994). For observing organs or embryos, fluorescence stereomicroscopes specially suited for observing GFPs are commercially available (Leica, hrfp:// www.leica.com/; Olympus, hrrp://www.olympus.co.jp/). By removing the objective lens from the fluorescence microscope, we could achieve a wider excitation beam for naked eye observation through an appropriate filter. For the simplest method of observation, a hand-held UV light (360 nm) works well to observe our green mice.

C. Fluorometry

It is very easy to measure the fluorescence in the tissue extracts using a fluorometer. The concentration of GFP in the solution can be measured by quantitative fluorometric assay using known amounts of recombinant GFPs (hrtp:// www.turnerdesigns.com/) even in the tissue homogenate (Chiocchetti et al., 1997). The fluorescence of muscle extract from our transgenic mice was detectable even after X 10,000dilution of a 10% homogenate (Ikawa et al., 19954.

16

Masahito Ikawa et al.

D. Analysis by Flow Cytometer

Because the excitation optimum for EGFP is close to 488 nm, cells from the EGFP-labeled cells were suitable for flow cytometric analysis. Multicolor labeling of cells could be done by combined use of GFP variants. wt-GFP and BFP are also applicable for flow cytometric analysis, if the cytometer has a laser that can excite in the violet region and is fitted with an appropriate filter.

E. lmmunostaining and Western Blotting

There have been reports that monoclonal and polyclonal antibodies against GFPs work well in immunohistochemical staining and western blotting (Naray Fejes Toth and Fejes Toth, 1996).

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totipotent embryonic stem cells expressing developmentally regulated lacZ usion genes. Proc. Natl. Acad. Sci. U.S.A.89,6721-6725. Rizzuto, R., Brini, M., De Giorgi, F., Rossi, R., Heim, R., Tsien, R. Y., and Pozzan, T. (1996). Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr. Biol. 6 , 183-188. Roderick, H. L., Campbell, A. K., and Llewellyn, D. H. (1997).Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors. FEBS Left. 405, 181-185. Sawin, K. E., and Nurse, P. (1996). Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein. Proc. Natl. Acad Sci. US.A. 93, 1514615151.

Scales, S. J., Pepperkok, R., and Kreis, T. E. (1997). Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action of COP11 and COPI. Cell 90, 1137-1 148. Sheardown, S. A., Findlay, I., Turner, A., Greaves, D., Bolton, V. N., Mitchell, M., Layton, D. M., and Muggleton, H. A. (1992). Preimplantation diagnosis of a human P-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo. Hum.Reprod. 7, 1297-1 303. Shelby, R. D., Hahn, K. M., and Sullivan, K. F. (1996). Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells. J. Cell Biol. 135,545-557. Shima, D. T., Haldar, K.,Pepperkok, R.. Watson, R., and Warren, G. (1997). Partitioning of the Golgi apparatus during mitosis in living HeLacells. J. Cell Biol. 137, 121 1-1228. Siemering, K. R.. Golbik, R., Sever, R., Haseloff, J.. Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S.. and Kricka, L. I. (1996). Mutations that suppress the thennosensitivity of green fluorescent protein. Chemiluminescent and bioluminescent reporter gene assays. Cum Biol. 6, 1653-1663. Subramanian, K.. and Meyer, T. (1997). Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores. Cell 89,963-971. Takada, T., lida, K., Awaji, T.,Itoh, K., Takahashi, R., Shibui, A.,Yoshida, K., Sugano, S., and Tsujimoto, G. (1997). Selective production of transgenic mice using green fluorescent protein as a marker. Narure BiorechnoL 15,458-461. Tarasova, N. I., Stauber, R. H., Choi, J. K., Hudson, E. A,, Czerwinski, G., Miller, J. L., Pavlakis, G. N., Michejda, C. J., and Wank, S. A. (1997). Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A. J. Biol. Chem. 272, 14817-14824. Wacker, I., Kaether, C., Kromer, A,, Migala, A,, Almers, W., and Gerdes, H. H. (1997). Microtubuledependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein. J. Cell Sci. 110, 1453-1463. Wang. H. G., Rapp, U. R., and Reed, J. C. (1996). Bcl-2 targets the protein kinase Raf-1 to mitwhondria [see comments]. Cell 87,629-638. Wang, S., and Hazelrigg, T. (1994). Implications for bcd mRNA localization from spatial distribution of exu protein in Drosuphila oogenesis. Narure 369,400-403. Ward, W., and Bokman, S. (1982).Reversible denaturation of Aequorea green-fluorescent protein: Physical separation and characterization of the renatured protein. Biochemistry 21, 12468-12474. Watsuji, T., Okamoto, Y.,Emi, N., Katsuoka, Y., and Hagiwara, M. (1997). Controlled gene expression with a reverse tetracycline-regulated retroviral vector (RTRV) system. Biochem. Biophys. Res. Commun. 234,769-773. Wiemer, E. A,, Wenzel, T.. Deerinck, T. J., Ellisman, M. H., and Subramani, S. (1997). Visualization of the peroxisonial compartment in living mammalian cells: Dynamic behavior and association with niicrotubules. J. Cell Biol. 136,71-80. Yano. M., Kanazawa, M., Terada, K., Namchai, C., Yamaizumi, M., Hanson. B., Hoogenraad, N., and Mori, M. (1997). Visualization of mitochondria1 protein import in cultured mammalian cells with green fluorescent protein and effects of overexpression of the human import receptor TomZO. J. Biol. Chem. 272,8459-8465. Zernicka Goetz, M., Pines, J., McLean Hunter, S., Dixon, J. P., Siemering, K. R., Haseloff, J., Evans,

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M. J., Siemering, K. R., Golbik, R., Sever, R., Haseloff, J., Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G.S., and Kricka, L. J. (1997). Following cell fate in the living mouse embryo. Mutations that suppress the thermosensitivity of green fluorescent proteion. Chemiluminescent and bioluminescent reporter gene assays. Developmen? 124, 1 133-1 137. Zhang, G., Gurtu. V., and Kain, S. R. (1996). An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochem. Biophys. Rex Commun. 227,707-71 I . Zhao, H.,Ivic, L., Otaki. J., Hashimoto, M., Mikoshiba, K.,and Firestein. S. (1998). Functional expression of a mammalian odorant receptor. Science 279,237-242. Zhuo, L., Sun, B., Zhang. C. L., Fine, A., Chiu, S. Y.,and Messing, A. (1997). Live astrocytes visualized by green fluorescent protein in transgenic mice. Dev. Biol. 187,36-42.

2 Insights into Development and Genetics from Mouse Chimeras John D. West Department of Obstetrics and Gynaecology University of Edinburgh Centre for Reproductive Biology Edinburgh EH3 9EW, United Kingdom

1. Introduction II. Mouse Chimeras as Experimental Organisms A. Production of Mouse Chimeras B. Production of Mouse ES Cell Chimeras C. Genetic Effects on the Composition of Chimeras D. Comparisons between Chimeras and Mosaics 111. Studies of Mouse Development with Chimeras A. Prospective Studies of Development B. Retrospective Studies of Development IV. Genetic Studies with Chimeras A. Quantitative Genetic Traits B. Phenotypic Analysis of Single Mutant Genes V. Studies of Developmental Genetics with Chimeras A. Cytogenetic Studies B. Sex Determination in Chimeras C. Genomic Imprinting D. Phenotypic Analysis of Mutant Genes and Genetic Knockouts VI. Concluding Remarks References

1. Introduction The production of the first experimental mouse chimeras was reported in the 1960s (Tarkowski, 1961; Mintz, 1962; Gardner, 1968) and once the techniques were established in other laboratories, chimeras were enthusiastically taken up as powerful analytical tools, particularly for studies of developmental biology (Mintz, 1971; Gardner and Papaioannou, 1975; McLaren, 1976; Le Douarin and McLaren, 1984). More recently, chimeras have often been relegated to providing a means for making “genetic knockout” mice via embryonic stem cell chimeras. The introduction of modern transgenic cell markers, however, has greatly increased the power of chimeras and this has rekindled an interest in their analytical

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uses. The growing realization that chimeras can provide powerful tools for the phenotypic analysis of abnormal genotypes (including those generated by genetic knockout technology) has led to a renaissance in experimentation with mouse chimeras. Consequently, they are likely to play a major role in studies of developmental genetics during the next few years. In this review I first overview the production and characteristics of mouse chimeras and then discuss some of the major contributions that they have made to mouse developmental biology and genetics. In the final section I focus on recent chimera studies which have provided insights into mouse developmental genetics.

I I. Mouse Chimeras as Experimental Organisms A. Production of Mouse Chimeras

A chimera is a composite multicellular organism in which different cell populations are derived from more than one zygote (McLaren, 1976). So, a chimera has two or more genetically distinct populations of cells. Mosaic animals differ from chimeras in that all of the cell populations arise from a single zygote. In the plant kingdom, however, no such distinction is made and the term “chimera” is used regardless of the number of zygotes involved (Tilney-Bassett, 1986; Szymkowiak and Sussex, 1996). According to the foregoing definition of a chimera, an individual comprising lineages derived from a normal zygote and an unfertilized cell (e.g., Strain et af., 1995) would be classed as a chimera rather than a mosaic. In this respect, the definition differs from some earlier versions (Anderson et ul., 1951 ; Ford, 1969). Mammalian chimeras are usually subdivided into primary and secondary chimeras. Secondary chimeras are formed when tissues are combined by a variety of tissue grafting or transplantation techniques from two or more adults or postimplantation embryos. secondary chimerism is usually restricted to one or a few tissues but the results may be dramatic as in the case of interspecies rat + mouse secondary chimeras resulting in rat spermatogenesis in a mouse testis (Clouthier et ul., 1996). Secondary chimeras can occur spontaneously, as in the case of blood chimeras arising by placental fusion and anastomosis of blood vessels in cattle twins. If cattle twins are of opposite sex, the female is a sterile, masculinized freemartin because anti-Mullerian hormone produced by Sertoli cells in the testes of the male twin affects the ovary and reproductive tract of the female twin (Vigier etal., 1984, 1987). Primary chimeras are formed at a very early stage of development so all body tissues may be involved. Rare cases of spontaneous human primary chimeras have been reviewed elsewhere (e.g., McLaren, 1976; Tippett, 1984) and more recent cases have been documented (e.g., Watkins et al., 1981; Zeilmaker et ul., 1983; Vandeleur and Zeilmaker, 1990; Sawai et al., 1994; Strain et al., 1995, 1998). One

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recently reported human intersex chimera was conceived by in vitro fertilization (IVF) procedures (Strain et al., 1998). This raises the question of whether the risk of producing human chimeras by spontaneous aggregation of dizygotic twin preimplantation embryos is higher after IVF than in natural dizygotic twinning. In principle, embryo aggregation could be facilitated if the embryos hatched early from their zonae pellucidae. IVF might induce early hatching either if the zona was accidentally damaged (e.g., during freezing and thawing for cryopreservation of embryos) or if it was intentionally breached (e.g., during micromanipulation procedures for intracytoplasmic sperm transfer (ICSI) or embryo biopsy for preimplantation diagnosis (Handyside and Delhanty, 1997); or to overcome the culture-induced effect of zona hardening to facilitate embryo hatching). In the reported case there is no mention of any of these procedures and it is still too early to judge whether IVF and related procedures increase the risk of producing chimerism. However they arise, most human chimeras are likely to go undetected unless they are formed from embryos with genetic dissimilarities that result in obvious phenotypic variegation (Zeulzer et al., 1964) or result in interXY chimera (see sexuality because of a preponderance of XX cells in an XX Section VB). For other mammalian species, primary chimeras may be produced experimentally. This is usually done by embryo aggregation or microinjection of cells into a blastocyst (Fig. 1) but various techniques have been used, including the following: ( I ) aggregating two or more cleavage-stage embryos (Tarkowski, 1961; Mintz, 1962), (2) aggregating cleavage-stage embryos with cultured cells (Stewart, 1982; Fuji and Martin, 1983), (3) microinjecting cells into the cavity of a blastocyststage embryo, where they become incorporated into the inner cell mass (Gardner, 1968, 1998), (4) microinjecting cells under the zona pellucida at the 8-cell stage (Rossant and Vijh, 1980;Thomson and Solter, 1988), (5) replacing the entire inner cell mass to produce a blastocyst reconstitution chimera (Gardner et al., 1973; Gardner and Johnson, 1973; Papaioannou, 1982), or (6) selectively replacing some cells at the morula stage (Gardner and Nichols, 1991). To produce aggregation chimeras, typically, two or more genetically distinct preimplantation-stageembryos are recovered from the reproductive tract and their zonae pellucidae are removed (usually by exposure to acidic Tyrode’s solution or pronase). The zona-free embryos are aggregated together in a culture drop, cultured overnight to the blastocyst stage, and surgically transferred to the uterus of a pseudopregnant female recipient (previously mated to a sterile male) for further development. Sometimes adhesion of the embryos is facilitated by various means, such as exposure of the embryos to phytohemagglutinin (Mintz et al., 1973; Pratt, 1987), making indentations in the culture dish (Nagy and Rossant, 1993), or using a multiwell plate which either has small V-shaped wells (Boland and Gosden, 1994) or can be briefly centrifuged (Mikami and Onishi, 1985). Injection chimeras may be made either with pluripotent cells isolated from a preimplantation-stage embryo (e.g., inner cell mass cells; Gardner, 1968, 1998) or with cells maintained

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Fig. 1 Left: Production of aggregation chimeras (Tarkowski, 1961; Mintz, 1962) either by aggregating two or more genetically distinct preimplantation-stage embryos or by aggregating one or more preimplantation embryos with cultured, pluripotent embryonic stem cells. Right: Production of injection chimeras (Gardner, 1968) by injecting pluripotent cells (either cultured embryonic stem cells or cells isolated from another embryo). The blastocyst consists of an outer layer of trophectoderm cells which envelopes the inner cell mass (epiblast plus primitive endoderm) and blastocyst cavity. See text for details.

in an undifferentiated state in culture (e.g., embryonic stem cells; see Section IIB). These cells are microinjected into the cavity of a genetically distinct blastocyststage embryo with a micromanipulator. The injected blastocyst is cultured briefly and then surgically transferred to the uterus of a pseudopregnant female recipient for further development. Aggregation chimeras are usually designated A ++ B, where A and B represent the genotypes or strains of the two aggregated embryos or cells. In some early studies they were referred to as “allophenic” or “tetraparental” mice but nowadays the term “chimera” (or “chimaera”) is used almost universally. Aggregation of two embryos means that the chimeric embryo is initially double the normal size but size regulation occurs soon after implantation and the chimeric pup is normal size at term (Buehr and McLaren, 1974).The A B convention may also be used to represent the genotype combination of injection chimeras but A+B is sometimes a more useful notation (where “A-B” implies the injection of A-type cells into a B-type blastocyst). Primary chimeras can also be produced by recombining different tissues of two blastocyst-stage embryos (Gardner et ul., 1973; Gardner and Johnson, 1973; Papaioannou, 1982; Papaioannou and Dieterlen-LiCvre, 1984; Gardner et ul., 1990). Typically, an inner cell mass of one genotype will be combined with a trophectoderm of another genotype to form a blastocyst reconstitution chimera. Although primary chimeras have been produced for several mammalian species, including interspecies combinations, mouse aggregation chimeras and injection chimeras have been most widely used. Coat and eye pigment (Figs. 2 and 7) provide convenient genetic markers for

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25 visualizing patches of clonally related cells in a primary chimera but other genetic markers have to be included to identify the contribution of the two genetically distinct cell populations to nonpigmented tissues. Electrophoretic variants of enzymes were the genetic markers of choice for studies with mouse chimeras in many early studies (Fig. 3) and are still widely used for both qualitative and quantitative analysis. Cytogenetic variants and tissue-specific histochemical markers (e.g., inherited enzyme activity polymorphisms) were also employed in early experiments but these have now been largely superseded by transgenic markers which can provide spatial information in many tissue types. Transgenic cells are typically identified in histological sections either by detection of reporter gene expression, such as B-galactosidase histochemical staining for lacZ expression (Figs. 4 and 8), or by DNA-DNA in situ hybridization (Fig. 5). Recent work suggests that it will also be possible to use green fluorescent protein as a lineage marker in chimeras (Ikawa et al., 1995;Takada et al., 1997; Zernicka-Goetz eral., 1997). The availability of these new transgenic lineage markers has vastly improved the power of chimeras as experimental tools. It is often useful to make chimeras that contain a cell population derived from a homozygous mutant genotype (m/m). Problems arise if the homozygous embryos, to be incorporated into a chimera, have to be derived from intercrosses between two heterozygotes (m/+ X m/+). This applies to embryos that are homozygous for a lethal gene or a gene that causes infertility, which are often the most interesting genotypes to study with chimeras. The required m/m embryos may sometimes be identified before the chimeras are produced but this is usually 2 . Mouse Chimeras

Fig. 2 A pigmented tf albino mouse chimera. Coat pigmentation provides an excellent marker for identification of the chimeras. Melanocyte clones migrate from the neural crest and often form visible stripes either side of the dorsal midline.

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A

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Fig. 3 Electrophoretic variants of glucose phosphate isomerase (GPII). Samples 2-5 are from Gpili'/Gpil" GpiIh/Gpilh chimeras and produced a mixture of GPI IA and GPllB allozymes (labeled A and B), which were visualized by electrophoresis and histochemical staining for GPII.

Fig. 4 Transgenic marker TgR(ROSA26)26Sor (Friedrich and Soriano, 1991). detectable by Il-galactosidase histochemistry for lncZ expression in an E15.5 day hemizygous transgenic nontransgenic (lacZ'/ cf /crcZ-/-) fetal mouse chimera. Hemizygous transgenic cells (+/-)express l m Z and stain blue. so patches of positive and negative cells can be seen in the surface tissues: (a) head; (b) body; (c) visceral yolk sac; (d) feet. Scale bar:4 mm in (a-c). 2 mm in (d). (M. A. Keighren, J. H. Flockhart. and J. D. West, unpublished.)

2. Mouse Chimeras

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not feasible. In most cases, all the available embryos are used to make chi+/+, m / + +/+, and meras and the different chimeric genotypes (m/m +/+ +/+) must be identified retrospectively. This may be difficult because +/+ chimeras contain m and + alleles. Breeding both m/m +-+ +/+ and m / + experiments can help to distinguish the chimeric genotypes if the chimera transmits the appropriate cell population. For example, albino offspring from albino (m/+ x m/+) pigmented +/+ chimeras mated to albino +/+ mice will all be from ( m / + x m/+) germ cells. If the chimera is m/m +/+, all of the albino offspring will be m/+ but if the chimera is m / + ++ +/+, 50% of the albino offspring will be m / + and 50% will be +/+; if the chimera is +/+ c-, +/+, all of the albino offspring will be +/+.

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Fig. 5 Transgenic marker TgN(Hbb-b1)83CIo(Lo, 1983), detectable by DNA-DNA in siru hybridization in a section of part of an eye from a pigmented, transgenic ++ albino, nontransgenic mouse chimera. Most of the hemizygous transgenic cells (with a single hybridization signal in their nuclei) are on the right of the photograph in the INL and ONL (e.g., arrows) and form a stripe across the neural retina. This plane of focus shows the majority of the hybridization signals but the cells are out of focus; the RPE is also out of focus. Abbreviations: Ch, choroid (outer dark pigmented layer); RPE, retinal pigment epithelium (inner pigmented layer); PR, photoreceptor cells; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (M. A. Keighren, J. H. Flockhart, and J. D. West, unpublished.)

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If two mutant alleles ( m ’and m 2 ) are available and the DNA sequences are distinguishable from each other and from the wildtype (+) allele, an alternative strategy is possible (Quinn er al., 1996; see section VDl). Embryos could be produced from (m’/+ x m 2 / + )crosses, permitting the four types of chimeras (m’/m2c-, +/+, m ’ / + t, +/+, + / m 2 +/+, and +/+ ++ + / + ) t o be distinguished by molecular techniques, such as the polymerase chain reaction or Southern analysis. Also, if DNA polymorphisms are available that have no adverse phenotype (e.g., “wildtype” alleles + I and + 2), three different classes of ( m / + I x m/+ I ) c-, 2/+ chimeras may be distinguished (m/m t-, + 2/+ 2, m/+ I + 2/+ 2, and + I/+ I t, + 2/+ 2). An alternative approach, gaining more widespread use, is to derive several embryonic stem cell lines from (m/+ x m / + ) embryos and then identify the homozygous m/m ES cell lines prior to the production of chimeras (Rashbass er al., 1991; Varlet et al., 1997).

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B. Production of Mouse ES Cell Chimeras

Chimeras were originally produced by combining two embryos but it is also possible to combine mouse embryos with in v i m cultured pluripotent cells by blastocyst injection (Brinster, 1974; Mintz and Illmensee, 1975; Papaioannou et ul., 1975), aggregation (Stewart, 1982; Fujii and Martin, 1983), or coculture techniques (Wood et al., 1993a, 1993b; Khillan and Bao, 1997). The usual aim of this approach is to make genetic changes in tissue culture cells, select in v i m for the mutant or transgenic cells, use these to make germ line chimeras, and transmit the altered genotype to mice. Such chimeras were first produced with embryonal carcinoma (EC) cells but the chimeras often developed tumors and, more critically, the EC cells failed to colonize the germ line (Papaioannou et al., 1978). This problem was overcome once techniques were developed to isolate embryonic stem (ES) cells from blastocysts (Evans and Kaufman, 1981; Martin, 1981). When injected into blastocysts or aggregated with morulae, ES cells produced a high rate of chimerism in tissues derived from the epiblast (fetal lineage), including the germ line, but colonized the extraembryonic primitive endoderm and trophectoderm extraembryonic lineages less frequently (Beddington and Robertson, 1989). The HPRT-deficient mouse was the first mutant produced by ES cell chimera technology because HPRT-deficient ES cells could be selected easily in culture (Hooper et al., 1987; Kuehn et al., 1987). Other selection strategies were devised subsequently (Mansour er al., 1988) and mouse ES cell chimeras are now widely produced as a means of making specific changes in the mouse genome, including genetic knockouts (Hooper, 1992; Joyner, 1993). This technique is having an important impact on many fields of biology and has been reviewed widely (Capecchi, 1994; Anonymous, 1996; Majzoub and Muglia, 1996; Nishimori and Matzuk, 1996; Ryffel, 1996) with lists of genetic knockouts, derived by this means, frequently updated in print and on the internet (Anagnostopoulos and Scharpf, 1997).

2. Mouse Chimeras

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C. Genetic Effects on the Composition of Chimeras

The composition of individual mouse chimeras of the same strain combination usually varies widely but chimeras of some strain combinations are consistently unbalanced so that one strain predominates (Mullen and Whitten, 1971; West and Flockhart, 1994). This may either reflect a generalized selective advantage of cells of one genotype or occur if those cells are preferentially allocated to the fetal lineage early in development. Within an individual chimera, the composition of different tissues is usually remarkably similar so that the composition of most organs in an adult chimera tends to be positively correlated (Falconer etal., 1981). Nevertheless, there are notable exceptions to this generalization: tissue-specific effects occur in some strain combinations which argue for genotype-dependent, tissue-specific selection pressures. For example, the skeletal muscles of 129 C57BL/6 chimeras were found to be predominantly derived from 129 strain cells (Peterson, 1979). Similarly, in AKR CBA-T6 chimeras, although both AKR and CBA-T6 were well represented in the coat and germ cells, AKR lymphocytes predominated (Tuffrey et al., 1973). Also, strain-specific, nonrandom anteroposterior differences have been reported for the composition of vertebral column (Moore and Mintz, 1972) and coat melanocytes (West and McLaren, 1976; Tachi et al., 1991). These are likely to result from developmental differences between the two contributing genotypes. Mintz (1970) introduced the concept of “SAM,” the statistical allophenic mouse, to describe the most likely bias in composition of different tissues (relative to the overall body composition) for a specific strain combination. For C3H C57BL/6 chimeras, C57BL/6 cells were favored in the heart and erythropoietic tissues, C3H cells predominated in the liver and mammary gland, but neither strain was favored in the kidney. The first observation predicted a temporal shift toward a predominance of C57BL/6 erythrocytes and such a shift was found in two out of the three studies addressing this issue (Mintz and Palm, 1969; Wegmann and Gilman, 1970; West, 1977). Other examples of temporal shifts in blood composition in chimeras and mosaics have been attributed to either random drift (Warner et al., 1977)or allele-specificselection (Ansell et ul., 1991; Kerner et al., 1995). In chimeras made between congenic strains of mice the blood composition may be more stable (Behringer et al., 1984).

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D. Comparisons between Chimeras and Mosaics

The similarity between the variegated patterns seen in the coats of chimeras (which are known to contain two genetically distinct cell populations) and Xlinked heterozygotes provided important evidence in favor of the single active X hypothesis (Lyon hypothesis: Lyon, 1961). This argued against the alternative complemental-X hypothesis, which proposed that both X chromosomes were active in each cell but gene expression was regulated so that total X-linked genetic

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activity was equal in XX and XY cells (Griineberg, 1967, 1969). It is now known that X chromosome inactivation occurs early in development. X-linked heterozygotes are, therefore, considered to be functional mosaics, with each cell only expressing one of the two available X chromosomes. The pattern of variegation in these X-inactivation mosaics is essentially equivalent to that seen in chimeras except that the mosaics are less variable in composition and seldom grossly unbalanced (Nesbitt, 1974; West, 1976a; Falconer and Avery, 1978). More recently, several cases of variegated patterns of gene expression in transgenic mice have been attributed to mosaicism, including that arising by position-effect variegation (Dobie et al., 1996, 1997). The variegated pattern of expression of a steroid 21hydroxylase/P-galactosidasetransgene in the mouse adrenal gland (Morley et al., 1996) was similar to the variegated patterns seen in rat chimeras (Iannaccone and Weinberg, 1987), suggesting mosaicism as a likely explanation.

111. Studies of Mouse Development with Chimeras Both prospective and retrospective studies with mouse chimeras have made important contributions to developmental biology (Gardner and Papaioannou, 1975; McLaren, 1976; West, 1978; Le Douarin and McLaren, 1984; Rossant, 1984, 1987, 1990; Ng and Iannaccone, 1992; Gardner, 1998).

A. Prospective Studies of Development

Donor cells from different sources have been incorporated into preimplantation chimeric embryos in various positions to test their prospective fate or prospective potency. The prospective fate of a cell is defined as what happens to it in normal undisturbed development whereas the prospective potency refers to the full range of developmental performances of which a cell is capable under any circumstances. Ideally cell fate would be studied by labeling one cell of an embryo and looking to see where its daughter cells end up later in development but experiments with chimeras have also been instructive and provided fate maps for preimplantation and early postimplantation mouse embryos. Early prospective studies with aggregation chimeras provided insights into the epigenetic control of allocation of cells to the trophectoderm and inner cell mass (ICM) lineages in preimplantation mouse embryos. Chimeras were produced by aggregating groups of whole, zona-free %cell stage embryos or groups of blastomeres from 4-cell and 8-cell embryos in different geometrical arrangements (Hillman et al., 1972; Kelly, 1977). Cells in different positions of the aggregate either were differentially labeled with tritiated thymidine or were homozygous for different alleles, encoding variants of glucose phosphate isomerase (GPI) (Fig. 3). The aggregates were cultured and either analyzed by autoradiography at the blas-

2. Mouse Chimeras

31

tocyst stage or transferred to pseudopregnant females and analyzed at E9.5 or after birth by GPI electrophoresis. These experiments demonstrated that the position of the cell in the aggregate affected its fate. Cells placed at the outside of the aggregate contributed to the trophectoderm but rarely contributed to the ICM, and so were excluded from the fetus and adult, whereas cells placed in the inside frequently contributed to the ICM so that chimeric offspring were often produced. Similar embryo disaggregation-reaggregation experiments were used to investigate the developmental potential of isolated blastomeres and showed that cells from 4-cell and 8-cell stage embryos were capable of contributing to all tissues of the conceptus (Kelly, 1975, 1977). Also, chimeras made by aggregating cells isolated from the outside of 16-cell stage embryos were capable of forming both ICM and trophectoderm derivatives (Rossant and Vijh, 1980; Ziomek et al., 1982), as were aggregates made entirely of inner cells (Ziomek et al., 1982). These experiments imply that at least some cells at the 16-cell stage remain totipotent. Evidence from ICM ++ morula and ICM c* ICM aggregation chimeras suggests that the early ICM retains the potential to differentiate into trophoblast (Rossant and Lis, 1979), although the experiments of Gardner and Nichols (1991), discussed below, imply that this is not the normal fate of ICM cells. Gardner and colleagues developed techniques for producing blastocyst reconstitution chimeras and injection chimeras and pioneered their use to study the fate and potency of cells in early mouse embryos. Blastocyst reconstitution chimeras were produced by replacing the ICM with a genetically distinct donor ICM in elegant experiments that demonstrated that the ectoplacental cone and trophoblast giant cells were derived from the trophectoderm rather than the ICM (Gardner et al., 1973). The embryo and extraembryonic membranes were analyzed together and were mostly of the ICM genotype; the small contribution from the trophectoderm was later attributed to the chorionic ectoderm. A later study (Papaioannou, 1982) supported these conclusions and demonstrated that the extraembryonic ectoderm and chorionic ectoderm were derived from the trophectoderm whereas parietal endoderm, like visceral yolk sac endoderm, was derived from the inner cell mass. More recently, groups of inner cells or outer cells were selectively replaced in decompacted late morulae by equivalent cells from genetically distinct morulae (Gardner and Nichols, 1991). These cell replacement chimeras were produced to test a previous claim that the ICM contributes cells to the overlying polar trophectoderm in normal development. E7.5, E8.5, and E9.5 conceptuses were mostly analyzed by GPI electrophoresis and, in those resulting from replacement of inner cells, trophoblast samples contained little or no donor-type GPI. The authors interpreted the small amount of donor GPI as tissue contamination rather than genuine chimerism, which argues against any role for the ICM as a source of stem cells for the overlying trophectoderm during normal development. In the blastocyst reconstitution chimeras and cell replacement chimeras, the chimeric embryos were of normal size and the donor cells directly replaced cells

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that were removed from the host blastocyst to allow the fate of donor cells to be investigated. When donor blastocyst cells are injected into an intact host blastocyst to produce injection chimeras, however, the total cell number is increased and the position of the donor cells in the host blastocyst may differ from their original position in the donor blastocyst. Strictly speaking, these experiments test aspects of the developmental potential of the injected cells rather than their undisturbed developmental fate. Injection of trophectoderm cells into host blastocysts did not produce chimeras but injection of single ICM cells from E3.5 blastocysts (Gardner and Papaioannou, 1975) or single epiblast or primitive endoderm cells from E4.5 blastocysts (Gardner, 1982) each contributed to chimeric conceptuses. Related experiments, spanning many years, have been reviewed recently by Gardner (1998). In summary, E3.5 ICM cells retained the ability to contribute to the fetus, amnion, yolk sac mesoderm, allantois, chorionic mesoderm, yolk sac endoderm, and parietal endoderm but not the trophoblast tissue, ectoplacental cone, extraembryonic ectoderm, or chorionic ectoderm, which are all trophectoderm derivatives. At E4.5 days, the two types of ICM cells (epiblast and primitive endoderm) contributed to different groups of tissues. Primitive endoderm cells were restricted to colonizing the yolk sac endoderm and parietal endoderm whereas the epiblast cells colonized the remainder of the tissues derived from the E3.5 ICM. This reflects the restriction in developmental potential of ICM cells that occurs between E3.5 and E4.5. Injection chimeras were also used in two series of experiments to test whether X chromosome inactivation had occurred by E3.5 (Gardner and Lyon, 1971) or E4.5 (Gardner et al., 1985). In each case, donor cells from female embryos were heterozygous for Cattanach’s translocation (Is 1Ct), which provides an X-linked pigment marker. If both X chromosomes were active, two distinct cell lineages (albino and black) would be founded by the injection of a single donor cell into a genetically distinct host blastocyst (pink eye, chinchilla). In each series of experiments, some chimeras were produced with all three types of coat pigmentation (two donor plus one host), implying that X inactivation was not completed by E4.5 in the epiblast lineage. Secondary chimeras were used to produce the first fate maps of early postimplantation mouse embryos. Groups of E7.5 donor epiblast cells, labeled with tritiated thymidine (Beddington, 1981, 1982) or carrying a lacZ transgene (Beddington ef al., 1989, 1991), were grafted into an equivalent position (orthotopic injections) in a host embryo and cultured for 36 h. These experiments revealed that posterior epiblast formed embryonic and extraembryonic mesoderm, distal epiblast formed embryonic mesoderm, notochord, and gut endoderm, whereas anterior epiblast usually formed neurectoderm and surface ectoderm. By varying the site of injection (heterotopic grafts), the same experimental design was used to investigate the developmental potential of E7.5 epiblast cells (Beddington, 1982). Distal and posterior epiblast usually conformed to the colonization patterns characteristic of their new locations but anterior epiblast tended to retain its preferen-

2. Mouse Chimeras

33

tial colonization of neurectoderm and surface ectoderm. The results for the distal and posterior epiblast show that cell fate can be modified by position so that the E7.5 epiblast is not a preexisting mosaic.

B. RetrospectiveStudies of Development

Retrospective analysis involves analyzing patterns of variegation seen in chimeras to deduce the nature of developmental events that occurred earlier. This is an attractive approach because it is technically simpler but the interpretation of the results is often controversial. The following description is largely confined to retrospective analysis of mouse chimeras but similar approaches have been used with a variety of mouse mosaic systems including X-inactivation mosaics (Nesbitt, 197 I ; Burton et al., 1982; McMahon et al., 1983; Wareham and Williams, 1986; Telfer et al., 1988), retroviral mosaics (Rossant, 1986; Soriano and Jaenisch, 1986; Price, 1987), somatic mutation mosaics (Kelly et al., 1989), and laacZ/lacZ mosaics (Nicolas et al., 1996; Mathis et al., 1997). Variegation may be analyzed qualitatively (presence or absence), quantitatively, or spatially; spatial analysis may be either qualitative or quantitative. This information has been used to try to infer lineage relationships, deduce the number of founder cells allocated to a tissue primordium, and evaluate the extent of cell mixing or pattern of growth during organogenesis. One example of a retrospective analysis with an unambiguous interpretation is the demonstration that multinucleated muscle fibers arise by cell fusion rather than nuclear division without cellular division (Mintz and Baker, 1967). Electrophoresis of a dimeric enzyme from most tissues of an AA c-, BB chimera revealed mixtures of AA and BB homodimers (Figs. 3 and 6). However, the presence of AB heteropolymer in skeletal muscle implied that cell fusion had occurred, producing a syncytium, containing both A and B monomers of isocitrate dehydrogenase (Mintz and Baker, 1967). This was later also demonstrated in the placenta using variants of glucose phosphate isomerase (West et al., 1995).

1. Analysis of Lineage Relationshipsby Retrospective Analysis Quantitative estimation of the composition of different tissues in a series of chimeras invites attempts to demonstrate statistically significantcorrelations between tissues. There are several examples where related tissues have different genotype compositions in mosaics or chimeras, suggesting that they have different founder cells. For example, the genotype composition of chimeras differs between left and right sides for Purkinje cells and large motor neurons of the facial nerve nucleus (Herrup et al., 1984a, 1984b). Also, there is variability among liver lobes in Xinactivation mosaics (Wareham and Williams, 1986). Paradoxically, it may be more difficult to demonstrate a close relationship between two tissues. For example, although several authors have drawn attention to highly significant positive

John D. West

34 Genotype

Monomers

Dlmers

Allozyme

1

A

B I _.

I

A AB B Fig. 6 Diagram showing the production of heterodimeric enzyme after cell fusion in a chimera containing two cell populations homozygous for different alleles (a and h) of a dimeric enzyme, such as isocitrate dehydrogenase or glucose phosphate isomerase. The expected distribution of nuclear alleles, protein monomers, and enzymatically active dimers is shown for mononucleated cells of each genotype (1 and 2) and a multinucleated syncytium formed by the fusion of homozygous a/a and h/b cells (3). AB heterodimer may be produced in these chimeric heterokaryons but not in the mononucleated cells in a chimeric tissue because neither mRNA nor monomers are exchanged between neighboring cells. A allozyme = AA homodimer; B allozyme = BB homodimer; AB allozyme = AB heterodimer.

correlations in composition between the left and right eyes (Deol and Whitten, 1972; Williams and Goldowitz, 1992), this does not provide evidence for a shared developmental lineage unless these tissues show weaker correlations with other tissues. In fact the composition of most tissues in a chimera tend to be positively correlated with one another (Falconer et al., 1981 ) so this requirement is not easily met. The analysis of midgestation chimeric conceptuses provides a case where correlation analysis confirms lineage relationships already established by prospective analysis. Tissue composition was found to be very significantly positively correlated among tissues within each of the three primary developmental lineages (epiblast, primitive endoderm, and trophectoderm) but correlations among these three primary lineages were much weaker (West et al., 1984, 1996).

2. Estimates of Numbers of Founder Cells by Retrospective Analysis Another use of retrospective analysis is to estimate the number of founder cells contributing to a tissue from the composition of tissues in a series of chimeras. The simplest question to ask is whether a structure is formed from a single clone of cells, in which case it should not appear variegated in chimeras. Clearly, be-

2. Mouse Chimeras

35

cause chimeras typically contain two genotypically distinct cell populations, the adult must be derived from more than one cell. Some adult chimeras, made by aggregating three genetically different 8-cell stage embryos, contained cells of all three genotypes (Markert and Petters, 1978). These chimeras have been widely cited as evidence that the whole fetus is likely to be derived from only three cells in a normal-sized preimplantation embryo. A more conservative interpretation is that when three whole embryos are aggregated together at least three cells contribute to the fetus. A later experiment also demonstrated that when four whole embryos are aggregated together at least four cells contribute to the fetus (Petters and Markert, 1980). It is clear that in multiembryo aggregates more cells are available to contribute to the fetus so, as Petters and Markert (1980) point out, the conclusions drawn from these experiments cannot easily be extrapolated to normal-sized (nonchimeric) embryos. The variegation in mouse chimeras is fine grained and almost all tissues examined are derived from more than one clone. Small structures that have been specifically tested and found to be polyclonal include somites (Gearhart and Mintz, 1972) and cortical somatosensory whisker barrels (Goldowitz, 1987). In contrast, hematopoietic foci in the neonatal liver (Rossant et al., 1986) and the submucosal glands of the trachea (D. Borthwick, personal communication) appear to be monoclonal in origin. The clonal structure of the epithelium of intestinal crypts changes after birth. At birth some intestinal crypts are polyclonal but one cell population is displaced during the first 2 weeks and the crypts are thereby “purified” (Ponder et a!., 1986; Schmidt et al., 1988). Thus, although the intestinal crypts appear to be monoclonal in adult chimeras and X-inactivation mosaics (Ponder et al., 1985; Griffiths et al., 1988), this does not reflect their polyclonal embryological origin. Having determined that more than one cell contributed to the tissue, the next problem is to try to determine how many cells were involved. Perhaps the most intuitive approach is to estimate the number of progenitor cells (n) from the smallest contribution found in a chimeric tissue, expressed as the fraction ’/”. In its crudest form, this estimate is based solely on the most unbalanced tissues in a series and is subject to error if, for example, the smallest identified contribution has been further reduced by cell death or cell selection (West, 1978). If the contributions to the same tissue in the other chimeras can be arranged in a quantal series (2/,,, ’/,,, etc.), this utilizes more of the available information but it still assumes that neither cell population has a selective advantage and the fit of variable biological data to a quantal series may be more apparent than real. This quantal approach has been used, for example, to estimate the number of founder cells contributing to the Purkinje cell layer (Wetts and Herrup, 1982) and the motor neurons of the facial nucleus ( H e m p et al., 1984a) but it has been criticized on statistical grounds (Mead et al., 1987). Other numerical analyses are based on the binomial theorem. The simplest of these relies purely on the qualitative difference between variegated and nonvariegated tissues. The frequency of nonvariegated tissues can be used to estimate the

John D. West

36

number of progenitor cells (n)because, from binomial theory, n progenitor cells would be expected to produce 2 X 1/2” nonvariegated individuals (Gandini et al., 1968). This argument was used to estimate that only 3 cells in an aggregate of two embryos produced the entire embryo because approximately 25% (2 X ‘h3) of a series of adult chimeras were not chimeric in any tissue (Mintz, 197 1). A related analysis relies on the quantitative estimates of the composition of tissues in a B chimeras. The predicted relationship between the number of series of A progenitor cells and the variation in proportions of type-A and type-B cells in the series of tissues is given by the equation n = p( 1 - p)/d, where p is the proportion of type-A cells, CI is the variance ofp, and n is the number of progenitor cells. This predicted relationship has been widely used to estimate the number of tissue progenitor cells allocated to a tissue primordium (e.g., Wegmann, 1970; Boland and Gosden, 1994) but is fraught with problems. This binomial approach rests on the unlikely assumptions that the primordial cells are drawn randomly from an equal mixture of A-type and B-type cells and that they survive and proliferate equally to contribute to the final tissue. When the tissue primordium is established, the pool of cells may be markedly skewed away from a 50 :50 composition, because A-type and B-type cells are unequally distributed among the embryonic and extraembryonic lineages or one has a selective advantage. Thus, the variation among tissues from different chimeras will reflect both the sampling events at tissue foundation and earlier sampling events. To minimize the effect of earlier sampling events, the analysis should be restricted to chimeras whose overall body composition is reasonably balanced (Russell, 1964; Boland and Gosden, 1994). Differences in proliferation between cells of different genotypes will also cause changes in the composition of the tissue after tissue foundation. Finally, if cell mixing is incomplete prior to tissue foundation, the sample of A-type and B-type cells allocated to the tissue is unlikely to be random, Thus, the calculated value of n would underestimate the number of founder cells because it would be closer to the number of clones of cells allocated to the tissue prirnordium than the actual number of cells involved. These issues have been discussed in more detail elsewhere (Lewis et al., 1972; McLaren, 1972; Falconer and Avery, 1978; West, 1978; Mead et al., 1987; Rossant, 1987).

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3. Qualitative Spatial Analysis of Cell Distributions in Chimeras Other uses of retrospective analysis require spatial information rather than simple qualitative or quantitative assessments of the tissue composition. Spatial markers can be used without numerical calculations to provide insights into the pattern of growth during organogenesis. This topic has been reviewed in detail elsewhere (Iannaccone, 1987; Ng and Iannaccone, 1992). Several studies have shown that relatively little cell mixing occurs between aggregation of two 8-cell stage embryos and the formation of a chimeric blastocyst (Garner and McLaren, 1974; Kelly, 1979; Dvorak et al., 1995) but by the end of gastrulation (E7.5) extensive

2. Mouse Chimeras

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Fig. 7 (a) The back of an adult pigmented albino chimeric eye, showing small irregularly distributed patches in the retinal pigment epithelium (RPE). (b) The front of an eye showing stripes of pigment in the RPE (top two thirds of the photograph) near where it meets the iris. The RPE is visible in this eye because the overlying choroid is largely unpigrnented.

mixing has occurred (Dvorak et al., 1995). This extensive mixing is consistent with quantitative evidence for positive correlations in contributions to most tissues of a chimera (Falconer et al., 1981). Most other studies have focused on individual tissues. albino chimeras revealed pigmented and albino Studies of adult pigmented stripes in the coat that implied the existence of two strings of melanocyte clones in the neural crest, either side of the midline (Tarkowski, 1964; Mintz, 1967; Wolpert and Gingell, 1970; Tachi, 1988). Subsequent work with a variety of genetic cell markers has revealed patterns of stripes in several other chimeric tissues including the neural retina (Fig. 5), adult retinal pigment epithelium, cerebral cortex, adrenal cortex and ovarian follicle (e.g., Mintz, 1971; Sanyal and Zeilmaker, 1977; Iannaccone and Weinberg, 1987; Goldowitz, 1989; Nakatsuji el af., 1991; Williams and Goldowitz, 1992; Boland and Gosden, 1994). In adult, pigmented t-* albino chimeras, the retinal pigment epithelium (RPE) is usually obscured by pigment in the overlying choroid but in some chimeras the choroid is predominantly unpigmented and this allows the RPE to be visualized directly in the intact eye (Fig. 7). Examination of intact eyes, whole mount prep-

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John D. West

Fig. 8 Transgenic marker TgR(ROSA26)26Sor (Friedrich and Soriano. 1991). detectable by 8-gdlactOSidaSehistochemical staining for lacZ expression in the corneas of eyes from an adult hemizygous transgenic ~1 nontransgenic (lacZ "- 0 lacZ ' ) chimera. Patches of stained (transgenic) and unstained (nontransgenic) cells form distinct stripes radiating from the center of the cornea. (M. A. Keighren, J. H. Flockhart. and J. D. West, unpublished.)

arations, and reconstructions of histological sections has enabled the patches of pigmented and albino cells to be visualized in the RPE of chimeric adult eyes (Mintz, 1971; West, 1976a; Sanyal and Zeilmaker, 1977; Schmidt et al., 1986; Bodenstein and Sidman, 1987b). This revealed small, randomly orientated patches at the bottom of the RPE (nearest the optic nerve head) but larger patches, arranged as radial stripes, nearer the equator of the eye (toward the ora serrata and iris). This distribution was neatly explained by a combination of computer modeling (Bodenstein, 1986) and mitotic analysis (Bodenstein and Sidman, 1987~). Computer simulation demonstrated that extensive cell mixing would occur during tissue growth if dividing cells were scattered throughout the tissue but not if they were confined to the edge of the growing tissue. Mitotic activity in the RPE was then shown to vary with age and radial position in a way that explained the observed distribution of patches in the chimeric eyes. Early in development (days El3 and E15), cell divisions occurred throughout the tissue but in older fetuses and postnatal pups mitotic activity in the RPE was concentrated at the periphery. Radial stripes have been visualized in the corneas of adult chimeras carrying a lacZ lineage (J. D. West, M. A. Keighren, and J. H. Flockhart, unpublished; Fig. 8). The aforementioned computer simulations of Bodenstein predict that mitotic activity in the developing cornea will occur mostly at the periphery of the cornea, throughout development. Even when stripes are not evident, differences in the extent of cell mixing may be apparent among different tissues. For example, the two cell populations are arranged differently in two extraembryonic membranes of midgestation chimeric conceptuses, being finely intermixed in the parietal endoderrn but separated into large patches in the visceral yolk sac (Gardner, 1984; Fig. 4c). In the intestinal

39 epithelium of adult chimeras, each crypt is composed entirely of one cell population which produces a larger scale pattern, based on the arrangement of whole crypts rather than individual cells (Ponder er al., 1985; Schmidt er al., 1985a). 2. Mouse Chimeras

4. Quantitative Spatial Analysis of Cell Distributions in Chimeras The size, shape, and distribution of patches of cells of the same genotype are all useful parameters for spatial analysis of chimeric tissues. The average size of a patch is a simple but useful parameter because it has the potential to provide information about the amount of cell mixing during tissue growth. Although the size of a patch of A-type cells in an A t-, B chimera is affected by the proportion ( p ) of A-type cells in the chimeric tissue, a correction can be applied to onedimensional measurements by dividing the mean patch length by 1/(1 - p ) (Roach, 1968; West, 1975). When the observed mean patch length is corrected, this provides a numerical estimate which is related to the extent of cell mixing, and early studies reported this corrected mean patch size as an estimate of the coherent clone size (West, 1976a, 1976b; Mullen, 1977b; Oster-Granite and Gearhart, 1981). The demonstration that coherent clones can vary in size (Schmidt et al., 1986) implies that it is now doubtful that the corrected mean patch length is an accurate estimate of a true biological clonal unit. However, the corrected mean patch length and the uncorrected median patch length (for the minor component in unbalanced chimeras; Schmidt er al., 1986) are both useful statistical parameters for comparing patch sizes in different groups of chimeras (West et al., 1997).Comparative analysis of this type has been largely neglected in recent years but it provides a useful means of demonstrating the extent of cell mixing during growth (West, 1976a) and could be useful for testing for genetic effects on cell interactions during development and growth of chimeras containing cells with abnormal genotypes. New techniques are needed to investigate the size, shape, and distribution of patches of cells in two and three dimensions. Morphometric techniques have been applied to the analysis of two-dimensional (2D) patches in the RPE of adult chimeras but it has proved difficult to correct the mean 2D patch size for the effects of different proportions of the two genotypes in the chimeric tissue (Bodenstein and Sidman, 1987a, 1987b). This means that summary statistics for comparative analysis of patch sizes remain unsatisfactory. However, the orientation and eccentricity of patches can be described adequately (Bodenstein and Sidman, 1987b) and patch shape can also be summarized by other simple indices (e.g., perimeter: area or area: volume ratios) or by considering their fractal dimensions (Mandelbrot, 1983). Fractal objects are complex irregular objects that have detail nested within detail and closer observation reveals more detail than is predicted on the basis of scale correction. This means that the perimeter of a fractal object can be described by a dimension that lies between 1 and 2. Small fractal dimensions would be expected when little cell mixing occurs but the available levels of nested

40

John D. West

detail would be limited by the finite size of the cells. In practice, this limitation may restrict the usefulness of fractal geometry for the analysis of patches in chimeric tissues but the feasibility of this approach is being explored by Iannaccone and his colleagues (Iannaccone, 1990; Ng and Iannaccone, 1992; Khokha et al., 1994). The distribution of patches in chimeric tissues has been analyzed with the Greig-Smith analysis of variance (Greig-Smith, 1952; Schmidt er al., 1985b) and the morphometric nearest-neighbor analysis (Aherne and Dunnill, 1982) might also be useful. If the genetic marker labels the entire cytoplasm of a cell, the spatial pattern may be visualized relatively easily. However, when a genetic marker provides a small, localized endpoint (e.g., Fig. 5 ) , it may be necessary to use morphometric techniques to identify patterns. This approach was used to identify stripes in ovarian follicles of transgenic wildtype chimeras after DNA in situ hybridization to detect the transgene in interphase nuclei (Boland and Gosden, 1994). The proportion of transgenic cells (p) was estimated along five randomly chosen radial lines (from the oocyte to the periphery of the follicle) and five concentric rings drawn at random intervals outward from the oocyte. The radial values of p were much more variable than the circumferential values, implying that alternating stripes of transgenic and wildtype cells radiated from the oocyte to the margin of the follicle.

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IV. Genetic Studies with Chimeras A. Quantitative Genetic Traits

Chimeras have been used in studies of several inherited quantitative traits. The general approach is to make chimeras from two stocks that differ for the quantitative trait, measure the trait in the chimera, and estimate the percentage contributions of the two parental stocks in a number of body organs. In three early studies of skeletal morphogenesis (Griineberg and McLaren, 1972), body size (Falconer et af., 1981), and behavior (Nesbitt, 1984), the measured values in chimeras were intermediate between those in the parent strains. This implies that these traits are controlled by a mixture of cells from both genotypes rather than a single clone of cells of like genotype. In another study (Dewey and Maxson, 1982), C57BL/6 DBA chimeras with balanced coat color composition were very variable in their susceptibility to sound-induced seizures (DBA strain mice are susceptible). This suggests that this phenotype might be controlled by a small number of cells, whose composition does not correlate with the general level of chimerism reflected by the coat color. Attempts to determine whether body size (Falconer et ul., 1981), and behavior (Nesbitt, 1984) were controlled by specific organs were made by calculating the covariance of the measured trait and the

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2. Mouse Chimeras

41

composition of different organ(s). Unfortunately this aspect of the analysis proved relatively unrewarding because the compositions of most organs in a chimera tend to be positively correlated with one another (Falconer et al., 1981). Also, different tissues within each organ could not be analyzed separately with the markers available at the time. Other studies of behavior using mouse chimeras have been reviewed elsewhere (Goldowitz, 1992) and the aggressive behavior of parthenogenetic +-+ normal chimeras is discussed briefly in Section VC, with genomic imprinting effects. There is also evidence of “vegetative heterosis” for body weight (Falconer et al., 1981), litter size (Mikami and Onishi, 1985), and hippocampal anatomy (Crusio et al., 1990) in chimeras (i.e., the measured value exceeds the quantitative range defined by the two parental genotypes). Unlike heterosis seen in F, hybrids (hybrid vigor), vegetative heterosis must involve interactions between cells of different genotypes in chimeric tissues.

B. Phenotypic Analysis of Single Mutant Genes

The early literature on mouse chimeras included the analysis of many single-gene effects and much of this work has been reviewed elsewhere (e.g., Mintz, 1974; McLaren, 1976; Russell, 1978). The availability of new transgenic in situ markers has opened up new possibilities for phenotypic analysis of mutant genes by “chimeric rescue analysis.” Consistent absence of mutant cells from a particular tissue in a series of mutant c* wildtype chimeras implies that the wildtype cells are unable to rescue the mutant cells because the gene acts cell autonomously in that tissue and that the cells require the gene to function normally. On the other hand, a reasonably high contribution of mutant cells, in the absence of a mutant phenotype, implies either that the gene is not needed in that cell type or that the surrounding wildtype cells are able to compensate for the defect in the mutant cells and rescue the mutant cells because the gene does not act in a cell autonomous way. Cell autonomous effects may operate in several ways to exclude mutant cells from a tissue, including cell selection, nonrandom allocation of cells to different developmental lineages, failure of cell migration, and failure of differentiation. Several examples of selection have been discussed elsewhere in this review, including selection against monosomic, parthenogenetic, or androgenetic cells (Sections VA and VC). Mild selective pressures may be difficult to detect except in the most actively dividing tissues (e.g., hematopoietic tissues; Section IIC) and, even there, it may not totally exclude the genetically compromised cells. At the other extreme are cases of very severe selection pressure, such as selection against monosomy 19 cells in chimeras (Section VA). Also, in mouse embryos heterozygous for an X; I6 translocation (X VX; 16V16), X-chromosome inactivation should produce two functionally distinct cell populations: cells with an inactive normal

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John D. West

X chromosome and cells with an inactive 16x translocation product. However, cells with an inactive 16x translocation appear to be rapidly eliminated, probably because inactivation of adjacent chromosome 16 regions results in partial monosomy 16 (Takagi, 1980). Selection may also account for the exclusion of tetraploid cells from the epiblast derivatives of 4n t-, 2n chimeras but nonrandom allocation of 4n cells may also play a role (see Section VA). Genetic abnormalities may also affect cell migration to the appropriate tissue; for example, p 1 -integrin-deficient hematopoietic stem cells fail to migrate from the visceral yolk sac to the liver (Hirsch er al., 1996). In many cases, loss of gene function will prevent differentiation and this is the most likely explanation for the exclusion of Pax6-deficient cells from the lens and nasal epithelium (see Section VDI). Chimeras incorporating mutants causing muscular dystrophy and retinal degeneration, respectively, were among the first produced to investigate the primary site of gene action. Although these are early studies and were limited by the lack of suitable cell markers that could be visualized in siru, they used chimeras in elegant ways to address questions that are still relevant today. These studies laid the foundation for many of the more recent developmental genetics studies with chimeras (discussed in Section VD) that have benefited from the availability of modern transgenic cell markers.

1. Phenotypic Analysis of a Gene Causing Muscle Degeneration Chimera experiments were used to investigate the site of action of the mouse dystrophia muscularis gene. Homozygotes for mutant alleles dy or dy2’ show a progressive weakness and paralysis and usually die by 6 months. The associated morphological abnormalities affect muscle fibers, interstitial tissue, and Schwann cells. Chimeras were made by aggregating normal (+/+) embryos and homozygous dy/dy or dy’/dyuembryos (Peterson, 1974, 1979). The muscle phenotype (normal or dystrophic) was assessed histologically and the muscle genotype (+/+, dy/dy, or mixed) was determined by enzyme electrophoresis, using variants of malic enzyme or glucose phosphate isomerase (see Fig. 3) that differed between the wildtype and dystrophic strains. Some muscle fibers were phenotypically normal but appeared to be entirely from the dy/dy strain, implying either that the primary site of dy gene action is outside the muscle fiber or that an intrinsic muscle fiber defect is rescued (Peterson, 1979). A second series of experiments focused on the Schwann cells as a possible site for dy gene action (Peterson and Bray, 1984). Chimeras were made by combining dyZJ/dy2’embryos with homozygous shiverer (shi/shi)embryos and Schwann cells were genotyped by immunocytochemistry to myelin basic protein (absent in shi/ shi but present in dy’/dyu Schwann cells). Both dy’/dyu and shi/shi Schwann cells were phenotypically normal, implying that an unknown nondystrophic (shi/ shi) cell type could rescue the abnormal phenotype of the dy’/dyu Schwann cells.

43

2. Mouse Chimeras

Later studies (Sunada et af., 1994; Xu et al., 1994) demonstrated that the dy locus encodes laminin 2 (also called merosin) and it has now been renamed Lama2. This protein is present in the extracellular matrix and is produced by muscle and Schwann cells. In the chimeras, wildtype cells will produce laminin 2 which may rescue the phenotype of the neighboring dy/dy muscle fibers and Schwann cells. An alternative explanation is possible for the rescue of the dy/dy muscle fibers. It is possible that the genotypically abnormal but phenotypically normal syncytial muscle fibers contained a few wildtype nuclei but these were below the detection limits of the methods available at the time (A. C. Peterson, personal communication). If so, the rescue of these muscle fibers could be mediated within the fiber and/or by neighboring cells.

2. Phenotypic Analysis Reveals Different Types of Retinal Degeneration Several genetic defects result in the degeneration of the photoreceptor cells, the nuclei of which lie in the outer nuclear layer (ONL) of the neural retina. Chimeras have revealed three different modes of action of retinal degeneration genes in the mouse and rat. Figure 5 illustrates the different layers of the neural retina in a chimera with no degeneration. Figure 9 illustrates examples of three genotypes that produce a similar phenotype (degeneration of the photoreceptors/ONL but not the inner layers) but are readily distinguishable by chimera experiments. It depicts the RPE (retinal pigment epithelium) and neural retina of five different types of adult mouse chimeras. They are all pigmented ++ unpigmented, so the RPE has patches of pigmented and unpigmented cells, but they differ for genes that cause degeneration of the ONL. Figure 9a represents a chimeric eye where both of the cell populations in the chimera are wildtype with respect to retinal degeneration (+/+ +/+) and the neural retina is uniformly of full thickness. Figure 9b shows the phenotype if both cell populations are homozygous for the mouse retinal degeneration, rd, gene (rd/rd c-, rd/rd). In this case the neural retina is uniformly thin because the ONL has degenerated. In the pigmented c-, albino eyes depicted in Figs. 9c-e the genotype of one cell population causes retinal degeneration whereas the other is wildtype. Homozygosity for each of the three mutant genotypes results in degeneration of the photoreceptors/ONL, similar to that shown in Fig. 9b for rd, but the three genotypes are readily distinguishable when incorporated into chimeras. Figure 9c represents a mouse chimera where one cell population is homozygous for retinal degeneration (rd/rd +I+). Several authors have reported patches of normal and degenerate photoreceptors/ONL in such chimeras. Studies of both pigmented rd/rd c-, albino +/+ and pigmented +/+ albino rd/rd chimeras showed that there was no spatial relationship between the patches of pigmented and albino cells in the RPE and the patches of degeneration in the ONL (LaVail and Mullen, 1976). The rd gene, therefore, acts in the neural retina and probably

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John D. West

Fig. 9 The multilayered neural retina lies underneath the retinal pigment epithelium (RPE) and contains three layers of nuclei (from the outside: photoreceptor cells with their nuclei in the outer nuclear layer; integrating neurons with their nuclei in the inner nuclear layer; optic tract cells with their nuclei in the ganglion cell layer). This arrangement is also shown in Fig. 5 . The diagrams (a-e) show the RPE and the outer nuclear layer (ONL) in different types of chimeras; for simplicity, the other layers (inner nuclear layer, ganglion cell layer, etc.) are shown as a single thin layer at the bottom of each diagram. All of the chimeras are pigmented unpigmented, so the RPE has patches of pigmented and unpigmented cells, but they differ for genes that cause degeneration of the photoreceptorslONL. (a) and (b) represent control chimeras and (c-e) depict chimeras where one cell population carries a gene causing degeneration of the photoreceptors/ONL. (a) Both cell populations in the chimera are wildtype with respect to retinal degeneration (+I+ +/+) and the neural retina is uniformly of full thickness. (b) Both cell populations are homozygous for the mouse retinal degeneration, rd, gene (rd/ rd rd/rd) so the neural retina is uniformly thin. (c) One cell population carries retinal degeneration (rd/rd +/+) and patches of normal and degenerate ONL are visible but these show no spatial relationship with the patches in the RPE. (d) One cell population is homozygous for rat retinal dystrophy (rdy), which causes retinal degeneration (unpigmented rdy/rdy pigmented +/+); the ONL degenerates only in regions adjacent to unpigmented RPE. (e) Mouse chimeras where one cell population carries a mutant pig rhodopsin transgene which causes retinal degeneration: uniform ONL of intermediate thickness.

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2. Mouse Chimeras

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in the photoreceptors themselves. It is now known that rd encodes for the 13 subunit of the rod photoreceptor cGMP-phosphodiesterase and it has been renamed Pdeb (cGMP-phosphodiesterase,p subunit). This enzyme depletes cGMP and mediates the conversion of the light energy (photons absorbed by rhodopsin) to neural impulses in the visual pathway (Bowes et al., 1990). As predicted by the chimera experiments, the gene is expressed in the photoreceptors. Retinal degeneration is also caused by another gene called retinal degeneration slow (rds)and rds/rds +/+ chimeras look identical to rd/rd +/+ chimeras, showing no spatial relationship between the patches in the RPE and the ONL (Sanyal et al., 1986). Again, the gene has now been identified and found to be expressed, as predicted, in the photoreceptors. The rds gene has been renamed peripherin 2 (Prph2)and encodes a protein located at the periphery of the disklike structures within the photoreceptors. Figure 9d illustrates a rat chimera where one cell population is homozygous for retinal dystrophy (rdy),which causes retinal degeneration. Experiments with unpigmented rdy/rdy t,pigmented +/+ rat chimeras showed that the ONL degenerated only in regions that were adjacent to unpigmented RPE (Mullen and LaVail, 1976). This elegantly demonstrated that, in this case, the primary defect was in the overlying RPE and not in the neural retina itself. In effect, the nonpigmented rdy/rdy cells in the RPE “murdered” the underlying neural retina cells. The deleterious effects of the rdy mutation can be ameliorated either by transplantation of normal RPE (Li and Turner, 1988) or by injections of basic fibroblast growth factor (bFGF) (Faktorovich et al., 1990). These observations suggest that the normal rdy gene produces a product which is secreted from the RPE and is required for survival of the adjacent photoreceptors. However, bFGF does not map near the rdy locus and the rdy gene product remains to be identified (M. M. LaVail, personal communication). Figure 9e represents a mouse chimera with one cell population that carries a pig rhodopsin transgene that causes retinal degeneration (Huang et al., 1993). In situ hybridization with a probe specific for pig rhodopsin RNA revealed the expected patchy distribution of transgenic and wildtype cells in the neural retina. However, instead of patches of degenerate and normal ONL, the chimeras had a uniform ONL of intermediate thickness, implying uniform degeneration of both wildtype and transgenic photoreceptor cells. As both wildtype and transgenic cells degenerate, the gene causing the degeneration must act nonautonomously and cell interactions are probably involved. One explanation, suggested by the authors, is that each photoreceptor cell releases atrophic factor but it must also take up this nutrient to survive (each cell contributes to and draws upon a common pool present in the retina). It’ the transgenic photoreceptors released less of the factor but required the normal amount, the pool would become depleted. Eventually the pool of nutrients would be insufficient and both wildtype and transgenic photoreceptor cells would die.

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@

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John D. West

3. Phenotypic Analysis of Other Single Genes Other genes have been analyzed in this way. Mutants that affect neural functions and coordination of movement include the mouse genes reeler (rl) and dystonia musculorum (dt). Once again chimera studies have shown that these genes act in different ways. Homozygous reeler mice show uncoordinated movement and have a small cerebellum, and the relative positions of the cortical layers are inverted. Early studies with rl/rl- +/+ chimeras predicted that the reeler gene acted extrinsically to migrating neurons (Mullen, 1977a; Terashima ef af.,1986). This was subsequently confirmed when reeler mice were found to be deficient in an extracellular matrix protein (designated reelin) that is expressed by Cajal-Retzius cells in the hippocampus during periods of neuronal migration (D' Arcangelo et al., 1995; Ogawa et al., 1995). Reelin is critical for the normal lamination of cortical neurons in the mammalian neocortex and is implicated in the control of cell migration in the cortex and cerebellum, and axonal growth and guidance (Del Rio et al., 1997). In contrast, chimera experiments involving the mutant dystonia musculorum (dt) identified neurons as the site of gene action. Homozygous dt/dr mice have uncoordinated limb movements associated with focal swellings and degeneration of peripheral and central sensory axons. Expression of a transgene in +/+ axons was used to distinguish them from dt/dt axons in dt/df +/+ aggregation chimeras and phenotypic abnormalities (swellings) were only found on the dr/df axons (Campbell and Peterson, 1992). This implied that the dt gene acted in the neurons themselves. The dt mutation has now been reclassified as an allele of dystonin (Dst'"),which is expressed, as predicted, in the nervous system (Brown et al., 1995). Analysis of other genes affecting the nervous system has been reviewed elsewhere (Rossant 1990; Ng and Iannaccone, 1992) and three examples of genes affecting development are discussed in Section VD.

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V. Studies of DevelopmentalGenetics with Chimeras A. Cytogenetic Studies

The more recent studies with chimeras involving cytogenetic anomalies have focused on fetal stages so this section has been included under Developmental Genetics. Trisomic (Ts) mouse or human embryos mostly die during postimplantation development and monosomic (Ms) embryos usually die before implantation. In contrast, human trisomy/diploid (Ts/2n) mosaics are often viable and frequently occur as confined placental mosaicism in humans (Kalousek and Dill, 1983; Gosden et al., 1994; Wolstenholme et ul., 1994). Several trisomies (Ts12,

2. Mouse Chimeras

47

Ts15, Ts16, Ts17, and Ts19) have been incorporated into mouse chimeras to test whether trisomic cells can be rescued in different tissues of the fetus or adult (Epstein et al., 1982; Cox et af., 1984; Epstein etal., 1984; Epstein, 1985; Fundele et al., 1985; Epstein, 1986). This showed survival of Ts cells in most tissues studied and provided evidence for modest selection against them in certain tissues of the fetus or neonate but, in most cases, the contribution to the extraembryonic tissues was not studied. A much stronger selection was apparent against monosomy 19 cells in E9.5 day Ms19 * 2n mouse chimeras (Magnuson et al., 1982) and few Ms19 cells would be likely to survive to term. At least one tetraploid t-,diploid (4n t-, 2n) mouse chimera has survived postnatally with a tetraploid cell contribution (Lu and Markert, 1980). Usually, however, the tetraploid cells fail to contribute to the fetus or other derivatives of the epiblast lineage (amnion, visceral yolk sac mesoderm, and allantois) but contribute well to the trophectoderm and primitive endoderm lineages (placenta, visceral yolk sac endoderm, and parietal endoderm of Reichert’s membrane) (Nagy et al., 1990; James et ul., 1995). This restricted distribution is apparent by E7.5 days and, despite evidence for preferential allocation of tetraploid cells to the mural trophectoderm lineage (Everett and West, 1996; P.-C. Tang, personal communication), it is probably largely attributable to cell selection (Everett and West, 1998). Preliminary results suggest that digynic triploid cells also show a nonrandom distribution in digynic triploid .++ diploid mouse chimeras, contributing little to the fetus (C. A. Everett, M. A. Keighren, J. H. Flockhart, and J. D. West, unpublished). Unlike the Ts and Ms chimeras so far studied, 4n t-, 2n chimeras provide an animal model for some types of human confined placental mosaicism. The poor ability of tetraploid cells to contribute to the fetus has also been elegantly exploited to maximize the contribution of ES cells to the fetuses of 4n-embryo cf 2n-ES cell chimeras (Nagy et al., 1990, 1993) and to analyze the Mash2 phenotype (Guillemot et al., 1994; see Section VD2).

B. Sex Determination in Chimeras When chimeras are made by randomly aggregating pairs of eight-cell stage embryos, 50% will be of mixed sex chromosome composition. The development of these XX t-, XY chimeras has been a source of interest to developmental biologists, geneticists, and reproductive biologists. Intuitively, one might predict that XX X Y chimeras would all develop as intersexes. Indeed, at fetal stages many do have ovotestes (Bradbury, 1987) but the ovarian tissue usually regresses so the postnatal gonad becomes a testis. In many other chimeric tissues the two cell populations are finely intermixed but the ovarian and testicular tissues in a fetal ovotestis are commonly spatially separated into testicular and ovarian domains. This may indicate that “sorting out” occurs during the aggregation of Sertoli cells

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John D. West

48

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to form testicular cords. Sex determination in XX t-, XY chimeras is unusual in that it includes a mechanism for converting this ovotestis into a testis. In a balanced strain combination, the majority of XX X Y chimeras develop as phenotypic males and the sex ratio is close to 3 males: 1 female (Mullen and Whitten, 197 1; McLaren, 1975, 1984).XX +-+ XY chimeras appearonly todevelop as females (or occasionally as intersexes) if the proportion of XY cells is low. However, among chimeras of unbalanced strain combinations, there are more XX t-, XY chimeras with a high proportion of X X cells (most are either XX>>XY or XY>>XX) so the sex ratio is closer to 1 : 1 (Mullen and Whitten, 1971). In this case, sex is determined by the predominating component. If strain-A predominates in most strain-A t-, strain-B chimeras, then the XX component will predominate in most (XX, strain-A) (XY, strain-B) chimeras (XX>>XY) but the XY component will predominate in most (XU, strain-A) (XX, strain-B) chimeras (XY>>XX). The occurrence of intersexes among XX XY chimeras has also led to the identification of a number of human chimeras (see Section IIA). The Sertoli cells of the testis and the follicle cells of the ovary are supporting cells derived from epithelial cells in the gonadal ridge. In XX t-, XY female chimeras a significant proportion of ovarian follicle cells can be XY (Burgoyne etal., 1988b; Patek et al., 1991). However, although XX XY male chimeras often have some XX Sertoli cells, the proportion is usually much lower than for other XX testicular cells (such as Leydig cells), which more closely reflects the XX contribution to nongonadal tissues (Palmer and Burgoyne, 1991; Patek et al., 1991). One possibility is that Sry (the Y-linked, testis-determining gene that encodes a transcription factor) acts cell autonomously in XY pre-Sertoli cells but a few neighboring XX prefollicle cells are somehow recruited into the Sertoli cell population even though they lack Sry. The current interpretation of sex determination in XX XY chimeras (P. S. Burgoyne, personal communication; McLaren, 199I ; Burgoyne and Palmer, 1992) is that, if there are sufficient XY pre-Sertoli cells, some testicular cords are formed and, in these cords, the germ cells enter mitotic arrest as prospermatogonia. In areas where there are insufficient pre-Sertoli cells, testicular cords fail to form and the germ cells enter meiosis to become oocytes. These oocytes are subsequently eliminated and the ovarian component regresses, thereby transforming the ovotestis into a testis. A viable oocyte is required for the survival of the surrounding granulosa cells, so death of the oocyte also ensures the death of the granulosa cells. Elimination of the meiotic oocytes is probably mediated by antiMiillerian hormone, which is produced by the Sertoli cells in the testicular regions of the ovotestis (Burgoyne et al., 1988a; Behringer et al., 1990). This interpretation is consistent with observations on the action of anti-Miillerian hormone on female reproductive organs both in culture (Vigier et al., 1987) and in transgenic mice that chronically express anti-Miillerian hormone (Behringer et al., 1990). In XX>>XY chimeras there are few XY pre-Sertoli cells so testicular cords do not form. Consequently, little or no anti-Miillerian hormone is produced and the oo-

-

--

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2. Mouse Chimeras

49

cytes survive and direct the prefollicle cells (and some pre-Sertoli cells) to form ovarian follicles rather than testicular structures.

C. Cenomic Imprinting

Elegant pronuclear transplantation studies have demonstrated that the maternal and paternal genomes are not equivalent, so implying the existence of genes that are differentially imprinted, depending on their parental origin (Barton et al., 1984; McGrath and Solter, 1984; Surani et al., 1984; Surani, 1986). Diploid parthenogenetic embryos (produced from a female gamete without participation of a male gamete), diploid gynogenetic embryos (e.g., male pronucleus replaced with a female pronucleus after fertilization), and diploid androgenetic embryos (e.g., female pronucleus replaced with a male pronucleus after fertilization) all fail to develop to term. Parthenogenetic and gynogenetic embryos are characterized by poor trophoblast growth. Blastocyst reconstitution chimeras were produced to test whether this is an intrinsic defect of the trophectoderm or a consequence of inadequate signaling from the inner cell mass (Barton et al., 1985). Reconstituted blastocysts comprising a normal inner cell mass in a parthenogenetic trophectoderm produced poor trophoblast growth but, in the reciprocal combination (parthenogenetic inner cell mass in a normal trophectoderm), the trophoblast grew normally. Phenotypic rescue by normal trophectoderm implied that poor trophoblast growth was an intrinsic defect of the parthenogenetic trophectoderm and that the paternal genome was needed for normal proliferation of trophoblast. Aggregation chimeras, incorporating transgenic markers, have also been produced to investigate the developmental potential of parthenogenetic and androgenetic cells beyond the time when the parthenogenetic and androgenetic embryos die. Parthenogenetic cells in parthenogenetic ++ normal and androgenetic cells in androgenetic ++ normal aggregation chimeras were initially present in all lineages at the blastocyst stage (Thomson and Solter, 1989) but later they showed a more restricted distribution. Parthenogenetic cells were excluded from the primitive endoderm and trophectoderm lineages and, although they survived in the fetus and other epiblast derivatives, they were gradually depleted in most tissues. They contributed well to some nonproliferating tissues, notably the oocytes and forebrain but not the hypothalamus/midbrain (Nagy et al., 1987; Fundele et al., 1989; Nagy et al., 1989; Paldi et al., 1989; Fundele et al., 1990, 1991; Allen et al., 1995; Bender et al., 1995). Parthenogenetic t* normal chimeras had small bodies but their brains were normal in size (or possibly enlarged) and male chimeras with a high parthenogenetic contribution in their brains tended to be more aggressive than normal (Allen et al., 1995). These studies suggest a role for imprinted genes in development of the central nervous system and the control of behavior. Androgenetic cells usually become confined to the trophectoderm lineage in

50

John D. West

postimplantation-stage androgenetic ++ normal aggregation chimeras but will contribute to the fetus if they are injected into normal blastocysts to make androgenetic-normal injection chimeras. Many features of androgenetic-normal chimeras are the reciprocal of those seen in parthenogenetic t-, normal chimeras. At the early fetal stage these chimeras were larger than normal and androgenetic cells tended to colonize those tissues that parthenogenetic cells failed to colonize (hypothalamus/midbrain and most proliferating tissues) (Fundele et al., 1995a, 1995b; Keverne et al., 1996). Striking embryonic growth enhancement also occurred when inner cell mass cells, with a paternal duplication of the distal region of chromosome 7 (PatDi7 cells), were injected into wildtype blastocysts to produce PatDi7 normal injection chimeras (Ferguson-Smith ef d.,1991). The authors suggested that rapid growth may be caused by overexpression of an imprinted gene, such as the insulin-like growth factor-2 gene (Igf2), which maps to the distal region of chromosome 7 and shows exclusive paternal expression.

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D. Phenotypic Analysis of Mutant Genes and Genetic Knockouts The current vogue for producing genetic knockouts attests to the power of the genetic approach to developmental biology. However, if the genetic defect causes death of the embryo or abnormal development of a whole organ, it will be unclear, from this gross phenotype alone, which tissues are primarily affected. Analysis of mouse embryos with an early-acting lethal null mutation will reveal the earliest critical time for normal gene expression but later roles and the range of tissues affected will be obscured. Sometimes these issues can be clarified by a chimeric rescue analysis where mutant and normal (wildtype) cells are combined in a mouse chimera to test whether the presence of the wildtype cells can rescue the mutant cells. As discussed in Section IVB, if the mutant cells are excluded from a specific tissue, this is likely to mean that normal gene function is required in that tissue. Rather than attempt to review this rapidly expanding topic comprehensively, I have selected three recent examples which illustrate how chimeras can reveal whether genes act in a cell-autonomous fashion and determine the developmental potential of mutant cells. These examples all make use of transgenic cell markers that are detectable either by DNA in situ hybridization or by P-galactosidase histochemistry to detect lacZ expression. The last two examples (Mush2 and nodd) also demonstrate how chimeras made with cells with a restricted tissue distribution can be exploited to determine the site of gene action. 1. Phenotypic Analysis of Pax4

The mouse Pux6 gene encodes a transcription factor and mutations in this gene are responsible for the small eye mutant phenotype. Chimeric rescue analysis has shown that mouse Pax6 acts cell autonomously in the lens and nasal placodes

51

2. Mouse Chimeras

(Quinn et al., 1996). Mouse embryos homozygous for the small eye (Pa~6~") mutation die soon after birth with severe facial abnormalities that result from the failure of the eyes and nasal cavities to develop. It is unclear from the mutant phenotypes which eye and nose tissues require functional Pax6 because eye and nasal development are totally disrupted in the homozygous embryos. Fetal mouse chimeras were made by aggregating wildtype embryos with embryos from matings between mice heterozygous for different small eye alleles x Pa~6~<"/+). The +/+ Pa~6~"Neu/Pax6sc~fetal chimeras were identified by PCR and the contribution of mutant Pax6Sev-Nru/Pax6Sr~cell~ was analyzed in tissue sections by in siru hybridization to a reiterated transgenic marker. The morphology of the optic cup was severely affected in these chimeras. Mutant cells were excluded from the normal retinal pigment epithelium and did not intermix with wildtype cells in other regions of the optic cup, suggesting that Pax6 affects the cell surface properties of these cells, causing the two genotypes to sort out. Even more strikingly, mutant cells were excluded from both the lens and nasal epithelium and both tissues were smaller (sometimes absent) in chimeras with high proportions of mutant cells. Since the wildtype cells were unable to rescue the mutant cells in these tissues, Pax6 must act cell autonomously. The observations that chimeras with high proportions of mutant cells either had no lenses or had small lenses composed entirely of wildtype cells suggest two possibilities (Fig. 10). First, mutant surface ectoderm cells could fail to respond to the signal (Fig. I0.4), explaining both the absence of mutant cells from the lens and its variable size. Alternatively, the small size of the lens could be a consequence of a low proportion of cells transmitting the signal (Fig. 10.3) and the lens would be entirely wildtype if mutant cells were somehow excluded from the overlying region of surface ectoderm (Fig. 10.5).

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2. Phenotypic Analysis of Mash2

Chimeras have also shown that Mash2 acts in the placenta and not the fetus (Guillemot et al., 1994). Mash2 (mammalian achaete-scute homolog 2) encodes a transcription factor that is expressed in the extraembryonic trophoblast lineage. Mash2 I-knockout mouse embryos die from placental failure at 10 days past coitum (placental spongiotrophoblast cells and their precursors are absent and chorionic ectoderm is reduced). Tetraploid Mash2 +'+ diploid Mash2 chimeras have been used to exploit the restricted colonization pattern of tetraploid cells and rescue Mash2 -/- fetuses. The chimeras were produced by aggreembryos + with morula-stage embryos produced gating 4-cell tetraploid Mash2 +I from ( M u ~ h 2 + / -X Mash2 +) intercross matings. It was predicted that, in diploid MashZ-/- chimeras, the tetraploid (wildthe tetraploid Mash2 +/+ type Mash2 +/+) cells would colonize the trophectoderm lineage (trophoblast) and extraembryonic endoderm lineage but be excluded from the fetus itself (see Section VA). This would produce a fetus composed entirely of mutant

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John D. West

52

Nonchimeric eyes

3. Sey"'eu/Sey optic vesicle cells fall to transmit

1. Normal +/+ eye

3)

Chimeric eyes

No lens Mvdopmnl

5. SepeuBey excluded from presumptive lens piacode

Fig. 10 Diagram illustrating several models for Pax6 function in the developing mouse eye that can be tested by phenotypic analysis with chimeras. ( I ) In the wildtype embryo the optic vesicle contacts the surface ectoderm and transmits a signal that induces the overlying surface ectoderm to form a lens placode which develops into the lens. (2) In the homozygous mutant embryo the lens placode fails to develop normally and neither the lens nor the rest of the eye forms. (3-5) Three models of Pux6 function in chimeras, showing the predicted composition of the lens. (3) Mutant optic vesicle cells fail to transmit the signal. (4)Mutant surface ectoderm cells fail to respond to the signal. ( 5 ) Mutant cells are excluded from the region of surface ectoderm overlying the optic vesicle. The results showed that mutant cells were excluded from the lens and the lens was small or absent in chimeras with high proportions of mutant cells (Quinn et al., 1996).This is consistent with (4)or a combination of (3) and ( 5 ) . See text for further details.

Mush2-I- (diploid) cells but, in some cases, the fetus would be supported by a significant contribution of wildtype Mush2 +I+(tetraploid) cells in the extraembryonic lineages. This expectation was fulfilled and about 15% of the viable chimeric pups at term were uniformly mutant Mash2 -I- (no Mush2 DNA identified). This neatly demonstrates that, normally, the lethality of Mush2 -I- conceptuses is a consequence of the absence of Mush2 in the trophectoderm (and/or the primitive endoderm) extraembryonic lineage. Together with other evidence, the viability of Mash2 +fetuses implies that Mash2 has no major role in the embryo +

53 itself but plays a critical part in the development of the mammalian trophoblast lineage. 2. Mouse Chimeras

3. Phenotypic Analysis of nodal The final example is an experiment that used chimeras to show that anterior embryonic structures depend on gene expression in an adjacent extraembryonic tissue. The mouse nodal gene encodes a member of the TGF-P family of secreted growth factors, which is expressed in both the epiblast (which produces the fetus) and the overlying primitive endoderm (which produces only extraembryonic endoderm). Homozygous nodal-deficient embryos (nodal -/-) fail to initiate primitive streak formation and so arrest at gastrulation without forming any mesoderm. Wildtype or nodal -IES cells (carrying a lacZ lineage marker) were injected into wildtype or nodal -/- blastocysts and the embryonic phenotype and the contribution of the injected ES cells were examined to test whether wildtype cells could rescue the mutant phenotype (Varlet et al., 1997; see Fig. 11). injection chimeras were small but gastruEmbryonic, +/+ ES cell-nodal -Ilation had occurred, indicating that the presence of wildtype cells had overcome the block to gastrulation. Use of the lacZ lineage marker indicated that chimeras with 10-30% wildtype cells were almost fully rescued in the posterior region but, despite good contributions of wildtype cells, the anterior structures were absent. Comparisons between +/+ ES cell+nodal-/- chimeras and the reciprocal nodal-/- ES cell++/+ chimeras (Figs. l l a , and llb) showed that anteriorly truncated embryos were only produced when nodal -/- cells were derived from the host blastocyst, not from the injected ES cells, despite good contributions of ES cell++/+ chimeric embryos. nodal -/- cells in nodal -IThe difference between these two groups of injection chimeras is accounted for by the previous observation that ES cells contribute well to the epiblast (fetal lineage) but not to the overlying primitive endoderm (see Section IIB). The anterior truncation only occurred when nodal -/- was introduced from the host blastocyst and so contributed to the primitive endoderm as well as the epiblast. This implies that normal development of the anterior structures depends on nodal expression in the overlying primitive endoderm rather than in the epiblast (fetal lineage) itself. The involvement of the primitive endoderm in specifying anterior embryonic structures is supported by other recent experiments (Thomas and Beddington, 1996).

VI. Concluding Remarks During the past 25 years, studies with experimentally produced primary chimeras have made many contributions to mouse developmental biology and genetics. The

ES cells (all lacZ+) _ _

~

Host blastocyst _ ~

Chimeric Chimeric egg cylinder Mastocyst (E6.5) _ _ _

Chimeric conceptus

(E10.5)

Genotype

Embryo phenotype

nodal

lac2

Small abnormal

ep: end:

+I+t,-1-1-

+I-t,-1-1-

Normal

ep: end:

+I+t,-1+/+

4-# -1-1-

+I+-P -1-

+u+ .eD

epe Normal (Control)

ep: end:

+I+t,+I+ +I-t,-1+/+ -1-

2. Mouse Chimeras

55

availability of new transgenic lineage markers during the past decade has significantly improved the power of chimeras for studies of cell mixing and organogenesis and the introduction of green fluorescent protein as an alternative transgenic lineage marker should further enhance this capability. The greatest impact of the new transgenic markers, however, has been to provide the means to analyze the developmental potential of genetically abnormal cells, including those with mutations (including genetic knockouts), cytogenetic abnormalities, or imprinting anomalies, as discussed in Section V. This increased analytical power accounts for the resurgent interest in using chimeras for phenotypic analysis. The analysis of Mush2 and nodal illustrate how the analytical power of chimeras can be enhanced by the incorporation of cells with a restricted developmental potential (tetraploid and embryonic stem cells, respectively). The restricted developmental potential of parthenogenetic cells, which survive preferentially in the forebrain and oocytes, or Pax6sev/Pan6se’cells, which are excluded from the lens and nasal epithelium, could be used in a similar fashion. The understanding of gene action during development requires analysis at both the genotypic and phenotypic levels. Recent advances in molecular genetics have provided effective approaches for genotypic analysis and chimeras provide a powerful approach to analysis at the phenotypic level. Chimeric rescue analysis is likely to play an increasingly important role in the phenotypic evaluation of numerous new mutations that are being produced by genetic knockout techniques. The use of chimeras for phenotypic analysis is also likely to be augmented by several recently developed techniques including laucZ/lacZ mosaics (Nicolas et ul., 1996; Mathis et al., 1997) and conditional mosaicism produced by Cre-LoxPmediated recombination (Betz et al., 1996).

Fig. 11 Diagram (redrawn from Varlet era/., 1997, with permission of Company of Biologists Ltd.) illustrating three groups of ES cell chimeras produced to demonstrate that normal function of the nodal gene is required in the primitive endoderm lineage to allow the epiblast lineage to produce a normal embryo. Both the ES cells and host blastocyst contributed to the epiblast but the ES cells made little or no contribution to the primitive endoderm or trophectoderm. (a) Wildtype +/+ ES cells (LacZ‘) injected into tiodd l mutant blastocysts produced chimeric embryos in which the primitive endoderm (end) was entirely nod&+ (host blastocyst genotype) and the epiblast (ep) was a mixture of wildtype (ES cells) and nodul (host blastocyst). After gastrulation these embryos appeared small and truncated, with anterior abnormalities. (b) Mutant nodal -I- ES cells (lacZ’) injected into wildtype blastocysts produced chimeric embryos in which the primitive endoderm was entirely wildtype (host blastocyst genotype) and the epiblast was a mixture of nodal-’- (ES cells) and wildtype (host blastocyst). After gastrulation these embryos appeared normal. (c) Control: wildtype +I+ ES cells ( / a c Z + )injected into wildtype blastocysts produced chimeric embryos in which the primitive endoderm was entirely wildtype (host blastocyst genotype) and the epiblast was also entirely wildtype (mixture of ES cells and host blastocyst). After gastrulation these embryos appeared normal. The diagram is shaded to distinguish nodal / (black) and wildtype (white); /acZ staining is not illustrated but was used to assess the composition of the chi , which were all of the /acZ+’- ES cell+lucZ-/- blastocyst combination. Abbreviations: end, ive endoderm lineage; ep, epiblast lineage.

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John D.West

Acknowledgments I thank Paul Burgoyne. Clare Everett, Anne McLaren, and Katrine West for helpful comments on different parts of the manuscript, Duncan Borthwick, Matthew LaVail, Alan Peterson, and Pin-Chi Tang for providing information, and Tom McFetters and Ted Pinner for help in preparing the figures. Most of all, I thank Anne McLaren for introducing me to mouse chimeras and encouraging me to write this review. I am also grateful to the Wellcome Trust (Grant 046359) and the Medical Research Council (Grant G9630132MB) for financial support for my own current work with chimeras.

References Aherne, W. A., and Dunnill, M. S. (1982). “Morphometry.” Edward Arnold, London. Allen, N. D., Logan, K., Lally, G., Drage, D. J., Norris, M. L., and Keverne, E. B. (1995). Distribution of parthenogenetic cells in the mouse brain and their influence on brain development and behavior. Proc. Nutl. Acad. Sci. U S A . 92, 10782-10786. Anagnostopoulos, A. V., and Scharpf, R. B. (1997). It’s a knockout! Trend. Genet. 13,499-500. Anderson, D., Billingham, R. E., Lampkin, G. H., and Medawar, P. B. (1951). The use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. Herediry 5,379-397. Anonymous. (1996). Delivering the knockout punch. Nurure Genet. 13,251-252. Ansell, J. D., Samuel, K., Whittingham, D. G., Patek,C. E., Hardy, K., Handyside, A. H., Jones, K. W., Muggleton-Harris, A. L., Taylor, A. H., and Hooper, M. L. (1991). Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse. Development 112,489498. Barton, S. C., Adams, C. A., Norris, M. L., and Surani, M. A. (1985). Development of gynogenetic and parthenogenetic inner cell mass and trophectoderm tissues in reconstituted blastocysts in the mouse. J. Embryol. Exp. Morphol. 90,267-285. Barton, S. C., Surani, M. A., and Norris, M. L. (1984). Role of paternal and maternal genomes in mouse development. Nature 311,374-376. Beddington, R. S. P. (1981). An autoradiographic analysis of the potency of embryonic ectoderm in the 8th day postimplantation mouse embryo. J. Embryol. Exp. Morphol. 64,87-104. Beddington, R. S. P. (1982). An autoradiographic analysis of tissue potency in different regions of the embryonic ectoderm during gastrulation in the mouse. J. Embryol. Exp. Morphol. 69, 265-285. Beddington, R. S., and Robertson, E. J. (1989). An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105,733-737. Beddington, R. S. P., Morgenstern, J., Land, H., and Hogan, A. (1989). An in siru transgenic enzyme marker for the midgestation mouse fetus and the visualization of inner cell mass clones during early organogenesis. Development 106,37-46. Beddington, R. S. P., Wschel, A. W., and Rashbass, P. R. (1991). Chimeras to study gene function in mesodermal tissues during gastrulation and early organogenesis. In “Postimplantation Development in the Mouse,” Ciba Found. Symp. Vol. 165. Wiley. Chichester. Behringer, R. R., Cate, R. L., Froelick, G. J., Palmiter, R. D., and Brinster, R. L. (1990). Abnormal sexual development in transgenic mice chronically expressing Mullerian inhibiting substance. Nuture 345, 167-170. Behringer, R. R., Eldridge, P. W., and Dewey, M. J. (1984). Stable genotypic composition of blood cells in allophenic mice derived from congenic C57BL16 strains. Dev. Biol. 101,251-256. Christ. B., and Fundele, R. (1995). Bender, R., Surani, M. A., Kothary. R.. Li, L. L., Furst, D. 0.. Tissue-specific loss of proliferative capacity of parthenogenetic cells in fetal mouse chimeras. Roux’s Arch. Dev. Biol. 204,436-443.

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3 Molecular Regulation of Pronephric Development' Thomas Carroll, john Wallingford, Dan Seufert, and Peter D. Vize2 Center for Developmental Biology Department of Zoology University of Texas Austin, Texas 78712

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Introduction Structure and Function Early Development Pronephric Patterning and lnduction A. Background B. Molecules Pronephric Tubules A. Background B. Molecules Pronephric Duct A. Background B. Molecules Glomus A. Background B. Molecules Conclusions References

1. Introduction The kidney is an essential organ that functions to filter the blood. It has been a favorite subject for developmental biologists for over 100 years. In the past 15 years, we have learned a great deal about the molecules that regulate its development. This review will describe the molecules believed to be involved in regulating one particular type of kidney, the pronephros, the simple embryonic kidney of lower vertebrates. The vertebrate kidney exists in three forms: pronephros, mesonephros, and I Key words: pronephros, pronephroi, tubules, duct, glomus. :Author to whom correspondence should be addressed.

Currrnr fiiprc.~m DeLd,Jppmenra/ Rinlogv, Vul. 44 Copyright 0 1999 by Academic Press. All rights of reproductionin any form reserved. (X)70-2 IS399 $25.00

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metanephros (for reviews, see SaxCn, 1987; Vize et al., 1997). Each kidney type derives its name from its position along the anterior-posterior axis as well as the developmental period in which it functions. The pronephros (first kidney) develops in the most anterior position of all three kidneys and is primarily used as an embryonic kidney in lower vertebrates, i.e., amphibia and fish. Whether or not pronephric tubules exist in higher vertebrates is still controversial. Pronephriclike structures have been described in the embryos of birds and mammals but they lack a glomus, the vascular source, and therefore may not be functional (SaxCn, 1987; Sainio et al., 1997). The mesonephros (middle kidney) is the functional embryonic kidney in higher vertebrates (birds and mammals) and the adult kidney in lower vertebrates. The mesonephros lies between the pronephros and the metanephros (last kidney), which is the adult, terminal kidney type in mammals, birds, and reptiles but which never forms in lower vertebrates. Early developmental biologists preferred the pronephros as a model system due to the ease with which amphibian embryos could be generated, reared, and manipulated. However, in the 1950s, in vitro culturing assays were developed which led to a shift in emphasis from the pronephros to the metanephros where it has remained since (Grobstein, 1955; Grobstein, 1956; Grobstein and Dalton, 1957). Over the past 15 years we have learned an enormous amount about the molecular regulation of metanephric development, mainly due to recent advances in mammalian molecular techniques, such as positional cloning and the ability to perform targeted gene ablation (for reviews, see Clapp and Abrahamson, 1993; St-Jacques and McMahon, 1996; Lechner and Dressler, 1997; Vainio and Muller, 1997). However, the metanephros has some inherent limitations which have restricted the scope of experiments one can perform and made the interpretation of some of the data difficult. Murine metanephroi do not begin to develop until 11.5 days post coitum, several days after the development of the mesonephros. The development of the metanephros depends on the normal development of the mesonephros as the mesonephric duct induces the metanephric mesenchyme (SaxCn, 1987). Because a number of the genes involved in the development of these two organs are the same, targeted ablation of genes believed to be involved in metanephric development usually also affect the mesonephros (Kreidberg er al., 1993; Shawlot and Behringer, 1995; Torres et al., 1995). Subsequently, metanephros development is affected but the defects may often be secondary, i.e., due to mesonephric defects. In addition, the metanephros has a complex architecture, which makes the observation of minor defects difficult. Another limitation is that the in vitro culture techniques that are in use are most useful in investigating the final stages of development, just prior to differentiation. The study of early inductive and patterning events in mice is extremely difficult. The rapid development of the pronephros and its simple organization make it ideal for the study of early events in nephrogenesis (Vize et ul., 1997). The recent advances in Xenopus expression screens and transgenic techniques (Kroll and Amaya, 1996) and the growing popularity and capabilities of the zebrafish

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model system (see Development, vol. 123, December 1996) now make the study of the molecular mechanisms of pronephric development much more feasible and attractive. As will be discussed, although the three different kidneys differ in their architecture and organization, many of the genes regulating their development appear to be the same. Understanding the molecular regulation of pronephric development will also help us understand metanephc development as well as help answer more fundamental questions such as why multiple kidney forms exist and how the complex metanephroi evolved from the simple pronephroi. This chapter will review what is known about the molecular regulation of pronephric development from early patterning and induction to differentiation. Because this is a relatively new field, most of the molecular mechanisms are speculative. Many of the genes proposed to be involved in pronephric development are so believed due to their expression patterns and the roles of their mammalian orthologs. Hopefully, the powerful Xenopus expression techniques and zebrafish genetics will be able to test these hypotheses and the next several years will see a great expansion in our understanding of this process. As the majority of the genes so far identified have been cloned in Xenopus, we will focus on this organism. In cases where orthologs have also been cloned in zebrafish or chicken, their expression patterns will also be discussed.

II. Structure and Function The pronephros, or head kidney, is considered the most primitive vertebrate kidney type because it contains only one nephron, the functional unit of the kidney (Vize et al., 1997). Mesonephroi usually have between 10 and 50 nephrons whereas metanephroi can have up to one million. All three kidney types are derived from the intermediate mesoderm which lies between the somites and the lateral plate. The intermediate mesoderm lying ventral to somites 3-6 will give rise to the pronephros in Xenopus (Nieuwkoop and Faber, 1967). The functional pronephros is made up of three subunits: the pronephric tubules, the pronephric duct, and the glomus (Fig. 1). The tubules and duct develop from the somatic layer of the lateral plate whereas the glomus will arise from the splanchnic layer. The glomus is responsible for the filtration of the blood. The filtrate from the glomus is dumped into a specialized portion of the body cavity, the nephrocoel, which in early stages is the dorsalmost portion of the coelom. Filtrate is then swept up by the ciliated nephrostomal funnels which link the pronephric tubules to the coelom. The tubules function to resorb all valuable nutrients and ions from the filtrate through a process of active transport. These molecules are then returned to the bloodstream through a blood sinus that surrounds the tubules. All molecules that are not resorbed by the tubules are swept down the pronephric duct and disposed of via the cloaca.

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sagittal view

transverse view

Fig. 1. Anatomy of the anuran pronephros. Left: Lateral view of a stage 35-37 Xenopus pronephros. The pronephros has three dorsal branches, two anterior and one posterior. Each branch is connected to a thinner tubule, or nephrostome, which extends under the overlying somite and is directly linked to the coelom (Vize et d ,1997).The three connecting tubules are linked to a broader common tubule which in turn is linked to the pronephric duct. The diameter of the common tubule is reduced at the point where it meets the duct. In transverse sections the glomus can be seen projecting into the coelom opposite to the pronephric tubules. The nephrostomes sweep fluids from the coelom into the pronephric tubules by ciliary action. D, dorsal; V, ventral; A, anterior; P, posterior; Lt, lateral; M, medial.

I 11. Early Development Even with simple anatomy and the extensive amount of investigation into the development of the pronephros, many of the embryological events leading up to differentiation have still not been clearly defined. This is in part due to previous investigations having taken place in a wide range of organisms which have slightly different modes of development and different developmental timing. Nonetheless, we have a general idea as to when these events take place (e.g., Fales, 1935) and with the assistance of molecular markers, we can normalize these events across species. It is generally believed that the specification of the pronephros is a result of early mesodermal patterning and occurs sometime during or shortly after gastrulation in Xenopus (Fales, 1935). Differentiation does not occur for several hours after the initial specification and continues well into swimming tadpole stages. What is occurring at the cellular level between the times of specification and differentiation is unknown but the cloning and characterization of genes expressed during this window should help to clarify this process. Because different genes are involved in each case, the development of each pronephric subunit will be dealt with separately. Most of the genes believed to be involved in pronephric development have counterparts involved in the development of the metanephros. Because in most cases the orthologs are expressed in structures that are homologous between the two different kidney types, it is very likely that orthologs found in the pronephros of other species such as zebrafish

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will be expressed in a manner that is similar to that in Xenopus. In cases where there is divergence in the expression patterns of orthologous genes, the deviations may be significant and may lead to insights into how the genes function and how the differences may have led to the morphological differencesbetween pronephroi or even between pro- and metanephroi.

IV. Pronephric Patterning and Induction A. Background

As the nephric system forms from mesoderm, the analysis of pronephric induction begins with the induction and patterning of the mesoderm. In the Xenopus neurula-stage embryo, dorsal mesoderm will form prechordal plate and notochord, whereas ventral mesoderm will form blood and mesothelium. In between these extremes are paraxial mesoderm, which will form somitic muscle, and the intermediate mesoderm, which in turn will form the urogenital system, including the pronephros. These lateral mesoderm types are patterned during gastrula stages from mesoderm that is initially either dorsal or ventral in character. A number of reviews cover the classical embryological data and the more recent Xenopus molecular data (Dawid, 1994; Heasman, 1997; Nieuwkoop, 1997; Stennard et al., 1997; Thomsen, 1997). During blastula stages, mesoderm is induced in the marginal zone by the vegetal endoderm. Most of the marginal zone is induced to form ventral mesoderm, and when explanted prior to gastrulation, will differentiate into blood and mesothelium. A small region of the marginal zone, about 60" of arc, is induced by the underlying dorsovegetal endoderm to form the Spemann organizer. This portion of the marginal zone will differentiate into notochord and muscle in explants. During blastula and gastrula stages, signals from the organizer will in turn dorsalize adjacent mesoderm to generate the intermediate mesoderm including the pronephros, which would form ventral mesoderm in the absence of such signaling (Dale and Slack, 1987). The pronephros may be induced directly by dorsalizing molecules from the organizer, establishing a region with pronephric specification, or organizer molecules may function to regulate the activity of ventralizing molecules, which at a specific level of expression establish the pronephric region. In the following section the expression of dorsalizing and ventralizing genes, along with genes activated by them, will be briefly reviewed. B. Molecules

1. Pan-Mesodermal Genes A number of genes are expressed throughout the marginal zone in pregastrula embryos. These genes are presumably being activated in response to general

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mesoderm induction and are probably involved more in the decision to become mesoderm than they are in subdividing the mesoderm. Examples of such genes include a number of T-box genes which are expressed in the entire marginal zone of the early gastrula (reviewed in Stennard et al., 1997). These genes can induce isolated ectoderm to form mesoderm. There is some variation in the character of mesoderm they induce, but in general this class of molecule is only capable of inducing more ventral type mesoderm and they are unlikely to be involved in the patterning of the pronephric region (Ryan et al., 1996).

2. Organizer-SpecificGenes A large number of genes have been identified which are expressed only in the dorsal marginal zone, or organizer, of gastrula-stage Xenopus embryos which may be involved in patterning the pronephric mesoderm. A number of these genes have the capacity to dorsalize mesoderm when they are ectopically expressed in regions which would otherwise form ventral mesoderm. Many of these encode secreted factors that act by antagonizing ventral signaling pathways. Chordin, noggin, and follistatin proteins all can bind and inhibit BMP-4 (reviewed in Stennard et al., 1997; Thomsen, 1997), whereas Xnr-3 encodes a distinct TGF-f3 family member. frzb (Leyns et al., 1997; Wang et al., 1997) and dickkopfl (Glinka et al., 1998) encode antagonists of the Xwnt-8 signaling pathway. As these factors are involved in patterning the lateral marginal zone, they may well be involved in the induction of the pronephros. However, to date, no direct link between any of these genes and pronephric specification has been established.

3. Ventrally Expressed Genes and the Ventralizing Pathway The BMP-4 signaling pathway ventralizes mesoderm (Graff et al., 1994; Suzuki et al., 1994). BMP-4 is a TGF-P related growth factor which can induce the expression of other ventrally expressed genes, including Vent-1 (Gawantka et al., 1995) and Vent-2 (VoxlPVI lXom) (Ladher el al., 1996; Onichtchouk et al., 1996; Schmidt et al., 1996). These genes play a role in the patterning of ventral mesoderm and are not simply markers of ventral specification, as ectopic expression of BMP-4, Vent- 1, or Vent-2 can ventralize dorsal mesoderm. Also, there is evidence that BMP-4 may even act as a morphogen, with different concentrations of BMP4 specifying different types of ventral mesoderm (Dosch et al., 1997). To date, however, no specific concentration of BMP-4, or combination of other ventral genes, has been identified which is capable of instructing mesoderm to adopt a pronephric fate.

4. Laterally Expressed Genes A number of genes which may function in lateral mesoderm patterning have also been identified. The myogenic regulatory factors XmyoD and Xmyf-5 are ex-

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pressed in the early gastrula marginal zone, with XmyoD being expressed in a ventrolateral band (Frank and Harland, 1991) and Xmyf-5 being restricted to a smaller region immediately adjacent to the organizer (Dosch et al., 1997). Overexpression of either of these factors is sufficient to induce muscle differentiation in nonsomitic tissue (Ludolph et al., 1994). Xwnf-8 is also expressed in much of the ventral and lateral marginal zone (Christian and Moon, 1993). The activity of Xwnr-8 is downregulated by an antagonist, sizzled, which is expressed in the ventralmost marginal zone (Salic et al., 1997). Other wnt antagonists are expressed in the organizer (see earlier). A combination of such antagonists may therefore establish a zone of Xwnt-8 activity in the lateral marginal zone and this may play a role in patterning both the somitic and pronephric mesoderm (Hoppler et a/., 1996). However, although such a role in somite patterning seems fairly clear, no direct relationship between these genes and pronephric patterning has yet been established, and none of the genes discussed in this section are specifically expressed in the pronephric precursors.

5. Genes Inducing the Pronephros The foregoing discussion indicates that none of the genes or gene products identified to date seem likely candidates as inducers of the pronephroi or as pregastrula markers of pronephric specification. However, shortly after gastrulation genes begin to be transcribed in patterns consistent with the position of the pronephric primordia. Thus, although we do not yet know what gene products are responsible for the induction of the pronephroi, some conclusions can be drawn about the tissue which may express these molecules. The pronephros normally develops adjacent to the anterior somites, and presumptive somitic tissue will form pronephric tubules if explanted and grown away from the influence of the notochord (Yamada, 1940). These cells therefore contain all of the information necessary to form pronephric tubules and may well be involved in relaying this information to the actual pronephric precursors. Transplanted pronephroi do not develop well when they are located at a distance from the normal subsomitic position (Fales, 1935), and Holtfreter (1933) identified a pronephric field surrounding the normal site of the pronephros which supports pronephric specification. Once again, the tissue necessary for pronephric growth and development may well be the anterior somites. Pronephroi can also form in ventralized embryos lacking head structures (Seufert and Vize, in preparation), indicating that more anterior tissues such as head mesoderm are unlikely to be the source of the inductive signal. Together, these data indicate that the dorsal mesoderm is a strong candidate as the source of the pronephric-inducing signal. We do not yet know the nature of this signal. However, we do know some of the genes which are early targets of these patterning signals and based on their expression can propose a model for pronephric induction and specification. In the following section the development of the pronephros and the role these genes play in this process will be described.

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V. Pronephric Tubules A. Background

The first morphological sign of the pronephros in the frog Xenopus laevis is a thickening of the intermediate mesoderm ventral to somites 3-5 at late neurula or early tailbud stages (Nieuwkoop and Faber stage 21, Fig. 2). This condensation is the primordia of both the tubules and the duct. Since tubulogenesis does not occur for several hours, the intermediate mesoderm can be referred to as a pretubular condensate at this stage of development. Nieuwkoop and Faber (1967) describe an internal segregation process that divides the pretubular condensate into tubule and duct components by stage 23. By stage 24, the pronephric thickening has extended posteriorly to somite 6 and is easily visible as a swelling that distends the overlying ectoderm. At stage 27 (late tailbud), the pronephros has achieved a distinct tubular organization although no lumen is present until stage 29/30 (posthatching). At this time the tubules begin to express the epitope for the antibody 3G8 (Fig. 2). It is also at approximately this stage that the individual connecting tubules first become visible as protrusions on the dorsal side of the pronephric condensation. The number of pronephric connecting tubules can differ from one species to another. Most teleosts, for example, have only one, whereas among the amphibia, urodeles have two and anurans have three. From stage 29 on, the tubules continue to elongate and coil but will retain their position ventral to somites 3-5. The pronephros becomes fully functional by stage 37 (Nieuwkoop and Faber, 1967), several hours after hatching (swimming tadpole). Beginning at stage 54, the pronephric tubules will undergo a process of atrophy and regression, their function having been replaced by the second amphibian kidney, the mesonephros.

Fig. 2. Temporal development of the anuran pronephros. n, notochord; 1.p.. lateral plate; d.a., dorsal aorta.

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B. Molecules 1. Lim-1 The earliest identified marker of pronephric specification, Xlim- 1 (Taira et al., 1994), encodes a Lim class homeodomain (for review, see Curtiss and Heilig, 1998). This gene has been identified in a wide range of organisms and its expression in the organizer, notochord, urogenital, and nervous systems is highly conserved (Barnes et al., 1994; Toyama and Dawid, 1997). For the purposes of this review, only the urogenital expression of Xlim- 1 will be further described. Xlim-1 transcription is activated in the lateral mesoderm of Xenopus during late gastrulation (stage 13), which probably corresponds to the time of pronephric specification. By stage 14, the lateral mesoderm expression has expanded into a belt which extends ventrally from each neural fold and encircles the embryo (Taira et al., 1994). This expression extends ventrally well beyond the tissue fated to form the pronephros. By stage 16, the more ventral expression is greatly reduced and Xlim-1 expression is mainly confined to the cells of the intermediate mesoderm. At early tailbud stages, Xlim- 1 expression resembles a teardrop on the side of the embryo, extending from somites 3-9 (Fig. 2). This staining pattern is enigmatic, as expression extends further caudally than one would expect if this were marking the condensing tubules or duct. The pronephric duct has not yet begun its caudal migration and so what exactly this expression pattern represents is unknown. One possibility is that this expression represents cells which have the potential to form pronephroi but which do not receive the appropriate signals to do so, and another is that the posterior Xlim-1 expression represents the future mesonephros. Previous work has shown that the mesonephros in urodeles is determined during the early tailbud stages (Humphrey, 1928). Xlim-1 could be playing a role in the specification of this structure also, a hypothesis supported by the Lim1 mutant phenotype in mice (see later). Eventually, Xlim-1 expression restricts to the differentiated tubules and duct and continues to be expressed there throughout pronephric development, with strongest expression in the nephrostomes. Xlim- 1 expression decreases in the duct as it becomes lumenized and is not expressed in the cells of the duct that migrate anteriorly from the cloaca (seeXpax-2). Zebrafish liml expression in the intermediate mesoderm and pronephros is analogous to that of Xlim- 1 (Toyarna and Dawid, 1997). The murine Lim-1 expression pattern corresponds closely to that of Xlim-1 (Barnes et al., 1994). Lim-1expression commences in the lateral plate mesoderm between late primitive streak and early neural fold stages (7.0 to 7.5 days post coitum). The expression pattern refines itself and at 9.0 days is seen only in the nephrogenic cords. As development proceeds, Lim-1 is expressed in the pronephric, mesonephric, and metanephric tubules. Besides the expression pattern, the strongest support for Lim- 1 playing a role in kidney specification is the fact that mice that lack functional Lim-1 do not have pro-, meso-, or metanephroi (Shawlot

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and Behringer, 1995). Unfortunately, such mice die early on in development due to other malformations and therefore the analysis of the kidney defect is limited. Although Lim is expressed extremely early in the development of the urogenital system in vertebrates, ectopic expression of Xlim-1 has little or no effect on the development of the frog pronephros (Taira et al., 1994; Carroll and Vize, unpublished observation). Thus Xlim- 1 alone cannot be responsible for committing cells to a pronephric fate and its activity must depend on other factors. Xlim-l protein may be made in an inactive form and only activated in a subset of the cells that express its message or it may require a cofactor to function. It has previously been shown by several groups that Lim proteins can bind to and synergize with a number of proteins (for review, see Curtiss and Heilig, 1998), including the lim domain binding factor, Ldb-1 (Agulnick et al., 1996; Bach et al., 1997), and the homeobox genes Otx (Bach et al., 1997) and POU (Xue et al., 1993; Bach et al., 1995; Lichsteiner and Tjian, 1995), and such cofactors may also be important in kidney development (see HNFs, Section VB3). 2. Pax-8

Another possible cofactor for Xlim- 1 is Pax-8. Pax-8 is a paired-type transcription factor (for review, see Dahl et al., 1997) which begins to be expressed in the intermediate mesoderm around stage 14 shortly after Xlim- 1 expression commences in the lateral plate. Pax-8 intermediate mesoderm expression during early stages has similar anterior-posterior boundaries to Xlim- 1 but does not extend as far ventrally (data not shown), presumably marking only the intermediate mesoderm. As development proceeds, the expression patterns of the two genes are refined and condensed until they are both expressed in a common domain within the intermediate mesoderm. It is possible that Xlim-1 and Xpax-8 are interacting directly and that only cells which contain both proteins are specified to form the pronephroi. The expression of P a x 4 early in pronephric development is quite surprising. Although Pax-8 is expressed in the pro-, meso-, and metanephric tubules and nephric duct of mice, targeted ablation of this gene in mice has no kidney phenotype (P. Gruss, personal commumication). The lack of a phenotype in mutant mice may be due to genetic redundancies between Pax-8 and Pax-2, another Pax gene expressed in the developing kidney (see later). However, Xpax-8 and Xpu-2 do not seem to be redundant in the amphibian pronephros as their expression patterns are both temporally and spatially distinct. From expression pattern data alone, one would expect that Xpax-8 plays a role in early specification or determination events of pronephric development whereas Xpax-2 plays a role in later differentiation. The role of Pax4 in kidney development would therefore be pronephros specific, with Pax-2 playing this role in meso- and metanephroi. Assuming that Pax-8 expression is the same in zebrafish and Xenopus, one might expect fish Pax-8 mutants to have an earlier, or more severe, kidney phenotype than Pax-2

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mutants. Pax-2 mutants, on the other hand, would be expected to express early specification markers and possibly some differentiation markers. It is also possible that in Pax-8 mutant mice the pronephros is indeed effected but that this went unnoticed because of its small size and early development. As the nephric duct can develop without the pronephric tubules (Holtfreter, 1944), such a defect need not interfere with the development of the later kidney forms. It is also possible that mice have lost the earliest stages of Pax-8 expression in the pronephros and it is because of this loss that higher vertebrates lack a functional pronephros (although the cause and effect may just as easily be reversed). In this case, the later expression of Pax-2 in the murine pronephros may substitute for Pax-8 and be sufficient to maintain normal mesonephric development. A closer look at the pronephric morphology in Pax-8 mutant mice or the expression of Pax-8 in the pronephroi of other organisms might help to explain some of these contradictions. As the pronephros differentiates, Xpax-8 expression closely resembles that of Xlim- 1 (Fig. 3). However, by stage 36, just prior to the stage when the pronephroi become functional, Xpax-8 expression is completely lost in the duct and expressed solely in the tubules. It is expressed at uniform levels throughout the tubules, unlike Xlim-1, which is expressed at high levels in the nephrostomes and low levels elsewhere.

3. HNF The HNFs (also known as LFl3s) are a family of winged helix transcription factors which contain POU and homeodomain-like motifs (Herr et al., 1988; for review, see Kaufmann and Knochel, 1996). Three HNFs are expressed in the pronephros, HNF-la (Weber et al., 1996), HNF-lp (Demartis et al., 1994), and HNF-4 (Holewa e? al., 1996). HNF-lp expression is detected in the intermediate mesoderm at stage 16 (midneurula) and possibly earlier (Demartis et al., 1994). It is expressed in both the tubules and the duct of the forming pronephros in a pattern that is quite similar to that of Xlim-1. HNF-la and HNF-4 are expressed much later in the development of the pronephric tubules and are not expressed in the duct. HNFs have been shown to homo- and heterodimerize, a process which is regulated at least in part by the cofactor DCoH (dimerization cofactor of homeoprotein of LFBl) (Strandmann and Ryffel, 1995). XDCoH is also expressed in the pronephros, although its early expression has not been characterized. HNF-4 mutant mice die early on due to gastrulation defects so the development of their kidneys could not be assayed (Chen et al., 1994). Targeted ablation of HNF-la results in mice with renal dysfunction but no gross defects in metanephric morphology (Pontoglio et al., 1996). The lack of a kidney phenotype may be due to functional redundancies with the other HNFs, particularly H N F - l p . Because H N F - l p is expressed in the pronephros several hours prior to the expression of the la or 4, one might expect that targeted ablation of this gene in the mouse would have the strongest kidney phenotype.

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HNFs may require additional factors to function in kidney development. HNFs contain a POU-like domain and Lim and POU genes have been shown to physically and functionally interact in a number of systems (Xue et al., 1993; Bach et a/., 1995; Lichsteiner and Tjian, 1995; for review, see Curtiss and Heilig, 1998). The only POU gene that has been shown to be expressed in the pronephros, Xlpou-2, is expressed too late to be involved in pronephric specification (Witta er al., 1995).The same is true for HNF-la and HNF-4. However, HNF-1p represents an excellent candidate for interaction with Xlim- 1. As already mentioned, early expression of HNF- 1p closely resembles that of Xlim- 1 in the pronephros, consistent with the possibility that these two proteins are interacting. There is also the possibility that there are other POU class proteins that have yet to be identified that are expressed in the pronephric precursors and that together with Xlim-I specify the pronephros. Xlim/HNF- I p coinjection experiments in Xenopus or zebrafish could easily test for genetic interactions whereas immunoprecipitations or yeast two-hybrid would be useful in testing for physical interactions as well as in finding additional factors. 4. WntandFrz

The earliest signs of the differentiation of the pronephros in Xenopus is a slight swelling ventral to somites 3-5 at stage 23. Just prior to this, several genes commence expression in the pronephric anlage. One member of the wnt family of secreted molecules, Xwnt-4, is expressed in the characteristic teardrop pattern of the intermediate mesoderm at stage 18 (McGrew et al., 1992, XMMR). Wnt-4 has been shown to be necessary for the mesenchyme to epithelial transition and formation of the pretubular aggregates in the metanephros (Stark et al., 1994). Its expression in frogs is consistent with it playing a similar role in the pronephros, with the caveat that the pronephroi form by segregation of cells from the intermediate mesoderm rather than by condensation from mesenchymal precursors. Initially, Xwnt-4 expression closely resembles the expression of Xlim- 1 and Xpux8 but soon expression intensifies in the anteriodorsal portion of the tubule anlage, presumably in the three connecting tubule primordia. As development continues, Fig. 3. Gene expression during pronephric development. (A) Xpax-8, stage 36. Within the pronephros, only the pronephric tubules are stained. (B) Xlirn-I, stage 36. Strongest staining is within the nephrostomes and the pronephric duct, with weaker staining in the pronephric tubules (compare to panel A). (C) Xwnt-4, stage 35. Strong staining is in the pronephric tubules, and weaker staining in the duct. (D) Xpax-2, stage 36. Pronephric pattern is very similar to that observed for Xlim-1 in panel B. (E) xWI,stage 30. This pattern corresponds to the forming glomus. (F) Double stain with antibodies 3G8 (blue) and 4A6 (red), stage 39.3G8 stains only the pronephric tubules (similar to Xpm-8 in panel A) while 4A6 stains the duct as it coils around and curves back toward the cloaca (compare to the duct stain of Xpax-2 in panel D). (G) X-re?. stage 28. Staining is strongest in the posterior portion of the migrating pronephric duct. The anterior portion of the duct (panels D and F) is only expressing low levels of X-re?.

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3. Molecular Regulation of Pronephric Development

79 Xwnt-4expression is maintained at low levels throughout the tubules and the duct but expression is much more intense at the tips of the tubules where the nephrostomes are forming (Fig. 3). Several genes have high levels of expression in the nephrostomes (or the connecting tubules immediately adjacent to the nephrostomes) at this stage: Xlim-1,Xwnt-4,and Xpax-2 (see later). These genes may mark the cells of the tubules that are actively dividing and therefore contributing to the elongation of the tubules. Zebrafish wnt-4is also expressed in the pronephros but its expression there has not been characterized in detail (Ungar et al., 1995). In mice, Wnt-11 is expressed at the caudal end of the elongating nephric duct (Kispert et al., 1996). Wnt-1 1 expression greatly increases in the region of the duct adjacent to the metanephric mesenchyme, the future ureteric bud. As the bud invades the mesenchyme, Wnt-1 1 is expressed at the distal tip of the ureteric bud but is absent from the ureter stalk. As the bud branches, Wnt- 1 1 expression splits in two and is expressed at the tips of each branch. The epithelialization of the metanephric blastema has been shown to rely on a signal from the ureteric bud. Previous reports have shown that the spinal chord, a tissue that expresses several wnts, can also initiate condensation of metanephric mesenchyme in explants. Although the current data only support a role for Wnt-l l in the branching of the ureteric bud, a role in the early induction of the metanephric mesenchyme cannot be ruled out. The expression pattern of Xwnt-I 1 in frogs is consistent with this gene playing a role in both induction and branching of the pronephros. Although its expression in the pronephros has not been characterized in detail, Xwnt-11 is expressed in the pronephric tubule primordia but has not been detected in the pronephric duct (Ku and Melton, 1993). Interestingly, Xwnt-11 is expressed in the somites, which as postulated earlier, may be the tissue responsible for the induction of the pronephroi. It is possible that Xwnt-11 expressed in the somite induces the expression of Xpax-8 (or the cofactor of Xlim-1) in the dorsalmost region of Xlim-1expressing cells initiating the specification of the pronephric kidneys. The limited diffusivity of the Wnt signal would prevent Xpax-8 from being activated more ventrally, thus determining where the kidney will form. One problem with this hypothesis is that Fales ( 1935) showed that transplants of prospective pronephros showed a decrease in size and differentiation the further posterior they were placed. This would suggest that if the necessary factor is coming from the somites, it is localized to the anteriormost somites, whereas Xwnt-1 1 is expressed evenly throughout all somites. This might be explained if Wnt receptor expression is localized to the pronephric region. It has recently been shown that members of the frizzled family of seven pass transmembrane receptor can act as receptors of the wnt proteins (Bhanot et al., 1996).In accordance with this, Xfrizzled-3 is expressed in the Xenopus pronephros (Shi et d.,1998).It is not known which wnt binds to Xfrz-3, but the expression pattern of Xwnt-4 shows extensive overlap with that of Xfrz-3,suggesting that it

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may be the ligand. Outside of the pronephros, Xwnt- 1 1 shows little overlap with Xfrz-3, suggesting that it may bind to different receptors at different sites, although Xwnt-4 and Xwnt-1 1 are members of the same class of wnts (Du et al., 1995). It is possible that otherfriuled hornologs will be expressed in the intermediate mesoderm and pronephros. The cloning of wnt receptors along with the findings that truncated wnts can act as dominant negatives (Hoppler et af., 1996) may help to better elucidate the role of these signaling molecules in kidney development. 5. Pax-2

The Pax-2 ortholog has been shown to be expressed in the pro- and mesonephroi of zebrafish (Krauss et af., 1991) and Xenopus (Heller and Brandli, 1997; Carroll and Vize, in preparation; Fig. 3) and in the pro-, meso-, and metanephroi of mammals (Dressler et ul., 1990; Asano and Gruss, 1992). In the pronephros, Xpax-2 expression begins at early tailbud stages when the intermediate mesoderm is just beginning to condense into the pronephric anlage, several hours after the onset of Xpax-8 and Xlim- 1 expression. Upon tubulogenesis, Xpax-2 is expressed throughout both the tubules and the duct, with the highest level of expression in the nephrostomal region, a pattern which is identical to that of Xlim-1 and shows some overlap with Xwnt-4. As was mentioned earlier, the early expressions of Xpax-8 and Xlim-1 closely resemble each other but diverge later on. At about the time when the expression of these two genes begins to diverge, Xpax-2 and Xlim-1 expression converges. It is possible that Xfim- 1 is interacting with Xpax-8 in the early stages of pronephric development and with Xpax-2 in the later stages. Xpax2 and Xfim- 1 may cooperate to maintain Xwnt-4 expression in the nephrostomal region. Pax-:! mutants in mice and zebrafish have similar urogenital defects (Torres et al., 1995; Brand et al., 1996). Targeted ablation of the Pax-2 gene in mice results in a failure of the meso- and metanephroi to develop although the metanephric mesenchyme does form (Torres et af., 1995). Because the nephric duct does not fully develop, it is difficult to say whether the failure of tubules to differentiate is due to a defect in the tubule mesenchyme or due to a lack of signals coming from the duct. The zebrafish noi mutant has been shown to be the result of mutation in the Pax-2 gene (Brand et uf., 1996). noi mutants lack both pronephric tubules and duct similar to the mouse mutant phenotype. Assuming that Pax-8 expression is the same in zebrafish as it is in Xenopus, these data indicate that Pax8 and Pax-2 are not redundant in the pronephros, consistent with their temporally distinct expression patterns. Thus, Pax-2 is playing a similar role in the development of these two different kidney forms. To date, little analysis of genes known to be involved in kidney development has been performed in either the murine or fish mutant embryos. It will be interesting to see the effects on a number of pronephric markers such as Xfim- 1, Pax-8,WT- 1 (see later), and Wnt-4.

3. Molecular Regulation of Pronephric Development

81 It has been suggested that the Pax-2, Pax-8, and WT-1 gene products may regulate each other’s activity. Murine WT-1, a zinc-finger transcription factor, has been demonstrated to be a powerful repressor of Pax-2 transcription, whereas Pax-2 and Pax-8 have been shown to activate WT-1 expression (Dehbi et al., 1996; Frazier et al., 1997). Since frog WT- 1 may only be activated in a small subset of Xpax8-expressing cells and does not overlap with Xpau-2 (see Section VII), it seems unlikely that these Pax genes are directly activating WT-1 in the pronephros. At stage 20, the frog ortholog of WT- 1, x WT- 1, commences expression in a pattern that closely resembles that of Xwnt-4 but is only in the adjacent, more interior cells that will eventually form the glomus (Carroll and Vize, 1996). Interestingly, xWT- 1 is not detected in the cells which will form the pronephric tubules as it is in the meso- and metanephros. This is surprising since WT-1 has been shown to be essential for the condensation of the meso- and metanephric tubules. It is possible that xWT- 1 has a slightly altered role in the development of the pronephros. We suggest that it is involved in reserving a group of cells for the glomus fate by inhibiting the expression of genes involved in tubulogenesis, such as Xpax-2 and possibly Xpax-8 (see later). Thus the biochemical function of xWT-1 is most likely the same between the different species. 6. FGFs and BMPs A number of growth factors have been shown to play a role in the development of the metanephros, including members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. Members of the FGF family and their receptors (FGFR) are expressed in the metanephric tubules and ureteric bud (Orr-Urtreger et al., 1993; Dono and Zeller, 1994; Nguyen et al., 1996). Fgf-2 can induce explanted metanephric mesenchyme to undergo the early events of nephrogenesis, including condensation and activation of WT-1;however, these condensates do not form epithelia (Perantoni et al., 1995). Although several FGF family members and their receptors have been identified in Xenopus (for review, see Isaacs, 1997), the characterization of the expression of these genes in later stages of development has not yet been described. To date, no member has been shown to be expressed in the Xenopus pronephros although the FGF receptor FGFR-4 is expressed in the teardrop pattern in late neurula stage intermediate mesoderm (C. Niehrs, personal communication), suggesting a possible role for the FGFs in early pronephric development. A similar situation exists for members of the BMP family. Several BMPs are expressed in the metanephros during early development and BMP-7 has been demonstrated to play an important role in nephrogenesis. Explanted metanephric mesenchyme cultured with BMP-7 undergoes normal epithelialization in the absence of the ureteric bud (Vukicevic et al., 1996). Additionally, BMP-7 mutant mice do not form normal metanephroi (Luo et al., 1995, see later). Once again,

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several BMPs have been cloned in Xenopus but their expression in the pronephros has not yet been determined.

7. Hedgehog Members of the hedgehog family of secreted proteins have been shown to be necessary for the development of a number of structures in a range of organisms (Heemskerk and DiNardo, 1994; for review, see Ingham, 1995; Perrimon, 1995, 1996). One hedgehog family member, banded hedgehog (bhh), is expressed in the pronephros at early tailbud stages (Ekker et al., 1995). Additionally, a member of the Hedgehog signal transduction pathway, Glil, has been shown to be able to activate members of the HNF family (Sasaki et al., 1997). bhh commences expression in the intermediate mesoderm too late to affect HNF-Ip but may play a role in its maintenance or in the activation of HNF-la or HNF-4 in the pronephros. Other hedgehog family members may be expressed at earlier stages in pronephric development and may play a role in its normal development (see Lechner and Dressler, 1997).

8. Cell Adhesion Molecules At the time of differentiation, the pronephric mesoderm condenses and forms an epithelium. As one would expect, this condensation is coincident with the onset of transcription of cell-cell adhesion genes. The first cell adhesion molecule known to be expressed in the pronephros was N-cadherin (Simmoneau et al., 1992). N-Cadherin is expressed in the condensing intermediate mesoderm beginning at stage 20 and continuing at least until tailbud stages. a-Catenin, a molecule which links cadherins to the cytoskeleton, is also expressed in the pronephric anlage at early tailbud stages (Schneider et al., 1993). Integrin a.6, an extracellular matrix receptor molecule, is expressed in the pronephric tubules and duct beginning at stage 26 and continuing into later tailbud stages (Lallier et ul., 1996). These genes are most likely involved in later differentiation events and represent good markers of the differentiated state. 9. 3C8

Another excellent marker of the differentiated tubules is the antibody 3G8 (Vize et ul., 1995). The epitope for 3G8 is unknown but is expressed in the lumen of the tubule epithelia beginning at stage 31 as well as in the otic vesicle. Although generated against Xenopus pronephroi, the 3G8 antibody cross reacts with zebrafish and axolotl pronephroi (I. Drummond, personal communication, data not shown) and may be useful in a wide range of organisms. Its one drawback is that because it recognizes a lumenal epitope, it is only a marker for pronephroi in which epithelialization has occurred.

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VI. Pronephric Duct A. Background

The pronephric duct has been extensively studied at t.: embryological level for many years. As this review focuses on the molecular aspects of pronephric development, only a few of these experiments will be discussed. The study of the pronephric duct has been concerned primarily with two questions. First, how does the pronephric duct rudiment form? The pronephric duct is often stated to arise as an outgrowth of previously formed pronephric tubules, which is not correct. Also, the dependency of metanephric development on inductive signals from the nephric duct often leads to the incorrect conclusion that the pronephric tubules develop in response to signals from the pronephric duct. In addition to being incorrect, these two statements are also contradictory. In reality, evidence from several systems indicates that the duct and tubules arise independently. For example, Holtfretter (1944) and Vize et al. (1995) demonstrated the independent origin of the pronephric duct by dissecting urodele or Xenopus embryos into anterior and posterior or dorsal and ventral halves prior to the formation of the pronephric rudiment. At later stages, they found that the pronephric duct was sometimes present in posterior or ventral halves of embryos that contained no pronephric tubule tissue whatsoever. Likewise, tubules were often found in anterior or dorsal halves in the absence of duct. This latter observation, along with the fact that the tubules are specified (as evidenced by Xpax-8 and Xlirn- 1 expression; see earlier) at least 12 h prior to when the duct forms, indicates that the tubules are not formed in response to inductive signals from the duct. The second key question concerning the development of the pronephric duct is how the duct anlage extends along the length of the embryo to join with the cloaca, and this issue has also been the subject of much investigation. The one anlage contains the precursors to both the tubules and the duct, with the duct precursors lying ventral and posterior to the tubule precursors. As the pronephric anlage forms and undergoes epithelialization, the ventroposterior duct begins to extend posteriorly along the ventral border of the somites. Vital dye staining (O’Connor, 1938) and injected lineage tracers (Lynch and Fraser, 1990) have demonstrated that the vast majority of the duct is derived from the pronephric anlage. Although it has previously been stated that the Xenopus duct arises by an alternative process, these papers convincingly demonstrate that this is not so. Some studies indicate that in Xenopus the pronephric duct does contain some cells derived from other, unmapped, sources (Fox and Hamilton, 1964; Cornish and Etkin, 1993). The origin of the recruited cells remains ambiguous, though the demonstration that at least some recruited cells are derived from the caudal neural crest may explain these observations (Collazo et ul., 1993). The two key questions concerning the development of the pronephric duct have thus been addressed fairly convincingly (for additional discussions, see Vize el al.

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(1997) and Fox (1963) and references therein). It will now be interesting to examine the molecular underpinnings of each event. The majority of this information will be in the form of extrapolation from other species and interpretation of gene expression patterns.

B. Molecules

The formation of the pronephric duct anlage is most likely achieved in much the same way as the formation of the pronephric tubule anlage. As discussed in Section IVA, the patterning of the pronephros begins with the patterning of the mesoderm during gastrulation. Many of the same genes that regulate the early patterning of the pronephric tubules are simultaneously expressed in the pronephric duct. For example, Xlim-1, Xpar-2, and Xwnr-4 are strongly expressed in both the tubules and duct throughout development. Differences in gene expression must be present as tubules and duct can develop independently by Xenopus stage 12.5 (Vize et al., 1995). The following discussion describes some genes which may be involved in the process of specifying duct versus tubule development.

1. gremlin One molecule which is differentially expressed in the duct, but not in the tubules, is gremlin, which encodes a member of the DAN family of secreted BMP inhibitors (Hsu et al., 1998). Expression of gremlin in Xenopus commences at the tailbud stages in the neural crest, and it is strongly expressed in the pronephric duct beginning at later tailbud stages. Expression of gremlin in both neural crest and the pronephric duct may suggest that the cells expressing gremlin are those derived from the neural crest (Collazo er al., 1993). Regardless of the origin of these cells, the differential expression of a secreted BMP inhibitor in the duct but not the tubules is intriguing. Many BMPs are expressed in the developing metanephros (Hogan, 1996) and BMP induces tubulogenesis in culture (Vukicevic et al., 1996). Selective inhibition of BMP activity by gremlin may then be central to establishingpronephric duct cell fate.

2. c-ret and Glial Cell Line-DerivedNeurotrophic Factor (GDNF) Another molecule which is expressed in the developing pronephric duct is the receptor tyrosine kinase, c-ref (Fig. 3). ret and its ligand, GDNF (Trupp et al., 1996; Durbec et al., 1996), play critical roles in the development of several tissues, including the metanephrickidney (reviewed in Robertson and Mason, 1997). GDNF encodes a member of the TGF-P superfamily, and in the metanephric kidney, GDNF expression is restricted to the uncondensed metanephric mesenchyme (Hellmich et al., 1996). The c-ref proto-oncogene encodes a tyrosine ki-

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nase which is a cellular receptor for GDNF and is expressed in the kidney in a pattern complementary to that of GDNF; that is, c-ref is expressed in the Wolffian duct and in epithelial cells of the growing ureteric bud (Pachnis et al., 1993). One of the key defects in both GDNF- and ref-null mice is severe renal agenesis resulting from a failure of ureteric bud formation (Schuchardt ef al., 1994; Moore et al., 1996; Pichel et al., 1996; Sanchez ef al., 1996), suggesting a prominent role for the GDNF/ret signaling system in kidney development. In mice lacking functional c-ref, the Wolffian duct forms and connects with the cloaca normally, but metanephric development is severely perturbed. In the majority of embryos, the ureteric bud never evaginates from the Wolffian duct, and in the few embryos in which buds form, these buds never reach the metanephric mesenchymal blastema. As the ureteric bud of the Wolffian duct is the source of terminal-inducing signals for the metanephric mesenchymal condensation (SaxCn, 1987), the mesenchyme of c-ref mutant mice degenerates by apoptosis during later embryogenesis (Schuchardt et al., 1996), a result attributed to the failure of the mesenchyme to receive the proper inductive stimulus (Koseki, 1993; Koseki et al., 1992). The critical function of c-ret/GDNF in the interaction between the ureteric bud and the metanephric mesenchyme may suggest a possible role for these molecules in the formation of the pronephric tubules. The fact that the duct forms in both cret and GDNF mutant mice indicates that although these genes are required for duct branching, they are not required for duct specification, migration, or differentiation. Given that no equivalent of ureteric bud branching occurs during pronephric or mesonephric development, it would be reasonable to predict that these genes would play no role in the morphogenesis of the simple kidneys. Paradoxically, c-ret is actually expressed in a pattern consistent with it playing a role in pronephric duct migration in Xenupus, and there is even indirect experimental evidence which supports such a role. X-ref is expressed most strongly in the tip of the elongating pronephric duct and only weakly in the pronephric tubules (Fig. 3, Carroll et al., submitted). The preliminary experimental data hinting at a requirement for X-ref signaling in duct migration will be discussed shortly.

3. limb deformity (M) The Id gene is alternatively spliced to produce several different proteins collectively known as formins (Woychik et al., 1990). During metanephric development in the mouse, several formins are expressed in the Wolffian duct and ureteric bud (Chan et ul., 1995), and mice homozygous for limb deformity mutations display renal agenesis resulting from a failure of ureteric bud outgrowth (Maas et al., 1994). However, metanephric mesenchyme from Id mutant mice is capable of condensing and forming normal metanephric epithelia when cocultured with normal spinal cord, consistent with a role for Id in the duct rather than in the mesenchyme. Moreover, metanephric mesenchyme from Id mutants will also condense

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in the presence of Id mutant spinal cord, indicating that the Id gene product is not required for the inductive event per se, at least when a heterologous inducer is used (Maas et al., 1994). Though no data are available concerning Id expression in amphibians, Id is expressed in the pronephric duct of the chick (Trumpp et al., 1992), a finding which suggests that it may well play a role in the development of the pronephric duct. 4. Emx-2

Another gene which could be speculated to play a role in the development of the pronephric duct is Em-2, a gene related to the Drosophila homeobox gene empty spiracles. Like c-ref, E m - 2 is expressed during mouse embryogenesis in the Wolffian duct as well as in epithelial cells of the growing and branching ureteric bud, but not in the uncondensed metanephric mesenchyme. Mice lacking E m - 2 exhibit renal agenesis resulting from a failure of the ureteric bud to grow and branch, and in v i m explant experiments indicate that, like c-ret, E m - 2 is required in the ureteric bud for further metanephric development (Miyamoto et al., 1997). Although no expression data are available for E m - 2 in the pronephros, these similarities suggest a possible role for E m - 2 in the development of the pronephric duct.

5. GPI-Linked Proteins in Duct Development and Guidance Several experiments, beginning with those of O’Connor (1940) and Holtfreter (1944) and recently extended by Steinberg and colleagues (reviewed in Drawbridge and Steinberg, 1996), demonstrated that guidance cues direct the migration of the pronephric duct from the pronephric anlage to the rectal diverticula. These guidance cues may consist of gradients of adhesion molecules. These guidance cues direct migration not only along the normal anteroposterior route of the migrating pronephric duct but along the dorsoventral axis as well as deflecting the duct ventrally (Holtfreter, 1944), or grafting pronephric anlage to more ventral positions will result in the duct migrating dorsally until it reaches the normal pronephric duct pathway. It will then turn posteriorally and continue migrating until it reaches the rectal diverticula (Holtfreter, 1944; Poole and Steinberg, 1982; Zackson and Steinberg, 1986, 1987). Additional guidance cues are provided by the epidermis which also influence migration in both the anteroposterior and dorsoventral directions (Drawbridge er al., 1995). Exactly what molecules are involved in this process is not known. Zackson and Steinberg (1989) hypothesized a molecular prepattern should exist which would correlate with the positional information which guides pronephric duct migration. They found that the glycophosphatidylinositol (GP1)-linked, membrane-bound enzyme alkaline phosphatase is active in a gradient of activity which correlates with the gradient of adhesiveness suggested by the aforementioned grafting ex-

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periments (Zackson and Steinberg, 1988). Beads soaked in the enzyme phosphatidylinositol-specific phospholipase C (PIPLC), which cleaves GPI-linked enzymes from the cell surface, and implanted into axolotl embryos along the pronephric duct migratory pathway strongly inhibit the activity of alkaline phosphatase in that region and effectively halt the migration of the pronephric duct (Zackson and Steinberg, 1989). Although it is possible that inhibition of alkaline phosphatase by PlPLC is responsible for the failure of pronephric duct migration, another likely candidate is the GPI-linked, high-affinity receptor for GDNF, GDNFR-a (see following section). 6. GDNFR-a

Although embryological evidence suggests that pronephric duct migration is guided by adhesion gradients, some expression data and a considerable amount of circumstantial evidence also suggest that GDNF and c-ret may play key roles in guiding its caudal migration. As mentioned earlier, mammalian c-ret is expressed in the tip of the growing ureteric bud, and GDNF expression in the mesenchyme seems to regulate the morphogenesis of the adult kidney. Xenopus X-ret is likewise expressed in the caudal tip of the elongating pronephric duct, presenting the possibility that GDNF expression along the duct pathway may regulate the migration of the pronephric duct. Such a mechanism may be similar to that by which GDNF and ret guide the migration of vagal neural crest cells to destinations in the enteric nervous system (Schuchardt et al., 1994; Moore et al., 1996; Pichel et al., 1996; Sanchez et ul., 1996) or how localized Steel guides the migration of c-kit positive cells. Two different groups (Jing ef al., 1996; Treanor ef al., 1996) have identified an additional high-affinity GDNF receptor, GDNFR-a, which is coexpressed with cret and is required for its activation by GDNF. GDNF appears to bind directly to GDNFR-a, which in turn associates with and activates c-ret . Interestingly, GDNFR-a encodes a GPI-linked cell surface protein, and PIPLC inhibits tyrosine phosphorylation by c-ref in response to GDNF in cultured cells (Treanor et al., 1996). As mentioned earlier, PIPLC-coated beads implanted on the axolotl pronephric duct migratory pathway strongly inhibit the elongation of the duct (Zackson and Steinberg, 1989). Furthermore, pronephric duct rudiment grafting experiments in which either the host embryo or the donor pronephric duct rudiment was treated with PIPLC have demonstrated that the GPI-linked enzyme cleaved by PIPLC is required not on the migratory pathway where alkaline phosphatase is present but on the surface of the migrating c-refpositive pronephric duct cells themselves (Thibaudeau et al., 1993). Moreover, although the expression pattern of Xenopus GDNFR-a has not been described in duct cells, c-ret expression data suggest that GDNFR-a will also be expressed there (Jing et al., 1996; Treanor et al., 1996). Finally, bathing of embryos in zinc chloride (an inhibitor of many enzymes,

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including some tyrosine kinases) at tailbud stages inhibits the elongation of the pronephric duct (P. Vize, unpublished observations). Although the anlage differentiates (and the duct-specific epitope 4A6 is expressed), the duct never extends posteriorly, a result consistent with a requirement for tyrosine kinase (c-ref?) signaling in the elongation of the pronephric duct.

VII. Glomus A. Background

Though the vascular component of the pronephros, the glomus (plural glomera), was described over 100 years ago, remarkably little experimental work has been done to investigate its development. In amphibians, the glomus develops as a fold in the splanchnic lateral plate which extends into the coelom (Figs. 1 and 2). This fold contains endothelial cells which migrate into the glomus from a remote source (Field, 1891). Heterotopic grafting experiments have shown that the folding of the splanchnic lateral plate and glomus formation are in response to inductive signals from the pronephric anlage. Transplanted pronephric anlage induce new glomera, and doubling the size of the pronephros by transplanting a donor anlage adjacent to that of the host results in a doubling of the size of the pronephros and a corresponding doubling of the size of the adjacent glomus (Fales, 1935). Although no experiments have as yet been performed to assess gene function in the developing glomus, the expression patterns of some genes in Xenopus glomera and the known function of genes in metanephric glomeruli allow us to make a few educated guesses concerning the genetics of glomus development. B. Molecules

1. Wilms’ Tumor Suppressor Gene, WTl Unlike a metanephric glomerulus, the glomus is not directly associated with nephric tubules. Instead, glomera filter fluid from the blood into the coelom, from which the fluid is swept into the pronephric tubules by cilia (Fig. 2). Thus, whereas the pronephric tubules arise from the somatic side of the intermediate mesoderm, the glomera arise instead from the splanchnic side (Field, 1891). This point is particularly intriguing in light of the fact that the Wilms’ tumor suppressor gene WTI, which plays a key role in the development of the metanephric tubules as well as the glomeruli (reviewed by Hastie, 1994), is expressed during pronephros development exclusively in the presumptive glomus (Carroll and Vize, 1996). W T I encodes a transcription factor with four zinc fingers, and mutations in WTI have been identified in Wilms’ tumors, pediatric tumors of the kidney (Hastie,

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1994; Coppes et al., 1995). In vitro,W T I is a powerful suppressor of transcription from several promoters (Madden et al., 1991 ;Drummond et al., 1992; Ryan et al., 1995) and may further regulate gene activity post-translationally by binding RNA (Larsson et nl., 1995; Caricasole et al., 1996; Kennedy et al., 1996). Mammalian WTZ is expressed at low levels in the condensing metanephric mesenchyme at early stages of kidney development (Armstrong et al., 1992; Mundlos et al., 1993). However, this low level of expression is clearly essential to metanephric development, as the metanephric blastema of mice lacking W T I degenerates by apoptosis early in development, and kidneys do not form (Kreidberg et al., 1993). At later stages, mammalian WTI expression is restricted solely to the podocytes of the developing glomeruli, suggesting an additional role for this gene in the development of the vascular component of the nephron. Consistent with a role for WTl in glomerular development, many human Wilms’ tumors resulting from mutations in WTl display glomerular nephropathy, a collapse of arteries in the glomeruli (Denys et ul., 1967; Drash et al., 1970). Unfortunately, the role of WT1 in glomerular development cannot be investigated in the WTl-mutant mice, as nephrons never develop due to the early apoptosis phenotype. Unlike mammalian WTI, Xenopus WTI (xWT1) does not appear to play a role in the development of the pronephric tubule complex, as expression of xWTl is not detected in these cells. Like W T I , however, xWTI is expressed in the vascular component of the pronephros (Carroll and Vize, 1996), presenting the possibility that the role for WTI in the development of the vascular components of the kidney may be the more ancestral, while this gene’s early role in mesenchymal condensation in the metanephros may be more derived. This expression pattern may also reflect the fact that pronephroi do not form from a mesenchymal precursor, and so may not require this early phase of WT1 expression. However, given the conserved expression in the vascular components of the kidney, studies of the function of xWTl in the amphibian pronephros may shed light on this gene’s pivotal role in the development of the renal vascular system. The tremendous amount of in vitro data concerning the activities of WT1 indicate potential roles for xWTl in pronephric development. WT1 is a powerful repressor of the Pax-2 promoter in vitro, and WTI expression in vivo correlates to a decrease in Pax-2 expression (Ryan et al., 1995). Furthermore, mammalian Pax8 is strongly upregulated in Wilms’ tumors (Poleev et al., 1992), suggesting that WTI may act to downregulate this gene in a manner similar to Par-2. In Xenopus, Xpux-2 is expressed strongly in the pronephric tubule and duct anlage at the same time that xWTl is expressed in the presumptive glomus (Carroll and Vize, 1996; Heller and Brandli, 1997; Carroll and Vize, in preparation). Moreover, like Xpax2, Xpnx-8 is expressed in the developing pronephric tubules (Fig. 3). These data suggest that the role of W T I in the glomus may be to prevent the expression of pronephric tubule-specific genes, such as the Pax genes, in the presumptive glomera. WTI expression may then be critical for reserving a pool of cells for the formation of the glomus in response to some later signal. That the glomus develops

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much later than the pronephric tubules and duct (Field, 1891; Nieuwkoop and Faber, 1967) is consistent with this hypothesis.

2. Vascular Endothelial Growth Factor (VEGF) The glomus forms from the splanchnic lateral plate mesoderm but is largely populated by vascular cells. Field (1891) hypothesized that these cells had migrated in from a remote source, and the expression of VEGF in the glomus (Cleaver er al., 1996) is consistent with his hypothesis. VEGF encodes an endothelial cell-specific mitogen with widespread roles in neovascularization (Keck et al., 1989; Leung et al., 1989; Carmeliet et al., 1996; Ferrara et al., 1996). In mice, VEGF is strongly expressed in the developing glomerular epithelium and is very likely involved in the formation of glomerular capillaries (Breier et al., 1992). Mice lacking VEGF die very early from widespread, severe defects in vascularization (Carmeliet et al., 1996; Ferrara et al., 1996). As such, the role of VEGF specifically in glomerular development cannot be assessed in these mice. Again, perhaps studies in the simple pronephroi will shed light on this important issue. Expression of VEGF begins in the Xenopus glomus at a rather late stage of its development, consistent with a role for this molecule in populating the glomus with the necessary endothelial cells. The source of these endothelial cells is most likely the Flk- l-expressing endothelial precursor cells lying just lateral to the glomus (Cleaver er al., 1997).

3. Platelet-Derived Growth Factor P (PDGF-P) and Its Receptor, PDGFR-P PDGF-P encodes a peptide growth factor expressed in many cell types during development, including endothelial cells (Raines and Ross, 1993), and PDGFR-P encodes one chain of the PDGF receptor tyrosine kinase (Kazlauskas, 1994). Both of these genes are expressed during mammalian embryogenesis in the metanephric glomerulus (Alpers er al., 1992), and loss of either PDGF-p or PDGFR-P in mice results in a complete absence of glomerular mesangial cells, endothelial cells which support the blood vessels (Leveen et al., 1994; Soriano, 1994; Hyink and Abrahamson, 1995). Although no expression data for PDGF-P or PDGFR-P are available for the pronephros, the glomerulus-specific phenotype in the kidneys of these knockout mice strongly suggests that these PDGFs may play a key role in the development of the glomus.

VI I I. Conclusions Although the role of many of the genes described herein in regulating pronephric development remains speculative, the fact that in examples tested to date both expression pattern and function of metanephric genes appear to be conserved in

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Model of inductive interactions patterning the pronephros.

pronephric kidneys, plus our knowledge of the timing and morphogenesis of pronephric induction and differentiation, allows the construction of a working model describing the patterning of the pronephros at a molecular level (Fig. 4). In this model the source of inducing molecules is inferred in part from the expression patterns of genes-for example, the endodermal induction of the ventral belt of Xlim- 1 expression-and partly from explant and embryological experimentation. In this model two inductive tissues are postulated. The first is the axial (notochordal) or paraxial (somitic) mesoderm, and the second is the deep endoderm underlying the presumptive pronephroi. Explanted somite can differentiate into pronephric tubules, and notochord can either directly suppress this event or indirectly suppress such differentiation by promoting or maintaining somitic specification (Ydmada, 1940). The signal coming from the deep endoderm is deduced from the Xlim-1 expression adjacent to this region. This staining extends far beyond the dorsal mesoderm. Following the establishment of the presumptive pronephros in early neurulae in the region in which the mesodermal (marked by

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Xpux-8 expression) and deep endodermal (marked by Xlim-1 expression) signals overlap, this region is subdivided into presumptive tubules and presumptive glomus by signals from the underlying endoderm in mid-late neurulae. Evidence in support of such a signal comes from the expression pattern of xWTl in the presumptive glomus. This gene has been demonstrated to be a potent repressor of transcription, so it may perform this function by directly downregulating target genes in the pronephric tubules. The final patterning event is proposed to be the further subdivision of the presumptive pronephric tubule region into presumptive tubule and presumptive duct domains, once again in mid-late neurula stages (Figure 4). Given the ability to easily manipulate the expression of each of these genes by mRNA injection in Xenopus embryos and the current zebrafish screens for developmental mutants, this model should be readily testable. It seems extremely unlikely that all of the genes required for pronephric patterning have already been identified, so developing a complete molecular pathway may not be possible until mutant screens near saturation. However, given the collection of molecular markers for each stage and each component of the pronephric kidney, experiments on the inductive interactions patterning the pronephros should be able to either support or disqualify this working hypothesis quite quickly. Once the sequence of inductive interactions has been established, we can go on to link the activity of specific genes to inductive signals and the responses they elicit.

Acknowledgments The authors are supported by NSF Grant IBN-963621 (P.V.), a Young Investigator Award from the National Kidney Foundation (P.V.), and the Center for Developmental Biology at the University of Texas, Austin.

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4 Symmetry Breaking in the Zygotes of the Fucoid Algae: Controversies and Recent Progress Kenneth R. Robinson, Michele Wozniak, Rongsun Pu, and Mark Messerli Department of Biological Sciences Purdue University West Lafayette, Indiana 47907

1. Introduction 11. Physiology of Fertilization 111. The Response to Light

IV. The Role of Calcium and Calmodulin in Photopolarization and Rhizoidal Growth A. Calcium B. Calmodulin

V. Cortical pH Gradients during Axis Formation and Rhizoidal Growth VI. Actin Microfilaments and Photopolarization VII. Axis Fixation VIII. The Signal Transduction Process for Photopolarization IX. An Opsin-like Photoreceptor in Pelvetia? X . A Speculative Model for Photopolarization XI. Summary References

1. Introduction The zygotes of various species of the fucoid brown alga Fucus have been used for the study of polarity for more than a century. Unlike the eggs of most organisms, Fucus eggs, as well as those of the closely related genus Pelveria, are apolar when they are released from the adult and fertilized. All eggs of multicellular animals have at least one axis preformed that is represented by the animal-vegetal gradient. In the case of frogs, for example, the animal hemisphere is densely pigmented and the vegetal hemisphere lacks pigment but is packed with yolk. The frog oocyte nucleus, the germinal vesicle, is displaced toward the animal pole and migrates to the animal pole during maturation into an egg. These obvious morphological gradients are accompanied by subtler molecular gradients. The mRNA for the TGF-P superfamily member Vgl is localized to the vegetal cortex of the oocyte and the Vgl protein appears to be involved in the induction of the Spemann’s I

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Organizer. In Drosophila, a complex pattern of localized maternal mRNA and localized activation of membrane receptors specify both the dorsal-ventral and anterior-posterior axes in the egg (Lawrence, 1992). Thus, in general, there is a considerable degree of pattern in the animal egg that arises during oogenesis in the ovary, making the study of the mechanism of patterning difficult. In contrast, Fucus and Pefvetia zygotes are spherically symmetrical. The nucleus is centrally located in the zygote and the many cytoplasmic inclusions are uniformly distributed. The first axis does not form until several hours after fertilization and is not visibly manifested for some hours after formation. The axis is expressed by germination, the localized outgrowth of the nascent rhizoid. First cell division is perpendicular to this axis, resulting in the formation of two highly unequal cells with different fates. These events occur over a period of hours in isolated cells developing in seawater. The experimenter can control the direction of the axes of a population of cells. Fucoid zygotes are highly sensitive to light and respond to unidirectional white light by forming an axis that parallels the light direction, with the rhizoid eventually appearing on the side of the cell farthest from the light source (see examples in Fig. 1). If cultured in the dark and in the absence of external gradients, a population of zygotes will form their rhizoidal axes randomly with respect to any coordinate system. This latter property is useful if one wishes to test the polarizing effect of a gradient of some substance. Another useful property of fucoid zygotes is their ability to stick themselves firmly to almost any substrate. The first task of the eggs when they are released into the swirling intertidal waters is to attach themselves to the rocks. The eggs are gravitationally dense, so they settle rapidly, and they begin to secrete a cell wall as soon as they are fertilized. Within about 5 h, they adhere tenaciously to the surface on which they rest. This simplifies many experimental manipulations, because the medium surrounding the cells can be changed without disturbing their orientation with respect to some imposed gradient. It is clear, then, that the fucoid system offers many advantages for the study of the genesis of polarity. The cells are relatively large, free-living, microinjectable, and available in large quantities. As a result of decades of research, much information about polarization of Fucus and Pefvetia zygotes has accumulated, and these cells remain a subject of continued investigation. Unfortunately, however, there is almost no agreement on the important facts of the developmental processes. There is disagreement about the role of Ca2+(both intracellular and extracellular) in photopolarization, the role of calmodulin, the role of the actin cytoskeleton, and the existence of “axis fixation” as a pregermination condition. Startlingly, even whether the zygotes germinate from their darkest point or brightest point in response to unidirectional light is controversial. It is the purpose of this review to present the (few) areas of agreement and to explore the areas of disagreement. We will offer an analysis of the controversies, as well as highlight some newer findings on the nature of the photoreceptor, which are too recent to have become controversial. In writing this review, we are indebted to a number of

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Fig. 1 (a) Pelvefia zygotes were allowed to settle on a grid of alternating transparent and opaque stripes and then illuminated from below (right) or grown in the dark (left). All of the light-grown cells developed their rhizoids on the side that was shaded from the light, that is, from the portions of the cells above the opaque stripes. The dark-grown cells developed rhizoids at random with respect to the grid. (b) The presence of 10 pM KN-93. an inhibitor of CaM kinase 11, inhibited germination of Pelvetin zygotes (right). Untreated controls are shown on the left. (c) Pelvetia zygotes were cultured in the continuous presence (left) and absence (right) of 10 pM nifedipine. No effects on polarity and growth are evident.

other reviews of the subject, including both recent and older ones (Jaffe, 1968, 1969; Goodner and Quatrano, 1993; Kropf, 1994, 1997; Quatrano and Shaw, 1997).

II. Physiology of Fertilization An interesting feature of the eggs of Fucus and Pelvefiu is their animal-like mode of sexual reproduction. Diploid plants (dioecious in some species, monoecious in

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others) release haploid eggs and motile sperm. The eggs lack a cell wall or any external coat; indeed, Fucus eggs within the oogonia can be readily induced to fuse with each other by raising the temperature slightly (Whitaker, 1931). Fertilization is relevant to the question of polarity because the zygotes will germinate in the absence of any external gradient and it is thought that the point of sperm entry might provide a sufficient organizing asymmetry as it does in Cystoseria (discussed in Jaffe (1969)). The first physiological indicator of fertilization is a depolarization of the membrane potential. The membrane potential of the unfertilized eggs of Pelvetiu fastigiata, Fucus vesiculosus, and Fucus ceranoides is about -60 mV, and it rapidly depolarizes to about -25 mV upon fertilization. Brawley (1991) has shown that this fertilization potential acts as a fast block to polyspermy, as it does in sea urchins and other animal eggs (Jaffe and Could, 1989). If the membrane potential was depolarized by current from an intracellular electrode, fertilization was prevented. The fertilization potential is caused, at least in part, by an increase in sodium permeability. If eggs were fertilized in seawater containing 10% of the normal Na ' concentration, the fertilization potential was slower and reached peaks about 10 mV more negative than in normal Na + seawater. In addition, fertilization in reduced sodium seawater resulted in a marked increase in polyspermic eggs (Brawley, 1987). In a voltage-clamp analysis, Taylor and Brownlee (1993) confirmed that the eggs of Fucus serratus are excitable but concluded that the inward current induced by depolarizing potentials was carried by Ca*', not Na +. Although the nature of this apparent discrepancy is not known, one clue may be found in the resistance of the eggs' membranes (R,) reported by the two groups. Brawley (1991) determined R, of unfertilized E vesiculosus eggs as 136 MQ whereas Taylor and Brownlee (1993) reported 5 1 MR for the similar-sized eggs of E serrutus. The lower resistance is similar to that of fertilized eggs, raising the possibility that the low-resistance eggs were partially activated by the impalement. Taylor and Brownlee (1993) used electrodes that had resistances of 20-60 MQ before beveling and 20-30 MQ after beveling, whereas Brawley (1991) used finer tipped electrodes with resistances of 70-90 MQ. An increase in the Ca2+levels of the eggs following fertilization was revealed by the injection of the calcium-sensitive photoprotein aequorin into a group of unfertilized E vesiculosus eggs (Robinson, 1990). However, that method did not allow information from single cells to be resolved or the magnitude of the increase to be quantified. Using dextran forms of the fluorescent Ca2+indicators Fura 2 and Calcium Green, Roberts et al. (1994) were unable to detect a change in Ca 2 + at fertilization in the large majority of the eggs, even though the eggs were found to be activated by other criteria. In the cases where an increase was detected, no wave of increased Ca2+was seen. Tracer flux measurements suggest that fertilization induces an influx of Ca2+from the external medium (Roberts et al., 1994), and the elevation of Ca2' by treating eggs with ionophore induces some of the events of fertilization (Brawley et al., 1976). Nevertheless, there is no evidence to

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support the idea that a long-lasting Ca2+trace is left to mark the site of sperm entry and thus to act as an organizing center for polarity in dark-grown zygotes.

111. The Response to Light One of the first facts established about the physiology of the fucoid zygotes is the ability of the unilateral illumination to regulate the direction of the rhizoid-thallus axis (Rosenvinge, 1889) as cited by Whitaker (1940)). In response to white light, the cells formed their rhizoids on the sides away from the light source. Hurd ( 1 920) showed that only light of wavelength less than 560 nm was effective in the establishment of polarity in F. injutus zygotes. It is important to note that the light signal need not be present continuously. If Fucus zygotes are exposed to a unidirectional light source of sufficient intensity during their photosensitive period (a few hours after fertilization) for an hour and then returned to the dark, the population is efficiently polarized (Whitaker and Lowrance, 1936). Whitaker (1941) extended these studies into the ultraviolet region and found that illumination at 280 nm was highly effective in inducing the rhizoids of E furcutus to form on “the least illuminated side,” that is, the side away from the light source. He also found that a beam of 280-nm light “which is incident on a single layer of Fucus eggs is completely extinguished,” whereas longer wavelengths were extinguished by 85-90%. In other words, the eggs absorb or scatter nearly all of the UV light that impinges on them within one cell diameter. Jaffe (1956, 1958) investigated the responses of both F. furcutus and P. fusrigiara zygotes to plane-polarized light. If the light was directed vertically from either above or below, the zygotes tended to form their rhizoids in the horizontal plane and parallel to the electric field vector of the light. One important conclusion of this work was that the absorption of visible light is mediated by a photoreceptor located in or near the plasma membrane and that the photoreceptor’s dichroic axis is aligned parallel to the membrane. Jaffe also found that about 50% of the cells exposed to polarized light formed double rhizoids; double rhizoids are normally exceedingly rare. In a given cell, the double rhizoids were on opposite sides of the cell and aligned with the light’s electric field vector. The implication of this result is that the polarity truly arises anew in the zygotes. If there were a preformed axis that was simply reoriented by the light, it would not be possible to produce double rhizoids by any means. The action spectra of both responses to polarized light, rhizoid alignment and twinning, showed a fairly broad peak centered at 450 nm; wavelengths above 550 nm were completely ineffective. It was explicitly assumed by Hurd, Whitaker, and Jaffe, as well as other investigators, that the densely pigmented fucoid zygotes responded to light by growing from their darkest region. Direct experimental support for this view is summarized by Kuhn (1971), who, in citing the work of Nienburg (1923-1930), says “[tlhe direction of light does not influence the polarity of the cell directly, but rather

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indirectly through the differences in light intensity in the parts of the cell. If one illuminates from below Fucus eggs or Equisetum spores on a plate which is half transparent, half opaque, those cells on the dividing line grow toward the dark part.” Due to the intense self-absorption of unilateral light, a zygote produces a steep illumination gradient between the membrane facing the light and the membrane on the other side and responds to that gradient by organizing an axis and growing a rhizoid from the darker side. This apparently universally held view has been challenged recently. Berger and Brownlee (1994) assert that “a mechanism of light focusing exists in Fucus zygotes.” They say that the side facing away from the light source is the one receiving the higher irradiance. In this view, Fucus zygotes focus unilateral light on the opposite pole and thus produce a rhizoid from the brightest region. This notion is reiterated in a subsequent publication (Love et al., 1997). There are two lines of evidence that cast doubt on the idea that fucoid zygotes focus light and thus grow from their brightest region. One is optical and the other, physiological. A cell growing in seawater is greatly constrained in the amount of light focusing that it can achieve. The index of refraction of seawater is about 1.34 and the index of refraction of the cytoplasm is unlikely to exceed 1.40. A ray that strikes the surface at a 45” angle (midway between the pole and the equator) will be deflected by only 2.4”. Very little focusing on the opposite side of the cell will result from these small deviations. This slight focusing effect must be viewed in the context of the obvious large degree of absorption and scattering across the cell. It has been shown that greater than 95% of the polarization-effective wavelengths of light are absorbed or scattered in one cell diameter by P. fastigiara zygotes (Robinson, 1996b). For the membrane of the opposite pole to be more brightly illuminated than that of the pole facing the light, focusing would have to increase the light intensity by more than 20-fold to overcome absorption and scattering. All of the light entering the illuminated hemisphere would have to be focused on a polar region having an area of less than 5% of the area of the opposite hemisphere. This does not seem possible with any physiologically achievable refractive index. The fucoid egg seems spectacularly poorly designed to act as a lens. The foregoing arguments are dependent on the optical properties of the cell, which are not fully known. The physiological data, however, are unequivocal. If P. fastigiata zygotes are grown on a grid of alternating transparent and opaque stripes and illuminated from below, many cells can be found that are approximately half-illuminated. These cells always germinate from the shaded region. Likewise, cells that straddle a transparent stripe always germinate from one of the two smaller shaded regions. Finally, cells that straddle an opaque stripe always germinate from the central shaded region, with the rhizoids growing parallel to the stripes. Examples of these results are shown in Fig. 1. It is difficult to escape the conclusion that the Pelvetia zygotes have a powerful tendency to germinate from their darkest region. The fact that they germinate from the pole facing away

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4. Symmetry Breaking in the Zygotes of the Fucoid Algae

from the source of unilateral light establishes that pole as the darkest region. As shown by Nienburg (1923-1930). the zygotes of F: vesiculosus respond in the same way as Pelvetia zygotes when half-illuminated, so this conclusion seems valid for the fucoids in general. Further evidence for this conclusion is found in the analysis of the response of Pelvetia zygotes to weak, unpolarized unilateral light by Jaffe (1958). He showed that the locus of rhizoid formation in a population of cells was centered between 90" and 135" from the light direction, that is, in a zone just beyond the equator on the shaded side. His analysis of the paths of light rays through the zygote clearly showed that this is a region of absolute darkness due to the refraction of the light (Fig. 2). This result is important because the analysis does not depend on any assumptions about light absorption or scattering. The cells formed their rhizoids at the darkest point. At higher light intensities, the locus of rhizoid formation

I II I

1

..

LCENTER 1

I II l l ,

Fig. 2 Model to show that a shallow subequatorial band in a unilaterally illuminated cell remains dark. Zone A is entirely bypassed, while zone B receives rays which have lost at least half of their energy by external reflection. Drawn for a spherical cell of refractive index n = 1.4 immersed in a medium, such as sea water, for whichn = 1.34. (Figure and caption reprinted, with slight modification, from Jaffe, 1958, with permission.)

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shifts toward 180". that is, directly away from the light, perhaps because the cells integrate the light intensity and grow from the center of the darkest region. A similar subequatorial response was seen when the cells were illuminated with plane-polarized light. Instead of forming their rhizoids exactly at 90" to the light direction (parallel to the electric field vector), the rhizoids were centered at 105", again coincident with the predicted region of absolute darkness.

IV. The Role of Calcium and Calmodulin in Photopolarization and Rhizoidal Growth A. Calcium

Perhaps no other aspect of the photopolarization process has been as controversial and uncertain as the involvement of Ca2+and the source of Ca2+for any cytoplasmic gradients. It is evident that exposure to unilateral light must leave a trace in the zygotic cytoplasm (or perhaps the plasma membrane) that persists for hours after the cell is returned to the dark. Whatever the physical nature of the trace, it must be able to convey information about the direction of the light; that is, it must exist as a gradient in the cytoplasm and be able to direct the locus of the germination region. The first direct evidence that Ca2+might be involved came from studies of 4sCa2+movements through the two halves of polarizing zygotes (Robinson and Jaffe, 1975). In those experiments, cells were forced into nearly perfectly round holes of a thin nickel screen. Every hole in a piece of screen containing 25,000 holes could be filled. As development proceeded, the cells glued themselves tightly into the holes, so the two sides of the screen could be isolated and steep gradients could be maintained across the screen. The zygotes were then polarized by directed light so that all of the developing rhizoids were on one side and the thalli on the other. In that way, Ca2+fluxes could be measured in the two parts of cells during the photopolarization process. The first measurements were taken at 6 h after fertilization, about 2 h after the light exposure began. The influx of Ca2+on the future rhizoidal sides was five times as large as the Ca2+influx on the future thallus sides. This difference in influx steadily declined as the time of germination approached, when there was no significant difference in influx. Unexpectedly, there was a marked difference in Ca2+efflux, with more Ca2+leaving the future thallus. This asymmetry also declined as germination approached. Another feature of these results was that the total influx and efflux did not change during the photopolarization process, suggesting the possibility that the number of open Ca2+channels and active pumps did not change but that they were redistributed in the plasma membrane. It was also interesting that the asymmetry in influx disappeared by the time of germination. The conclusion drawn from the nickel screen experiments was that photopolarization of Pelvefia zygotes involves the formation of an intracellular gradient of

4. Symmetry Breaking in the Zygotes of the Fucoid Algae

109 Ca*+,with elevated Ca2+at the future rhizoidal site. As a direct test of this notion, cells were then grown in the dark near a localized source of calcium ionophore so that the cells were exposed to a gradient of ionophore during development. The zygotes showed a strong tendency to form their rhizoids on the sides exposed to the higher concentration of ionophore (Robinson and Cone, 1980). While supportive of the Ca2 gradient hypothesis, those experiments were hardly definitive as fucoid zygotes are known to respond directionally to gradients of a number of external factors, including temperature, pH, K +,nearby eggs, nearby adult algae, and flow (Jaffe, 1968). Kropf and Quatrano (1987) examined the requirement for extracellular Ca2+ during photopolarization. They exposed F. distichus zygotes to unidirectional light from 8 h after fertilization to 15 h after fertilization in varying concentrations of external Ca2+.Only small effects on photopolarization were reported. About 80% of the cells formed their rhizoids on the sides away from the light source in seawater with a pCa of 10. In the usual way of calculating polarization (% rhizoids growing away from light source - % growing toward light source (Nelson and Jaffe, 1973)), this represented a polarization of about 60%, whereas cells grown in normal Ca2+seawater (pCa 2) were fully polarized. One shortcoming of this study was that about 70% of the cells failed to germinate following the prolonged low-Ca2+treatment, so the conclusions were based on the surviving 30%. In later experiments with P.fustigiutu, stronger effects of reduced extracellular Ca2+on photopolarization were seen (Robinson, 1996a). In that study, zygotes were given shorter orienting light pulses with correspondingly shorter exposure to Ca2+-freeseawater, so that cell survival was high. Exposure to directed light in Cazt-free seawater (an artificial seawater containing 4 mM EGTA and no added Ca2+)for 90 or 150 min abolished photopolarization, whereas controls exposed in normal Ca2+seawater were 61 and 78% polarized, respectively. If the cells were pretreated with Ca2+-freeseawater for an hour before light exposure to remove cell wall calcium, a remarkable negative photopolarization of about 25% was observed; that is, the rhizoids tended to form toward the light source. It was also shown that photopolarization was markedly increased if the zygotes were exposed to light in seawater containing only 10% of the normal Caz+ of 10 mM. It is known from earlier experiments that the net influx of Ca2+is increased in 1 mM Ca2’ seawater as a result of greatly increased Ca2+ permeability (Robinson, 1977), which may explain this apparently paradoxical result. In any case, these results showed that external CaZ+is required for photopolarization of Pelvetiu zygotes during brief polarizing light pulses. This is not in conflict with the earlier results of Kropf and Quatrano (1987), who used much longer light pulses and obtained substantial photopolarization in the absence of extracellular Ca2+.The longer light treatment appears to impose a long-lasting asymmetry that is not directly dependent on Gal+ entry. We suggest that Ca2+entry during the photopolarization process is part of a positive-feedback loop that considerably enhances the sensitivity of the system but can be dispensed with if the polarizing signal is +

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sufficiently strong. In this view, changes in Ca2+influx are not the immediate consequence of nonuniform illumination, but a later result of localized light stimulation. This suggestion may explain the recently reported failure of Ca* free seawater to inhibit photopolarization of F: serratus zygotes (Love et ul., 1997). They used a 3-h light pulse of 3.6-fold more intense light than Robinson (1996a), so the total quantum dose was four- to eightfold greater. Alternatively, it may be that there are real differences between Fucus and Pelvetia in this regard. In any case, the issue of the requirement for external Ca2+during photopolarization remains controversial. Despite considerable effort, attempts to detect the putative Ca2+ gradients during the photopolarization process have met with little success. Berger and Brownlee (1993) used ratiometric confocal imaging of Calcium Green/SNARF in an attempt to visualize the Ca*+distribution during photopolarization of F. serrutus. In only one case were they able to get an injected zygote to develop on a normal schedule. However, it did exhibit higher Ca2+at the future rhizoidal site. In other cases, germination was delayed by up to 40 h. One difficulty with fluorescent imaging of fucoid zygotes is their intense autofluorescence, especially when excited in the blue and UV wavelengths. To overcome autofluorescence, it is necessary to achieve a relatively large cytosolic concentration of the indicator, compared to the amount required for imaging pollen tubes, for example. The Ca2+-sensitivedyes can act as Ca2+buffers, and it is known that buffers at a high enough concentration and an appropriate binding constant will inhibit germination (Speksnijder et al., 1989). We have directly observed this effect in this laboratory. Another problem is that the photopolarization process itself is highly sensitive to blue and UV light, so repetitive imaging can disturb the process being studied. Our solution has been to use the longest excitation wavelength indicator currently available, Calcium Crimson (CC), in conjunction with confocal microscopy. We find that there is a window of cytoplasmic concentration of CC that gives an adequate signal above autofluorescence but does not interfere with the normal course of development. Furthermore, the polarity of the cells is not affected by the 568-nm exciting light. Unfortunately, we have not found a suitable Ca2+-insensitivedye that can be coinjected and simultaneously imaged with CC and thus used for true ratiometric imaging. As a Ca2'-insensitive marker of dye distribution, we inject other zygotes with Rhodamine B (RB); these are not injected with CC. We then average the CC fluorescence and the RB fluorescence over several cells during photopolarization and calculate a ratio of the two averages. We have validated this method by also imaging the known Ca*+ gradient in the tip of the growing rhizoid. The method has revealed the formation of a Ca*+gradient within an hour of exposure to unilateral blue light, well before a rhizoid is evident. The Ca? + is highest at the site of future germination. In these experiments, only cells that germinated at the normal time were included in the analysis. The Ca2+gradient is largest 2-3 h after exposure to the polarizing light was begun and actually declines after that. By the time of germination, no Ca*+ + -

4. Symmetry Breaking in the Zygotes of the Fucoid Algae

111 gradients can be detected (unpublished work of R.P. and K.R.R.). This somewhat awkward method of constructing ratiometric images of the Ca2+distribution works because the Pelvetiu cells are quite perfectly spherical and individuals of uniform size can be selected. As noted earlier, one interpretation of the nickel screen results was that the localized entry of Ca Z’into Pelvetia zygotes is regulated not by the coordinated and localized opening and closing of Ca2+channels but by the redistribution of those channels. Evidence apparently supportive of this idea has been published (Shaw and Quatrano, 1996). They used a fluorescent derivative of dihydropyridine (FL-DHP) (Knaus et ul., 1992) to label putative Ca2+channels on the surface of F. distichus zygotes and to follow the redistribution of the label during photopolarization. They found that the fluorescent label was uniformly distributed initially but became asymmetrical in response to unilateral light. The fluorescence accumulated on the shaded side prior to germination and the asymmetric fluorescence could be relocalized to a different region by changing the direction of the light. Their interpretation of those results was that the FL-DHP labeled calcium channels in the plasma membrane and tracked the rearrangement of the channels during photopolarization. There are two aspects of this work that undermine acceptance of this conclusion. First, there is no evidence that fucoid zygotes possess DHP receptors. Germination of the zygotes is quite sensitive to the entry of Ca2+; they will not germinate in Ca’+-free seawater (Kropf and Quatrano, 1987). Ca2+ gradients and germination are also affected by a variety of Ca2+channel blockers, including lanthanides, verapamil, methoxyveraparnil, and diltiazem (Robinson, 1996a; Brownlee and Pulsford, 1988). However, germination and growth of fucoid zygotes are completely insensitive to high concentrations of either FL-DHP or another dihydropyridine, nifedipine (Robinson, 1996a; Shaw and Quatrano, 1996) and nifedipine does not affect tip-focused Ca2+gradientsin the Fucus rhizoid (Brownlee and Pulsford, 1988). Examples of Pelvetiu zygotes grown in 10 pM nifedipine are shown in Fig. I . Love et ul. (1997) report that nifedipine does inhibit photopolarization in F. serratus, but they attribute the effect to an inhibition of release of Ca’’ from intracellular stores, not to the blockages of plasma membrane channels. There is no evidence to support the suggestion that fucoid zygotes’ plasma membranes possess the L-type Ca2+channels that are the target of dihydropyridines. The second objection to the conclusion of Shaw and Quatrano (1996) concerns the actual images that they show. It is well understood that fluorescent labeling of the surface of a cell results in a bright ring of fluorescence when median sections of the labeled cells are viewed. This phenomenon is especially pronounced in confocal microscopy, but it has long been apparent in ordinary fluorescence microscopy (Albertini rt ul., 1977). Examples of this phenomenon in Fucus can be found in the study by Catt et uf. (1983) of the distribution of lectin receptors on eggs of F. serrutus. This is in marked contrast to the images of Shaw and Quatrano (1996). For example, their Fig. 4 shows 12 time-lapse images of four

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FL-DHP-labeled cells during the photopolarization process. No hint of peripheral brightness is seen in any of the cells in any of the images. The cells are uniformly bright across the entire plane in the early images before the label begins to accumulate on one side; likewise in Fig. la. This distribution is characteristic of a ligand that has labeled cytoplasmic, not plasma membrane, components. The localization of the FL-DHP label that is shown (e.g., their Fig. 3) is punctate, suggesting that the label is compartmentalized into vesicles in the cell and then transported to the germination site. While interesting, this result tells us little about plasma membrane Ca2+channels and their mobility. In summary, the role of Ca2+and Ca2+gradients in the photopolarization process remains obscure. While there is general agreement that Ca2+is somehow involved, there is little agreement about the source of the Ca2+(internal or external) or the spatial arrangement of putative Ca2+ gradients. Definitive answers await appropriate imaging of the Ca2+distribution during photopolarization. The recent use of CC as a probe for Ca2+in these cells offers hope that this thorny question may soon be resolved.

B. Calmodulin Given the apparent involvement of Caz+at some level in these processes, it has been of interest to know what the target of Ca2+gradients might be. Brawley and Roberts (1989) demonstrated that Pelveria and Fucus zygotes contained both calmodulin and calmodulin-binding proteins, as well as other calcium-binding proteins. We have recently confirmed this result for Pelvetiu zygotes (R.P. and K.R.R., unpublished). The natural-product calmoddin inhibitor ophiobolin A (Leung er ul., 1984, 1985, 1988) is a potent inhibitor of germination of Pelvetiu zygotes (Robinson, 1996a). At a concentration of 300 nM, this fungal toxin completely blocked germination, whereas its noninhibitory analog 3-anhydroophiobolin A had no effect on germination or growth at 10 pM. Surprisingly, when 300 nM of ophiobolin A was present during a brief photopolarizing light pulse, the sensitivity of the cells to light was increased. A 45-min light pulse was three times as effective in polarizing a population of zygotes in the presence of ophiobolin A as in the inhibitor's absence. This suggests the possibility that calmoddin acts somewhat distally to the light stimulation and may be a later effector of Ca2+gradients. The fact that the cells germinate in the dark indicates that they have some polarity that is independent of light (discussed earlier) and that faint polarity may exist early in development. In this view, a brief light pulse would have to compete with this randomly oriented polarity. Ophiobolin A may erase this competing polarity without interfering with the formation of a lightinduced axis. Another, less specific, calmodulin inhibitor, W-7, has also been tested for its effects on germination and polarity. In one case, it was found to inhibit germination of Pelveriu zygotes at concentrations of 5-10 pM but to be toxic to the cells

113 at higher concentrations (Robinson, 1996a). If present at 7 pM during a polarizing light pulse, W-7 had no effect on polarization. Higher concentrations of W-7 (50 pM and above) killed felvetia zygotes in a 90-min exposure. In contrast, Love et a f . (1 997) reported that 100 pM W-7 did not harm E serratus zygotes when present during a 3-h light treatment, but did abolish photopolarization. Apparently, felvetia and Fucus zygotes differ greatly in their sensitivity to W-7. Love et af. (1997) injected recombinant calmodulin into E serratus zygotes after the cells were exposed to unilateral light and reported that it enhanced photopolarization. They also reported that fluorescently labeled calmodulin localized to the presumptive germination site. We have microinjected antibodies to both recombinant maize calmodulin and Dicryostelium calmodulin into felvetia zygotes (R.P. and K.R.R., unpublished). We find that the antibodies block germination when injected alone but are not inhibitory when coinjected with excess authentic calmodulin. The minimum inhibitory concentration of the Dictyostelium antibodies in the zygotes was 0.44 pM. Unfortunately, this treatment is not reversible so it has not been possible to test the effects of the antibodies on photopolarization. The more difficult experiment of first injecting antibodies, then pulsing the cells with unidirectional light, and finally injecting calmodulin to (perhaps) allow germination has not yet been done. We have used these same antibodies to identify calmodulin in felvetia zygotes by immunoblotting. In summary, it is generally agreed that fucoid zygotes require active calmodulin for germination, but the role of calmodulin in the formation of the light-induced axis is unclear. What might be the target of active calmodulin in fucoid zygotes? We have one clue from our recent work. We find that felvetia zygotes are quite sensitive to KN-93, which is an inhibitor of the multifunctional Ca*+/calmodulin-dependent kinase, CaM kinase 11. The drug completely inhibits germination at a concentration of 10 pM (Fig. 1) and effects on germination and growth can be detected at concentrations as low as 1-5 pM. 4. Symmetry Breaking in the Zygotes of the Fucoid Algae

V. Cortical pH Gradients during Axis Formation and Rhizoidal Growth Kropf et af. (1 995) have reported that there is small but definite pH gradient formation during the photopolarization of felvetia zygotes. The magnitude of the difference between the cortex on the side away from a source of unilateral light and the opposite pole was 0.02-0.04 pH units during axis formation and jumped to about 0.07 pH units after germination. This is consistent with an older report that dark-grown zygotes respond to an imposed external pH gradient by germination on the low-pH side (Whitaker, 1938). One caveat, however, is that felvetia zygotes regulate their cytosolic pH quite rigorously and changing external pH between 6.2 and 9.2 results in intracellular pH changes of less than 0.2 pH units (Gibbon and Kropf, 1993). Thus, Whitaker's finding may not have been due to an intracellular pH gradient resulting from the applied extracellular gradient. If

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cytoplasmic pH gradients were capable of directing the rhiziod-thallus axis, one would expect strong red light to polarize the cells due to pH gradients produced by photosynthetic gradients. However, if Pelveria zygotes were half-illuminated by red light, no effect on the direction of the axis was detected (Robinson and Cone, 1980). A pH gradient in the same direction was reported in the growing rhizoidal tip; that is, the rhizoid tip was relatively acidic compared to the base of the rhizoidal cell (Gibbon and Kropf, 1994). Both photopolarization and germination were inhibited if the cytosolic pH was clamped outside a permissive range. The existence and requirement for a pH gradient, with the growing tip being acidic, may be unique to the Pelvetia zygote. Parton et al. (1997) were unable to detect pH gradients in growing fungal hyphae, pollen tubes, and fern rhizoids. In view of these more recent negative findings, it will be important to confirm the existence and requirement for pH gradients in fucoid development.

VI. Actin Microfilamentsand Photopolarization The one area of broad agreement among Fucus researchers is that actin microfilaments are involved in photopolarization-or so we thought until recently. In a review specifically focused on the role of the cytoskeleton, Kropf (1 994) stated “All stages of cell polarization, except mitosis, are inhibited by cytochalasins and are therefore dependent on F-actin.” The basis for this view began with back-toback papers published in 1973 from two laboratories (Nelson and Jaffe, 1973; Quatrano, 1973). Nelson and Jaffe (1973) determined the effects of cytochalasin B (CB) on both photopolarization and germination of Pelvetia zygotes. Germination was completely inhibited by 7 pM CB, but photopolarization was considerably less sensitive, requiring about 100 pM for half-inhibition of the percent polarization in response to light pulses of various durations. Interestingly, an axis, once formed, could not be erased. If the cells were exposed to light for 1, 2, or 3 h, and then treated for 2 h with 200 pM CB, the percent photopolarization (which ranged from 48 to 92%) was identical to that of controls that were not exposed to the drug. Quatrano (1973) reported very similar results for Fucus disfichus zygotes. He found that 100-200 pM CB reduced the percentage of cells responding to a 2-h light pulse from 97 to 52%. He also reported that a lightinduced axis could not be obliterated by CB. The first localization studies of F-actin in fucoid zygotes, using a fluorescent derivative of phalloidin, NBD-phallacidin, indicated that an asymmetric distribution of F-actin could not be detected until shortly before germination (Brawley and Robinson, 1985).That study was hampered by the fact that the emission spectrum of the NBD fluorophore overlapped significantly with the most intense autofluorescence of the cells, so careful quantitation of F-actin distribution was not possible. Brawley and Robinson (1985) also found that cytochalasin D (CD) greatly reduced the inward ionic current at the growing rhizoid and eliminated the

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pulsatile component of the current. CD also prevented the development of the inward current that precedes germination. These results suggest that the actin cytoskeleton plays a role in the maintenance of the localized distribution of ion channels. In an important study, Kropf et al. ( 1 989) first demonstrated the presence of actin in fucoid cells by biochemical means. Two-dimensional electrophoresis showed that there are at least three isoforms of actin in E distichus. Using rhodamine-labeled phalloidin, they too found that asymmetric distribution of F-actin could not be detected during the photopolarization process, but was first seen shortly before germination. Interestingly, they observed that actin filaments were not depolymerized by cytochalasins. Whatever the mechanism of F-actin localization at the presumptive rhizoid, the stability of actin filaments may explain the failure of cytochalasins to abolish an axis induced by unilateral light. Kropf et ul. (1989) also found that asymmetries in F-actin could be detected in darkgrown cells that were polarized by the presence of another zygote nearby, so actin’s involvement is not limited to light-driven events. The localization of F-actin to the rhizoidal pole is not related to the distribution of actin mRNA. Bouget et al. (1996) have mapped the movement of actin mRNA and found that it is initially uniformly distributed but localizes to the thallus pole shortly before germination where it remains until the time of first cell division. These movements of mRNA appear to be dependent on F-actin itself as they are inhibited by CB. The redistribution of actin mRNA before germination mirrors the movement of total mRNA (Bouget et al., 1995). A contrary view of the role of F-actin in photopolarization has been presented by Love et al. ( 1 997). They report that three drugs that inhibit F-actin polymerization, CB, CD, and Latrunculin B, have no effect on photopolarization of E serratus zygotes. The concentrations of cytochalasins that they used overlapped with those used by Nelson and Jaffe (1973) and Quatrano (1973). In view of the differing results, we have begun to reinvestigate this matter. We have found that CD is an effective inhibitor of photopolarization by a 90-min light pulse that polarizes control Pelvetiu zygotes by 60-65%. The CD-treated cells were polarized to half the extent of controls at a concentration of 20 pM,indicating that CD is considerably more effective in this regard than CB (Fig. 3). We have not yet tested Latrunculin B in this assay, but we expect that it will be effective at yet lower concentrations. Given the disagreement between Love et al. (1 997), Quatrano (1973), Nelson and Jaffe (1973), and our present results, it will be important to resolve this issue. It seems most unlikely that E sert-atus is different from both E distichus and P. ,fu.stigiutu in this fundamental matter.

VII. Axis Fixation A widely accepted feature of the process of polarity formation and expression in fucoid zygotes is the notion that overt germination is preceded by the rigid

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Fig. 3 Effect of cytochalasin D on photopolarization of Pelveria zygotes. The zygotes were grown in the dark until 6 h after fertilization and then exposed to a 90-min light pulse (50 pmol photonslmY s) in the presence of various concentrations of cytochalasin D. In each experiment, 200 cells in each of two dishes were scored by determining the locus of the rhizoid with respect to the direction of the light. The % polarization (% rhizoids arising on side away from light - % rhizoids arising on side toward light) was calculated for each dish. The relative polarization was calculated by dividing the % polarization of the treated cells by the % polarization of controls. The cytochalasin was dissolved in DMSO,which was found to have no effect by itself on polarization. The controls were polarized by 60-65% by this light treatment. The points shown in the graph are the averages of three experiments and the standard errors are indicated. Cytochalasin D was half-inhibitory at 20 pM.This concentration is 5- to 10-fold lower than the half-inhibitory concentration reported previously for cytochalasin B (see text).

fixation of an axis induced by light or other means. A clear statement of this idea was framed by Goodner and Quatrano (1993): “ . . .just before rhizoid emergence, unidirectional light cannot reverse a previously established axis-i.e., the polar axis is fixed.” The experimental basis for “axis fixation” as an independent step that precedes germination and can occur in the absence of germination rests on a single paper (Quatrano, 1973). It was reported that if germination of Fucus zygotes was delayed, by either elevated external osmolarity or protein synthesis inhibition, beyond the time when the zygotes normally germinate, a previously imposed axis could not be redirected by a second light from a different direction. The second light treatment was begun as soon as the inhibitor was washed out. Thus, it appeared that the axis formed in response to the first light became fixed in place independently of overt germination. Jaffe ( 1990) showed that if Pelvetia zygotes are cultured in the dark in hyperosmotic seawater, the cells remain photopolarizable for as long as 5 days; that is, they did not “fix” an axis during that period. Furthermore, he found that lightpolarized cells could have their axes reversed by a second light in the opposite direction after germination if the cells were exposed to the second light in the

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presence of slightly hypertonic medium. The original rhizoidal outgrowth aborted and the new rhizoid was formed on the opposite side. He concluded that the ‘commitment’ to outgrowth localization can be so easily delayed that it cannot be usefully thought of as being controlled by an internal clock.” The idea of rigid specification of polarity in the zygote by an internal clock that runs independently of germination is central to the notion of axis fixation as defined by Quatrano (1973). Subsequently, it was shown that if zygotes were exposed continuously to unilateral light in the presence of an osmotic block to germination for hours after unblocked cells had germinated, the axis could be easily and completely reversed by reversing the direction of light 2 h before removing the osmotic block (Robinson, 1996a). Apparently, no irreversible fixation of the axis occurs on any schedule related to the time of fertilization or axis formation. It was also found that zygotes grown in the dark in the presence of an osmotic block to germination were fully responsive to unilateral light 1 day after fertilization. It is our conclusion from the foregoing experiments that the developmental axis of the zygote remains plastic for many hours or days, if germination and normal development are impeded. At some point after germination, however, simple reversal of the light direction is not capable of reversing the rhizoid-thallus axis, although that point with respect to other developmental events such as degree of rhizoid extension and cell division is not known. Interactions with the cell wall seem to be important in maintaining the differentiated state. In an elegant series of experiments, Berger ef al. (1994) used laser microsurgery to selectively destroy rhizoid or thallus cells of early embryos. The descendent of the remaining cells generally maintained their differentiated state, but if a rhizoidal process came in contact with the cell wall of an ablated thallus cell, it ceased tip growth and began to develop as a thallus. Thus, at some time after germination, information is embedded in the wall in a stable, nondiffusible form that can specify fate and is normally involved in maintaining polarity. These experiments emphasize the plastic nature of the polar axis in the fucoid algae. Polarity arises in a graded way involving a number of steps, each acting through a spatial gradient. No rigid, irreversible specification of the axis occurs until quite late in development, well after germination. “

VIII. The Signal Transduction Process for Photopolarization As discussed earlier, a relatively brief exposure to unilateral blue light is sufficient to polarize fucoid zygotes. Experiments with plane-polarized light show that photoreceptor molecules are in or immediately adjacent to the plasma membrane. Somehow, activation of the receptors on one side eventually leads to germination on the opposite side. Calcium appears to be involved in this process, but not immediately. We have recently tested a number of signal transduction molecules for

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their possible involvement in the phototransduction process. Our idea was that whatever the photoreceptors were coupled to, the signaling molecules must appear as gradients in the cytoplasm. Either inhibition of the pathway or flooding the cell with the signaling molecule in order to swamp light-generated gradients should interfere with photopolarization. From this initial screen, only one reagent inhibited photopolarization, the cell-permeant, dibutyryl form of cyclic GMP (cGMP). This result encouraged us to measure cGMP changes during the photopolarization process. Using radioimmunoassay (RIA), we found that Pelvetia zygotes at 6 h after fertilization contained about 0. I pM cGMP but no detectable levels of CAMP (Robinson and Miller, 1997). In response to blue, but not red, light the cGMP levels rose to about twice those of dark-grown controls in about 90 min. The guanylyl cyclase inhibitor LY 83583 did not affect basal levels of cGMP but abolished the light-induced increase. LY 83583 also completely eliminated photopolarization at a concentration of 10 pM when present during a 90-min light pulse. If the zygotes were exposed to gradients of dibutyryl cGMP in the dark, using the nickel screen method described earlier, it was found that the cells showed a modest tendency to form their rhizoids on the sides exposed to lower concentrations. These results suggest that changes in cGMP are closely associated with polarizing light stimulation and that gradients of cGMP may be part of a cytosolic signal that leads to further epigenesis of polarity. There is evidence from other studies that cGMP is important in higher plant light responses. Chua and his collaborators have used the aurea mutant of tomato, which lacks functional phytochrome, to dissect the signal transduction pathway in phytochrome signaling. The hypocotyl cells of the mutant seedling do not develop chloroplasts or synthesize anthocyanin when exposed to light. Their strategy was to inject various signal transduction components into the mutant cells and assay for the normal phytochrome responses. They found that G-protein activation led to both chloroplast development and pigment synthesis and that calcium and calmodulin could induce only immature chloroplasts (Neuhaus et al., 1993). The injection of nonhydrolyzable cGMP analogs caused the biosynthesis of anthocyanins, and if both the Ca2+/ calrnodulin and cGMP pathways were activated, mature chloroplasts developed as well (Bower et al., 1994). However, they have not yet reported actual changes in cytosolic cGMP concentration in response to phytochrome activation in the tomato system.

IX. An Opsin-like Photoreceptor in Pelvefia? The finding that cGMP increases in response to polarizing light and that the increase is necessary for the development of a light-directed axis in Pelvetia zygotes draws attention to the parallel with the vertebrate visual system. The photoreceptor in vertebrate vision is a seven-pass transmembrane receptor, rhodopsin.

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Rhodopsin’s chromophore is I 1-cis-retinal, which is attached to the apoprotein opsin by forming a Schiffs’ base. Upon absorption of a photon, the retinal is isomerized to the all-trans form and the rhodopsin molecule is activated. Activated rhodopsin then activates a heterotrimeric G-protein, transducin, which in turn activates a cGMP-specific phosphodiesterase. The resultant drop in photoreceptor cGMP causes plasma membrane Na channels to close and a hyperpolarization of the membrane potential. In view of the involvement of cGMP in Pefvetia photopolarization, we have begun to consider the possibility that a rhodopsin-like photoreceptor molecule is involved in light sensing. As a first step, we have found that considerable amounts of retinal can be extracted from Pefvetia zygotes, as determined by mass spectroscopy (Robinson et al., 1998). The levels of retinal were quite high, about 4 X 10’ molecules per cell. Retinal is found only in association with opsin-like proteins, so the implication is that Pelvetia zygotes have substantial amounts of an opsinlike photoreceptor molecule in their plasma membranes. If so, we would anticipate that the opsin is coupled to a G-protein that regulates a guanylyl cyclase. The functional presence of opsin-like proteins is well established in green algae. Phototaxis by Chfumydomonas is dependent on retinal (Foster et af., 1984). Recently, a retinal-binding protein, chlamyrhodopsin, has been isolated and sequenced, and it turns out not to be a typical seven-pass transmembrane receptor (Deininger et af., 1995); instead it has only four putative transmembrane domains. Deininger et af. (1995) suggest that chlamyrhodopsin may be a light-regulated ion channel. Nevertheless, it appears that light modulates heterotrimeric G-proteins in the green algae (Calenberg et al., 1998); thus the signal transduction pathway for chlamyrhodopsin may be similar to that for other opsins. Retinal has also been isolated from Paramecium (Tokioka et af., 1991) and tomato (Lorenzi et ul., 1994), so rhodopsin-like photoreceptors may be widespread in nonvisual light perception. In vertebrates, an opsin named melanopsin has been identified in Xenopus melanocytes, and mRNA for this protein was found in the brain, iris, and the inner nuclear layer of the retina as well (Provencio et af., 1998). It appears that opsins play important roles in nonvisual light responses in animals. It will be exciting to see if fucoid zygotes possess a rhodopsin-like photoreceptor, and if so, how that protein is coupled to the photopolarization response and how it is related to other nonvisual rhodopsins. +

X. A Speculative Model for Photopolarization While key components of the photopolarization process are not known, and others are controversial, it is nevertheless useful to attempt to construct a model in order to identify testable elements and stimulate experiments. The aim of the model is to suggest a mechanism whereby a light gradient can eventually cause germination on the shaded, darker side (but see the earlier discussion concerning the contro-

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versy about even this point). To make the model, we necessarily must make assumption to fill in gaps, but those aspects of the model will be clearly identified. Certain features of this model were presented in an earlier publication (Robinson and Miller, 1997). A diagrammatic view of the model is shown in Fig. 4. The first step in the polarization process must be the graded activation of plasma membrane photoreceptors. While the nature of the photoreceptors is not known and need not be specified for the model, we assume that they are rhodopsin-like proteins. The end point is assumed to be the creation of an oppositely directed Ca2+gradient. As discussed earlier, there is some evidence to suggest that C a Z + channels are not coordinately opened and closed at the respective poles of the zygote but rather are redistributed during the photopolarization process. This may occur by a diffusion-trapping mechanism. Specifically, we assume that CaZ+ channels are immobilized by links to the cytoskeleton and that an increased level of cGMP somehow severs those links, perhaps by activating a kinase or a phosphatase. The CaZ+channels in the membrane adjacent to the cytoplasmcontaining light-elevated levels of cGMP would then be free to diffuse and would move toward the other side of the zygote where cGMP was low. As cGMP is a small molecule that can diffuse readily through the cytoplasm, we assume that a significant gradient of cGMP can be maintained only by the continuous increased production on the illuminated side and an active phosphodiesterase uniformly distributed in the cytoplasm. Once the CaZ+channels reached the region where cGMP was low, the immobilizing links to the cytoskeleton would be reestablished, resulting in the accumulation of Ca2+channels on the shaded side. This would lead to the beginning of a cytosolic Ca2+gradient. It is critical to identify possible positive-feedback elements, as such feedback is necessary to explain the sensitivity of the system to weak external gradients and to assure that germination occurs at only one site. There is evidence from studies of the vertebrate photoreceptor that increased Ca'+ both inhibits guanylyl cyclase and activates phosphodiesterase (Koutalos and Yau, 1996). If this were true in the fucoid zygote, the appearance of elevated Ca2+on one side and reduced Ca2+on the other would lead to greater mobility of Ca2+channels on the low-Ca2+side and less mobility

Fig. 4 A speculative model for light signal transduction in the polarization of fucoid zygotes. (A) A section of plasma membrane is depicted in the dark state. C a 2 +channels and photoreceptor proteins are uniformly distributed and the channels are anchored by cytoskeletal elements. The unstimulated photoreceptors are shown in yellow. Inactive guanylyl cyclase is shown in green; these molecules may be soluble or membrane associated. (B) In response to unilateral light, the photoreceptors are activated (red) and then activate, via unknown mechanisms, the guanylyl cyclase molecule (now shown in red also). This leads to an increase in cGMP on the illuminated side (blue shading) and the dissolution of the links between the cytoskeleton and the Ca2+channels. (C) The Ca2+channels are now free to diffuse away from the illuminated region. (D)Ca2+channels are trapped on the shaded side. where cGMP is low, and a Ca?' gradient begins to form (red shading). This continues in the absence of a continuing light signal.

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121 on the high-Ca?' side, so diffusion-trapping of Ca2+channels would be enhanced. One strength of this model is that it explains the relatively slow appearance of a Ca2+gradient across the cell and the experimentally observed conservation of calcium influx. Diffusion of channels through 25% of the circumference of a zygote will require an hour or so, depending on the diffusion coefficient of the unconstrained channel. This is also consistent with the average time required to polarize a population of cells with unilateral light. A weakness of the model is its inability to explain the relatively slow increase of cGMP in response to light. While some change is detectable within 10 min of illumination, the increase continues for about 2 h. This continued increase might be attributed to the positivefeedback loop postulated earlier, but a sharper initial increase in cGMP in response to light would be expected if that is the primary effect of illumination.

XI. Summary Despite its many advantages as an experimental system for the study of the epigenesis of polarity, it is obvious that the fucoid zygote also presents many problems. The development of polarity proceeds largely independently of direct gene action and thus may be considered a problem in cellular physiology. Ca2+appears to play an important role in the process, but the optical properties of the zygotes (opacity and autofluorescence)hamper the use of modern methods of visualizing the distribution of Ca2+and other ions. Likewise, other approaches, such as injection of fluorescent-labeled G-actin, in order to study the dynamics of actin filaments, are subject to the same limitations. It may be that the application of two-photon microscopy will enable experimenters to avoid some of these problems. This technique uses excitation wavelengths that are twice the wavelength of maximum absorption by fluorophores, and sufficient photon density for absorption is achieved only in a thin section. The fucoid zygotes are considerably more transparent to longer wavelengths, so attenuation of the exciting light and autofluorescence should be significantly reduced. Perhaps we will then be able to see further into these opaque cells. Another problem concerns the use of different species and genera. This may be unavoidable; for example, those of us who are land-locked tend to rely on Pelvetin, as it travels and stores better than the various species of Fucus and is less seasonal. Our colleagues fortunate enough to work near the ocean prefer to use the species that are locally available. Nevertheless, it is important to be careful about cross-genus and cross-species generalizations. While it is unlikely, based on what we know, that there are fundamental differences in physiological mechanisms among species, there may be small but still important differences in details. Obviously, investigators should directly compare results in more than one species whenever possible. The area of greatest disagreement, perhaps, concerns the mechanism of polarity

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formation, as opposed to its overt manifestation, germination. Are Ca2+and actin involved or not? Assuming Ca2+is involved, is the source internal or external? One basis for the different findings may be the differences in the strength of the polarizing signal provided to the zygotes. Clearly, the cells have powerful mechanisms for amplifying a faint asymmetry and developing an axis in response to an external signal. Furthermore, the fucoids generally develop in the intertidal zone and thus must be adapted to meeting the challenge of a widely varying external environment. They may have alternate mechanisms for responding to unilateral light. We have adopted the approach of presenting the cells with a fairly weak light signal-the minimum required to induce a considerable degree of organization of a population of zygotes. We then determine the effects of various inhibitors on photopolarization. One advantage of this approach is that it has allowed us to find treatments that increuse the sensitivity of the zygotes to light, something that would not be possible if the untreated controls were fully polarized. Some of the differences between our results and those of others may be related to their use of a stronger light stimulus. It may be that if given a strong stimulus, a sufficient trace is left in the cells so that they can organize an axis when an inhibitor is removed. Careful consideration of this point may help to reconcile apparently contradictory findings. Despite these difficulties, the fucoid zygotes are likely to continue to be an important experimental system. Technology, including the development of more specific inhibitory reagents, may allow some of the shortcomings of the system to be overcome, and careful consideration of experimental conditions may resolve some of the points of disagreement.

References Albertini, D. F. , Berlin, R. D., and Oliver, J. M. (1977). The mechanism of concanavalin A cap formation in leukocytes. J. Cell Sci. 26,57-75. Berger, F., and Brownlee, C. (1993).Ratio confocal imaging of free cytoplasmic calcium gradients in polarising and polarised Fucus zygotes. Zygore 1,9-15. Berger, F., and Brownlee, C. (1994).Photopolarization of the Fucus sp. zygote by blue light involves a plasma membrane redox chain. Plunr Physiol. 105, 519-527. Berger, F., Taylor, A., and Brownlee. C. (1994).Cell fate determination by the cell wall in early Fucus development. Science 263, 1421-1423. Bouget, F.-Y., Gerttula, S., and Quatrano. R. S. (1995).Spatial redistribution of poly(A)+ RNA during polarization of the Fucus zygote is dependent upon microfilanients. Dev. Bid. 171,258-261, Bouget, E-Y., Gerttula, S., Shaw, S. L., and Quatrano, R. S. (1996).Localization of actin mRNA during the establishment of cell polarity and early cell divisions in Fucus embryos. P l m t Cell 8, 189-201. Bowler, C., Neuhaus, G., Yamagata. H.,and Chua, N.-H. (1994).Cyclic GMP and calcium mediate phytochrome phototransduction. Cell 77,73-8 1, Brawley, S. H. (1987). A sodium-dependent, fast block to polyspermy occurs in eggs of fucoid algae.

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Reevaluating Concepts of Apical Dominance and the Control of Axillary Bud Outgrowth Carolyn A. Napoli Department of Plant Sciences University of Arizona Tucson, Arizona 85721

Christine Anne Beveridge Department of Botany The University of Queensland Brisbane, Queensland 4072, Australia

Kimberley Cathryn Snowden School of Biological Sciences University of Auckland Auckland, New Zealand

I. Introduction and Overview 11. Plant Architecture and Meristem Potential

A. Meristems and Shoots B. A Definition of Bud Outgrowth Potential C. Main Shoot Zonation and Axillary Bud Outgrowth: A Morphological View 111. Apical Dominance

IV. Use of Induced Mutations to Study Axillary Bud Outgrowth V. Molecular and Genetic Approaches for Understanding Bud Outgrowth A. Recent Gene Isolation B. Hormonal Modulation Using Transgenic Approaches C. Genetic Approaches for Studying Axillary Bud Outgrowth

VI. Conclusions and Perspectives References

A large amount of diversity of architectural form is found among flowering plants, and an important aspect of this diversity is the wide variation, ranging from simple to complex, found among branching patterns in plant shoot systems. Historically, the control of bud outgrowth has been attributed to the presence of a growing shoot apex. The term “apical dominance” is used to indicate that the shoot tip exerts an inhibitory control over proximal axillary buds. Through decapitation and/or hormone manipulation experiments, this inhibition has been attributed to the phytohormones auxin and cytokinin. Recent studies with mutants demonstrating increased branching indicate important additional roles for organs apart from those in the shoot tip and for signals other than cytokinin and auxin. This chapter provides a critical review of

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branching with an emphasis toward bud outgrowth in a developmental context. This review provides a detailed synopsis of physiological, genetic, and molecular studies and approaches for the investigation of branching regulation in plants. 0 1999 Academic Press

1. Introductionand Overview The shoot systems of angiosperms include a primary axis initiated during embryogenesis with the progressive addition of lateral shoots initiated after seed germination by the process of lateral branching. Lateral branches are derived by the growth and expansion of axillary buds that develop from new meristematic centers that originate in the axils of leaves (Steeves and Sussex, 1989).In this manner, each shoot is derived from the activity of a shoot apical meristem, and the development of a branched shoot system is dependent on the activity of these apical meristems. However, for plants demonstrating little or no branching, either an axillary meristem does not form or, more commonly, a meristem forms but growth and expansion arrests or slows down, resulting in a truncated and dormant, or very slow growing, axillary bud. Some plants show differential expression of branching, and particular nodes on a main shoot axis are more likely than others are to have a branch. In some instances, plants will lack branch development during one phase of growth, e.g., vegetative development, but branches will develop during the reproductive phase. This differential expression of branching can be considered in terms of the developmental potential of the axillary bud (Stafstrom, 1995). In this chapter, the term “developmental potential” is used to indicate there is an intrinsic ability or tendency of a bud to become suppressed or to continue to grow into a branch. As a prelude to considering the control of this outgrowth potential, it is important to consider the biological function of axillary buds. Axillary bud function can be considered as either a renewal of growth, in the case where a shoot apex is lost, or a supplement to existing apices (Stafstrom, 1995). For example, a renewal of growth occurs through innovation buds at the base of perennial plants after the plant dies back during winter dormancy (Weberling, 1989). Axillary buds also replace growing shoots after the apex is damaged or removed through disease, herbivore grazing, or pruning or when the shoot apical meristem differentiates into determinate organs as part of a normal developmental sequence (Stafstrom, 1995). Supplemental shoots are important for increasing the total number of leaves during vegetative growth or to display flowers during the reproductive growth phase. Plants have evolved to be receptive and responsive to environmental stimuli. The mature form of a plant is an expression of a number of genetically controlled traits, many of which have been constantly shaped by environmental stimuli or which require environmental cues, e.g., an appropriate photoperiod for floral induction. Hillman (1984) lists 14 different treatments, including shoot apex re-

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moval, which promote the growth of axillary buds; so clearly, axillary bud outgrowth, as a developmental potential, is influenced by environmental conditions. The term “apical dominance” arose from the well-known observation that removal of a shoot apex usually allowed the outgrowth of one or more dormant axillary buds. From experiments coupling shoot removal by decapitation with the addition of auxin and cytokinin, a model has been developed with these hormones as the signals controlling axillary bud outgrowth. We will explore these concepts further in this chapter, and one important objective is to examine critically the term “apical dominance.” Because the concept of apical dominance has evolved from decapitation studies, it is appropriate to ask how applicable is this concept of a dominant apex during growth and development of nondecapitated plants. Is the apical bud the main regulator of bud outgrowth in intact plants or do other plant parts have considerable importance? We discuss a number of approaches that enable the use of intact plants in the study of axillary bud outgrowth. These approaches include the use of mutant plants that have an altered branching phenotype and mutant or transgenic plants with altered levels of, or responses to, the plant hormones auxin and/or cytokinin. The relative merits and recent results from these approaches are discussed.

II. Plant Architecture and Meristem Potential Bell (1991) makes the case for the constructional organization of plants to be considered in terms of the potential, position, and time of activity of shoot apical meristems. Inherent in that statement is the idea that branch position or geometry alone is not sufficient to account for the enormous diversity of form exhibited among angiosperms. Other developmental programs relating to shoot architecture, e.g., onset or timing of shoot growth, differential growth rates of internodes, and the alternate forms of shoot construction (monopodial versus sympodial development), are equally important in establishing a particular architectural pattern (Bell, 1991). In this review, we are considering one aspect of plant form, whether or not an axillary bud will develop into a growing and expanding shoot and thereby contribute to the mature form of the plant. Our discussion is limited to herbaceous plants and many of the concepts discussed herein on dormancy and shoot growth are not necessarily applicable to trees.

A. Meristems and Shoots

A recent issue of “The Plant Cell” (Vol. 9, No. 7, 1997) provides a series of articles that are excellent sources of information on meristem and shoot development. Of particular interest to the subject matter in this chapter are discussions on

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shoot apical and axillary meristem development during vegetative development (Kerstetter and Hake, 1997), plant cell division (Jacobs, 1997), and organ formation during vegetative growth (Clark, 1997). Evans and Barton (1997) provide an additional review on meristem development. Consequently, we will concentrate on a few features of meristem and shoot development that are important aspects of axillary bud outgrowth. Vegetative shoots of flowering plants are constructed of reiterated modules consisting of an internode and a node with one or more leaves and associated axillary buds (Steeves and Sussex, 1989; Stafstrom, 1995). The cells of each module or phytomer are derived through the mitotic activity of an apical meristem, a small number of undifferentiated and actively dividing cells located at the apex of a shoot or stem. The shoot apical meristem is the ultimate source of all cells of the shoot, but it is by no means the direct source of all the cells comprising a shoot (Steeves and Sussex, 1989). After the initiation of leaf primordia, a considerable amount of cell division and enlargement takes place within the subapical region. This growth constitutes the expansion phase of development and serves to expand the individual primordia as well as the internode of each phytomer (Steeves and Sussex, 1989).Whereas ontogeny usually proceeds without the imposition of dormancy for the vegetative main shoot axis, this is not always the case for those shoots that arise as lateral shoots in axillary positions. This discussion focuses first on axillary meristem development during the vegetative growth phase and then contrasts axillary meristem ontogeny during the reproductive phase. Three points concerning axillary meristem and bud development are important. The first point relates to timing and concerns when an axillary meristem is initiated in comparison to the ontogeny of the subtending leaf. Axillary meristem development is first detected most frequently in the axil of the second or third leaf from the apex (Esau, 1977). By the time of axillary meristem initiation in the leaf axil, the leaf is well into the expansion phase of growth. This delayed timing leads to the second important point, which is the relative position, i.e., subapical or apical, of axillary meristem ontogeny in relation to leaf ontogeny. Leaf primordia arise directly from the morphogenetic region on the flank of the shoot apical meristem. In contrast, axillary meristem ontogeny begins outside the apical region in a subapical region. In this manner, axillary meristem development is associated more with a leaf than with the shoot apical meristem. The final point concerns the relative amount of growth and expansion that occur for an axillary bud. Many axillary buds on a vegetative shoot enter a state of dormancy after a small number of leaves are initiated and before significant growth and expansion has taken place in the individual components. The situation is very different after a plant gains reproductive competence. Newly formed axillary meristems demonstrate a marked precocity and acceleration (Lyndon, 1990). In this case, an axillary meristem appears within the same time interval (ptastochron)as the subtending leaf, and the new meristematiccenter arises directly in the morphogenetic region on the flank of the shoot apical meri-

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stem. Axillary buds developing during the reproductive phase of growth are not necessarily subjected to dormancy and growth is sustained until the mature form of the shoot is attained. As pointed out earlier, the action of shoot apical meristems in axillary positions on vegetative axes is often curtailed by the imposition of dormancy after a certain number of leaf primordia develop. The resulting structure is called a bud and the extent to which the bud grows reflects the degree to which leaves and internodes expand relative to their mature appearance. In this review, the term “bud” is used in a generic sense to indicate an axillary meristem and an undefined number of reiterated, but unexpanded, modules, whereas a branch indicates significant shoot expansion in terms of both leaves and internodes. If the development of the lateral branch proceeds to the point where a flower or inflorescence develops, the branch can be further described as a paraclade (Weberling, 1989).

B. A Definition of Bud Outgrowth Potential

Using the garden pea Pisum sutivum, Stafstrom and co-workers (Stafstrom and Sussex, 1992; Stafstrom et ul., 1993; reviewed in Stafstrom, 1993, 1995) have defined four different stages of bud development including dormancy and growth as well as two transitional states, dormancy to growth and growth to dormancy. Bud growth is initiated by decapitation and the outgrowth potentials of the larger, principal axillary bud and the smaller, accessory bud were studied. Using a probe to a pea ribosomal protein gene (rpL27),Stafstrom and Sussex (1992) have determined that this gene is induced in axillary buds within 1 h of apical decapitation. This is an extremely early indication of growth potential that occurs well in advance of visible growth detected 8 h after decapitation. Whereas the principal axillary bud continues to grow after decapitation, the accessory bud undergoes a growth-to-dormancy transition. The expression of rpL27 diminishes to basal levels in the smaller accessory bud but expression continues in the apical meristem of the growing, principal bud. These stages are presented diagrammatically in Fig. 1 and depict possible ways by which a bud achieves a particular developmental potential. The diagram also includes the formation of a bud from an axillary meristem. Genes that function to control axillary bud outgrowth may act at any of the stages depicted, starting from the control of axillary meristem initiation itself. This type of molecular study is important because it provides a basis for understanding the regulation bud outgrowth, in terms of both timing and events that occur between bud release and bud dormancy. These results indicate a rapid response to decapitation-induced bud release, minutes for the induction rather than the hours before bud growth and expansion becomes measurable. The transitional stages are important because they provide the means to explain the dynamic molecular and physiological events, both known and unknown, underlying the growth and dormancy stages of bud development.

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Sustained Growth

Sustained Dormancy Fig. 1 Developmental stages of bud outgrowth extrapolated from Stafstrom (1993).As described in the text, meristem initiation precedes and is required for formation of an axillary bud ( I ) . In most situations the bud then passes (2) to a transition stage (3 and 4), intermediate between dormancy ( 5 ) and sustained growth (6). Perhaps under some circumstances, newly formed buds may transit immediately to sustained growth (2'). As reviewed in Stafstrom (1993). to achieve sustained growth, a dormant bud is induced ( 5 ) to pass through a transition stage (3) and if not subsequently inhibited (4) will pass to a sustained growth stage (6). Buds may cycle many times in the transition phase before becoming fully dormant or undergoing sustained growth.

C. Main Shoot Zonation and Axillary Bud Outgrowth: A Morphological View

Different areas on a plant, named enrichment, inhibition, and innovation zones (Weberling, 1989), display differential expression of axillary bud potential. These morphological terms are used infrequently and thus may be unfamiliar terms. The enrichment zone is the region where the inflorescence develops and often this represents the most highly branched part of a plant. This is not surprising as the inflorescence shoot system displays the flowers, supports the developing fruits, and relates to the overall reproductive success of the plant. The inhibition zone occurs proximal to the enrichment zone. Axillary buds in this zone may show little or no outgrowth prior to the onset of flowering; however, paraclades often develop in a basipetal fashion into this zone as the inflorescence continues to develop acropetally. In perennial plants, an innovation zone lies proximal to the inhibition zone, at the basalmost position on the shoot axis, and represents a source of dormant buds that provide for a renewal of the complete branching system. Branching at this innovation zone is often favored in species such as Coleus and tomato (Cline, 1991) as well as petunia (Napoli, 1996). These zonal patterns have been

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described for whole-plant morphology and are not dependent on decapitation to induce branching. The morphological zones bear a resemblance to growth phase or maturity zones as reviewed by Poethig (1990). The innovation zone, in the basalmost position, represents a juvenile state. The inhibition zone lies in the region where the adult stage would occur and the enrichment zone is the reproductive phase. Other equivalent terminology commonly used in the literature is basal branching for innovation branching and aerial branching for enrichment branching. Section VC3b discusses the influence of photoperiod and photoperiod response flowering genes and their relationship to basal and aerial branching in Pisum sativum. As previously pointed out, a plant demonstrating no or limited axillary bud outgrowth during the vegetative phase can produce a highly branched inflorescence during the reproductive stage. A number of standard arrangements of branched inflorescences are recognized and described in detail in Weberling (1989). Although an appropriate photoperiod may be required in order to induce reproductive competence, the absolute arrangement of inflorescence branches and flowers cannot usually be altered by environmental control. This is probably a consequence of the importance of the underlying genetic program and the subordinate role of environment in the control of axillary bud potential during branch formation in inflorescences.Many of these branched inflorescencesdevelop in the presence of a growing shoot apex.

111. Apical Dominance Apical dominance is defined broadly as the inhibitory control of the shoot apex over the outgrowth of lateral buds (Bangerth, 1989; Cline, 1991, 1994; Tamas, 1995). A more precise definition includes the young leaves and growing tissues near the apex. Depending on the plant, young leaves and stems have been reported to demonstrate a greater influence on apical dominance than the shoot apex. In these cases, the effect of shoot apex removal is often only manifested after young leaves and internodes are no longer replaced (e.g., Hosokawa et al., 1990). The apices of plants have been classified as expressing different levels of apical dominance, according to the degree of axillary bud outgrowth. Typically, complete or strong apical dominance is used to imply that macroscopic buds do not form or that buds are arrested very early in development. This is in contrast with partial or weak apical dominance where a number of buds are released to form lateral shoots. In plants exhibiting weak apical dominance, such as Phaseofus vufgaris, axillary bud growth continues at a slow rate in the intact plant (Hillman, 1984). Specific examples depicting the usage of this terminology can be found in Cline (1996, 1997) and Tamas (1995). It is important to stress that studies on apical dominance often employ decapitation as the means to compare and contrast dormant versus growing axillary

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buds. In addition to the stress imposed by wounding, decapitation also induces a number of specific responses, e.g., xylem sap flow rate changes, sink strength alterations, and changes in hormone and assimilate transport. Furthermore, decapitation removes a region of hormone synthesis, including both auxin and gibberellins and perhaps other as yet unidentified signals. Thus, it is reasonable to ask if decapitation-induced axillary bud outgrowth and nondecapitation-induced outgrowth, i.e., basal branching in the presence of an intact apex, operate through the same programs. Quite likely, these programs overlap to some extent and the apical portion of the plant plays a major role. Approximately 60 years ago, the phytohormone auxin was shown to substitute for a decapitated shoot tip by sustaining considerable inhibition on a bud (Thimann and Skoog, 1934; Thimann, 1937). Subsequent work in Thimann’s laboratory showed exogenous cytokinin treatment could reverse the inhibitory effect of auxin (Wickson and Thimann, 1958; Sachs and Thimann, 1964). Sachs and Thimann (1967) were perhaps the first to suggest that a ratio of cytokinin to auxin may be important for apical dominance. In the model, high auxin levels originating in the shoot apex inhibit lateral bud outgrowth. Removal of the apex decreases this auxin supply while cytokinin synthesis continues and/or increases (Bangerth, 1994) to a level that promotes growth of the lateral bud. Many authors, including subsequent work from Thimann’s laboratory (Russell and Thimann, 1990), argue for an indirect, rather than a direct, role for auxin action in axillary bud suppression, an argument that is supported by two lines of reasoning (reviewed in Cline, 1994). First, auxin levels in axillary buds should decrease after decapitation prior to or at the time of bud release, but they do not. For example, Gocal er al. (1991) found a fivefold increase in auxin. Second, results from “W”-shaped bean by Snow (1937) and two-shoot pea by Morris (1977) show inhibitory effects caused by auxin application occur acropetally, whereas auxin is known to move mostly basipetally. Bangerth (1989) hypothesizes that auxin levels in buds do not need to decrease to allow release, because the basipetal transport of auxin down a dominant or main shoot acts to reduce the flow of nutrients and cytokinins to axillary buds or a subordinate shoot (see Morris and Johnson, 1990). Cytokinins have repeatedly been shown to promote axillary bud release and/or subsequent growth in intact plants (Pillay and Railton, 1983; Semenuik and Griesbach, 1985). Bangerth and colleagues have shown that when plants are decapitated, induced bud release is correlated with increased cytokinin concentrations in the stem and/or xylem sap (Bangerth, 1994; Li et al., 1995). A very high dose of auxin applied to the stump partially prevents this increase in cytokinin and reduces bud release. Given the importance of cytokinins in promoting lateral bud outgrowth, this class of hormones may be a candidate for a primary or secondary signal in apical dominance. Cline (1994) argues for a secondary role in which cytokinins interact with auxin. Turnbull er al. (1997) have provided perhaps the first study which correlates endogenous bud cytokinin levels with release over the first few hours following decapitation. They found that a measurable increase in

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cytokinin concentration occurs in the bud just after, rather than before or during, initial measurable bud growth. So there is reason to question whether cytokinin is the first stimulus promoting branching in decapitated plants. In this case, studies are required to determine if the results reflect a limitation of the available experimental approaches or whether this is the real sequence of events. It is possible that important changes in cytokinin levels prior to bud release may occur in only a small percentage of cells in the bud or for only a very short period of time. The importance of auxin and cytokinin, as having major roles as signals controlling axillary bud outgrowth, stems from decapitation studies. Where do we stand today on understanding the role of these hormones, or other hormones, in controlling nondecapitated bud outgrowth? Section VB describes recent approaches with intact transgenic plants with altered hormone levels. While much data from these studies provide support for the auxinkytokinin ratio theory on the control of branching, they also demonstrate that these hormones may not be sufficient to account for branching under all circumstances in intact plants, particularly during the juvenile growth stage. The possibility that hormones other than auxin and cytokinin contribute to the regulation of branching has been suggested by a number of authors (Blake et al., 1983; Russell and Thimann, 1990; Gocal et al., 1991; Stafstrom, 1993). Some genetic evidence for at least one other signal has been provided by studies with the rmsl mutant of pea (Beveridge et al., 1997b; Section VC). Nevertheless, the very real possibility of additional hormone-like substance production in the shoot tip is sometimes overlooked. Indeed, if an inhibitory signal is produced in apical tissues, the removal of the source of this signal by decapitation would partially account for the high quantities of exogenous auxin required and for the often incomplete or even impossible inhibition of branching in auxin-treated decapitated plants (Cline, 1996; Kotov, 1996).

IV. Use of Induced Mutations to Study Axillary Bud Outgrowth Recently there has been increased interest in using induced mutations to gain an understanding of axillary bud outgrowth potential, and mutants represent an ideal way to tie both development and physiology together. This is especially true for induced mutations in one genetic background, as this allows for a comparison of two plants, wild type and mutant, that differ at only one genetic locus. The caveat for mutant analysis is that the loss of a single gene product can result in a number of pleiotropic effects and these may complicate an understanding of the main biological function of the wild-type gene. Or, the mutation may reveal a new function that, while important for the expression of the mutant phenotype, bears no relationship to the biology of the wild-type plant. However, the value of a mutant is that it represents a means toward understanding complexity in a stepwise fashion. The importance of mutants in the elucidation of complex physiological processes

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in plants has been discussed in a number of reviews (King, 1988; Estelle, 1992; Reid, 1993).Rothenberg and Ecker (1993) provide a comprehensive review on the importance of mutant analysis for understanding plant hormone action. A mutant-based approach allows for a study of the genetic basis of lateral branching. The ideal phenotypes for comparative studies are ones where mutant and wild type differ only in branch number and in a quantitative or even qualitative manner. If the intent were to avoid imposing preexisting models on branch development, i.e., the involvement of hormones, it would be best to carry out an unbiased screen whereby axillary buds undergo release without external manipulation. Ideally, the best mutants would not be altered in fertility, and the vegetative and reproductive phases would be unaltered in comparison to wild type. In reality, this mutant phenotype may not exist for a number of reasons. First, hormones are likely to be important for some aspect of bud outgrowth suppression or promotion and changes in hormonal balances may not have a neutral effect. Second, lateral branches bear leaves, causing increased shoot/leaf area, shading, and sites for seed and fruit production. The outcome of these factors alone may mean that ideal mutations for the study of bud outgrowth may have a number of downstream effects that are manifested as pleiotropic alterations in organ growth and development. The other type of mutant that would be ideal for testing current theories of branching would be one where the primary loss of function is in an auxin or cytokinin biosynthesis, metabolism, or signal transduction pathway, thus enabling a genetic test for the involvement of these hormones. Auxin- or cytokinin-deficient mutants may be difficult to isolate because, as stated by Reid (1993), (1) null mutations are likely to be lethal, (2) there may be duplication of genes and/or pathways, and (3) there is lack of accurate information on the appearance of these mutants. In carrying out an analysis of phenotypes, it is important to have detailed descriptions of phenotypic traits. The terms “reduced apical dominance,” “increased branching,” and “lack of apical dominance” are frequently found in the literature as traits associated with mutant phenotypes (reviewed by Cline, 1997). In some instances, the assessment is not accompanied by supporting documentation such as detailed comparisons to the wild-type growth habit. The problems associated with the concept of “bushiness” have been examined by Cline (1997), who illustrates the manner by which certain plants, especially dwarf plants, can present an illusion of increased branching. Degrees of compaction (internode length), length of branches (long versus short), and differences in node number on a stem can influence a perception of bushiness. As the total node number affects the number of potential sites for branching, the node of flower initiation can influence the branching phenotype. Finally, an often-overlooked part of a phenotypic analysis, especially in terms of branch formation, is what constitutes a lateral branch. Axillary buds that can undergo transient growth and expansion cycles (see Fig. 1) may become dormant even after attaining a significant length and yet be-

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fore realizing the full outgrowth potential. The description of a branch is potentially subjective but this is easily addressed by applying a strict definition as to what constitutes a lateral branch for each species. In the end, the best way to measure branching is in lateral length at each node and over more than one time point or developmental stage, so as to account for growth rate and delayed dormancy.

V. Molecular and Genetic Approaches for Understanding Bud Outgrowth Molecular approaches require that isolated and cloned sequences are available to study some defined aspect of lateral branching or apical dominance. Genes that are important for meristem action may be valuable for studying axillary bud outgrowth, given the importance of the shoot apical meristem, both as a source of cells that lead to axillary bud development and as a dominating structure. The question is whether these genes are part of a primary signal transduction system controlling dormancy, thus relating directly to development, or whether the genes are part of a more generalized growth process. Isolated genes with important roles in shoot apical meristem development and maintenance are available and are being characterized in a number of laboratories, e.g., homeodomain-containing genes such as the family of Knotted and Knotted-like genes (Evans and Barton, 1997; Kerstetter and Hake, 1997). While these genes are important for understanding shoot apical meristem development, at this time they do not provide insight into understanding a molecular basis for the different developmental potentials associated with axillary bud outgrowth. This will change in the future as our knowledge of plant development continues to increase and connections are made between the expression of these genes in the meristem of the bud and signals from other parts of the plant. In trying to understand the importance of these genes, it is worth pointing out that the control of bud outgrowth can be manifested in several ways. For example, so-called dormant axillary meristems are not necessarily organs that are completely devoid of metabolic activity (Stafstrom and Sussex, 1988). Phuseolus vulguris demonstrates weak or incomplete apical dominance and axillary bud outgrowth proceeds at a slow rate in the presence of the shoot apex (Hillman, 1984). In this case, growth in the axillary bud is not arrested but is slowed down considerably. A. Recent Gene Isolation

Two genes deemed important by virtue of mutant phenotypic traits relating to axillary bud outgrowth or initiation, the Teosinte brunched gene (Doebley et al.,

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1997) and the tomato Lateral suppressor gene (Schmidtt et al., 1997), respectively, have been recently isolated and will be reviewed herein. It is beyond the scope of this chapter to review the extensive subject of auxin-related or auxinresponse gene expression. This i s an exciting area of research, and genetic and molecular studies with Arabidopsis are providing a considerable amount of information on the regulation of auxin as well as auxin transport and plant responses to auxin (Hobbie, 1998). Our understanding of auxin and its role in plant growth is increasing at a fast pace, as witnessed by the recent isolation of the AXRl (Leyser et al., 1993) and AXR3 genes (Rouse et al., 1998), discussed in Section VCl. The recent report on the isolation of a putative cytokinin receptor, CKI1 (Kakimoto, 1996), may provide insight into cytokinin signal transduction. However, at this time a connection relating these hormone genes to axillary bud outgrowth potential is not complete. This will change in the future as more information becomes available linking the developmental and physiological aspects of axillary bud outgrowth to signal transduction pathways involving auxin and cytokinin. 1. Teosinte branched1 Maize and its wild ancestor, teosinte, demonstrate contrasting architectural features, including differences for the number of lateral branches, the length of the branches, and the type of reproductive structures borne on the branches (Doebley et al., 1997). A mutation at the maize Tbl locus results in a proliferation of tillers (basal branches) instead of the normal dormant buds and in this way affects the developmental potential of the bud and converts the architecture of maize to a teosinte-like appearance. The mutation does not alter the total node number on maize nor does it extend the upper range of nodes that normally produce branches on the main stem. The node position of the uppermost branch in the mutant is the same on wild type, and this uppermost node represents the position where a short, ear-bearing branch forms on maize. Comparative analyses between maize and teosinte suggest that in maize Tbl acts as a repressor of axillary shoot growth and regulates the sex of the inflorescences terminating the shoots (Doebley et al., 1995). Thus, there is an additional effect of the mutation relating to the floral determination. The Tbl gene has been isolated by transposon tagging (Doebley et al., 1997). Northern analysis using RNA from a variety of plant tissues shows Tbl is expressed only in ear husks and axillary primordia, both the inflorescence and shank (branch) regions. The maize wild-type Tbl allele is expressed higher than the teosinte Tbl allele in shank tissue. As maize axillary primordia are more repressed in growth than teosinte primordia, higher gene expression correlates with a repression of growth. The coding sequence of Tbl shares two short regions of homology with three Arabidopsis ESTs as well as the Cycloidea gene from Antirrhinum majus (Doebley et al., 1997) and a putative nuclear localization signal indicates Tbl may have a regulatory role in transcription (Doebley et al., 1997).

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A mutation in Cyc shows a phenotype in flowers only (Luo et al., 1996), and there

is no known connection to axillary bud outgrowth. Doebley et ul. (1997) proposed the following model for Tbl in maize evolution. Tbl is functional in teosinte and is usually expressed in axillary meristems on the secondary branches. The teosinte allele controls the conversion of axillary meristems into ear primordia. Tbl is not normally expressed in axillary meristems on the main shoot and bud outgrowth results in elongated tassel-tipped branches. Maize represents an allelic variant of Tbl where the gene is expressed in the axillary meristems on the main shoot with the result that bud outgrowth is suppressed. In the strict sense, to say that this gene functions in apical dominance requires that an apical portion of the plant regulates Tbl expression and thereby controls the developmental potential of the axillary bud. Would decapitation be a useful approach to test this? It may, but an early caveat mentioned, that decapitation-induced branching may not be the same as the developmental program operating in nondecapitated plants, is important to keep in mind. Decapitation studies are physically difficult experiments to carry out in maize because of the monocot growth habit, and the effect of decapitation on Tbl expression has not been carried out at the present time (J. Doebley, personal communication). One last comment about Tbl relates to the pleiotropic effect for floral determination in the tbl mutant. Section VC3b discusses the influence of photoperiod and photoperiod response flowering genes on basal branching.

2. lateral suppressor The recessive luterul suppressor (Is)mutant of tomato has three prominent phenotypic traits, a loss of axillary meristem development in some leaf axils, absence of petals, and reduced fertility (Williams, 1960). A study by Szymkowiak and Sussex (1993) focused on the petal phenotype, but their results have significance for the axillary meristem defect. They constructed a periclinal chimera consisting of a genetically recessive Is L1 layer and genetically wild-type L2 and L3 layers. The chimera produced normal petal primordia in the second whorl, but most importantly for the subject of this chapter, axillary meristems developed at all vegetative axils (Szymkowiak and Sussex, 1993). Thus, a functional Ls gene is required in only certain cell layers of the meristem. This has implications that some type of signaling is occurring between the cell layers. Immunolocalization studies indicated significantly less cytokinin accumulation in apical and axillary buds for the mutant than for the wild-type isogenic parental line (Sossountzov et al., 1988), thus indicating a relationship between diminished cytokinin and impaired axillary bud outgrowth. However, increased cytokinin production, mediated by an introduced ipt transgene, failed to induce bud formation on the ls mutant (Groot et al., 1995). It is interesting to note that the ls mutation does not affect all axillary meristems on the plant. Whereas meristems do not form in axillary positions on the vegetative main shoot of Is mutants, axillary

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meristems giving rise to sympodial shoots develop on the reproductive shoot systems (Szymkowiak and Sussex, 1993; Schumacher et al., 1995). This differential expression of the mutant effect in different types of axillary meristems is intriguing, and gene expression studies on Ls should prove to be very interesting. The Ls gene has been isolated using a map-based cloning approach (Schmidtt et al., 1997), but details on the isolation of the gene and molecular analyses have not been published at this time.

B. Hormonal Modulation Using Transgenic Approaches

Four bacterial genes are used in transgenic experiments as a way to modulate internal levels of plant hormones. In Agrobacrerium tumefaciens, the genes iaaH (indoleacetamide hydrolase) and iaaM (tryptophan monooxygenase) represent a two-step biosynthetic pathway, not commonly utilized by plants, but which nevertheless cause the synthesis of IAA in transgenic plants (Romano and Klee, 1993). The bacterial gene iaaL (indole-3-acetic acid lysine synthetase), isolated from Pseudomonas savastanoi, catalyzes the conjugation of free auxin, rendering it biologically inactive, and therefore may cause a reduction in free auxin levels (Romano et al., 1991; Spena et al., 1991). Finally, cytokinin levels can be increased with the ipt (isopentenyl transferase) gene from A. tumefaciens, which catalyzes a committed step in cytokinin biosynthesis (e.g., Barry et al., 1984; Medford et al., 1989). Currently there is no isolated gene, either bacterial or plant, which has been used to reduce cytokinin production in plants. The rolC gene from A. rhizogenes induces a range of pleiotropic traits in transgenic plants, including increased branching (reviewed by Hamill, 1993; Coenen and Lomax, 1997). However, alterations in lateral branching with rolC are not always consistent ( e g , Nilsson et al., 1993; Faiss et al., 1996). Although rolC appears to encode a 0-glucosidase that was thought to hydrolyze and potentially activate certain cytokinin glucosides (Estruch er al., 1991a), more recent results indicate that the role of rolC may not be that simple (Faiss et al., 1996). Faiss et al. (1996) suggest that there may be a number of possible substrates for rolC and put forward a hypothesis regarding low molecular weight oligosaccharins. Meyer et al. (1995) have reported the presence of a rolC homologous sequence in tobacco (troZC), indicating the possibility of an ancient transfer of DNA from A. rhizogenes. Northern analysis shows trolC expression is localized in young shoot and leaf tissues, and the accumulation of trolC mRNA in cultured leaf tissues was strongly downregulated by auxin and induced by cytokinin (Meyer et al., 1995). Future studies on the function of this gene should be interesting. The advantage of the transgenic approach over the exogenous manipulation of hormone supply is that problems associated with hormone uptake are prevented or minimized. The disadvantage of this approach is the limited number of appropriate plant promoters whereby hormone biosynthesis can be controlled and re-

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stricted to particular tissues at specific stages of development, although this problem will be reduced with future progress on promoter isolation. There are numerous reports on the use of bacterial transgenes known to change auxin or cytokinin levels and there are a number of reviews dedicated entirely to transgenic plants expressing auxin and cytokinin. For example, Synkova et al. (1997) list and briefly summarize 33 different publications on ipt transgenic plants or cultures. Recent reviews on the subject include Klee and Estelle (1991), Klee and Romano ( 1 994), and Klee and Lanahan ( 1995). We will describe results most pertinent to branching regulation and highlight developmental aspects that have been too frequently overlooked.

1. Modulation of Auxin Levels Transgenic plants with reduced free auxin levels have been produced containing the iaaL gene fused to the cauliflower mosaic virus (CaMV) 35s promoter (Romano et al., 1991). These plants showed increased branching particularly at aerial nodes induced during the onset of flowering. The differences in bud growth were not apparent until the vegetative to floral transition, even though the iaaL plants had 19-fold less auxin at a juvenile stage (6 nodes; Romano et al., 1991). Consequently, reduced auxin levels may be important for bud growth, rather than release, and therefore may be most important at the onset of flowering in tobacco, when aerial bud release commences in both wild-type and transformed plants. An additional important outcome of the work is that an induction of bud release in juvenile plants caused by decapitation might not act primarily through the decreasing auxin levels per se unless decapitation causes a greater than 19-fold reduction in free auxin level. Alternatively, a rapid drop in auxin level or transport may be more important than the absolute auxin level at a given time. An up to 10-fold increase in auxin level caused by transformation of the iaaM and iaaH genes into tobacco resulted in the inhibition of axillary bud growth at aerial nodes during the period of bud release in comparable wild-type plants (Sitbon et al., 1992). This effect of increased auxin on bud inhibition was later shown to act independently from ethylene (Romano et al., 1993). Decapitation promoted bud elongation in wild-type plants within 7 days but did not promote bud release in iaaM iaaH plants during a period of more than 2 months (Sitbon et ul., 1992). Based on the foregoing results, it is tempting to suggest that reduced auxin levels do not promote bud release, whereas high auxin levels may inhibit bud release.

2. Increases in Cytokinin Levels Among the first ipt plants produced with roots were those transformed with the maize heatshock (hsp70)-promoter controlling expression of ipt (Medford el ul., 1989; Smigocki, 1991). The report of Medford et al. (1989) is perhaps

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the most widely quoted source of evidence that endogenous cytokinins promote branching. The nonheatshocked hsp70-ipr plants described by Medford et al. (1989) contained 3-fold and 7-fold higher zeatin riboside and zeatin riboside 5-monophoshate levels than control plants and had increased growth of axillary buds at all nodes. No additional architectural changes were observed after cytokinin levels were increased still further by heatshock. However, it was apparently only subsequent growth and not bud release which was promoted in hsp70-ipt plants. Furthermore, bud growth at juvenile nodes of hsp70-ipt and wild-type plants appeared minimal, compared with upper nodes, despite a number of other phenotypic alterations and despite high cytokinin levels at the juvenile stage (Medford et al., 1989). The architecture of hsp70-ipr plants is therefore similar to that of free-auxin reduced transgenic 35s-iaaL tobacco plants (Romano et af., 1991; C. P. Romano, personal communication). The data therefore support a role for constitutive changes in cytokinin and auxin levels in influencing bud growth under late stages of whole-plant development in transgenic tobacco, but less so for juvenile plants. A number of studies have used the ipt gene in combination with regulatory sequences that provide a considerable degree of specificity in the site or induction of gene expression (e.g., in fruits, Martineau er af., 1994; senescence regulated, Gan and Amasino, 1995; induced by tetracycline application, Faiss et al., 1997; induced by copper treatment, McKenzie et al., 1998) or by somatic activation (Estruch et al., 1991b). Hewelt er al. (1994) and Eklof et al. (1996) have generated plant material useful for branching studies by introduction of promoterless ipt constructs into tobacco plants. In comparison with wild-type plants that undergo slow bud release and subsequent growth at aerial nodes at the time of flowering, some of the ipr plants have increased basal bud growth before flowering, whereas others have increased basal and aerial bud growth after the onset of flowering (Hewelt et al., 1994). Detailed correlative and molecular analyses are yet to be performed, but the differences observed to date have been ascribed to insertion of the transgene into regions of the genome that provides different promoters for the ipt gene. As a result, either the timing of cytokinin induction may differ among transformants or the cytokinin is being produced in different tissues.

3. Auxin and Cytokinin Interactions and Homeostasis in Transgenic Plants Transgenic plants derived from crosses between iuaM iauH and ipr plants produce an intermediate phenotype (Klee and Estelle, 199I). Although decapitation did not promote bud release in auxin-overproducing iaaH iaaM tobacco plants, a high concentration of a synthetic cytokiniri applied to the bud of these plants was able to stimulate bud release (Sitbon et af.. 1992). These results for tobacco transgenic plants are consistent with the 1967 auxinkytokinin ratio theory of Sachs and Thimann and the recent adaptation of it by Bangerth (1994; Li et al., 1995). How-

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ever, as we stated earlier, they do not account for the comparatively low extent of bud release and/or growth in juvenile plants. Zhang et al. (1995, 1996) have shown that auxin application also decreases cytokinin levels and ipt expression in cytokinin-overproducing plants (see also Palni et al., 1988). Eklof et al. (1997) have performed a series of analyses of both auxin and cytokinin in various transgenic and wild-type plants, while taking into account differences in growth rates and developmental stages. In so doing, they have found that intact auxin-overproducing tobacco iaaM iaaH transgenics had even lower levels of major cytokinins than did wild-type plants. Consequently, the inhibition of bud growth in iaaH iaaM plants may be partly due to a decrease in cytokinin level (Eklof et al., 1997). Eklof et al. (1997) also found an overall decrease in free-auxin levels in stems and leaves of cytokinin-overproducing ipt plants with different cytokinin levels. Studies which show the opposite trends (e.g., Binns et al., 1987; Smigocki and Owens, 1989) have not used intact plants. Consequently, the trends shown by Eklof et al. (1997) indicate that the lack of bud outgrowth in intact juvenile ipt plants is probably not due to an increase in auxin level. In addition to those which refer to the role of auxin, a number of recent studies with ipr plants have indicated that cytokinin levels are subject to homeostatic regulatory mechanisms (e.g., Eklof et al., 1996; Redig et al., 1996). It has become apparent that increased cytokinin levels activate cytokinin oxidase and that a fall in cytokinin level is followed by a decrease in cytokinin oxidase activity (Motyka et al., 1996). This metabolic power is also demonstrated by a study in rose, whereby cytokinins in the shoot have a halflife of approximately 1 day (Dieleman et al., 1997). Cytokinin homeostasis may be very important considering that cytokinin increases as low as a doubling may be sufficient to induce changes in morphology in ipr transgenics (Wang et al., 1997a, 1997b). The induction of ipt in individual buds does not lead to bud release at other nodes (Faiss et al., 1997), indicating either that cytokinin is not easily transported between all tissues, or that the catabolism is so effective as to isolate any transient or localized oversupply. There is currently some debate as to whether or not changes in the quantity of cytokinin exported from the roots even have any effect on shoot morphology (Beveridge et al., 1997a; Faiss et al., 1997; McKenzie et al., 1998). A resolution will be achieved when we have all the information on fluxes and levels of cytokinin to and within the shoot and gene expression studies from the shoot and root of ipt-transformed plants grown under normal conditions.

C. Genetic Approaches for Studying Axillary Bud Outgrowth

The remainder of this chapter focuses on three different model plants and examines the potential or the realized utility of these plants for studying the genes that control axillary bud outgrowth. The three plants included are Arabidopsis

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thaliana, Petunia hybrida, and Pisum sativum, and the omission of other model plants reflects not on their applicability for these studies but on space limitations. Arabidopsis is an extremely important model plant for molecular biology and rnolecular genetics, and the impending completion of the genomic sequencing project ensures the continued importance of this plant as a model genetic organism and as a biological and genetic resource. The utility of petunia and pea has been clearly established through a series of unique mutations and combined with phenotypic and physiological characterizations.

1. Arabidopsis The development of Arabidopsis has been described in detail (Medford et al., 1992; Bowman, 1994). The vegetative phase is brief, consisting of a rosette form, and the reproductive stage allows for a proliferation of flowers (Hensel et al., 1994). Arabidopsis development is monocarpic; i.e., the plant dies after a single reproductive effort. This developmental pattern results in a drive toward reproduction at the expense of vegetative growth, such that, after floral induction in Arabidopsis, the indeterminate inflorescence rneristem partitions lateral floral primordia without subtending bracts. Floral determination occurs in a basipetal wave commencing with the shoot apical meristem and continuing in the axillary meristems on the paraclades. This process results in a bidirectional inflorescence development in which flower development proceeds acropetally and paraclade (branch) development starts within the axils of the last formed, youngest vegetative leaves and continues basipetally to the older leaf axils (Hernpel and Feldman, 1994). In this manner, lateral branching is associated with reproductive competence, beginning in the inflorescence and developing progressively downward to the vegetative zone within the rosette. That is, bud outgrowth does not occur in Arabidopsis plants until reproductive competence has been obtained. An association of branching with photoperiod response genes and other flowering-time genes and reproductive competence has been observed in pea and is an important consideration for the classification of mutants (Section VC3b). Furthermore, Hensel et ul. (1994) have shown that the development of inflorescence branches in male sterile lines is associated with interactions between existing fruits and inflorescence meristems such that inflorescence branches proliferate. A close examination of leaf axils shows that, at least for some ecotypes, obvious development of axillary meristems is not present during the rosette stage of growth (Grbic and Bleecker, 1996). Furthermore, no obvious dome of meristematic cells is observed until after the transition to flowering (Grbic and Bleecker, 1996), at which time axillary meristems develop in the basipetal direction, as described earlier. In the case of the Schizoid (shz)mutant, the loss of the shoot apical meristem through necrosis of the main stem results in the premature development of axillary meristems (Medford et al., 1992).

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a. Mutants with Altered Hormone Response. Given the importance of auxin and cytokinin in decapitation studies, a study of mutants having altered hormone levels, alterations in transport, sensitivity, or responses could provide insight into the roles of these hormones in controlling bud outgrowth. Arabidopsis mutants selected on the basis of altered hormone responses and that have clearly defined differences in branch number (with position of branches also noted), maintain the same node number as wild type, and are male and female fertile would be extremely valuable in studying the control of lateral branching. A problem with the analysis of plant hormone resistant mutants is that they often show resistance to more than one class of hormone (e.g., Leyser et af., 1996). Furthermore, a lack of response to an applied hormone provides no direct evidence for an interaction between the wild-type gene product and the hormone. It is becoming more apparent that it is not always possible to examine developmental responses to hormones in isolation, because of the combinations and interactions of plant hormone action. The recessive axrl and axr4 and the dominant axr2 and semidominant axr3 mutants are well-characterized mutants selected on the basis of resistance to auxin supplied exogenously (see review by Leyser (1997) and references therein). The axrl mutation, which perhaps defines the best candidate at present for a gene involved in an early stage of auxin signal transduction (Leyser, 1997), has a pleiotropic phenotype, with small rosettes, small crinkled leaves, and shortened petioles, as well as a much greater number of inflorescences than wild type (Estelle and Somerville, 1987). An analysis of the cloned AXRl gene shows similarities exist with a ubiquitin activating enzyme (Leyser et af., 1993), but further analyses are yet to shed light on the role of ARXl in the control of axillary bud growth. Detailed analysis of the inflorescence branching has shown that the number of inflorescences arising from the rosette of the plant (i.e., primary, basal inflorescences) is similar to wild type, but uxrl has a much greater total number of inflorescences, with some allelic variation (Lincoln et al., 1990). It has not been established whether this increase in inflorescences is from an increase in orders of branching (secondary, tertiary, etc.) or a greater number of lateral branches on the primary inflorescences. In contrast with axrl, mutants in the axr3 gene have been described as having increased apical dominance because of a reduction in the number of inflorescences over wild-type controls (Leyser et al., 1996). AXR3 has been cloned and is a member of the AUX/IAA auxin-response gene family (Rouse et af., 1998). Consequently, the semidominant mutation, implying a gain of function, is suggested to confer, for example, increased auxin-response gene product stability. Further studies are required to substantiate this hypothesis. Other auxin mutants such as axr2, axr4, and auxl do not appear to greatly affect the degree of branching (Timpte er al., 1994; Hobbie and Estelle, 1995; Timpte et af., 1995). However, a mutant that can suppress the phenotype of axrl has been identified (sarl, Cernac et af., 1997). The sarl mutation on its own does not have any obvious effects on branching but results in a decrease in branching when in

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combination with the axrl mutation (Cernac et al., 1997). Future challenges lie in demonstrating whether and how these genes directly control auxin responses such as signal transduction and whether the effects on branching are direct or indirect consequences of auxin action. Another interesting class of auxin mutants are those that may be defective in the polar transport of auxin. These include pinl, lopl, fir3 and pisl (Okada et al., 1991; Carland and McHale, 1996; Fujita and Syono 1997; Ruegger et al., 1997). The pinl, lopl, and pisl mutants have no reported increases in lateral bud outgrowth, whereas the fir3 mutant does have an effect on inflorescence branching (Ruegger et al., 1997). The fact that not all of these putative auxin transport mutants are affected in their branching would argue that polar auxin transport is unlikely to be the primary signal controlling apical dominance in plants. However, further studies are required to correlate auxin transport with bud outgrowth in these mutants, with a view to the possibility that there are a number of basipetal auxin transport pathways (Kotov, 1996; Fujita and Syono, 1997).

b. Other Mutants with Branching Phenotypes. Mutants showing increased basal (early vegetative phase) branching, even in terms of additional leaf formation from axillary meristems, have generally not been reported for Arubidopsis. One exception is the waldmeister (wam) mutant that shows a number of morphological differences from wild type during floral as well as vegetative development (Felix er al., 1996). Most interestingly from the perspective of this review is that the plant has multiple rosettes. The increased number of rosettes during vegetative development might be interpreted as a change in the ontogeny of axillary meristem development. It appears that the meristems developing in this plant are forming ectopically, indicating that a change in apical dominance of wam plants is probably not the cause for the increased number of rosettes. A more detailed analysis of the morphology of the plant would be of great interest to determine whether all additional rosettes are formed in nonaxillary positions. The revoluta (rev) mutant has pleiotropic phenotypic changes with effects in the production of lateral organs, including leaves, axillary meristems, and floral meristems (Talbert et al., 1995). Axillary meristems develop rarely in the cauline leaf axils, even when the main inflorescence shoot is decapitated. The authors did not invoke a role for apical dominance but instead focus on aspects of axillary meristem development. Although pleiotropic, this mutant could be valuable toward understanding the role of leaf development and/or growth in influencing axillary meristem development because the size of leaves appeared to be inversely related to the development of the axillary meristem. The altered meristem program (ampl-I) mutant is a photomorphogenetic mutant that has been described as having decreased apical dominance (Chaudhury et al., 1993). Interestingly, ampl-I seedlings have increased levels of cytokinin in comparison with the wild-type parent, although comparisons have not been

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made at the onset of bud release. It is difficult to determine if the increased branch number is dependent on increased cytokinin because of two other phenotypic traits. First, the umpl-I mutant has a greater number of rosette nodes and leaves are produced at a faster rate than in wild type. So, ampl-I has increased sites for branch formation in the basipetal wave of branch induction. Second, ampl-I has decreased male and female fertility, a trait that induces increased inflorescence branches (Chaudhury et al., 1993). Other alleles of ampl have been isolated and are known by gene designations such as p t (primordiu timing), cop2 (constitutive photomorphogenesis), and hpt (haupting)(Conway and Poethig, 1997). A mutant that would be extremely interesting to isolate from Arabidopsis is one that shows basal branching (formation of new rosette leaves from axillary positions) during the vegetative phase of development. It is possible that it would take a lesion in more than one gene to achieve this change in development. It is interesting in this respect that some mutants with delayed flowering time, which have a prolonged vegetative phase, can eventually develop the basal axillary meristems (Grbic and Bleecker, 1996). 2. Petunia hybrida

V26, an inbred line of the horticultural plant Petunia hybridu Vilm, has been the subject of a mutational analysis of plant architecture (Napoli and Ruehle, 1996). A series of recessive mutations have been induced by ethyl methanesulfonate (EMS) that increase the total number of lateral branches formed during the vegetative growth phase. The vegetative, monopodial main shoot axis of V26 shows distinct zones constituting an innovation zone and an inhibition zone (Napoli, 1996). If cultural and environmental conditions are conducive for bud outgrowth, axillary buds situated at leaf nodes 3 -9, counting acropetally from the cotyledonary node, sustain growth and grow out as branches, or more specifically as paraclades. Environmental conditions that suppress paraclade development include crowding, nutrient deficits, water deprivation, and long daylengths. For the inbred line V26, paraclades do not form below leaf node 3 and buds at these nodes either enter a state of sustained dormancy or remain in the transition zone and grow very slowly. Leaf node 3 as the first node for branch development is constant and not altered by environmental conditions. However, this trait can be modified by mutations (Napoli, 1996). Three loci have been identified in V26 that control the outgrowth potential of axillary buds (Napoli, 1996; Napoli and Ruehle, 1996). Decreased apical dominance (Dud)genes function to suppress bud outgrowth potential at the basalmost nodes on the vegetative monopodial shoot axis and mutations at any of these loci result in multibranched phenotypes distinct from wild type. Figure 2 shows an unbranched V26 wild-type plant and each dud mutant. This discussion focuses on the dadl mutant. In contrast to leaf node 3 for the parental line, branching commences from each cotyledonary axil for dadl (Napoli, 1996). The dadl mutant

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Fig. 2 Comparison of wild-type and mutant phenotypes: (A) wild-type V26; (B) sym dadl-I; (C) dud2-I; (D)dad3.

has increased branch development in both basipetal and acropetal directions as well as extensive second order branching, a trait completely absent in the wildtype parent. Axilly-y bud outgrowth is seen for the primary bud as well as accessory buds. The mutant has several pleiotropic features, e g , late flowering, mild chlorosis, and increased production of adventitious roots at the base of the shoot axis. The extension of branching in the acropetal direction is not dependent on late flowering or increased node development (Napoli, unpublished results). An isogenic, EMS-induced mutation in V26 (sympodiul mutant) converts the parental line to an early flowering phenotype (Napoli and Ruehle, 1996). The double mutant sym dudl (shown in Fig. 2B) has the same number of nodes on the main shoot axis as wild-type V26, but the total number of primary branches on the double mutant is the same as for dudl (Napoli, unpublished results). To test the effect of grafting on dud mutants, a procedure was devised using axenically grown young petunia seedlings with the grafts aligned on MurishigeSkoog medium (Napoli, 1996). Graft unions commence healing within 48 h after

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alignment, and grafted plants can be removed to the greenhouse in several weeks. Three types of graft constructions are used to test for graft-transmissible substances (Fig. 3). Hypocotyl grafts are combinations assembled by severing seedlings within the hypocotyl region (Fig. 3A). Hypocotyl interstock grafts are made when a seedling is severed within the hypocotyl region and a small fragment of interstock, usually a hypocotyl section, is inserted between the rootstock and scion (Fig. 3B). Internode-interstock grafts are constructed using a seedling severed in an internode region, and a hypocotyl fragment is inserted between the rootstock and scion (Fig. 3C). Self-grafted plants for all three types are indistinguishable from nongrafted plants. A dadl rootstock has no effect on branching in a wild-type scion, but a wild-type rootstock converts a dadl seedling to a “near” wild-type appearance (Napoli, 1996). For hypocotyl interstock grafts, a dadl interstock inserted into the hypocotyl region of a wild-type seedling produces no changes in the wild-type branching pattern. However, a wild-type interstock inserted into the hypocotyl region of a dadl seedling is sufficient to convert the mutant scion to a “near” wild-type appearance (Napoli, 1996). The results for hypocotyl interstock grafts are shown in Fig. 4 (adapted from Fig. 6, Napoli, 1996). In this case, the plants were grown under crowded conditions to emphasize the phenotypic differences between wild type and mutant. Whereas crowding usually suppresses vegetative branching in the wild type, first-order branching cannot be suppressed in the mutant (Napoli, 1996). In the case of the internode interstock grafting, a wild-type interstock inserted into the internode of a dadl seedling converts the shoot above the graft union to wild type but the nodes below the interstock branch freely (Napoli, unpublished results). Thus, the phenotypic reversal is unidirectional. There is a circumstance when a wild-type rootstock does not restore a dudl scion to wild type. The appearance of adventitious roots above the graft union on the stem of a dudl scion correlates with branch development on a dadl mutant scion, despite the presence of a wild-type rootstock or interstock fragment (Napoli, 1996). These results demonstrate that roots or interstocks (hypocotyls)

A

B

C

Fig. 3 Graft constructions used for petunia: ( A ) hypocotyl grafting; (B) hypocotyl interstock grafting; (C) internodal interstock grafting.

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Fig. 4 Results of hypocotyl interstock grafts: (A) dudl-1 nongrafted; (B)dudl-l plant with a dudl-I interstock fragment; (C) dudl-1 plant with a wild-type interstock fragment; (D)wild type nongrafted; (E) wild-type plant with a wild-type interstock; (F) wild-type plant with a dudl-1 interstock. Figure adapted from Napoli ( 1996). Copyright American Society of Plant Physiologists. used with permission.

have an inhibitory effect on branching. Two hypotheses are suggested for mechanisms whereby wild-type roots or interstocks function to suppress branching in a dud1 scion or dadl roots promote branching. First, either wild-type roots or an interstock export a compound that suppresses branching in the mutant scion. Alternatively, a putative branch-promoting substance, transported from dud1 roots, is required to promote branching. Either this branch-promoting compound would be the substrate for the DAD1 gene product and accumulate as a result of the recessive mutation or this putative branch-promoting compound could result as a derepressed product of a pathway regulated by the Dad1 gene. Hypocotyl grafting supports either hypothesis. A wild-type rootstock transports a branch-suppressing compound to a dadl mutant scion and this is sufficient to

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suppress branching. The production of a branch-promoting compound requires mutant dadl roots. This compound is not produced in the dadl,,,,, wild typerwtclock graft combination until mutant roots develop above the graft union, an event that correlates with the induction of branching. Hypocotyl interstock grafting also supports both hypotheses. For the hypocotyl interstock combination dadl,wt,,ockwild type ,n,rr,luk dadl,,,,,, the explanation for the first hypothesis is the same as the hypocotyl grafts, except the branch-suppressing compound has to be produced in the wild-type interstock fragment. The internode interstock grafting indicates that this putative compound is transported only acropetally. For the second hypothesis, either the putative branch-promoting compound is not transported through the wild-type interstock or this compound is metabolized within the interstock. Again, the development of mutant roots above the interstock is sufficient to deliver the putative branch promoting compound to the scion. The proof of either of these hypotheses may require the isolation of the Dad1 gene, a process currently being attempted using transposon tagging. 3. Pisum sativum

Garden pea has been the most commonly used species for studies on branching, mostly because of its easily defined qualitative branching response. Pea is also a source of a large range of developmental mutants, many of which have been thoroughly investigated over many years (reviewed by Murfet, 1985, 1989; Murfet and Reid, 1985, 1993; Reid et al., 1996; Ross et al., 1997). Consequently, the understanding of development in pea is already considerably advanced in areas including photoperiod responses, light responses and flowering (most recently reviewed by Weller et al., 1997), internode elongation, and hormone physiology. a. Growth Habit. The shoot architecture of pea consists of a monopodial axis reiterated by an indeterminate shoot apical meristem. Leaf nodes 1 and 2, counting acropetally from the cotyledonary node, bear a scale leaf whereas all other nodes bear a compound leaf and stipules. The leaf axil at each vegetative node contains a principal axillary bud and two or more accessory buds. Wild-type peas referred to in this review are tall and photoperiod-responsive cultivars such as Torsdag, Parvus, and Weitor, rather than the dwarf TCrkse, Solara, and Paloma cultivars or the day-neutral, early flowering Alaska cultivar. Under many conditions, vegetative buds remain dormant and reach only a few millimeters in length throughout ontogeny. Therefore, comparisons of branching in pea are often qualitative and quantitative, and branching can be described in terms of bud outgrowth. Bud outgrowth potential is therefore the potential of a bud to undergo release and subsequent growth. If bud release does occur at a given node, the principal bud forms a shoot, whereas accessory buds remain dormant. Promotion of flowering results in the formation of solitary axillary floral meristems in the leaf axil rather than axillary vegetative meristems.

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b. Photoperiod Response Genes and Branching. As in most plants, the garden pea achieves a particular growth habit through a complex interaction among genotype and environmental influences. Studies comparing wild-type pea plants under different photoperiods have revealed a correlation between daylength and the tendency for branching at basal nodes (Floyd and Murfet, 1986; Murfet and Reid, 1993). Simply stated, short days delay flower initiation and increase the outgrowth potential of buds at basal nodes, whereas long days promote flower initiation and decrease basal outgrowth potential. Short days alone are often not sufficient to induce basal branching in tall wild-type plants, because other influences, such as resource availability, light quantity, plant spacing, and other growth-related factors are also important determinants of bud outgrowth. A reduction in internode length on dwarf (le) gibberellin-deficientbackgrounds is correlated with increased branching, perhaps due to increased resource availability (Floyd and Murfet, 1986; reviewed by Murfet and Reid; 1993). Figure 5 , kindly provided by Prof. I. C. Murfet, shows that the realization of basal branching potential in plants of a photoperiod le genotype is nevertheless dependent on photoperiod. One or more elongated basal branches are present in the 9-, 12-, and 15-h photoperiods, whereas branching was completely inhibited under continuous light.

Fig. 5 Effect of photoperiod on basal lateral outgrowth in dwarf (le) pea. From left to right, dwarf cv. Paloma plants are shown grown under a 9-, 12-, 15-, or 24-h photoperiod. All plants received a photoperiod of 9 h of daylight followed by darkness or a weak incandescent light extension (3 prno1.m ?.s-' at pot top). This photo was kindly provided by M. J. Gregory and 1. C. Murfet.

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The photoperiod response in pea is conferred by the joint presence of the dominant genes Sn, Dne, and Ppd (recently reviewed by Weller et al., 1997). Mutations at any of the aforementioned loci confer a largely day-neutral, early flowering phenotype, including a concomitant reduction in basal branching potential and node of flower initiation. Decapitation can provide a means to observe the branching potential in, for example, nonbranched isogenic wild-type and dne plants. In short days, decapitation above node 10 of adult plants caused the outgrowth of basal nodes of wild-type plants only and the simultaneous promotion of bud outgrowth at the uppermost nodes of both genotypes (C. A. Beveridge, unpublished data). The different basal outgrowth between short-day-grown wild-type and dne plants may be explained by developmental and/or physiological differences because morphological differences, such as bud size before decapitation and main shoot length, were not apparent. The response also demonstrates that the outgrowth potential at basal nodes of short-day-grown wild-type plants remains high even after the juvenile phase. Photoperiod, acting through the photoperiod response genes (Sn, Dne, and Ppd), also affects aerial branch development (Murfet and Reid, 1985; Floyd and Murfet, 1986; Arumingtyas et al., 1992; Murfet and Reid, 1993). By influencing flowering node, the photoperiod genes influence the number of aerial nodes available for vegetative bud outgrowth. Under short photoperiods, where flowering is late, wild-type plants often produce a number of lateral branches immediately below the flowering node. However, the influence of the photoperiod genes on aerial bud outgrowth can be uncoupled from floral initiation in pea. This has been shown using mutations that prevent flowering but do not affect photoperiod response, such as veg (Reid and Murfet, 1984). Aerial branching in veg plants occurs just prior to the node of flower initiation in comparable wild-type plants. Similarly, aerial branching in sn veg double-mutant plants occurs just prior to the node of flower initiation in comparable early flowering sn Veg plants. A similar effect can be observed by modifying photoperiod response gene activity in late flowering gi (reduced production of floral stimulus; Murfet and Reid, 1993; Beveridge and Murfet, 1996) and L t d plants (reduced sensitivity to floral induction; Murfet, 1985; Murfet and Reid, 1985). The promotion of axillary bud outgrowth in veg, gi, and Lf-d plants in inductive environmental conditions (long days) is manifested as a bushy, compact appearance that often includes the release of one or two accessory lateral buds per aerial node (Reid and Murfet, 1984; Murfet, 1985; Murfet and Reid, 1985; Murfet and Reid, 1993; Beveridge and Murfet, 1996). This phenotype is quite different from the relatively normal phenotype during bud release in these mutants under noninductive conditions. The bushy phenotype under inductive conditions is correlated with suppressed Sn Dne and Ppd activity during the absence of normal flower development (Weller et ul., 1997) or, in other words, with the attainment of reproductive competence without floral induction and/or development. The question remaining is how does photoperiod control bud outgrowth under conditions of

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normal flower production apart from through limiting the number of nodes available for branching. The photoperiod response genes appear to control the level of a graft-transmissible substance and may regulate resource allocation within the shoot (reviewed by Weller et al., 1997). Taylor and Murfet (1994) and Weller et al. (1997) propose that the photoperiod genes also control the supply of floral stimulus to the apical meristem. According lo this hypothesis, the photoperiod gene system in pea may control both flowering and branching through assimilate and hormone partitioning toward or away from apical and axillary meristems. The significant effects of various pea flowering mutants on bud outgrowth demonstrate the potential control that flowering mutants can have on bud outgrowth and how easy it may be to classify a mutant as a branching mutant when the wildtype gene acts primarily on the flowering system. Weller er al. (1997) list similarities between flowering control systems of pea and Arabidopsis and, as suggested in Section VI, comparisons between these and other species should lead to exciting advancements in the future.

c. Growth Habit and Branching Genes. Several specific genes controlling branching have been identified through mutagenesis in pea. A total of 26 mutants, defining five Ramosus (Latin for having many branches) loci, have been isolated (Blixt, 1976; Apisitwanich et al., 1992; Arumingtyas et al., 1992; Rameau et al., 1997; Symons and Murfet, 1997). This high number of alleles per locus indicates that mutagenesis for the Ramosus phenotype may be saturated. Having been selected on the basis of a branched phenotype alone, the ramosus mutants have few (if any) phenoty pic characters which are not simply caused by increased branching or underlying hormonal regulatory changes (Fig. 6; Beveridge et al., 1994, 1996, 1997b). The relatively nonpleiotropic phenotype of ramusus plants under glasshouse conditions is important because it indicates that the functions of the Ramosus genes are likely to be more closely related to the regulation of branching than to other processes (see Section IVA). Furthermore, the rammus mutants are probably the largest range of phenotypically and physiologically described nonpleiotropic branching mutants from any species. Lateral bud outgrowth for ramosus mutants usually occurs during both juvenile and adult growth phases and in two zones, basal and aerial, but can also occur at nodes in between (Arumingtyas et al., 1992; Beveridge et al., 1994; Stafstrom, 1995; Beveridge et al., 1996, 1997b). When branching occurs, the principal bud in ramosus plants grows out to form a shoot, whereas accessory buds usually remain dormant. In contrast, accessory bud release also occurs in rmsl rms2 double-mutant plants (Beveridge et al., 1997b). Similar to wild type, branching in ramosus plants is affected by photoperiod such that basal branching is greatest in mutant plants grown in short days and may even be inhibited in continuous light (Arumingtyas et al., 1992; Stafstrom, 1995). Removal of basal laterals from rms2 plants increases outgrowth of aerial buds, indicating that the total mass of

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Fig. 6 Rarnosus mutant and wild-type plants. From left to right, plants are cv. Weitor, rmsl-2. cv. Torsdag, rms4-I. rmsZ-l, and rms.3-2 and were grown under an 18-h photoperiod. Mutant rmsl-2 was derived from cv. Weitor, whereas the remaining mutants were derived from cv. Torsdag.

branches supported by a plant may be limited by a homeostatic control mechanism (Beveridge et ul., 1996).

d. Grafting Studies. The rumusu~mutants can be grouped (Table I) according to whether branching is promoted because of altered levels of grafttransmissible substances (Figs. 7 and 8; Beveridge et al., 1994, 1996, 1997b). Rmsl and Rms2 appear to control graft-transmissible substances produced in the

Table I Classifications of ramosus MutantsU Mutant Observation

rnis I

rms2

rms3

rms4

Increased bud outgrowth due to graft-transmissible substance Increased shoot auxin level Decreased xylem sap cytokinin

Yes Yes Yes

Yes Yes No

No Yes Yesb

No No Yes

~~

~

~~~

~

~

~~

~

~~

~

"The runiosus mutants can be grouped according to whether or not they (i) appear to have increased bud outgrowth due to altered levels of graft-transmissible substance(s), (ii) have increased auxin levels in the shoot, and (iii) have decreased xylem sap cytokinin concentrations (Beveridge et u/., 1994, 1996, 1997a. 1997b). hUnpublished data.

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Fig. 7 Reciprocal grafts between wild-type and rmsl-2 seedlings. Plants shown from left to right are wild-type scions grafted to wild-type and rmsl-2 rootstocks and rmsl-2 scions grafted to wild-type and rmsl-2 rootstocks. Seedlings were grafted immediately after germination (day 6 or 7). Nodes above node 5 or 6 are shown. Basal lateral branches formed only on rmsl-2 self-grafts and were removed at the juvenile stage. Pods can be observed at the uppermost 6 or less nodes. The plants were grown under an 18-h photoperiod.

root and shoot whereas Rms3 and Rmsl appear to act mostly or solely in the shoot. Grafting also indicates that expression of the Rmsl and Rms2 genes in either roots or shoots is sufficient to inhibit branching. In contrast, Rms3 and Rms4 may inhibit branching by controlling synthesis, transport, or metabolism of substances in, or

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rmsl

ms2

ms3-1

ms3-2

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ms4

Scion Genotype Fig. 8 Branching in reciprocal grafts among ramn.w mutant and wild-type plants. Reciprocal grafting studies from experiments described by Beveridge er al. (1994, 1996, 1997b) are represented along with new additional graft combinations, provided by C. A. Beveridge, among m l - 2 , rms3-2, and r m d - I compared with grafts with cvs. Weitor and Torsdag rootstocks. For each scion genotype, the values presented are percentages of total lateral lengths of mutant self-graft combinations and combinations with mean total lateral lengths that were significantly different from comparable mutant selfgrafts within all experiments are shown with an *. Studies that repeated experiments using the same or different mutant alleles and comparable wild types are presented as averages. Bud release and/or subsequent growth in wild-type scions was not significantly increased when grafted with mutant rootstocks of any genotype (Beveridge er al., 1994, 1996, and 1997b and data not shown). n.d., not done.

arriving in, the shoot or by controlling hormone reception or signal transduction in the shoot. Reciprocal grafts between rmsl and rms2 seedlings indicate some type of interaction between Rmsl and Rms2 (Fig. 8; Beveridge et al., 1997b) because bud outgrowth was significantly reduced in all mutant scions of rmsl, m s 2 , and wildtype reciprocal graft combinations, except for rms2 scions grafted to rmsl rootstocks. This interaction will be discussed later in relation to xylem sap cytokinin levels in Section VC3h. In contrast, reciprocal grafts among all other genotypes indicate that mutant rootstocks act as wild-type rootstocks (or cause an even greater inhibition) on bud outgrowth in other mutant scions (Fig. 8; Beveridge et al.. 1996, 1997b). Consequently, Rms3 and Rms4 may act downstream of Rmsl and Rms2 or on one or two totally different signaling pathway(s). Double-mutant analyses may help distinguish between these possibilities, although such studies are difficult if the double-mutant phenotype is not additive, because all the phenotypes are otherwise similar. e. Hormone Studies: Auxin. According to Beveridge et al. (1994, 1996, 1997b), branching in ramosus mutant plants does not appear to be caused by reduced auxin (indole-3-acetic acid) levels in tissues proximal to axillary buds

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at stages during bud release (see Stafstrom, 1993). Rather, the mutants may be separated according to whether they contain elevated (rmsf, rms2, and rms3) or normal (rms4) endogenous auxin levels (Table I). The cause of the elevated auxin is not clear at this stage but may be related to a feedback mechanism intended to reduce further bud release. If feedback regulation of auxin is shown to be a part of branching regulation, the observation that rms4 is the only mutant that contains normal shoot auxin levels and appears to act primarily in the shoot may provide some clue for the mode of Rmsl action (Beveridge et al., 1996). Immunolocalization of auxin or auxin analyses using minute quantities of tissue are required to confirm whether auxin is compartmentalized differently in ramosus plants in such a way that low auxin levels might be causal of bud release. f. Hormone Studies: Cytokinin. The rumosus mutants can also be classified according to xylem sap cytokinin content (Table I). Xylem sap cytokinin concentrations are reduced 95-98% in rmsl and rmsl (Beveridge et al., 1997a, 1997b) and rms3 plants (C. A. Beveridge, unpublished data) but not in rms2 plants. The implications of this observation for rms2 plants are discussed in Section VC3h. Preliminary analyses have not revealed similar differences in cytokinin levels in mutant and wild-type shoots (C. A. Beveridge, P. Walton, and S. Morris, unpublished data). This indicates that for rmsf, rms3, and rmsl very low quantities of root-sourced cytokinin are sufficient to maintain cytokinin levels in the shoot or that, as Chen et al. (1985) reported, cytokinins are also synthesized in the shoot of pea. The hypothesis that low xylem sap cytokinin content in rmsl, rms3, and rms4 plants is a downstream consequence of branching induction, rather than a primary effect of mutations, is discussed in Section VC3h.

g. Hormone Studies: RMSl and a Novel Substance. As described in Section VC3d, Rmsl appears to regulate the level of a graft-transmissible substance that moves from root to shoot (Fig. 7; Beveridge et al., 1997b). However, rmsl plants contain significantly reduced, rather than elevated, xylem sap cytokinin levels. Furthermore, branching in rmsf plants does not seem a consequence of reduced levels of auxin. The simplest explanation for these results is that wild-type rootstocks cause a restoration in the level of a hormone-like, graft-transmissible substance other than auxin or cytokinin (Beveridge e?al., 1997b). It is not known whether this substance inhibits or promotes bud release. Based on the ramosus phenotype and the action of known phytohormones (Creelman and Mullet, 1997; Kende and Zeevaart, 1997, and references therein), it seems likely that the substance influenced by rmsl is novel. Further studies should be designed to confirm if the rmsf mutation alters the level of an obscure cytokinin that is regulated differently from other, well-known cytokinins.

h. Hormone Studies: Shoot-to-Rootand Root-to-ShootSignals. Evidence from reciprocal grafts between rmsl and wild-type plants (Beveridge e? al.,

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1997a) indicates that the shoot controls cytokinin export from roots. In reciprocal graft combinations, rms4 scions cause wild-type rootstocks to export very low levels of cytokinin, whereas wild-type scions cause rms4 rootstocks to export wild-type concentrations of cytokinin. Therefore the reduction in xylem sap cytokinin level in rms4 plants is due to an effect of the rms4 mutation exerted in the shoot and infers the existence of a graft-transmissible shoot-to-root signal. Although Rmsl, Rms3, and Rms4 genes appear to affect different parts of the branching regulation system, mutations in these genes all result in reduced cytokinin export from the roots. This observation, together with the graftingxytokinin studies, provides evidence indicating that the shoot-to-root signal is part of a feedback mechanism induced at a relatively late stage of bud release, at least after action of rmsl, rms3, and rms4. Bangerth (1 994) and Li et al. (1995) put forward a hypothesis, based on studies of auxin applications to decapitated plants, that cytokinin concentration in the xylem sap is downregulated by the polar transport of auxin. However, as suggested for rms4 by Beveridge et al. (1997a), the shoot-to-root feedback signal that regulates cytokinin export from the roots in ramosus plants is probably not associated with auxin transport in the shoot, because rmsl, rms3, and rms4 mutant shoots each appear to have normal basipetal auxin transport and have equivalent or higher than wild-type auxin levels (Beveridge er al., 1996, 1997b). More importantly, although it is possible that rms3 and rms4 block auxin reception in the shoot, this would better account for increased rather than decreased xylem sap cytokinin concentrations according to the auxin-cytokinin hypothesis (Bangerth, 1994; Li et al., 1995). Apart from having the highest shoot auxin levels, mutant nns2 plants differ significantly from rmsl, rms3, and rms4 plants because rms2 xylem sap cytokinin concentrations are not reduced (Beveridge et al., 1997b). If the reduced xylem sap cytokinin level in other rumosus plants is due to a feedback downregulation process caused by an early stage of branching induction as suggested by Beveridge et al. (1997a, 1997b), it may be highly relevant to the regulation of branching that rms2 plants may not have this capacity. Major evidence for rms2 causing a block in xylem sap cytokinin downregulation includes the observation that cytokinin concentration in the root-sap of rmsl rms2 double-mutant plants was comparable to that of rms2 and wild-type plants rather than to that of rmsl plants. That is, the near wild-type xylem sap cytokinin concentration in rmsl m 2 plants indicates that the decrease in xylem sap cytokinin content which occurs in rmsl plants may be prevented by the rms2 mutation (Beveridge et al., 1997b). These data in isolation also indicate that the sequence of gene action in the roots may be Rms2 before Rmsl. Further evidence that rms2 blocks production of a shoot-to-root signal comes from reciprocal grafting studies. The possibility that Rmsl and Rms2 act on different biochemical pathways is consistent with the additive phenotype of rmsl rms2 double-mutant plants (Beveridge et al, 1997b), although this does not provide proof as the mutations

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may be leaky. Reiterating the results from grafting studies described earlier, rms2 scions grafted with rmsl rootstocks had a fully branched phenotype, whereas plants with the reciprocal graft combinations (rmsl scionlrms2 rootstock) had a significantly reduced branching phenotype (as do all other combinations of rmsl or rms2 scions with wild-type or other mutant rootstocks; Fig. 7). These grafting results, taken together with the additive double-mutant phenotype and xylem sap cytokinin levels, can be reconciled in terms of unidirectional transport of gene products (Beveridge et ul., 1997b). That is, the Rms2 gene may regulate shoot-toroot signal(s) which control xylem sap cytokinin content and Rmsl activity, whereas Rmsl may regulate a root-to-shoot signal that controls the release of buds from dormancy.

i. Hypothesis for Branching Control by Rumosus Genes. The best hypothesis on the current data from the ramosus mutants is that the rumosus phenotype is largely controlled by a root-to-shoot signal other than a major cytokinin, regulated by Rmsl. Rms2 influences a shoot-to-root signal that regulates Rmsl activity and cytokinin export from the roots. The identity of these signals is unknown, but they may represent novel phytohormone-like signaling molecules which differ significantly from known phytohormone groups in that their action appears to be relatively specific. Rms3 and Rmsl appear to act largely in the shoot and may control sensitivity, activity, or compartmentalization to/of signal(s) which arrive at the bud.

VI. Conclusions and Perspectives One important point of this chapter is that concepts concerning apical dominance and axillary bud outgrowth potential may need to be reevaluated if we consider more than decapitation-induced branching and examine bud outgrowth potential in a developmental context. Furthermore, we advocate reserve when using the term apical dominance as a growth suppression mechanism for intact plants, unless the apical bud is shown with certainty to be the sole or major regulator of axillary bud outgrowth. Placing bud outgrowth in a developmental context has importance for decapitation, mutant, and transgenic studies examining effects on bud outgrowth. It is important to emphasize that the apex has a dynamic role in development and is responsive to a number of signals, of endogenous and exogenous origin, and patterns of growth can be altered accordingly to these changing signals. Obviously, the shoot apex is important in these processes, but the question remains whether the apex is the only component controlling these patterns of growth. Thus, the regulation of bud outgrowth occurs in the context of plant ontogeny, and although the discrete steps remain unknown, we can see that development

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proceeds in accordance with a number of stages. Bud outgrowth may, for example, include the following sequence: bud initiation, transition phase to dormancy, dormancy, transition phase to growth followed by sustained growth (reviewed by Stafstrom, 1993; also see Fig. 1). The utility of these stages is that we can place different regulators or influences into a sequence or system and therefore allow some understanding of the relative importance and interaction of each. Bud formation first depends on the organization of a meristematic region and the initiation of leaf primordia. Currently, we do not understand the stimuli regulating bud outgrowth, but our knowledge of the identity of genes required for meristem initiation is increasing, e.g., the Knotted and Knotted-like class of genes (Evans and Barton, 1997; Kerstetter and Hake, 1997), the Ls gene (Schmidtt et al., 1997). and Tbl (Doebley et al., 1997). The intrinsic ability or tendency of a dormant bud to undergo the transition to growth is regulated by the interplay of a number of variables. Perhaps the most important of these are the age of the bud relative to its initiation, photoperiod response genes such as those from pea and hence photoperiod, and the zone of the stem in which the bud develops (i.e., basal versus aerial branching). The number of nodes formed by a branch before reproductive conversion of the branch is also usually associated with the zone of the stem in which the bud developed. However, whether or not the outgrowth potential is realized depends on a range of variables, including environmental influences such as light, nutrients and apex loss, and timing of reproductive conversion. It is possible that cytokinin and auxin as well as branching genes such as Dad, Ramosus, and Tbl (Section V) act at this comparatively late stage of bud outgrowth or, alternatively, act earlier, controlling the intrinsic ability or tendency of dormant buds to undergo the transition to outgrowth. Changes in endogenous auxin and cytokinin levels in intact transgenic plants are not always associated with bud outgrowth and therefore may be more important for decapitated plants, specific ontogenetic stages (e.g., at the onset of flowering), and/or buds already at the growth transition stage (Section VB 1,2). However, the possibility exists that pulses of hormone, or small but localized changes in hormone levels, may be instrumental in controlling bud outgrowth (Sections I11 and VB2). Large changes in hormone-altered transgenic and hormone-treated decapitated plants may represent a general overriding of the developmental plan of bud outgrowth seen in intact or nontransformed plants. This is demonstrated for many ipt transgenics by effects not common during normal plant development, including the production of epiphyllic or ectopic shoots in plants transgenic for the ipt gene (Estruch et al., 1991b; Hewelt et al., 1994; Faiss et af., 1997; McKenzie et af., 1998). An advantage of the mutant approach in biology is that complexity can be studied in a stepwise fashion. Through mutations leading to a small number of phenotypic alterations, we can observe the outcome of perturbations to developmental control. Although a clear understanding of the gene product may not always be evident, we can observe that genetic control exists and can evaluate the

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importance of the gene and its possible role(s) in development. The petunia and pea branching mutants are interesting and informative because these recessive mutations uncover novel responses and mechanisms underlying the regulation of bud outgrowth in wild-type plants (Section VC). When these processes are altered in the mutants by the loss of structural or regulatory genes, sustained bud outgrowth proceeds at nodes that normally would have had buds in a stable, reversible state of dormancy or in a transition state. It is also evident from interstock grafting with petunia that organs other than the apex, such as hypocotyls, may be important not just in the transport of these signals but also potentially in regulatory roles. Grafting studies and hormone analyses with rumosus mutant and wild-type pea plants indicate the presence of a root-to-shoot signal not accounted for by cytokinins in the xylem sap and a shoot-to-root signal not accounted for by auxin. By way of these mutants, we are uncovering potential new ways in which branching can be induced. The next step is to isolate these genes and determine how they are controlled in wild-type plants to suppress branching. One of the most difficult problems facing plant biologists lies in the enormous complexity inherent in biological systems. The developmental and physiological control of plant architecture is certainly one of the more complex systems,because the mature form of a plant is the sum total of growth processes occurring at different stages of development. Trewavas ( 1986) writes, “every plant constituent forms part of a network whether it be a molecule, cell, tissue, organ or whole plant. The critical property of a network is that external or internal effects on one or more constituents do not take place in isolation but through linkage are experienced throughout the network.” This is very pertinent to bud outgrowth and is a problem to varying degrees in all approaches for studying biology. The solution for gaining insight into such a complex problem requires multifaceted approaches including but not limited to (1) the continuing use of traditional physiology studies, (2) the incorporation of new technologies such as molecular biology, (3) the use of a number of model plants with different growth habits, (4)the continuing use of genetic perturbations, including induced mutations and transgenes, and ( 5 ) integration of developmental, molecular, and physiological studies employing both intact and decapitated plants. The sum total of knowledge gained from these approaches will enable us to chip away at the complexity and ultimately understand how plants grow and develop a shoot system.

Acknowledgments The authors would like to thank Drs. Bart-Jan Janssen, Catherine Rameau, and Colin G . N. Turnbull for reviews of the manuscript and helpful discussions. We thank Dr. Erin Irish for helpful discussions on meristem and shoot development and Dr. John Doebley for discussions regarding Teosinle branchedl. Work on the petunia mutants is supported by NSF Grant No. IBN9507082 to C.A.N.Work on the pea mutants by C.A.B. is funded by an Australian Research Council Fellowship.

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6 Control of Messenger RNA Stability during Development Aparecida Maria Fontes, Jun-itsu Ito, and Marcel0 Jacobs-Lorena Department of Genetics School of Medicine Case Western Reserve University Cleveland, Ohio 44106

1. Introduction 11. Regulation of mRNA Stability in Plants

Regulation of mRNA Stability in Caeriorhabditis elegans Regulation of mRNA Stability in Drosophila Regulation of mRNA Stability in Xenopus Regulation of mRNA Stability in Avians A. a1 (I) Collagen Gene Expression B a 2 (I) Collagen Gene Expression C. 01 (11) Collagen Gene Expression VII. Regulation of mRNA Stability in Mammals VIII. Conclusions and Prospects References 111. IV. V. VI.

1. Introduction A fundamental goal in developmental biology is to understand the mechanisms that regulate gene expression during the development of a fertilized egg to an adult organism. While regulation of gene activity at several levels, such as transcriptional, RNA processing (splicing), and translational, has received considerable attention, regulation of mRNA stability has not been investigated to the same extent. It is becoming increasingly evident that differential regulation of mRNA stability is crucial for normal embryonic development. In most species, the initial steps of embryonic development progress in the absence of transcription and depend on a store of mRNAs accumulated during oogenesis. For example, structural genes such as those encoding ribosomal or cytoskeletal proteins are actively transcribed during oogenesis and their products stored in the egg. This strategy obviates the need for massive gene expression during the rapid series of cell divisions that occur during early embryonic development. Most of the abundant mRNAs encoding these proteins remain intact in the oocyte until the end of oogenesis. How is this mRNA silenced after fertilization? Interestingly, in Xenopus the corresponding mRNAs are rapidly degraded soon after egg activation (Weiss er al., Currenr TcJpicsin Develupmerinrl Biology. V~JI. 44 Copyright 0 1999 hy Academic Press. All rights of reproduction in any form reserved. 0070-2153/99 $25.00

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1981; Surdej et al., 1994), whereas in Drosophila the full complement of the maternal ribosomal protein mRNAs (which account for -7% of the total message population) are kept intact in the embryo but in a translationally repressed state (Kay and Jacobs-Lorena, 1985). The basis for the selective degradation of ribosomal protein mRNAs in Xenopus is not understood. Specific translational repression of ribosomal protein mRNAs in Drosophila is due in part to depletion of initiation factor activity in the early embryo (Hongo and Jacobs-Lorena, 1991) and is mediated by sequences in the 5’UTR (Pate1 and Jacobs-Lorena, 1992). Fertilization signals the translational activation of many stored maternal mRNAs encoding developmentally important proteins, such as those derived from the Drosophila gap and segmentation genes, mouse pattern-forming genes, and genes encoding regulators of cell division. Expression of these genes is often transient and restricted in space. Transient expression requires a rapid means of turning off gene expression and instability of both mRNA and protein is critical for rapid silencing. Given that most mRNAs share the same cytoplasm, on what basis does the embryo or the differentiating cell distinguish between stable and unstable mRNAs? This is in part due to specific nucleotide sequences (cis-acting sequences) that target mFtNAs for degradation. Such cis-acting sequences have been identified for a relatively large number of unstable mRNA species. As a rule, mRNAs are inherently stable and specific cis-acting sequences act as destabilizing elements. This conclusion is derived from experiments in which removal of the destabilizing sequence from an unstable mRNA stabilizes it (demonstrating that the cis-acting element is necessary for instability) and experiments in which insertion of that sequence into a normally stable mRNA destabilizes it (demonstrating that the element is sufficient for instability). The alternative possibility, that stable mRNAs have sequences that actively promote their stabilization, occurs rarely (Wang er al., 1995; Weiss and Liebhaber, 1995). While a reasonable amount of information is available about cis-acting elements, not much is known about trans factors. One example is the iron-binding protein (IRP). IRP recognizes a short sequence-the iron regulatory element (IRE)-that has the potential for forming a strong secondary structure. The IRE is found in the 5’UTR of the ferritin mRNA, where it regulates its translation, and in the 3’UTR of the transferrin receptor mRNA, where it regulates its stability. When iron is scarce, the IRP has a high affinity for the IRE of the transferrin receptor mRNA and protects it from endonucleolytic attack. Conversely, when the iron concentration increases, it binds to the IRP and changes its conformation. This conformation change reduces the affinity of the IRP for the IRE, making the transferrin receptor mRNA more susceptible to degradation (Constable et al., 1992; Schlegl et al., 1997). A number of mammalian mRNAs, such as those encoding proto-oncogenes and cytokines, have in their 3’UTR short AU-rich sequences that mediate mRNA degradation (reviewed by Chen and Shyu, 1995). A family of proteins, the AUBPs (AU-rich region binding proteins), that bind with high affinity to these AU- or U-rich sequences have been characterized (DeMaria er al., 1997; reviewed by Ross, 1995, and by Rajagopalan

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and Malter, 1997). These AUBPs have been implicated in the process of mRNA degradation but the exact role that they play has not been elucidated. Another example of protein-mRNA interaction is the poly(A)-binding protein (PABP) (Sachs et al., 1986). Some studies are indicating that Pablp has multiple functions, including the stimulation of translation, the inhibition of decapping, and influencing the rate of deadenylation (Caponigro and Parker, 1995). Most available information about mechanisms that regulate mRNA stability comes from model systems, such as yeast or cultured cell lines. A few general mechanisms emerged from these studies. A biphasic mechanism has been proposed whereby mRNA degradation begins with the shortening of the 3’ poly(A) tail and is followed by the degradation of the body of the message. This mechanism has been reported for several mammalian mRNAs such as the short-lived proto-oncogenes c-myc and c-fos (reviewed by Beelman and Parker, 1995; Ross, 1996). Several yeast mRNAs have also been shown to decay via a biphasic pathway. Poly(A) tail removal is the first step in the degradation of the yeast PGKl and MFA2 mRNAs. Removal of the 5’7-methylguanosine cap ( m 7 G cap) is the second step in the degradation of mRNA, followed by decay of the body of the message (reviewed by Caponigro and Parker, 1996; LaGrandeur and Parker, 1996). Another pathway for mRNA decay that bypasses the need for removal of the poly(A) tail has been proposed for the iron-regulated transferrin receptor mRNA, insulin-like growth factor I1 mRNA, and apolipoprotein mRNA. In these cases, degradation is initiated by an endonucleolytic cleavage event within the body of the message, followed by exonucleolytic degradation of the fragments (reviewed by Ross, 1995). Alternatively, mRNA decay may be regulated by expression of antisense RNAs as reported to bFGF mRNA in Xenopus (reviewed by Knee and Murphy, 1997). In this case, the RNA duplex is assumed to be modified by a specific enzyme (DRADA) and then targeted to rapid degradation by doublestranded RNA-specific RNases (Kimelman and Irschner, 1989). Some mRNAs, such as c-myc mRNA may be degraded by more than one pathway (biphasic and endonucleolytic cleavage), depending on the growth status of the cell (Swartwout and Kinniburgh, 1989). These examples suggest that there are a limited number of pathways that degrade mRNAs; however, a myriad of cis- and trans-acting factors may be involved in regulation of these degradation pathways. It is also important to consider that mutations of critical determinants of mRNA instability can lead to diseased states, such as cancer, thalessemia, and Alzheimer’s disease. Aberrant accumulation of growth factor, globin, or amyloid protein precursor mRNAs, respectively, may significantly contribute to the development of these disorders (reviewed by Carter and Malter, 1991). This review focuses on the regulation of mRNA stability in embryonic development and cell differentiation. Discussion of earlier findings and related topics may be found in previously published reviews (Belasco and Brawerman, 1993; Green, 1993; Surdej et al., 1994; Ross, 1995; Caponigro and Parker, 1996; Ross, 1996; Rajagopalan and Malter, 1997).

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II. Regulationof mRNA Stability in Plants The mechanisms, extent, and significance of post-transcriptional regulation of gene expression during plant embryonic development are only beginning to be addressed. Several studies suggest that there may be similarities to animal embryogenesis. Translational activation and changes of mRNA stability of stored or “maternal” mRNAs seem to be as important in plant as in animal embryogenesis (reviewed by Green, 1993). Stored mRNA was analyzed in the fern Marsilea vestifa (Kuligowski et al., 1991) and in Fucus (Masters et al., 1992). Evidence for the regulation of mRNA stability in soybean comes from the observation that in late embryos a mRNA encoding a 15-kDa seed protein declines by more than 50fold, whereas transcription rates are reduced by only 10% (Walling et al., 1986). Plant embryos develop through a series of morphologically identifiable stages, beginning with the globular stage and proceeding through the heart, torpedo, and mature stages (reviewed by Thomas, 1993; Zimmerman, 1993). emb-f mRNAs encode a class of ubiquitous angiosperm proteins of unknown function that accumulate to high abundance in mature seeds (e.g., the wheat Em protein and the cotton Lea D19 protein). In siru hybridization to sectioned carrot embryos demonstrated that up to the globular stage emb-l mRNAs are uniformly distributed. Subsequently, in heart stage embryos, emb-f mRNA begins to show an asymmetrical distribution, with higher levels detected in the peripheral regions (Wurtele et al., 1993). This transition from symmetrical to asymmetrical distribution is reminiscent of Drosophifa hunchback and caudal, whose mRNAs are believed to form a gradient by selective degradation only in the posterior or in the anterior regions of the embryo, respectively (see Section IV). Transmitting-tissue specific (TTS) mRNAs encode glycoproteins involved in the promotion of pollen tube growth (Wang et al., 1993; Cheung et al., 1995). Three classes of TTS mRNAs have been identified: TTS itself, p- I ,3-glucanase, and MG15 (Wang et al., 1996). Northern blot analysis with RNA from pollinated and unpollinated styles showed that pollination induces differential mRNA poly(A) tail-shortening. p- 1,3-glucanase and MG 15 mRNA abundance declines rapidly following poly(A) removal, as is commonly observed in plant and animal cells. However, TTS mRNA abundance and translatability remain high even after poly(A) tail shortening (Wang et al., 1996). This is in contrast to a number of maternal mRNAs in animal embryos, where the translational activation during oogenesis and embryogenesis is promoted by poly(A) tail extension (Ahringer and Kimble, 1991, for C. elegans fem-3; Salles er al., 1994, for Drosophila bicoid; Riviera-Pomar et al., 1996, for Drosophila caudal; Wreden er al., 1997, for Drosophifa hunchback). How TTS mRNAs are specifically stabilized remains to be elucidated. The rbcS (ribulose- 1,5-biphosphate carboxylase) mRNA encodes an enzyme which catalyzes the initial fixation of atmospheric carbon dioxide into carbohydrates. It comprises approximately 50% of the soluble protein of leaves and is

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present at much lower levels in other tissues (Wanner and Gruissem, 1991). Initial analysis by nuclear run-on transcription assays and Northern blotting in tomatoes showed that differential expression of this transcript in seed, leaves, and fruit is due to control at the level of both mRNA transcription and stability (Wanner and Gruissem, 1991). Subsequently, a more detailed study of the decay pathway in soybean seedlings and in transgenic petunia leaves showed that mRNA decay initiates with endonucleolytic cleavage events (Thompson et al., 1992; Tanzer and Meagher, 1995). By using competition experiments for inhibition of endonucleolytic activity, four major degradation sites were mapped within the coding region of this transcript. Analysis of these sequences showed no significant similarities or obvious sequence motifs. However, all cleavage sites are GU-rich and computer folding of these four major degradation sites of the rbcS transcript predicts similar hairpin structures (Tanzer and Meagher, 1995). Evaluation of the significance of either the GU richness or the local secondary structures in directing endonucleolytic cleavage will require further experimentation. Note that hairpin structures are also predicted for the histone mRNA, transferrin receptor mRNA, ferritin mRNA (Belasco and Brawerman, 1993), and the putative instability element (FIE3) of the Drosophilafushi-turuzu mRNA (Riedl and Jacobs-Lorena, 1996). Abscisic acid (ABA) occurs universally in higher plants and acts as an endogenous regulator of gene expression during embryonic development. ABA abundance increases in the early embryo and declines late in embryogenesis. ABA may be required to keep the embryonic program and prevent precocious germination (Galau et al., 1987; Garciarrubio et al., 1997). In cultured barley embryos ABA regulates expression of the bifunctional a-amylaselsubtilisin inhibitor (BASI) gene. Nuclear run-on assays combined with Northern blot analysis indicate that ABA increases the stability of BASI mRNA without changing its transcriptional activity. Moreover, inhibition of protein synthesis stabilized the BASI mRNA which led Liu and Hill, (1995) to suggest that an unknown short-lived protein protects the message. However, an alternate possibility must also be considered, namely that ribosome transit along the mRNA triggers mRNA decay. While poly(A)-binding proteins (PABP) play a role in the regulation of mRNA stability and translation in yeast and mammalian cells (Bernstein et al., 1989; Jacobson and Peltz, 1996), their role in plant cells remains to be determined. A family of PABP genes from Arubidopsis thaliana has been partially characterized (Belostotsky and Meagher, 1993). One of these genes, PAB5, is developmentally regulated since gene expression is limited to pollen and ovule development and to early embryogenesis. Belostotsky and Meagher (1996) have shown that PAB5 can partially restore PABP function to a PABP-deficient yeast strain. While PABS could rescue the poly(A)-shortening and translational initiation functions of mutant yeast, it could not restore the deadenylation-dependent decapping. These experiments suggest that PABPs may also possess the ability to regulate mRNA stability and translation during plant sexual development and early embryogenesis (Belostotsky and Meagher, 1996).

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111. Regulationof mRNA Stability in Caenorhabdifiselegans The two best studied cases of mRNA stability regulation during C. elegans development are fem-3 and p a l - ] . The fem-3 (feminization) gene regulates sex determination (reviewed by Hodgkin, 1990), whereas the pal-1 (posterior alae) gene is required for posterior patterning (Waring and Kenyon, 1990). The primary sex-determining signal in C. elegans is the ratio of the number of X chromosomes to the number of sets of autosomes (the X/A ratio). XO individuals develop as males and XX individuals develop as self-fertile hermaphrodites. Hermaphrodites are somatically females, but they briefly produce sperm before making oocytes during the fourth larval stage. The adult hermaphrodite germline tube contains sperm proximally and oocytes distally. Expression of fem-3 directs spermatogenesis and must be negatively regulated to allow the switch to oogenesis to occur. Loss-of-function fem-3 mutants produce only oocytes, no sperm, whereas gain-of-function (gf) fem-3 mutants produce only sperm and never switch to oogenesis (Ahringer and Kirnble, 1991). There has been considerable progress in the understanding of the mechanism of fem-3 mRNA destabilization during the fourth larval instar. (1) In wild-type worms, the poly(A) tail of fem-3 mRNA shortens during development whereas in fem-3(gf) mutants, poly(A) tail increases in length (Ahringer and Kimble, 1991; Ahringer et al., 1992). (2) Seventeen of 19fem-3(gf) mutations are single-nucleotide changes that map to a five-nucleotide stretch in the center of the 3’UTR. This sequence was named PME, for point mutation element (Zhang et al., 1997), and is a candidate for a cis-acting element that controls fem-3 mRNA poly(A) length and stability. (3) RNAs containing the PME bind specifically to a factor (FBF, for fem-3 binding factor) present in worm extracts. The FBF gene was cloned. Interestingly, theJbf mRNA increases in abundance at the same time the poly(A) tail of fem-3 mRNA shortens and the spermatogenesis-to-oogenesisswitch occurs (Zhang et al., 1997). (4)Overexpression of a RNA harboring fem-3 3’UTR sequences masculinizes the nematode germ line, consistent with the titration of a repressor of the wild-type fem-3 gene (Ahringer and Kimble, 1991). Thus, jbf appears to negatively regulate fem-3 by binding to its PME. Absence of fem-3 protein allows oogenesis to occur. Iffbf protein is titrated away with.fem-3 PME sequences,fem-3 cannot be silenced and the switch of spermatogenesis to oogenesis fails to occur. The destabilization of fem-3 mRNA by binding of the FBF protein is reminiscent of the destabilization of the hunchback mRNA through the interaction of the Purnilio protein with the nanos-response element (NRE) in the 3’UTR of mRNA (see Section IV). Interestingly, the FBF and Pumilio proteins share similar features. The RNA-binding domain of both proteins consists of a stretch of eight tandem repeats with a similar conserved core consensus sequence. Furthermore, comparison of individual repeats in FBF and Pumilio reveals a conserved pattern from repeat to repeat (Zhang et al., 1997). In hermaphrodites each adult germline

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tube contains sperm proximally and oocytes distally. FBF restricts expression of fem-3 mRNA so that sperm are only made in the-most proximal region of the C. elegans germ line, whereas Pumilio limits expression of hunchback mRNA to the anterior portion of the Drosophila embryo. In the absence of EBF, the spatial pattern of sexual cell fates is lost and a uniform field of sperm is found instead. Similarly, in the absence of Pumilio, the spatial pattern of hunchback expression is lost and it becomes uniformly distributed throughout the embryo (Zhang et al., 1997). The pal-1 mRNA is provided maternally and zygotically and encodes a membrane receptor that is required in C. elegans for posterior development (Waring and Kenyon, 1990; Yandell et al., 1994). Whole-mount in situ hybridization to early embryos indicated that pal-I transcripts are uniformly distributed in 1- and 2-cell embryos as well as in about half of the 4-cell embryos. In the remaining half of the 4-cell embryos and in older embryos, pal-1 mRNA is more abundant in the posterior region. Because zygotic pal-1 mRNA synthesis begins only after the 24-cell stage, the asymmetric distribution ofpal-1 RNA cannot be due to transcriptional regulation and is most likely caused by differential RNA stability. The distribution of the PAL-1 protein during embryogenesis was analyzed by immunostaining. The earliest stage at which the protein was detectable is the 4-cell stage, and then only in the two posterior blastomeres. At subsequent developmental stages, PAL-I was only detected in the descendants of the two posterior blastomeres. After the 24-cell stage, when zygotic pal-1 expression is activated, PAL-I protein abundance increases in the posterior part of the embryo (Hunter and Kenyon, 1996). Formation of the pul-1 mRNA gradient in early embryos appears to involve the degradation of the transcripts specifically in the anterior region. Degradation is thought to follow translational repression mediated by the binding of the MEX-3 protein to the pal- 1 3’UTR (Hunter and Kenyon, 1996). This model is supported by the following observations. ( I ) Embryos were injected either with a hybrid [lacZ coding::pal-I 3’UTRJ RNA or with a control lac2 RNA (lacking pal-1 sequences). Injection of the hybrid RNA resulted in p-galactosidase activity only in the posterior region, whereas injection of control RNA resulted in uniform pgalactosidase activity (Hunter and Kenyon, 1996). (2) The mex-3 gene encodes a putative RNA-binding protein that is preferentially localized in anterior blastomeres. In mex-3 mutant embryos the PAL- 1 protein is ectopically expressed. (Draper et al., 1996). (3) When the [lacZcoding::pal-13’UTRI RNA was injected into mex-3 mutant embryos, no gradient of P-galactosidase activity was detected (Hunter and Kenyon, 1996). This is in contrast to injections in wild-type embryos [see ( I ) ] . Binding of MEX-3 protein to the pal-I 3’UTR remains to be directly demonstrated. (4) The degradation of pal-1 mRNA is also dependent on smg-3 function since in smg-3 mutant embryos pal-I mRNA is expressed ectopically (Hunter and Kenyon, 1996). The C. elegans smg genes are required for the degradation of untranslated mRNAs or mRNAs with premature stop codons (Pulak and Anderson, 1993).

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IV. Regulation of mRNA Stability in Drosophila The initial steps of Drosophila embryogenesis are directed by maternally expressed genes. Many of these genes, such as bicoid, are involved in anteriorposterior and dorsal-ventral axis determination. Another maternal gene, string, is required for early nuclear divisions. The earliest zygotically-expressed genes belong to the gap group of genes, which function in pattern formation in relatively broad, but spatially restricted, regions of the embryo. The hunchback and caudal genes belong to this class (both of these are expressed both zygotically and maternally). Shortly after gap gene expression, the pair-rule group of genes is activated. These genes, of which fushi tarazu (ftz) is an example, are required for the formation of every other embryonic segment. The bicoid gene encodes a homeobox-containing transcription factor that is essential for the determination of anterior embryonic structures (Berleth et al., 1988). bicoid transcription is strictly maternal. The mRNA localizes to the anterior tip and remains untranslated during oogenesis. When translation is activated at fertilization, the protein forms a gradient, the concentration being highest at the anterior tip and decreasing posteriorly as the protein diffuses and gets degraded. (Berleth et al., 1988; Driever and Nusslein-Volhard, 1988). In ovaries the bicoid mRNA has a 70-nucleotide-long poly(A) tail. After fertilization the poly(A) tail length increases, peaks at 140 nucleotides at around 1-1.5 h, and then progressively shortens to 70 nucleotides (Salles et al., 1994). Thus, translational activation of bicoid mRNA appears to be regulated by cytoplasmic polyadenylation, a common mechanism for activating maternal mRNAs at fertilization (reviewed by Richter, 1996; Wickens et al., 1996). The stability of the bicoid mRNA is developmentally regulated. bicoid mRNA abundance is constant until cellularization of the blastoderm (about 2.5 h after fertilization) and then decreases abruptly and disappears (Surdej and Jacobs-brena, 1998). Since the gene is not transcribed in embryos, this must mean that the bicoid mRNA is stable during the initial stages of embryogenesis and that message degradation is activated at the cellular blastoderm stage. Message degradation temporally coincides with the activation of zygotic transcription, suggesting a connection between the two events. In contrast, there seems to be no relationship between bicoid mRNA translation and stability (Surdej and Jacobs-Lorena, 1998). In ovaries, the bicoid mRNA is stable for up to 12 days in retained oocytes of virgin females. The message is also stable for many hours in oviposited (and activated) unfertilized eggs where the bicoid mRNA is fully translated. Thus, bicoid mRNA is stable both when translated (in activated unfertilized eggs, during the first 2 h of embryogenesis) and not translated (during oogenesis, in retained eggs). This is unlike many other rnRNAs, whose translation status seems to be critical for control of mRNA decay. For instance, many mRNAs are stabilized by addition of inhibitors of protein synthesis, as is the case for the Drosophila frz mRNA (Edgar et al., 1986), for the mammalian proto-oncogenes c-myc and c-fos (Green-

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berg et a[., 1986), or when a premature stop codon is introduced in the c-myc transcript (Cole and Mango, 1990). The cis-acting sequences required for the developmentally regulated destabilization of the bicoid mRNA have been mapped. This was done by analysis in transgenic flies of hybrid genes containing bicoid mRNA sequences fused to those of a stable mRNA (Surdej and Jacobs-Lorena, 1998). One major bicoid instability element (BIE) was identified in the 3' UTR. The BIE lies within 43 nucleotides immediately following the stop codon. It is in close apposition to, but distinct from, the nanos-response element, which mediates translational silencing by the Nanos protein (Wharton and Struhl, 1991).The BIE contains a UUUCAUU motif which is present in many developmentally important genes but the significance of this sequence has not been established (Surdej and Jacobs-Lorena, 1998). The hurichback gene is expressed in ovaries and embryos but either maternal or zygotic expression is sufficient for normal development (Lehmann and NiissleinVolhard, 1987). It encodes a zinc-finger transcription factor and is required for differentiation of head and thoracic structures (Tautz et af., 1987). Embryos derived from heterozygous parents were used to follow the fate of the maternal hunchback transcripts in the absence of zygotic transcription. In situ hybridization revealed that at around the 8th nuclear division the distribution of the hunchback transcripts changes from uniform to an anterior-to-posterior gradient in one-quarter of these progeny (the presumed hunchback - homozygous mutants) (Tautz et al., 1987). By the 14th nuclear division (after which the blastoderm cellularizes and the major zygotic activation occurs) maternal hunchback transcripts are not detected anymore. The hunchback mRNA is not translated in ovaries. The protein can be observed for the first time just before pole cell formation (-9th nuclear division) when the protein forms an anterior-to-posterior concentration gradient. Full establishment of the anterior expression domain begins during nuclear divisions 11-12, coinciding with the zygotic expression of the hunchback mRNA. The Hunchback protein expression can no longer be detected at the beginning of gastrulation (Tautz, 1988). The asymmetric distribution of Hunchback protein is a consequence of the complementary activities of the Bicoid and Nanos proteins. Bicoid activates zygotic transcription of the hunchback gene only in the anterior part of the embryo, whereas Nanos blocks the translation of maternal hunchback mRNA only in the posterior (Tautz, 1988). The maternal nunos mRNA is localized exclusively at the posterior tip of the egg. Similarly to the bicoid mRNA, translational activation of the naizos mRNA occurs soon after egg deposition to produce a posterior-to-anterior gradient of Nanos protein (Wang and Lehmann, 1991; Wang et al., 1994). By blocking the translation of the uniformly distributed maternal hunchback mRNA, the Nanos protein effectively creates an anterior-to-posterior gradient of Hunchback protein (Tautz and Pfeifle, 1989). Translational regulation of the hunchback nlRNA by Nanos is mediated by two cis-acting sequences (GUUGU and AUUGUA) mapped to the 3'UTR and

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collectively called the nanos-response element (NRE) (Wharton and Struhl, 1991). Accordingly, either mutations in the nunos gene or alterations of the hunchback NRE result in the ectopic translation of hunchback mRNA in the posterior half of the embryo. Posterior hunchback expression causes a switch to anterior fate and the complete lack of abdominal segments (Tautz and Pfeifle, 1989; Lehmann and Niisslein-Volhard, 1991; Wharton and Struhl, 1991). In addition to Nanos, translational repression of hunchback also requires the product of the pumilio gene. In contrast to Nanos, the Pumilio protein is distributed uniformly throughout the embryo (Macdonald, 1992). In embryonic extracts the Pumilio protein and a separate 55-kDa protein can bind specifically to NRE sequences, even in the absence of the Nanos protein (Murata and Wharton, 1995). Furthermore, it was shown that ( I ) the RNA-binding domain of Pumilio (termed PUM-HD) corresponds to a 334-amino acid region of the 158-kDa Pumilio protein that is conserved in animals, plants, and fungi (Zamore et al., 1997); and ( 2 ) Nanos binds RNA with high affinity in vitro,but with little sequence specificity (Curtis et al., 1997). RNA-binding activity is associated with two C-terminal metal-binding domains of Nanos. Disruption of these domains abolishes Nanos translational repression activity in vivo. (Curtis et al., 1997). Together, these data suggest that Pumilio and/or other RNA-binding factor(s), but not Nanos, initially recognize the NRE. Nanos might then be recruited, through a combination of protein-protein and protein-RNA interactions, to assemble a highly specific complex (Curtis et al., 1997). Recent evidence indicates that translational repression of the hunchback mRNA is mediated by the shortening of the poly(A) tail. The developmental polyadenylation profile of the hunchback mRNA is similar to that of the maternal bicoid mRNA. In oocytes the hunchback mRNA has a poly(A) tail of approximately 30 nucleotides. As translation is activated between 0.5 and 1.5 h after egg deposition, the poly(A) increases to approximately 70 nucleotides. Embryos lacking Nanos or Pumilio protein have a longer poly(A) tail relative to wild type ( I 00 nucleotides in nanos embryos versus 70 nucleotides in wild-type embryos). However, if Nanos protein is made to be expressed throughout the embryo (nunos""'),the hunchback mRNA is not further polyadenylated after fertilization and remains at a length of 30 nucleotides. This indicates that nanos activity either blocks polyadenylation or promotes deadenylation. To distinguish between the two alternatives, one of the processes needs to be selectively disrupted. Accordingly, a hunchback mRNA lacking a polyadenylation element but containing a 60-nucleotide poly(A) tail was synthesized in v i m and injected into embryos. This mRNA was deadenylated in the presence of nanos activity but not in its absence, indicating that nanos promotes active deadenylation of hunchback mRNA (Wreden et al., 1997). The caudal gene encodes a homeobox-containing transcription factor that is essential for the specification of the abdominal segments (Mlodzik and Gehring, 1987). The gene is expressed during oogenesis and embryogenesis and, unlike

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hunchback, both maternal and zygotic expression is required for normal embryonic development (Macdonald and Struhl, 1986). At fertilization the caudal transcript is uniformly distributed through the egg. The transcripts appear to be associated with nuclei and surrounding cytoplasm but excluded from the yolk. During migration of nuclei toward the periphery of the embryo around the 9th mitotic division, the cytoplasm comigrates with the nuclei, carrying with it the maternal transcripts. At this point there is no apparent difference in caudal transcript distribution along the anteroposterior axis. During the syncytial blastoderm (9th to 13th nuclear divisions) there is a striking change of caudal transcript distribution with the formation of a concentration gradient with a peak at the posterior of the embryo. The mechanism of gradient formation cannot be deduced from these data. However, specific degradation of the transcripts at the anterior pole may be involved. This is suggested by in situ hybridization data with a caudal probe that showed that during gradient formation the number of silver grains decreased at the anterior pole while they remained constant at the posterior pole (Mlodzik et al., 1985, Mlodzik and Gehring, 1987). The accumulation of the Caudal protein during early embryonic development was analyzed by immunofluorescence (Mlodzik and Gehring, 1987; Rivera-Pomar et al., 1996). The protein is not detected in embryos up to 1-1.5 h after fertilization. Just prior to pole cell formation the Caudal protein begins to accumulate in the posterior of the embryo and at the syncytial blastoderm stage the protein is easily detectable in a posterior-to-anterior concentration gradient. The Bicoid protein has been implicated in the direct translational repression of caudal mRNA and in the formation of the posterior-to-anterior Caudal gradient. In vitro cross-linking experiments of caudal mRNA to nuclear proteins from wildtype and bicoid mutant embryos have shown that the Bicoid protein can bind specifically to a 120-nucleotide caudal mRNA sequence termed Bicoid binding region (BBR). The BBR spans the 3' end of the caudal protein coding region plus the first nucleotides of 3'UTR (Rivera-Pomar el al., 1996). The functional significance of the in vitro interaction between Bicoid and the caudal mRNA was assessed by cotransfection experiments of Drosophilu tissue culture cells with a mixture of a plasmid encoding the Bicoid protein plus reporter genes containing or not caudal 3'sequences. In these assays, chimeric mRNAs containing the BBR sequence were translated sevenfold less efficiently than control mRNAs without BBR. These results suggest that the Bicoid protein suppresses the translation of BBR-containing mRNAs and are consistent with previous observations that in embryos lacking bicoid activity, the caudal gradient fails to form and becomes evenly distributed along the embryo (Mlodzik and Gehring, 1987). Similar conclusions were reached by Dubnau and Struhl(1996), although these authors identified a somewhat different 3'UTR regulatory sequence. This sequence, named BRE, for Bicoid-response element, lays within a 553-nucleotide stretch of the caudal 3'UTR (Dubnau and Struhl, 1996, 1997; Chan and Struhl, 1997). The

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BBR/BRE and the NRE appear to have similar functions: while the BBR/BRE mediates translational repression of caudal by Bicoid, the NRE mediates translational repression of hunchback by Nanos. Although it is not clear how degradation of the caudal mRNA is controlled, one may speculate that caudal mRNA degradation in the anterior of the embryo is a direct consequence of Bicoid binding and translational inhibition. Thefushi rurazu (ftz) gene encodes a homeobox-containingtranscription factor that is essential for the development of every other larval segment (Kuroiwa et al., 1984; Laughon and Scott, 1984). In sifu hybridization to early embryos revealed that low levels of evenly distributedftz transcripts are first detected after the 9th nuclear division (about 90 min postfertilization). Shortly thereafter, theftz mRNA is distributed in a broad band between 15 and 65% egg length (where 0% is the posterior tip). Just before cellularization, this pattern is resolved into three broad stripes followed shortly thereafter by the formation of the “mature” pattern of seven evenly spaced stripes. Expression of frz in seven stripes is short-lived and noftz mRNA is detectable by 4-5 h of development (Hafen er ul., 1984; Yu and Pick, 1995). The FTZ protein is first detected at the cellular blastoderm stage in a seven-stripe pattern and is no longer detected by midgastrulation, which is consistent with the presumed instability of the FTZ protein (Kellerman et al., 1990). At later stages of embryogenesis, theftz mRNA is again expressed, but this time only in selected neurons and in the hindgut (Carol1 and Scott, 1985; Krause et al., 1988). mRNA instability is probably crucial for the formation of the seven-stripe pattern offtz expression. In support of this assertion is the observation that the FTZ protein activates the transcription of its own gene by a positive feedback loop. Control of stripe formation occurs primarily at the transcriptional level: stripes form by cessation of transcription in interband regions coupled to the rapid turnover of the ftz gene products. Considering that ftz is broadly expressed early in embryogenesis and that the protein can activate the transcription of its own gene, it is easy to understand the need forftz mRNA and protein to be both highly unstable.ftz mRNA half-life was measured by injecting a-amanitin into embryos (Edgar et al., 1986). The estimated half-life was 14 min at early stages of expression and then decreased to about 6 min at cellular blastoderm. This makesftz mRNA one of the shortest lived mRNAs among metazoans. The importance offtz mRNA instability during the embryogenesis is further supported by the analysis of the dominantftz mutant, f t z Y This mutation truncates the gene and removes destabilizing elements from the message (see following text). InftzRp‘mutants the RNA persists longer than in wild type and results in a recessive lethalftz phenotype (Kellerman et al., 1990). Sequences required for the destabilization of the ftz mRNA have been mapped by germline transformation of chimeric genes containing ftz mRNA sequences joined to sequences of a stable mRNA and measuring the stability of the corresponding hybrid mRNAs in transgenic embryos (Riedl and Jacobs-Lorena, 1996).

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Two destabilizing elements were identified in the ftz mRNA: one within the 5’ one-third of the message and the other within a 200-nucleotide region at the end of the 3’UTR (the latter termed FIE3 forftz instability element in the 3’UTR). Further deletion analysis identified a 68-nucleotide sequence within the FIE3 that is essential for function (Riedl and Jacobs-Lorena, 1996). The FIE3 has some interesting features: (1) An RNA structure folding program predicts a stem-loop structure. (2) It contains a conserved six-base WUUGUA (where W is A or U) sequence that is also present in the 3’UTR of many other unstable early zygotic mRNAs, including hunchback. Further experiments are needed to understand how the FIE3 promotes ftz mRNA degradation. The fact that cycloheximide stabilizes thejiz mRNA (Edgar et al., 1986) suggests that the degrading activity depends on the synthesis of an unstable protein or that transit of ribosomes along the message is necessary. Interestingly, there is some evidence that the activity that degrades jiz mRNA is developmentally regulated.ftz is a zygotically expressed gene and is normally not expressed during oogenesis. However, when a hybrid frz gene was expressed in ovaries of transgenic flies, the mRNA was completely stable. Upon fertilization or upon in vitro egg activation, the same hybrid transcripts were destabilized (Riedl and Jacobs-Lorena, unpublished). Further experiments are required to determine whether the ovary is an exception, and thefrz mRNA degrading activity occurs in most tissues, or whether this activity is developmentally regulated and restricted to early embryogenesis. The string gene encodes a conserved Cdc25 type tyrosine phosphatase that is a regulator of mitotic initiation in the yeast S. pombe (Edgar and O’Farrell, 1989). String triggers mitosis by dephosphorylating (and thus activating) the Cdc2 mitotic kinase (Edgar et d., 1994a). The active form of this kinase triggers mitotic events such as nuclear envelope breakdown, chromatin condensation, and spindle formation (reviewed by Murray and Kirschner, 1989). In Drosophila, nuclear divisions up to cellularization of the blastoderm are supported by maternally provided string gene product. After this stage, further cell division depends on the regional string expression from the zygotic genome (Edgar and O’Farrell, 1989). Embryos lacking string undergo no mitotic divisions after mitotic cycle 13 and arrest at the G2/M boundary of the 14th cycle, just prior to the first mitosis that requires zygotic transcription (reviewed by Reed, 1995). In situ hybridization and Northern blot experiments indicate that the abundance of the string mRNA does not change from fertilization to cycle 13 and that it is degraded abruptly during the first 30 min of the cycle 14 interphase (Edgar and O’Farrell, 1989). Studies with transcription inhibitors suggested that string transcription is negligible prior to cycle 14, indicating that the early string mRNA is all maternal (Edgar and O’Farrell, 1989). In contrast to the mRNA, which is stable until cycle 14, analysis by immunoblotting showed that the abundance of String protein is very low at fertilization, rises during the first 8 cycles, and then declines gradually (Edgar et al., 1994b). string transcription begins later in the interphase of cycle 14 (25-35 min before the first cells enter mitosis 14) and is limited to

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small patches of cells (the mitotic domains) that are destined to divide. Thus, postblastoderm divisions are anticipated by regional bursts of zygotic string transcription. Since other components of the mitotic cell cycle regulatory apparatus (cdc2, cyclins A and B) are present in excess at this time and since the string message and protein are short-lived, transcription of string is rate-limiting for the postblastoderm divisions (Edgar and O’Farrell, 1989, 1990; Reed, 1995). In an attempt to map genes that control the degradation of maternal string mRNA, embryos were generated that lack one of the major Drosophila chromosome arms. In every case, the maternal string mRNA was degraded on schedule (Myers et al., 1995). One interpretation of this finding is that more than one gene is required for activation of mRNA degradation (or that redundant genes exist) and that they reside in different chromosomes. Alternatively, the factors controlling maternal string mRNA degradation are not zygotically expressed but are maternally derived. To distinguish between these two hypotheses, permeabilized early embryos were incubated in the presence of the transcription inhibitor aamanitin. The degradation of maternal string mRNAs was unaffected by the drug, indicating that degradation of maternal string mRNA is dependent of maternal genes (Myers et al. 1995). However, Edgar and Datar (1996) had results that contradicted this conclusion. The latter group tracked string mRNA stability by injecting a-amanitin, instead of incubating permeabilized embryos, and found that string mRNA persists at a high level for at least 2 h after the time the mRNA is normally degraded. These results support the model that postulates the need for zygotic transcription to activate string RNA degradation. One possible explanation for the discrepancy between the results of Myers et al. and Edgar and Datar is that the embryo permeabilization procedure used by Myers et al. did not inhibit transcription rapidly or completely enough to block the activation of RNA degradation. By injecting a-amanitin the stabilization of string mRNA was highly reproducible but required that a-amanitin be injected throughout the embryo and before cycle 6. When a-amanitin was injected during cycle 13, after the onset of zygotic transcription, degradation of the string mRNA occurred as in controls, and string mRNA was virtually undetectable at 1 h after injection. These results indicate that the transcription of a gene(s) critical for degradation of maternal string mRNA occurs between cycles 6 and 13 (Edgar and Datar, 1996).

V. Regulation of rnRNA Stability in Xenopus Among the best studied cases of regulation of mRNA stability during Xenopus embryonic development is a class of maternal mRNAs, denoted Eg, which are deadenylated and released from polysomes after fertilization but are degraded only later, during the blastula stage. The maternal Egl gene encodes a protein highly homologous to the cell cycle regulators cdc2 of S.pombe (65%) and cdc28 of S. cerevisiae (64%). Egl tran-

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scription is strictly maternal. However, the EGl protein is expressed only transiently. It first appears during oocyte maturation and is degraded after fertilization (Paris et af., 1991). Northern blot analysis demonstrated that Egl transcripts are polyadenylated and stable during oogenesis. After fertilization the RNA is deadenylated and released from polysomes. However, deadenylated Eg 1 mRNA remains stable until the midblastula transition (the time of major transcriptional activation of the zygotic genome), at which time it is rapidly degraded (Paris and Philippe, 1990). Therefore, as is the case for the Drosophifa bicoid mRNA (Surdej and Jacobs-Lorena, 1998), there is no relationship between Egl mRNA translation and stability since it is stable both when translated (in oocytes) and not translated (during early embryogenesis). The apparent temporal uncoupling between deadenylation and degradation is of particular interest because it may shine light on the functional relationship between the two processes. For instance, deadenylation and degradation could require different cis-acting elements. Moreover, the delayed degradation of Eg 1 mRNA could be triggered by synthesis or activation of a factor that is absent or inactive in the early embryo. Another possibility is that deadenylation is sufficient to trigger degradation but that at least one component of the degradation pathway is absent or limiting during early cleavage stages. To examine sequences that promote embryonic deadenylation of Egl mRNA, chimeric polyadenylated mRNAs containing different regions of 3’UTR Egl were synthesized and injected into fertilized eggs and the poly(A) tail length of the hybrid mRNAs was measured 1.5 and 3.0 h later. These experiments identified two sequences required for embryonic deadenylation. One is 80 nucleotides long and is situated upstream of the CPE and the other sequence consists of the message’s terminal 8 nucleotides (Stebbins-Boaz and Richter, 1994). Therefore the postfertilization deadenylation of the Eg 1 mRNA is a sequence-specific process. The maternal Eg2 gene encodes a protein with sequence similarity to Ser/Thr protein kinases (Audic et af., 1997). The Eg2 mRNA displays the same characteristic polyadenylation-deadenylation behavior as the Egl mRNA. The Eg2 mRNA is polyadenylated and translationally activated during oocyte maturation and then deadenylated and translationally repressed after fertilization. The Eg2 transcript remains stable for 6 h after fertilization and is then rapidly degraded at the midblastula transition (Paris and Philippe, 1990). As for the Egl mRNA, Eg2 mRNA deadenylation activity is a sequence-specific process. Deletion of a 17-nucleotide fragment from the 3‘UTR switched the behavior of a chimeric RNA from deadenylation to adenylation (Simon et al., 1993; Simon and Richter, 1994). The sequence has been named EDEN, for embryo deadenylation element, and has a number of U(A/G) repeats. A cis-acting deadenylation element with similar characteristic has also been reported for an1998). other Eg mRNA (Eg5; Paillard et d., A possible candidate for a factor that directs Eg-specific deadenylation has been identified. This is the Eg-specific binding factor p53/p55 that is detected as a doublet in UV cross-linking experiments (Legagneux et af., 1992). Deletion of the

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aforementioned 17-nucleotideEDEN from the Eg2 3’UTR abrogated the binding of the factor as well as the deadenylation of the chimeric RNA in embryos (Bouvet et ul., 1994). The p53/p55 protein also binds to the Egl 3’UTR (Legagneux et al., 1992) and to the cis-acting deadenylation element of Eg5 mRNA (Paillard et al., 1998). Recently, the gene encoding the p53/55 factor was cloned and termed EDEN-BP (embryo deadenylation clement-binding protein). This doublet (p53/ p55) contains three putative RNP-type RNA recognition motifs (Paillard et al., 1998). Together, these observations suggest that EDEN-BP binds to a specific sequence within 3‘ UTR of Eg mRNAs and promotes the deadenylation in the embryo. Audic et al. (1997) also investigated cis-acting sequences required for the degradation of the Eg2 mRNA at midblastula transition. The authors showed that ( I ) chimeric RNAs were degraded only if they had been previously deadenylated; (2) RNAs containing either the globin or the chloramphenicol acetyl transferase (CAT) coding sequence but no poly(A) tail, and devoid of a polyadenylation site or deadenylation element, were degraded at a rate similar to that of the maternal Eg2 mRNA; and (3) an RNA lacking a poly(A) tail but containing a polyadenylation element was protected from degradation due to in vivo polyadenylation. These experiments indicate that deadenylation is sufficient to cause sequence-independent (globin, CAT) degradation at midblastula transition. Thus, the degradation of the Eg2-derived RNAs does not appear to require any specific sequence information other than that necessary to achieve deadenylation. The Xlhbon2B gene has a homeodomain with 98% identity to the Drosophila Antennapedia class homeodomain (Muller er al., 1984). It also has sequence similarity to cyclins (Howe et al., 1995; Howe and Newport, 1996).Xlhbox2B mRNA abundance is high during early oogenesis and decreases to a low level at the end of oogenesis. The message is not detectable at early stages of embryogenesis and becomes detectable again after the gastrula stage (Muller et al., 1984; Wright er ul., 1987). Interestingly, the Xlhbox2B mRNA is degraded during oogenesis by endonucleolytic cleavage within the 17-nucleotide ACCUACCUACCCACCUA sequence in the 3’UTR (Brown et al., 1993). An endonuclease that binds to this region was partially purified from Xenopus oocytes (Brown et al., 1993; see also Surdej er al., 1994).

VI. Regulationof mRNA Stability in Avians The regulation of two classes of mRNA stability during avian development will be reviewed here: basic fibroblast growth factor (bFGF) and collagens. bFGF regulates growth and differentiation of a variety of cell types in the embryo and also in cell cultures. For example, FGFs and their receptors are required for gastrulation, for the regionalization of the posterior mesoderm, and for the patterning of the limb, midbrain, and otic vesicle (Borja et al., 1996). Expression of bFGF during chick embryonic development is complex, three

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classes of transcripts having been recognized: (1) the authentic bFGF sense transcript, (2) at least three RNAs generated by alternative splicing of exon 1 (alttranscripts), and (3) two antisense transcripts. Analysis of expression of the three classes of transcripts during development revealed that in adult chickens the abundance of bFGF sense and antisense RNAs is inversely proportional, in agreement with the proposed negative regulation of bFGF mRNA stability by antisense transcripts (Borja et al., 1993). The collagen genes encode a family of proteins sharing common structural features. Collagens are the basic building blocks of extracellular matrices and basement membranes in practically every tissue of the body. Sequence comparisons identify different types of collagens. Type I is the most common and has been extensively analyzed. Type I collagen consists of two subunits, a 1 and a2, each encoded by a different gene (al(1) and a2(I), respectively). Type I1 collagen is a homotrimer of al-type collagen protein chains encoded by the al(I1) gene (Mayne and Burgeson, 1987; Pallante er al., 1996). The process of long bone formation during chicken embryogenesis involves cell differentiation and remodeling of the extracellular matrix. As mesenchymal cells differentiate into chondrocytes, the synthesis of type I1 collagen is activated and that of type I collagen is repressed (Thorogood and Hinchlife, 1975; von der Mark, 1980; Kimura et af., 1985; Schmid and Linsenmayer, 1985). A cell culture system has been developed to study gene expression during differentiation. When embryo tibia chondrocytes are dissociated and cultured in plastic dishes, they adhere to the dishes and dedifferentiate into mesenchymal cells. As the cells dedifferentiate, collagen synthesis switches from type I1 to type I (Castagnola et al., 1988). Dedifferentiation can also be induced by treatment with 5-bromo-2’-deoxyuridine (BrdU) (Askew et al., 1991; Farrell and Lukens, 1995). Conversely, when dedifferentiated cells are transferred to suspension culture on agarose-coated dishes, they revert to the chondrocyte phenotype and resume type I1 collagen synthesis (Dozin et al., 1990). Investigation of al(I), a2(I), and al(I1) gene expression in this in vitro system has shown that all three genes are regulated both at the transcriptional and at the mRNA stability levels. However, the stability of different mRNAs is regulated by different mechanisms. al(1) transcript stability is regulated by expression of antisense RNAs, a2(I) by choice of an alternative transcription initiation, and al(I1) by selective mRNA stabilization (Dozin et al., 1990; Farrell and Lukens, 1995; Pallante et al., 1996).

A. al(1) Collagen Gene Expression

Initial nuclear run-on assays revealed that differentiated chondrocytes actively transcribe the a1(I) gene. This was surprising since these cells do not synthesize type I collagen. The run-on assays had initially been carried out with doublestranded probes. Subsequent analysis with single-stranded probes revealed that both the sense and the antisense strands were transcribed (Farrell and Lukens,

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1995). Furthermore, these studies showed that the antisense RNA is synthesized only in chondrocytes (which do not synthesize type I collagen) and not in cells that synthesize type I collagen such as in intestine, heart, kidney, brain, liver, lens, and retina cells and dedifferentiated cells (after BrdU treatment). Northern blot analysis identified at least two antisense transcripts larger than 10 kb (Farrell and Lukens, 1995). Although the complete structure and function of these antisense transcripts remain to be determined, their specific expression in chondrocytes strongly suggests a role in downregulation of al(1) gene expression. The antisense transcripts could act by enhancing the rate of degradation and/or by reducing the translatability of the al(I) mRNA (Farrell and Lukens, 1995).

B. a2(I) Collagen Gene Expression

As for the al(1) gene, nuclear run-on assays indicated that the a2(I) gene is transcribed in chondrocytes, even though no type I collagen is synthesized. However, in this case regulation seems to occur through the use of alternative promoters. Chondrocytes utilize an alternate downstream promoter that results in the synthesis of a transcript (named alternative mRNA) that is truncated at the 5’ end. This message is chondrocyte specific and does not encode a collagen protein because of a change in the translational reading frame (Pallante et al., 1996). The alternative message is less stable (-3-h half-life) than the authentic a2(I) collagen mRNA (-9-h half-life). While most of the sequences of the authentic and alternative mRNAs are common, the reason for the decreased stability of the alternative transcript is not understood. It could be due to instability-conferring elements in the sequence unique to the alternative RNA or to a premature stop codon created by the frame shift.

C. al(I1) Collagen Gene Expression aI(I1) mRNA is barely detectable in mesenchymal cells and abundant in differentiated chondrocytes (Kravis et al., 1985). This is in agreement with the sharp increase in al(I1) gene transcription during in vitro differentiation of chondrocytes (Castagnola et al., 1988). The stability of the al(I1) mRNA was measured in the same cell culture system by blocking transcription with actinomycin D. While the al(I1) mRNA was relatively unstable (-5-h half-life) in undifferentiated mesenchymal cells and in early differentiating cells, it was quite stable (-30-h half-life) in fully differentiated chondrocytes (Dozin et al., 1990). These data suggest that stabilization of the al(I1) mRNA is a relatively late event during chondrogenesis. Taken together, induction of l(II) collagen mRNA expression seems to occur by transcriptional activation at early stages and by both, transcriptional activation and mRNA stabilization, at later stages of differentiation.

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189 Changes of collagen mRNA half-life have been described in other systems, although the effect was not correlated with the process of differentiation but rather triggered by exogenous signals. For example, transforming growth factor-(j was shown to stabilize al(1) collagen mRNA in confluent mouse 3T3 fibroblasts (Penttinen et al., 1988) and cortisol was shown to reduce the half-life of al(1)and a2(I) collagen mRNAs in human skin fibroblasts (Hamalaien et al., 1985).

VII. Regulation of mRNA Stability in Mammals In mammals not much information is available on the regulation of mRNA stability during development. This may be in part because development occurs in utero (making external observation or manipulation difficult) and in part due to the limited availability of sufficient amounts of research materials. One critical developmental event during early embryogenesis is the switch from maternal to embryonic control of gene expression, designated as MZT (maternal to zygotic transition). The MZT marks the time of major transcriptional activation from the zygotic genome. Also, it is during (and to some extent prior to) MZT that a considerable proportion of stored maternal mRNAs are degraded. The MZT is conceptually similar to the midblastula transition in Xenopus or to the transition from maternal to zygotic gene expression in Drosophila. In the mouse, MZT occurs 24 h postfertilization, at the G2 phase of the two-cell stage (Flach et al., 1982). However, degradation of maternal mRNAs starts during oocyte maturation and continues after fertilization, and by the mid-two-cell stage, most of the maternal poly(A)-containing mRNA has been degraded (Piko and Clegg, 1982). This decrease in maternal mRNA coincides with a fourfold increase in the rate of mRNA synthesis directed by the embryonic genome (reviewed by Telford et al., 1990). In mammals, balanced expression of X-linked genes is achieved by the random inactivation of one of the female X chromosomes during early embryonic development (reviewed by Heard et al., 1997). X-chromosome inactivation requires the expression of the Xisr gene from the inactive X chromosome’s X-inactivation center. The Xist RNA is 15 kb long, is nuclearly localized, and is devoid of any long open reading frame. Female embryonic stem (ES) cells contain two active X chromosomes (Xa), one of which is inactivated upon differentiation. Prior to X inactivation, Xist is expressed at low levels from both (Xa)s in female cells and from the single Xa in male cells. However, in differentiated female ES cells or in female somatic cells Xist is expressed only from the inactive X chromosome (Xi). During the process of differentiation there is an intermediate stage at which Xist is expressed preferentially (but not exclusively) from the inactive X chromosome. Thus, X inactivation appears to involve the following pattern of Xist expression: low-level biallelic expression in cells prior to differentiation, a transient stage of differential biallelic expression at the time when one X chromosome is chosen for

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inactivation,and, finally, a stable pattern of high-level monoallelic Xist expression in somatic cells. That activation of Xist expression is achieved by the stabilization of Xist transcripts at the Xi chromosome rather than by the stimulation of its transcription is supported by the following recent evidence: (1) Nuclear run-on assays which compared transcription rates between cells with a high level of Xist expression (such as female somatic cells) and cells with a low level of Xist expression (such as male ES cells) indicated that Xist mRNA is transcribed at similar rates in all cell lines (Panning ef al., 1997). (2) RNA slot blot analysis on the same population of cells after treatment with the transcriptional inhibitor actinomycin D showed that the abundance and half-lives of unspliced Xis? RNA are similar in female fibroblasts and male ES cells. Yet, these cells differ dramatically in the accumulation and stability of the spliced Xist RNA: the spliced RNA is present in female cells after 12 h of actinomycin D treatment, whereas it could not be detected in male cells after 2 h of drug treatment. Furthermore, the abundance of spliced Xisr RNA level in different ES cell fractions was measured by RT-PCR. As expected, Xist RNA was not detected in either the cytoplasmic or poly(A)selected fractions of female fibroblasts and was abundant in the nuclear and chromatin fractions. In male ES cells, Xisr RNA was detected only in the chromatin fraction and only if large amounts of RNA were assayed suggesting that Xisr transcripts are not transported out of the nucleus (Panning et al., 1997). (3) Using RNA fluorescent in situ hybridization, Xist RNA was detected in both X chromosomes of ES cells and following differentiation, accumulation of the transcript on the Xi occurs prior to silencing of the Xa (Sheardown et al., 1997). Taken together, these experiments suggest that the active X chromosome somehow retains the ability to rapidly degrade its own Xist RNA, while the Xisr transcripts are stabilized on the other X chromosome. Stable Xist RNA associates with its X chromosome of origin where it in some way establishes an inactive chromatin state. Failure of Xist to accumulate on the Xa would preserve a transcriptionally active state (reviewed in Kuroda and Meller, 1997). In general, mechanisms that regulate transcript stability are thought to involve active transcript destabilization whereas in the case of Xisr, active stabilization seems to be involved (recall that the Xist transcripts are initially rare and expressed from both chromosomes and that at differentiation the transcript accumulates in one but not in the other chromosome). Differential stability could be explained either by availability of a regulatory factor or by changes in transcript structure. Both Panning et al. (1997) and Sheardown et al. (1997) attempted to determine whether the primary structure of the stable and the unstable Xist RNAs is the same. Neither group was able to detect any differences between the two, although this is a difficult question to answer with certainty using RT-PCR. One model has been proposed that postulates the involvement of Xist RNA stabilization factors (Panning ef al., 1997). According to this model, three distinct activities are required to regulate the transitions of Xis?expression during X inactivation: (1) factors that both stabilize Xis?RNA and promote its association with the inactive X chromosome, (2) factors that block

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access of the Xist RNA-stabilizing factors to the active X chromosome, and (3) a mechanism, such as DNA methylation, that silences Xist expression from the active X chromosome. These three activities would be induced upon differentiation of pluripotent embryonic cell types and the production of the blocking factor would be transient (Panning et al., 1997). The mammalian Hox genes encode a homeobox-containing transcriptional factor with a helix-turn-helix motif. The proteins are highly conserved, with mammalian and Drosophila orthologous homeodomains often showing greater than 95% amino acid identity (McGinnis and Krumlauf, 1992). Three of these genes, Hoxd-3, Hoxa-11, and Hoxb-3, have been found to give rise to endogenous antisense transcripts (Swiatek and Gridley, 1993; Bedford efal., 1995; Hsieh-Li etal., 1995; reviewed in Knee and Murphy, 1997). Here, we discuss the Hoxa-11 gene. Hoxa-11 is involved in the specification of both appendicular (limb) and axial regions of the body (Small and Potter, 1993). Hoxa-11 mutants show posteriorization of the 13th thoracic vertebra to form a first lumbar vertebra and anteriorization of the sacral region by one segment. In addition, the ulna and radius of the forelimb and tibia and fibula of the hindlimb are dramatically malformed, with extensively altered contour shapes (Hsieh-Li et al., 1995). Investigation of the genomic organization and sequence of Hoxa-11 led to the finding that the antisense strand gives rise to a complex pattern of splicing, suggesting a possible regulatory role for the antisense transcript (Hsieh-Li et al., 1995). Four differentially spliced antisense transcripts were identified, each containing ORFs and having significant overlap with the 5' end of the Hoxa-11 sense transcript. The abundance of ORFs and multiple splicing alternatives suggest that some of the Hoxa-11 antisense transcripts encode protein. By in situ hybridization to limbs (whole mount and serial sections) it was shown that antisense RNAs increase in abundance in regions where sense RNAs diminish in abundance (Hsieh-Li et al., 1995). This pattern of expression is suggestive of a role of the antisense RNA in the regulation of the Hoxa-11 sense transcript stability. The stability of the neurofilament (NF) transcripts may also be regulated during development (Schwartz et al., 1994). The neurofilament protein is the major cytoskeletal component of neurons (reviewed by Lee and Cleveland, 1996). Transgenic mice that overexpress neurofilament protein display motor neuron pathology similar to amnyotrophic lateral sclerosis (Morrison and Hof, 1997). The neurofilament protein is composed of three subunits-light (NF-L), medium (NF-M), and heavy (NF-H)-each encoded by a distinct gene. The three genes are differentially expressed during development. In general, NF expression is low throughout embryonic development and rises in postnatal neurons, coincident with axon enlargement and myelination. A rapid increase in NF-L mRNA abundance normally occurs at postnatal day 2. When transgenic mice were created that carried a NF-L transgene that removed most of the 3'UTR sequences [termed NF-L(-)I, upregulation of the NF-L(-) gene occurred much earlier, at embryonic day 18. In the same mice, temporal

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expression of the wild-type NF-L gene [NF-L(+)] was unaffected. Meanwhile, mRNA stability was measured in cells in culture. RNase protection assays combined with actinomycin D treatment indicated that in cells transfected with the NF-L(-) or the NF-L( +) genes, the NF-L( -) transcript was relatively stable ( 1824 h estimated half-life) whereas the NF-L( +) transcript was considerably less stable (6 h estimated half-life). These results suggest that the 3’UTR has a role in destabilizing the NF-L mRNA. The mechanism by which the 3’UTR confers mRNA instability during embryonic development but not in postnatal development is not understood, though it is likely to involve developmentally regulated destabilizing factors (Schwartz et al., 1995). In humans hemoglobin synthesis undergoes a number of specific switches during embryonic and fetal development that serve to adapt the growing organism to the changing environment. Cower I(<2&2),Portland(~2y2),and Cower II(a2~2) hemoglobins are produced in the embryo, HbF(a2y2) is produced in the fetus, and HbA(a2P2) is produced in the adult (Hofman et al., 1995).The murine and human a-globin gene clusters contain a single embryonic <-globin gene and two coexpressed a-globin genes: a1 and a2. The expression of these genes is developmentally regulated. At the transition between the embryonic and the fetal stages (6-8 weeks in humans, 8.5-10.5 days in mice), a switch occurs from I;- and a-globin coexpression to exclusive a-globin expression. This event is known as <-globin gene silencing. While transcriptionalregulation is important, the full silencing of <-globingene expression also requires sequences in the transcribed region (Liebhaber et al., 1996). Human <-globin mRNA is less stable than a-globin mRNA in transgenic mice. When the 3’UTR of the two mRNAs was swapped, the a-globin mRNA containing the 105-nucleotide <-3’UTR was significantly less stable than authentic a-globin mRNA (Russell et al., 1998). Thus, the globin 3’UTR contains determinants of mRNA stability. a-Clobin is one of the most stable mRNAs known and this is believed to be in part due to the formation of a protective ribonucleoprotein complex in the 3’UTR termed the -complex (Kiledjian et al., 1995).The 3‘UTRs of the a- and <-globin mRNAs contain an identical pyrimidine-rich tract, except that the <-3’UTR is interrupted by a single purine residue. Russell et al. (1998) have shown that this single nucleotide change leads to a reduction of the efficiency of a-complex assembly. Moreover, the decreased affinity of a-complex formation in vitro correlated with the presence of a shorter <-globin poly(A) tail in the haemopoietic cells of the transgenic mice. These results suggest that <-globinmRNA 3’UTR has a lower affinity for a-complex formation, subjecting the unprotected &-globinmRNA to accelerated deadenylation and degradation in adult erythroid cells. The relative inefficiency of the <-globin mRNA a-complex assembly is likely to be less important in embryonic erythroblasts because they contain less competitor a-globin mRNA. Antisense RNAs have been implicated in the regulation of mRNA stability for several mammalian genes, including basic fibroblast growth factor (bFGF) and

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193 some homeobox genes (reviewed by Knee and Murphy, 1997). bFGF is a highly conserved mitogen involved in the regulation of a variety of developmental processes, including mesoderm induction, neurite outgrowth, differentiation, angiogenesis, and wound repair (Basilico and Moscatelli, 1992). The bFGF gene contains three exons which give rise to multiple polyadenylated sense and antisense transcripts in all species examined so far, including human, rat, chicken, and Xenopus (reviewed in Knee and Murphy, 1997). In rats, the sense and antisense transcripts overlap by at least 425 bp at their 3’ and, which is similar to the structural organization in Xenopus (Knee et al., 1997). To address the possible involvement of antisense RNA expression in the regulation of bFGF expression during development, the pattern of sense and antisense bFGF expression was examined in embryonic, fetal, and postnatal rat tissues. In general, the abundance of the sense and antisense transcripts was inversely related. In the developing brain, the 6.0 kb bFGF sense transcript increased in an age-dependent manner from days 3-30 of postnatal development, whereas the 1.5 kb antisense transcript decreased to nearly undetectable level over the same developmental period (Li et af., 1996). These results suggest that abundance of bFGF sense mRNA is negatively regulated by antisense transcripts. It is possible that sense mRNA stability is regulated by the formation of dsRNA hybrids, as postulated for bFGF transcripts in Xenopus oocytes (Kimelman and Kirschner, 1989). Alternatively, antisense RNA may inhibit gene expression at the level of transcript processing, transport from the nucleus, translation, or indirectly by stimulation of dsRNA-dependent serinekhreonine protein kinase (Li er af., 1996). Moreover, the rat bFGF antisense RNA is polyadenylated and contains an open reading frame encoding a predicted 35-kDa protein (Knee and Murphy, 1997). Thus, it is possible that the bFGF antisense has multiple roles, serving both as a regulator of sense RNA expression and as a template for protein synthesis. Although the possible function of this hypothetical protein is currently unknown, its deduced amino acid sequence contains a conserved sequence motif characteristic of the MutT-related family of NTPases, which may be involved in DNA repair (Volk et al., 1989; Koonin, 1993). Albumin is one of only a few known cases of mRNA degradation by endonucleolytic cleavage. Albumin is an abundant liver-specific message. The message is very stable in adults but unstable in fetal liver. S1 nuclease protection assays with mouse liver RNA suggested that albumin mRNA is cleaved at four internal sites as it is degraded. Moreover, the abundance of these degradation intermediates gradually decreases as a function of development such that in adults, these degradation intermediates are not detectable (Tharun and Sirdeskhmukh, 1995). The four cleavage sites were mapped and determined to be located within the coding region of albumin mRNA. Analysis of these sequences revealed the presence of the CCAN,-,CUGN,-,CGAU motif at three of the sites. A computer search for homology comparison with specific cleavage sites reported for other genes (such as apo 11, histone, Xenopus homeobox, IGFII, and groa mRNA) showed no significant similarities (Tharun and Sirdeshmukh, 1995). Further

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characterization of these cleavage sites should prove to be useful for the identification and characterization of the putative nuclease, whose activity appears to be developmentally regulated.

VIII. Conclusions and Prospects Considerable progress has been made toward the understanding of how mRNA stability is controlled during development. In many cases cis-acting sequences have been identified and a connection between length of the poly(A) tail or the translational status of the RNA has been established. However, no unifying pathway for mRNA degradation can be deduced from these studies. Rather, the results indicate that the mechanism of action varies for different mRNAs and different organisms. Cis-acting elements that mediate mRNA degradation are frequently located in the 3’ untranslated regions (UTRs) of mRNAs. In some cases, these regulatory elements appear to activate RNA degradation through deadenylation, as for the Drosophila bicoid and hunchback mRNAs, the C. elegansfern-3 mRNA, the Xenopus for Eg2 mRNA, and the plant TTS mRNAs. In case of the Eg2 mRNA, deadenylation is required (and sufficient) for mRNA degradation but deadenylation and degradation are temporally uncoupled. Temporal uncoupling of poly(A) tail shortening and mRNA degradation was also observed for the BhB 10 mRNA in B. hygida fourth-instar salivary glands (Fontes et al., 1998). In other cases, destabilizing elements act without affecting deadenylation, apparently by activating endonucleolytic cleavage as in the case of the Xenopus Xlhbox2 mRNA and the mammalian albumin mRNA. Yet a different mechanism of mRNA degradation appears to require the synthesis of an antisense RNA, as for bFGF and collagen mRNAs in avians and bFGF and Hoxa-11 mRNAs in mammals. The case of the Xist RNA is unique, since RNA degradation appears to be controlled not only as a function of sex and developmental stage but also, in the same nucleus, as a function of which chromosome (active or inactive X) the RNA is associated with. For many mRNAs the translational status seems to be critical for the control of mRNA decay as evidenced by the stabilization of some mRNAs by a premature stop codon. In other cases inhibitors of protein synthesis block mRNA decay and this may mean either that ribosome transit on the specific mRNA is necessary or that degradation depends on the continuous synthesis of an essential transfactor. In some cases, such as for the Drosophila bicoid or the Xenopus E g l mRNAs, the translational status does not appear to be the primary signal for mRNA degradation since these mRNAs are stable both when translated (in activated unfertilized eggs or in oocytes, respectively) or not translated (in retained eggs or during the first stages of embryogenesis,respectively). Some progress has recently been made in the identification of trans-acting factors that recognize regulatory sequences in the target RNA. Drosophila and

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C. elegans genetics have been instrumental in the identification of such factors and some interesting insights regarding the similarities and differences between these organisms have emerged. For instance, the FBF protein which binds to the 3'UTR of the C. elegansfern-3 mRNA shares homology with hmilio, which binds to the Drosophila hunchback mRNA. The RNA-binding domain of both proteins consists of a stretch of eight tandem repeats with a similar conserved core consensus sequence. In both cases, binding of the protein to the mRNA causes translational repression and RNA destabilization in the posterior region of the embryo. Further, the C. elegans pal-] mRNA and the Drosophila cad mRNA are specifically degraded in the anterior region of the embryo and degradation correlates with translational repression. The pal-] mRNA is recognized by the MEX-3 protein and the cad mRNA by the Bicoid protein. While the two proteins do not share homology, it will be interesting to learn which other factors are involved in RNA recognition and degradation. Also, future research may reveal whether the regulatory circuitry that is beginning to be elucidated for Drosophila and C. eleguns has been conserved in mammals, and thus open new perspectives on how the mammalian embryo progresses through the preimplantation period. Despite the significant progress made, much remains to be learned. One important subject for future research is the elucidation of the nature of the ribonucleoprotein complex formed at the sites of the cis-acting sequences. As mentioned previously, individual proteins have been isolated that recognize certain mRNAs. However, it is likely that the proteins do not act individually, but as a complex. The isolation and characterization of interacting proteins is a direction that promises to bring significant progress to our understanding of mRNA decay. Another important aspect about which hardly any information is available is the nature of the nucleases that function in mRNA decay. Not only do these nucleases need to be identified, but it will be interesting to know how their activity is modulated in development. Thus, a solid foundation has been laid and we can foresee significant progress in the next few years.

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7 EGF Receptor Signaling in Drosophila Oogenesis Laura A. Nilson Department of Molecular Biology Princeton University Princeton, New Jersey 08544

Trudi Schupbach’ Howard Hughes Medical Institute Department of Molecular Biology Princeton University Princeton, New Jersey 08544

1. Overview 11. Introduction

A. Dro.sophi/aOogenesis and Axial Polarity B. Role of the Egfr in Oogenesis Spatial Regulation of Egfr Activation A, Localization ofgurken RNA B. Translational Regulation of gurken C. Posttranslational Regulation of Gurken Activity Response of Follicle Cells to Egfr Activation A. Downstream Targets of the Egfr in the Follicle Cells B. Determination of Multiple Cell Fates within the Dorsal Follicular Epithelium C. Dorsal Patterning along the AP Axis D. Model: Initial Signal from Germline and Refinement within the Follicular Epithelium Determination of Embryonic DV Polarity by Local Egfr Activation A. Role of the Ras Pathway in Embryonic DV Polarity Determination B. Negative Regulation of Ventral Embryonic Cell Fates by Egfr Activity Summary References

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1. Overview The Drosophila homolog of the vertebrate epidermal growth factor receptor (Egfr) is required for cell fate determination in multiple processes during development, including embryogenesis, imaginal disk development, and oogenesis (for I

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review, see Perrimon and Perkins, 1997; Schweitzer and Shilo, 1997). A paradigm for Egfr function in these various contexts has emerged in which the Egfr is broadly expressed, but Egfr activation is spatially restricted. Regulation of Egfr activation is achieved either by localized ligand expression or by localized activation of a uniformly expressed ligand precursor. In the ovary, the Egfr is expressed throughout the somatically derived cells of the egg chamber, but its activity is regulated by the localization of its ligand in the underlying germline. Localized Egfr activation functions first in early oogenesis to establish the anterior-posterior axis of the developing egg chamber and then in midoogenesis to define the dorsalventral axis. Thus the same signaling process polarizes both axes of the developing egg. Determination of egg chamber polarity is essential both for the proper specification of eggshell morphology and for the patterning of the embryo that will develop within. Here we review the spatial regulation of Egfr activation in oogenesis and consider possible mechanisms for localization of its ligand. We then discuss the downstream targets of the Egfr and how these may contribute to the observed pattern of follicle cell fates generated in response to localized Egfr activation. Finally, we consider the process by which determination of dorsal-ventral polarity within the follicular epithelium regulates the establishment of the dorsalventral axis of the embryo.

11. Introduction A. Drosophila Oogenesis and Axial Polarity

The Drosophila ovary consists of a bundle of tubular structures called ovarioles, which contain linear arrays of developing egg chambers (Figs. 1A and 1B). Egg chamber development begins at the anterior end of the ovariole in a distinct region known as the germarium, where germline and somatic stem cells are located. Within the germarium, a germline stem cell divides asymmetrically to produce a cystoblast, which then divides four times to form a 16-cell cyst. Cytokinesis is incomplete in these divisions, and the cells of the cyst remain interconnected by cytoplasmic bridges called ring canals. Only one of these cells becomes an oocyte, and the remainder become nurse cells, which provide RNAs and proteins to the oocyte through the ring canals. The somatic stem cells divide to give rise to the follicle cells, which, in the posterior region of the germarium, envelop an individual germline cyst in a single-layered epithelium to form the egg chamber. Later in oogenesis, the follicle cells provide yolk to the developing oocyte and secrete the eggshell. Based on morphological criteria, oogenesis has been divided into 14 developmental stages, where stage 1 describes a newly formed egg chamber w i h n the posterior of the germarium and stage 14 refers to the most mature egg chambers, located at the posterior end of the ovariole (for a review of oogenesis,

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Fig. 1 Morphology of the Drosophila ovary and egg. (A, B) Confocal images of ovary stained with rhodamine-phalloidin to highlight the actin cytoskeleton. (A) A single ovary is composed of about 15 ovarioles. In this photograph, the germaria are located at the top and the most mature egg chambers at the bottom. Normally the ovarioles are more closely aligned, with the germaria together at the anterior tip of the ovary, but this ovary has been teased apart to better display its organization. Bar is approximately 200 pm. (B) Magnification of an individual ovariole (compare size to ovariole in (A)). Each ovariole is composed of a series of developing egg chambers. The germarium is to the far left, and a midstage egg chamber is to the far right. Each egg chamber is composed of a cluster of interconnected germline cells, 15 nurse cells, and an oocyte, surrounded by an epithelium of somatically derived follicle cells. The oocyte is positioned at the posterior of each egg chamber (right) and grows throughout oogenesis, supplied with RNAs and proteins from the nurse cells, which later degenerate. The follicle cells also degenerate at later stages (not shown), after secreting the eggshell. (C) Dark-field micrograph of a mature egg, dorsal view; anterior is to the left (compare size to mature egg chambers in (A)). Anterior eggshell structures include the micropyle, where the sperm enters (projection at far anterior), two dorsal respiratory appendages, and the operculum, where the larva exits (between the micropyle and dorsal appendages). The surface pattern is derived from the imprints of the overlying follicle cells, which secrete the eggshell. (For a review of oogenesis, see Spradling, 1993.)

see Spradling, 1993). The mature egg exhibits clear asymmetry along both the anterior-posterior (AP) and dorsal-ventral (DV) axes (Figs. 1C and 2A). The anterior and posterior poles are marked by distinct eggshell structures, with a micropyle and operculum at the anterior and an aeropyle at the posterior, and the dorsal side is distinguished by two prominent anterior respiratory structures, the dorsal appendages. DV asymmetry is also evident in the shape of the egg, where the ventral side is curved and the dorsal side is more flat. The polarity of the AP and DV axes, as reflected in the structure of the mature egg, is established during oogenesis. The AP polarity of the developing egg chamber is visible very early in oogenesis, when the oocyte assumes a posterior position within the germline cluster, and later in oogenesis it is evident in the localization of certain RNAs within the oocyte and in spatially restricted gene expression

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within the follicular epithelium. DV polarity is first apparent in midoogenesis, with the repositioning of the oocyte nucleus from its initially central posterior location to a new, acentric position along the anterior circumference. The side of the egg chamber closest to the oocyte nucleus will become the dorsal side of the egg chamber. The dorsal follicle cells adopt a distinct pattern of cell fates, and later two dorsolateral subpopulations migrate to give rise to the dorsal appendages. In addition to its role in production of the eggshell, the follicular epithelium also contributes DV patterning information important for the establishment of the DV axis of the future embryo.

B. Role of the Egfr in Oogenesis

The requirement for the Egfr in polarity determination during oogenesis was first recognized through the study of two genes, gurken and torpedo, that are required for DV patterning of the eggshell and embryo (Schiipbach, 1987). Females homozygous for mutant gurken or torpedo alleles are sterile and produce eggs in which the position of the dorsal appendages is shifted dorsally so that a single appendage of reduced length is found at the dorsal midline (Fig. 2B). In extreme cases, this single appendage is severely reduced or absent (Fig. 2C). This eggshell phenotype reflects an expansion of ventral pattern elements at the expense of more dorsal elements, or a “ventralization” of the pattern. Because the eggshell is secreted by the follicle cells, this ventralized phenotype reflects a corresponding shift of cell fates within the follicular epithelium and can also be detected in the ovary using molecular markers for follicle cell fate along the DV axis. Furthermore, this ventralized eggshell phenotype is accompanied by ventralization of the embryo, which also exhibits an expansion of ventral cell fates, indicating that gurken and torpedo are also required for proper establishment of the DV axis of the future embryo. Analysis of mosaic females demonstrated that the gurken gene is required in the germline, even though gurken mutants display defects in patterning of the

Fig. 2 Eggshell phenotypes. (A) Dark-field micrograph of a wild-type egg, lateral view; dorsal is at the top, and anterior is at the left. Two dorsal appendages are located just lateral to the dorsalmost, appendage-free region. (B) Weakly ventralized egg. The dorsalmost region has been eliminated, and the appendages have shifted dorsally and fused to yield a single thin appendage. (C) Strongly ventralized egg. No dorsal appendages are present. Note the presence of a second micropyle, an anterior structure, at the posterior (asterisks). This duplication indicates a failure to determine AP, as well as DV, polarity. (D) Dorsalized egg. The dorsalmost, appendage-free region is expanded, and the appendages have shifted laterally. (E)Dorsalized egg. The dorsalmost region is further expanded, and the appendages have expanded and fused ventrally. Maternal genotypes: A, wild type; B, gurken“”21;C, gurkenffif4#; D, wild type plus four transgenic copies of the gurken gene; E , f s ( I ) K I O !

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follicular epithelium. This finding, and the previous demonstration of a germline requirement for fs(1)KIO (KIO),which is also required for eggshell patterning, indicated that DV patterning must involve a transfer of spatial information from germline to soma (Wieschaus et al., 1978; Wieschaus, 1979; Schiipbach, 1987). In contrast, torpedo is required in the soma, presumably in the follicle cells. This somatic requirement for torpedo suggested a role in the reception of the postulated patterning signal from the germline. Furthermore, the requirement for torpedo in establishing the DV pattern of the embryo indicated that spatial information is also transmitted from the follicle cells back to the future embryo and that therefore intercellular communication between the germline and follicle cells must be bidirectional (Schiipbach, 1987). Molecular analysis of mutant alleles revealed that torpedo encodes the Drosophilu homolog of the vertebrate epidermal growth factor receptor (Egfr), a receptor tyrosine kinase (RTK) known to regulate cell proliferation and differentiation in response to extracellular signals (Price e? al., 1989; Schejter and Shilo, 1989). The Egfr consists of an extracellular ligand binding domain, a single hydrophobic transmembrane region, and a cytoplasmic domain with tyrosine kinase activity (Livneh et al., 1985; Wadsworth et al., 1985; Schejter et al., 1986; Schejter and Shilo, 1989). The finding that torpedo encodes an RTK was consistent with the genetic evidence suggesting that torpedo functions in a process of intercellular communication, acting in the follicular epithelium to receive patterning information from the germline. The gurken gene was a good candidate for a germline ligand for torpedo, since gurken is required in the germline and females mutant for gurken or torpedo display similar oogenesis phenotypes. In fact, molecular analysis of the gurken gene revealed that it encodes a protein with homology to the transforming growth factor (TGF)-a family of vertebrate growth factors (Neuman-Silberberg and Schiipbach, 1993). Since TGF-a is a known ligand of the vertebrate EGF receptor, this finding suggested that gurken is a potential Egfr ligand in the Drosophila ovary. The predicted Gurken protein consists of an extracellular domain with a single EGFlike motif, a putative membrane-spanning region, and an intracellular domain. Although both transmembrane and secreted forms of TGF-a can act as ligands (for review, see Massagut, 1990), it has not yet been determined whether Gurken functions as a membrane-localized Egfr ligand or is processed to a localized secreted form. Whereas the Egfr is expressed in all follicle cells (Kammermeyer and Wadsworth, 1987; Sapir et al., 1998), gurken has a strikingly asymmetric pattern of localization (Neuman-Silberberg and Schupbach, 1993, 1996) (Fig. 3). The gurken RNA and protein are localized to the dorsal anterior corner of the developing oocyte in midoogenesis, coincident with the first morphological signs of dorsoventral polarity. Mutants in which Gurken protein is reduced or absent display a ventralized phenotype, whereas mutants where Gurken is present but mislocalized

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Fig. 3 Distribution of the Egfr and Gurken in midoogenesis. Egfr and Gurken proteins were detected using specific antisera. The Egfr is detected in all follicle cells (top). Gurken is localized to the dorsal anterior corner of the underlying oocyte, adjacent to the oocyte nucleus (bottom). From Sapir et al. (1998, Fig. IC, p. 193), with permission from the Company of Biologists Ltd.

to the entire anterior circumference of the oocyte display a “dorsalized” phenotype, with the expansion of dorsal follicle cell fates at the expense of more ventral fates (Figs. 2D and 2E). Moreover, overexpression of gurken during oogenesis, in females with extra copies of the gurken gene, results in gurken mislocalization and yields a dorsalized phenotype (Neuman-Silberberg and Schupbach, 1994; see Fig. 2D). These mutant phenotypes, and the dorsal anterior localization of this putative ligand, suggest that localized activation of the Egfr within the follicular epithelium by the Gurken protein induces dorsal follicle cell fates. Interestingly, although gurken homozygotes display reduced viability, they do not exhibit the defects in other tissues observed in torpedo mutants, suggesting that gurken function is restricted to oogenesis and that gurken may be an ovary-specific Egfr ligand. Taken together, these results suggested a model for the establishment of the DV polarity of the developing egg chamber. The dorsally restricted distribution of the Gurken protein, a putative Egfr ligand, within the oocyte presumably results in Egfr activation in a dorsal subpopulation of follicle cells. This localized activation of the Egfr determines dorsal follicle cell fates, generating asymmetry within the follicular epithelium and thereby defining its DV axis. In addition to determining the DV pattern of the eggshell, establishment of DV polarity within the follicular epithelium is required for embryonic DV patterning. Thus the spatial information generated by Gurken-Egfr signaling must be later conveyed back to the oocyte to establish the DV axis of the future embryo, indicating that the communication between germline and follicle cells is bidirectional.

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In addition to their role in establishment of DV polarity, further analysis of the gurken and Egfr mutant phenotypes revealed that these genes are also required for determination of the AP axis of the egg chamber (Gonzhlez-Reyes et al., 1995; Roth et al., 1995). In loss-of-function gurken or Egfr mutants, markers normally restricted to certain anterior follicle cells are expressed in follicle cells at the posterior of the egg chamber, suggesting that these cells have failed to assume the proper posterior cell fate and instead have assumed a default anterior fate. This duplication of anterior fates can result in the formation of a second micropyle, an anterior eggshell structure, at the posterior pole (see Fig. 2C). As in DV patterning, the intercellular communication defining AP polarity is bidirectional. Early in oogenesis, the Gurken-Egfr signal is transmitted from the germline to the soma to determine posterior cell fates and proper AP polarity of the eggshell. In addition, these posterior follicle cells convey back to the oocyte a signal required for the correct repolarization of the microtubule cytoskeleton in midoogenesis (Ruohola et al., 1991). The nature of this signal is poorly understood but appears to require the function of mago nashi and DCO, which encodes the catalytic subunit of the Drosophila protein kinase A, in the germline (Lane and Kalderon, 1994; Micklem et al., 1997; Newmark et al., 1997). Thus the determination of AP polarity by Gurken-Egfr signaling is required for proper organization of the oocyte microtubule cytoskeleton, and this in turn determines the localization of certain RNAs within the oocyte as well as the position of the oocyte nucleus. In early oocytes the microtubule cytoskeleton is polarized such that the minus ends are concentrated at the posterior of the oocyte in a mictotubule organizing center (MT0C)-like structure (Theurkauf et al., 1992; Clark et al., 1997). In midoogenesis this posterior MTOC disappears and the microtubule minus ends become concentrated at the anterior margin of the oocyte. This reorganization is associated with the movement of the oocyte nucleus to the anterior cortex of the oocyte, to the future dorsal side. gurken is localized adjacent to the oocyte nucleus and also assumes a dorsal anterior localization. The polarity of the microtubule cytoskeleton is reflected in the localization of certain other RNAs within the oocyte. For example, bicoid RNA is localized to the anterior and oskar RNA is localized to the posterior. In gurken mutants, the defect in posterior follicle cell fate determination results in improper cytoskeletal reorganization. The posterior MTOC persists, resulting in a “bipolar” microtubule organization, which leads the mislocalization of oskur RNA to the middle of the oocyte and bicoid RNA to both ends. In addition, the anterior migration of the oocyte nucleus is often defective, though not in all cases, resulting in its localization to the posterior or middle of the oocyte (Gonzhlez-Reyes et al., 1995; Roth er al., 1995). Interestingly, in these egg chambers gurken remains associated with the oocyte nucleus and is therefore also mislocalized. Thus the determination of AP polarity by Gurken-Egfr signaling is required for proper positioning of the later GurkenEgfr signal determining DV polarity.

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signal 10 follicle cells IAP)

signal back to m y t e

microtubule reorganization

signal 10follicle cells

IDV)

Fig. 4 Determination of AP and DV polarity by Gurken-Egfr signaling.Early in oogenesis, the Egfr is activated in a posterior subpopulation of follicle cells, inducing them to assume a posterior fate (dark gray), instead of a default anterior fate (light gray). This signaling event determines the AP axis of the egg chamber and is required to convey a signal back to the oocyte that results in the repolarization of the microtubule cytoskeleton in midoogenesis. This cytoskeletal reorganization leads to the relocalization of the oocyte nucleus (circle) and Gurken (gray crescent in oocyte) to the anterior of the oocyte, where Gurken-Egfr signaling determines dorsal follicle cell fates and the DV polarity of the egg chamber.

Taken together, these results suggest that localized Egfr activation acts at two spatially and temporally distinct steps to determine the AP and DV axes (Fig. 4; for review, see Lehmann, 1995; Ray and Schupbach, 1996). In early oogenesis, Gurken-Egfr signaling determines posterior follicle cell fates, thereby establishing the AP polarity of the eggshell. A signal back to the germline is required for the later reorganization of the oocyte microtubule cytoskeleton, which in turn is required for the localization of certain RNAs and the anterior migration of gurken and the oocyte nucleus in midoogenesis. At this stage, a second Gurken-Egfr interaction determines dorsal cell fates in the overlying follicular epithelium. This interaction establishes the DV pattern of the follicular epithelium, which both determines eggshell polarity and provides spatial information that will later define the DV axis of the future embryo. In addition to its function in the establishment of the AP and DV axes of the developing egg chamber, the Egfr is also required for the formation of the follicular epithelium and the maintenance of its epithelial character. In gurken or Egfr mutants, some of the egg chambers contain an abnormal number of germline cells or exhibit gaps in the follicular epithelium, suggesting a defect in epithelium formation during the envelopment of each germline cluster (Goode et al., 1996b). The neurogenic genes brainiuc and egghead exhibit similar mutant phenotypes, including ventralized eggshells, and brainiac interacts genetically with gurken (Goode et al., 1992, 1996a, 1996b; Rubsam et al., 1998). Interestingly, some follicle cells in brainiac and egghead mutants exit the epithelium and accumulate in multiple layers, further suggesting a role in epithelial maintenance (Goode et a!., 1996a). This phenotype is also observed in ovaries mutant for mago nashi or for both Egfr and holdup, suggesting a possible role for these genes in the maintenance of epithelial integrity as well (Micklem et al., 1997; Rotoli et al., 1998).

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brainiac, egghead, and mago nashi are required in the germline, while holdup is required in the soma. brainiac encodes a putative secreted protein with homology to glycosyltransferases (Goode et al., 1996b; Yuan et al., 1997), egghead encodes a novel secreted or transmembrane protein (Goode et al., 1996a), and mago nashi encodes a novel nuclear protein (Micklem et al., 1997; Newmark et al., 1997). These genes may function with gurken and the Egfr in a germline to follicle cell signaling process regulating epithelium formation and integrity, perhaps through a role in adhesion between these two cell types (Goode et ul., 1996a, 1996b). Though intriguing, the function of the Egfr in the formation and maintenance of the follicular epithelium, and the relationship between epithelial integrity and follicle cell fate determination, are not well understood and will not be considered further here. In this review we discuss the processes occurring upstream and downstream of the Egfr in oogenesis that mediate both localized activation of the Egfr and its ability to determine follicle cell fates. Since Egfr activation is regulated by the localization of its germline ligand, gurken, we review the requirements for gurken RNA localization as well as accumulating evidence suggesting that gurken is also subject to translational, and possibly post-translational, regulation. We also consider the downstream effectors of the Egfr in oogenesis and the mechanisms by which they mediate determination of the observed pattern of follicle cell fates along the DV axis. Finally, we discuss the issue of how the pattern of follicle cell fates determined by local activation of the Egfr by Gurken generates the spatial information that is later required for determination of the DV axis of the embryo.

111. Spatial Regulation of Egfr Activation The Egfr is broadly expressed and is required for multiple processes in embryogenesis, imaginal disk patterning, and oogenesis (for review, see Ray and Schupbach, 1996; Freeman, 1997; Schweitzer and Shilo, 1997). Egfr function in these various contexts seems to be regulated by the spatial restriction of its ligand. Multiple Egfr ligands have been identified and exhibit some degree of tissuespecific expression or requirement. Three of these, Gurken (Neuman-Silberberg and Schiipbach, 1993), Spitz (Rutledge et al., 1992), and Vein (Schnepp et al., 1996), positively regulate Egfr activity, whereas Argos appears to function as an inhibitory ligand (Freeman et al., 1992b; Schweitzer et al., I995a). Interestingly, the mechanism of localization appears to be different for different Egfr ligands. For example, Spitz is uniformly expressed but only active in a restricted region, presumably by localized processing to an active form (Schweitzer et al., 1995b; Golembo et ul., 1996a). In contrast, Gurken activity is spatially restricted by the localization of its transcript (Neuman-Silberberg and Schupbach, 1993). In this section, we consider the current understanding of the mechanisms by which

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the gurken RNA is localized and the possible role of translational and posttranslational regulation of the Gurken protein.

A. Localization of gurken RNA

The gurken transcript is first detectable early in oogenesis, in the germarium (Neuman-Silberberg and Schiipbach, 1993). The gurken RNA is localized to the oocyte throughout oogenesis and, in early egg chambers, is enriched at the posterior cortex of the oocyte. By midoogenesis, after a transient phase of localization throughout the oocyte and at the anterior margin, the gurken transcript becomes localized to the dorsal anterior cortex of the oocyte, between the oocyte nucleus and the oocyte plasma membrane. This localization persists until later stages of oogenesis. Other examples of localized RNAs involved in polarity determination in Drosophila include bicoid, oskar, and nanos. bicoid is localized to the anterior of the oocyte and is required for the determination of anterior embryonic structures, whereas oskar and nanos are located at the posterior and are required for abdomen formation (for review, see St. Johnston, 1993). For these RNAs, the 3' UTR is necessary and sufficient for proper localization, and certain cellular factors have been identified that interact with these RNAs to mediate their localization (for review, see St. Johnston, 1995; Gavis, 1997). The mechanism of gurken localization, however, is not well understood. In females with multiple copies of a gurken transgene, levels of gurken expression are increased and gurken RNA is partially mislocalized along the anterior circumference of the oocyte. These females produce dorsalized eggs, suggesting that there exists a saturable cellular machinery for localization of gurken mRNA within the oocyte (Neuman-Silberberg and Schiipbach, 1994). However, no cis-acting sequences responsible for gurken RNA localization have been identified. In addition, although gurken RNA is mislocalized in a number of mutants, revealing candidates for trans-acting factors mediating gurken localization, the process remains poorly understood. Among the genes required for wild-type gurken localization, K10 and squid fall into a distinct class. In K10 or squid mutant egg chambers, gurken RNA is not restricted to the dorsal anterior corner, as in wild type, but instead is localized to the entire anterior circumference of the oocyte (Neuman-Silberberg and Schupbach, 1993). As a result, activation of the Egfr by gurken is not restricted dorsally, resulting in expansion of dorsal follicle cell fates around the DV axis and the production of dorsalized eggs. These eggs exhibit dorsal appendages that, instead of being restricted to the dorsal side of the eggshell, are either expanded and shifted laterally, suggesting an expansion of the dorsalmost region of the chorion, or expanded such that dorsal appendage material surrounds the anterior pole of the egg (Figs. 2D and 2E; Wieschaus et al., 1978; Kelley, 1993).

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This mutant phenotype indicates that KIO and squid are required for gurken RNA localization. In principle, the accumulation of gurken at the anterior margin of the oocyte could be a consequence of increased levels of gurken RNA, resulting in saturation of the localization machinery. The level of gurken RNA in squid mutants has not been reported, but no increase in gurken expression is observed in KIO mutant ovaries (Cohen and Serano, 1995; Neuman-Silberberg and Schiipbach, 1993; Neuman-Silberberg and Schiipbach, unpublished data). In addition, genetic data demonstrating that the phenotype of a weak gurken allele is not improved in the absence of K1O function also provide evidence against a role for the wild-type K10 protein in downregulation of gurken RNA levels (Serano et al., 1995). These results suggest that K10 may play a more direct role in gurken mRNA localization. The K10 protein is localized to the oocyte nucleus and is first detected in early oogenesis (Prost et al., 1988; Serano and Cohen, 1995). The predicted KIO protein contains a helix-turn-helix (HTH) motif, a well-characterized prokaryotic DNA binding domain, suggesting that K10 may regulate transcription of genes required for gurken localization (Prost et al., 1988). However, targeted mutagenesis of this domain within the KIO protein and in vivo analysis of the function of the resulting mutants suggest that this motif may not represent a functional HTH domain, and it is unclear whether KIO binds DNA (Cohen and Serano, 1995). Thus, although the mutant phenotype clearly indicates a role for KIO in gurken RNA localization, its function in this process remains unresolved. The squid gene encodes one of the major Drosophila heterogeneous nuclear RNA-binding proteins, or hnRNP proteins (Kelley, 1993; Matunis et al., 1994). These proteins bind to heterogeneous nuclear RNA, or pre-mRNA, and are involved in mRNA biogenesis (for review, see Dreyfuss et al., 1993). Given the genetically defined role for squid in the localization of gurken RNA, this homology is intriguing and suggests that the Squid protein could interact directly with the gurken transcript. The Squid protein is found in the nurse cell nuclei and in the oocyte nucleus, where it colocalizes with the karyosome (Matunis et al., 1994). The squid mutant phenotype and this nuclear localization could suggest that Squid might regulate pre-mRNA processing to generate a form of gurken transcript that is competent for localization; however, no alternative species of gurken RNA have been detected (Neuman-Silberberg and Schiipbach, 1993). Squid is also detected in the oocyte cytoplasm, concentrated near the posterior in the early oocyte and at the anterior and posterior cortex in midoogenesis, suggesting that Squid could function in the cytoplasm to mediate gurken localization. Interestingly, some hnRNPs have been shown to shuttle between the nucleus and the cytoplasm (PiRol-Roma and Dreyfuss, 1992), prompting speculation that Squid might function by delivering the gurken RNA to a cytoplasmic anchor. Analysis of other mutants displaying DV patterning defects has identified additional genes required for localization of gurken RNA. Unlike K10 and squid,

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however, these genes are required for multiple aspects of oogenesis, and their role in gurken localization appears to be indirect. For example, gurken RNA and protein are mislocalized to the anterior margin of the oocyte in the ovaries of females mutant for cappuccino or spire. Consistent with this distribution, these females produce dorsalized eggs and embryos, although this phenotype is variable and ventralization is also observed (Manseau and Schiipbach, 1989; Neuman-Silberberg and Schiipbach, 1993; Emmons et al., 1995). In addition to an effect on gurken, embryos from homozygous cappuccino or spire females exhibit abdominal segmentation defects and lack pole cells, indicative of a defect in posterior RNA localization (Manseau and Schupbach, 1989; Manseau et al., 1996). The variable effect on dorsoventral polarity and the mutant embryonic phenotypes suggest that cappuccino and spire play a more general role in the organization of the egg chamber. In fact, the primary defect in egg chambers mutant for cappuccino and spire seems to be in the organization of the oocyte cytoskeleton. Studies of microtubule distribution in mutant egg chambers revealed a premature reorganization of tubulin filaments into long bundles around the oocyte cortex, coincident with a premature onset of streaming of the oocyte cytoplasm (Theurkauf, 1994; Emmons et al., 1995). These events normally occur at a later stage of oogenesis, after DV polarity has been determined. These mutant phenotypes suggest that cappuccino and spire regulate this cytoskeletal reorganization. It has been proposed that the premature streaming observed in cappuccino and spire mutant egg chambers disrupts the localization of patterning components such as gurken and oskar and therefore could be the underlying cause of the mutant phenotypes (Theurkauf, 1994). However, this interpretation is not entirely straightforward, because no demonstrable difference has been observed in the speed or timing of streaming between cappuccino alleles exhibiting strong or weak defects in DV patterning (Emmons el al., 1995). Molecular analysis has shown that Cappuccino is a member of a group of proteins related to vertebrate formins (Emmons et al., 1995). These proteins have been implicated in actin-mediated processes, such as cytokinesis and polarized cell growth, and may function at least in part by recruiting and directly binding to profilin, an actin-binding protein (for review, see Frazier and Field, 1997). Mutations in chickadee, which encodes Drosophila profilin (Cooley et al., 1992), yield some of the same phenotypes as mutant cappuccino alleles, including premature streaming of the ooplasm, and cappuccino and chickadee interact in a yeast twohybrid assay (Manseau et al., 1996). Furthermore, disruption of the actin cytoskeleton during midoogenesis with cytochalasin D also induces premature ooplasmic streaming. Taken together, these results suggest that there is an interaction between the actin and microtubule cytoskeletons, either direct or indirect, such that a change in the actin cytoskeleton induces an effect on microtubule organization. Thus the observed microtubule defects may reflect a primary role for Cappuccino in regulation of the actin cytoskeleton (Manseau er af., 1996).

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Like cappuccino and spire, midstage egg chambers mutant for maelstrom also exhibit premature bundling of oocyte microtubules and early ooplasmic streaming (Clegg et al., 1997). These egg chambers exhibit AP and DV patterning defects, consistent with a disruption of Gurken-Egfr signaling. In addition, in earlier stages gurken RNA is not localized to the posterior of the oocyte, as in wild-type egg chambers, but is instead localized to the lateral or anterior margins. Mislocalization of bicoid and oskur RNA is also observed in these stages, suggesting that maelstrom is required for the subcellular localization of multiple RNAs, perhaps via a direct or indirect role in organization of the microtubule cytoskeleton. The interpretation of effects on the microtubule cytoskeleton is potentially complicated by the early role of gurken in AP axis determination. As described earlier, gurken activity is required early in oogenesis for determination of posterior follicle cell fates. These cells, in turn, convey back to the oocyte a signal required for the correct repolarization of the microtubule cytoskeleton in midoogenesis. In gurken mutants, this cytoskeletal reorganization is defective, resulting in an altered microtubule distribution. Thus, in principle, defects in microtubule organization could arise either through a direct effect on cytoskeletal organization or as a result of a defect in Gurken-Egfr signaling. However, the premature bundling of cortical microtubules present in cappuccino, spire, and maelstrom mutant egg chambers is not observed in gurken mutants (Clegg er al., 1997), suggesting that the defective microtubule cytoskeleton of cappuccino, spire, and maelstrom mutants is not simply a consequence of an effect on gurken. Another gene required for gurken RNA localization is orb. Like cappuccino and spire, females homozygous for the orbmerallele produce eggs with fused or absent dorsal appendages and embryos that have variable dorsoventral patterning and abdominal segmentation defects and lack pole cells (Christerson and McKearin, 1994). In orb mutant egg chambers, gurken RNA is not localized to the dorsal anterior corner but is instead mislocalized to the anterior margin (Christerson and McKearin, 1994; Roth and Schupbach, 1994). Unlike the distribution in K10 and squid mutants, gurken RNA is found throughout the anterior cytoplasm in orb mutants rather than being restricted to the cortex (Roth and Schiipbach, 1994). In addition, oskar RNA is detected throughout the oocyte cytoplasm in orb mutants, instead of at the posterior pole, consistent with the observed abdominal segmentation defects (Christerson and McKearin, 1994). These pleiotropic effects suggest that orb is required for the localization of multiple RNAs. The Orb protein contains predicted RNA recognition motifs (Lantz et al., 1992), suggesting that Orb may interact directly with RNA and thereby mediate the localization of gurken and other transcripts. In addition, Orb is a homolog of a Xenopus CPEB protein, which binds to the 3’-UTR of certain mRNAs in the Xenopus oocyte and regulates their translation by mediating cytoplasmic polyadenylation, suggesting a possible role for Orb in translational regulation (Hake and Richter, 1994). In fact, levels of Gurken protein are reduced in orb mutants

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(Neuman-Silberberg and Schupbach, 1996; see Section IIIB). These observations have led to the proposal that Orb may function to both localize certain RNAs within the oocyte and to regulate their translation (Christerson et al., 1995). In summary, a number of genes required for the correct localization of gurken RNA have been identified, and for some of these there is considerable molecular and functional data. However, the mechanism by which gurken RNA is localized to the dorsal anterior corner of the oocyte remains unclear. Some of these factors, such as Squid and Orb, may bind directly to the gurken transcript, whereas others, such as Cappuccino, Spire, and Maelstrom, may function indirectly, through organization of the oocyte cytoskeleton. Finally, although localization sequences have been found in the 3’-UTRs of other localized RNAs, little is known about the cis elements required for gurken localization.

B. Translational Regulation of gurken Within the developing oocyte, the Gurken protein is also spatially restricted, and its distribution closely resembles the RNA localization pattern (Roth et al., 1995; Neuman-Silberberg and Schupbach, 1996). Gurken protein is first detected in the germarium and is localized to the developing oocyte throughout oogenesis. In early egg chambers, most of the Gurken protein exhibits a punctate staining in the ooplasm, suggestive of localization to the secretory apparatus and consistent with the prediction that Gurken is an exported protein. By midoogenesis, Gurken is localized exclusively to the dorsal anterior cortex and is colocalized with membrane-associated F-actin (see Fig. 3). This pattern of localization is consistent with the proposed role for gurken in determination of posterior follicle cell fates in early oogenesis and dorsal follicle cell fates in midoogenesis. This correspondence between RNA and protein localization suggests that Gurken protein localization is determined largely by the localization of the gurken RNA. Other localized RNAs, such as bicoid, oskar, and nanos, are regulated at the level of translation, presumably to ensure their expression at the right place and time (Gavis and Lehmann, 1994; for review, see Macdonald and Smibert, 1996). Accumulating evidence suggests that gurken expression is also translationally regulated. Analysis of mutants exhibiting DV patterning defects similar to those seen for gurken mutants has identified candidate positive regulators of gurken translation. These genes include aubergine, encore, maelstrom, okra, orb, vasa, and the spindle group (spindle-A, spindle-B, spindle-C, spindle-D, and spindle-E/homeless) (Christerson and McKearin, 1994; Wilson et al., 1996; Clegg et al., 1997; Ghabrial et al., 1998; Gonzhlez-Reyes er al., 1997; Hawkins et al., 1997; Styhler et al., 1998; Tomancak et al., 1998). Mutant females produce ventralized eggs, suggesting that these genes are required for Gurken-Egfr signaling. However, in most cases the levels of gurken RNA appear relatively normal.

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In contrast, Gurken protein levels are decreased in each of these mutants. Although an effect on the stability of the Gurken protein has not been ruled out, these genes appear to regulate translation of gurken RNA. Most of these mutants also exhibit defects in gurken RNA localization, although for the most part these defects are mild. In egg chambers mutant for encore, maelstrom, orb, okra, spindle-B, or spindle-D, gurken RNA is often detected along the anterior of the oocyte in addition to its normal dorsal anterior localization (Christerson and McKearin, 1994; Clegg et al., 1997; Gonzdez-Reyes et al., 1997; Hawkins et al., 1997; Ghabrial et al., 1998). However, these defects in gurken RNA localization are not sufficient to account for the ventralized phenotypes observed in these mutants. In fact, anterior mislocalization of gurken would be expected to result in dorsalization, as in KIO and squid mutants, instead of ventralization. Thus the observed mutant phenotypes appear to result from a defect at the level of the Gurken protein, rather than the gurken RNA. Moreover, the observation that gurken RNA is mislocalized in a number of mutants affecting Gurken protein levels suggests a possible connection between translation and localization. For example, gurken translation could be required to maintain localization of the gurken RNA. Alternatively, these genes may be required for the translation of other proteins required for gurken RNA localization. The levels of certain other proteins in the oocyte are also decreased in most of these mutants, suggesting that their putative function in translational regulation is not specific for gurken. For example, aubergine, orb, and vasa have been implicated in the translation of oskar as well as gurken. This interpretation is perhaps most straightforward in the case of aubergine. In aubergine mutants, levels of oskar RNA are normal but Oskar protein levels are substantially decreased (Wilson et al., 1996). Mislocalization of oskar RNA is also detected in later stage aubergine mutant oocytes, consistent with the finding that Oskar protein is required to maintain oskar RNA localization at the posterior of the oocyte (Rongo e f al., 1995). Egg chambers mutant for orb or vasa also exhibit a diffuse mislocalization of oskar RNA, and levels of the short isoform of Oskar protein are reduced in vasa mutants, suggesting that these genes regulate the accumulation of Oskar as well as Gurken (Christerson and McKearin, 1994; Markussen et al., 1995; Rongo et al., 1995; Styhler et al., 1998). Similarly okra, spindle-B, and spindle-D, in addition to their putative role in gurken translation, are also required for the accumulation of wild-type K10 protein levels in the oocyte nucleus (Ghabrial et al., 1998; see later). Moreover, this defect in K10 accumulation may account for the defect in gurken RNA localization observed in these mutants. Thus these genes are not specific for translation of gurken and may function in a more general mechanism of translational regulation. The only known candidate for a negative regulator of gurken translation is a conserved RNA-binding protein, Bruno, that binds to specific elements (BREs) in the 3’-UTR of oskar and represses translation of unlocalized oskar RNA (Kim-

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Ha et al., 1995). Bruno is encoded by the arrest gene, and genetic experiments have shown that decreased levels of arrest can enhance oskar activity, providing in vivo evidence for negative regulation of oskar translation by Bruno (Webster et al., 1997). Since the gurken 3’-UTR contains also BRE sequences, it has been proposed that Bruno may also negatively regulate gurken translation. Bruno has been reported to bind to gurken RNA in vitro and to colocalize with gurken RNA in the ovary, but a role for Bruno in regulation of gurken translation has yet to be demonstrated (Kim-Ha et al., 1995; Webster et al., 1997). The molecular nature of some of these genes suggests a possible role in translational regulation through direct interaction with RNA. As already described, the Orb protein is homologous to the Xenopus CPEB protein and therefore has been proposed to regulate translation by interacting with the gurken RNA and mediating poly(A) elongation (Christerson et al., 1995). The vasa and spindle-E/ homeless genes encode members of a family of DEAD box mRNA helicases (for review, see Schmid and Linder, 1992), suggesting a model in which these proteins regulate translation of gurken, and possibly other messages, by binding directly to RNA (Hay et al., 1988; Lasko and Ashburner, 1988; Liang et al., 1994; Gillespie and Berg, 1995). Vasa is similar to eukaryotic initiation factor 4A (Hay et al., 1988; Lasko and Ashburner, 1988), and a yeast protein related to Vasa appears to play a role in translational initiation (Chuang et al., 1997; de la Cruz et al., 1997). However, although Vasa has RNA binding activity in vitro, a direct interaction between Vasa. or Spindle-E/Homeless, and gurken RNA has not been demonstrated. In contrast, Bruno binds both gurken and oskur RNA (Kim-Ha et al., 1995). Interestingly, Bruno has been shown to interact with Vasa in vitro (Webster et al., 1997). Although vasa is required for oskar translation independent of the BRE elements in the 3’-UTR (Rongo et al., 1995), indicating that Vasa does not act solely through Bruno, this interaction could reflect a role for Bruno in blocking the ability of Vasa to initiate translation of unlocalized transcripts (Webster et al., 1997). Alternatively, these proteins could interact with transcripts of other genes that are then required for gurken translation. The recent cloning of the okra and spindle-B genes provides evidence for an additional level of translational control (Ghabrial et al., 1998). As described earlier, mutations in these genes result in reduced or undetectable levels of Gurken protein, suggesting a positive role for these genes in regulation of gurken translation. Interestingly, the molecular nature of these genes suggests that translation of gurken, and perhaps other RNAs, is regulated by events occurring in the oocyte nucleus. okra is the Drosophila homolog of the yeast RAD54 gene, which encodes a DNA helicase, and spindle-B encodes a RADSl-like protein. These proteins are both involved in double-strand break repair, which is required in meiosis for recombination and chromosome disjunction, and these processes are impaired in okra and spindle-B mutants. Mutations in the spindle-D gene, which has not been molecularly characterized, yield similar defects. Given that the accumulation

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of Gurken protein is greatly reduced in these mutants, it has been proposed that progression through the meiotic cell cycle is required for translation of a specific set of messages at certain stages of oogenesis. Thus meiotic checkpoints may exist that act as a clock within the oocyte, coupling certain developmental events to the meiotic cell cycle. Since other steps of oogenesis proceed normally, such as oocyte growth and the uptake of yolk, such a mechanism would operate independently of the regulation of certain other processes. Analysis of some of these mutants has also revealed that gurken translation may be differentially regulated at different stages of oogenesis. Although Gurken protein is present throughout oogenesis in wild-type ovaries, certain combinations of mutant vasa or okra alleles appear to dissociate early (AP) and late (DV) gurken signaling, possibly revealing temporal differences in the regulation of gurken protein levels (Ghabrial et al., 1998; Tomancak et al., 1998). A range of eggshell phenotypes is observed in these allelic combinations, including some eggs exhibiting a posterior micropyle but normal dorsal appendages. This phenotype suggests that the first step of gurken signaling, the determination of posterior follicle cell fates, was defective but that later, in midoogenesis, gurken activity was sufficient for the determination of dorsal follicle cell fates. In the combination of vasa alleles where this phenotype is most penetrant, Gurken protein is often absent from egg chambers in early oogenesis but present in midoogenesis, consistent with this eggshell phenotype. Such a distribution of gurken protein has also been reported for maelstrom mutants, where Gurken protein levels are reduced or absent in the early stages of oogenesis but return in midoogenesis (Clegg et al., 1997). These results suggest that gurken translation may be regulated differently at different stages or that different levels or activity of these regulatory factors are present over time. Alternatively, it is possible that early and late gurken RNA is derived from different sources, perhaps the nurse cells and oocyte nucleus, and may therefore be differentially regulated. Taken together, these results identify a number of genes that are required for the accumulation of wild-type levels of Gurken protein and may function in the translation of gurken RNA. The nature of the proteins encoded by these genes suggests that translation of gurken requires both factors that interact directly with gurken RNA and progression through the meiotic cell cycle. Since the distribution of Gurken within the oocyte is important for proper dorsal follicle cell fate determination, these factors might regulate gurken translation to ensure that only properly localized gurken RNA is translated, as appears to be the case of oskar and nanos (for review, see Curtis et al., 1995). This hypothesis is somewhat complicated, however, by the observation that mislocalized gurken RNA is translated in KIO and squid mutants. Alternatively, these factors could be more generally required for translation. The specific mutant phenotypes observed could suggest that these genes are required for translation of only a certain subset of RNAs within the germline.

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C. Posttranslational Regulation of Gurken Activity Mutations in cornichon result in abnormal localization of the Gurken protein, suggesting that gurken may also be regulated at the posttranslational level. Females mutant for cornichon produce eggs and embryos with phenotypes identical to those produced by gurken and Egfr mutants, suggesting that comichon is required for Gurken-Egfr signaling (Roth et af.,1995). In comichon mutant egg chambers, gurken RNA localization and protein accumulation appear normal but localization of the Gurken protein is defective. Although some Gurken protein is localized to the oocyte membrane adjacent to the oocyte nucleus, as in wild type, a relatively high level of Gurken also accumulates in the oocyte cytoplasm. This abnormal Gurken distribution, together with the observation that cornichon function is required in the germline, suggests a possible role for comichon in posttranslational regulation of the Gurken signal. It is possible that the abnormal Gurken distribution is a secondary consequence of a cytoskeletal defect caused by the defective AP axis determination observed in cornichon mutants. Alternatively, a more direct role for cornichon might involve regulation of the maturation or transport of Gurken through the secretory pathway. Although the biochemical function of Cornichon is unknown, the protein is predicted to be highly hydrophobic and possibly membrane-associated,consistent with such a hypothesis.

IV. Response of Follicle Cells to Egfr Activation As described earlier, the mutant phenotypes and molecular characterization of the gurken and Egfr genes suggest a model for axial patterning during oogenesis in which interaction of a localized germline ligand with a receptor in the overlying follicle cells determines cell fates. This signal specifies both the AP and DV polarity of the developing egg chamber, acting in early oogenesis to specify posterior follicle cell fates and in midoogenesis to determine dorsal fates. In this section we consider the events that occur in the follicle cells in response to Egfr activation, including the pathways that transduce the initial signal and the potential mechanisms by which localized Egfr signaling yields the pattern of cell fates observed within the follicular epithelium, thus establishing the pattern of the mature chorion.

A. Downstream Targets of the Egfr in the Follicle Cells

Studies of vertebrate RTKs have provided considerable evidence for a general mechanism of signal transduction (for review, see Pazin and Williams, 1992; Schlessinger and Ullrich, 1992). Binding of a ligand to the RTK extracellular

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domain induces receptor dimerization, and the resulting proximity of the intracellular kinase domains allows phosphorylation in trans on tyrosine residues in the receptor, creating binding sites for receptor substrates which subsequently may be themselves phosphorylated. This event initiates transduction of the extracellular signal to the nucleus, resulting in changes in gene activity or expression that mediate the cellular response. RTK activity in various organisms and multiple cellular contexts leads to activation of the Ras pathway, in which conversion of p21 ‘“dRas1 from the inactive GDP-bound form to the active GTP-bound form results in activation of a protein kinase cascade involving Raf, MEK, and MAPK proteins (for review, see McCormick, 1994). In Drosophilu, the Ras pathway functions downstream of multiple RTKs in various developmental processes, including the Egfr in oogenesis (for review, see Perrimon, 1994; Wassarman et al., 1995). Similar to the maternal Egfr mutant phenotype, females homozygous for hypomorphic alleles of Rasl, D-raJ; or D-mek lay ventralized eggs, indicating that the activity of these genes is required for Egfr signaling (Brand and Perrimon, 1994; Hsu and Perrimon, 1994; Lu et al., 1994; Schnorr and Berg, 1996). Conversely, gain-of-function alleles of D-ruf or Rasl induce dorsalization, indicating that activation of these components is sufficient to activate the Egfr signaling pathway (Brand and Perrimon, 1994; Lee and Montell, 1997). Gap1 also functions downstream of the Egfr in the follicular epithelium, but Gap1 loss-of-function alleles result in dorsalized phenotypes, indicating that, as in other systems, GapZ acts as a negative regulator of Egfr function (Chou et al., 1993). In addition to these Ras pathway components, the nonreceptor protein tyrosine phosphatase Corkscrew is also required downstream of the Egfr for dorsoventral patterning during oogenesis (Perkins et al., 1996). Like the Ras pathway, corkscrew is required throughout development and functions downstream of various RTKs in multiple developmental processes. An allelic combination of corkscrew that allows adult viability results in defective dorsal follicle cell fate determination and the production of ventralized eggs, and a reduction of corkscrew function enhances an Egfr loss-of-function phenotype. These results suggest that corkscrew acts in the follicular epithelium as a downstream effector of Egfr activity. A number of transcription factors have been identified that function downstream of the Egfr and are involved in patterning of the follicular epithelium. For example, pointed encodes two members of the Ets family of transcription factors, PointedP1 and PointedP2, that share a conserved Ets DNA binding domain (Klambt, 1993; for review, see Wasylyk et al., 1993). Expression of both pointedPZ and pointedP2 in the follicular epithelium is induced by Gurken-Egfr signaling and both are required for dorsal follicle cell fate determination (Morimot0 et ai., 1996; see Section IVB3). In addition, Egfr activation regulates expression of a member of the Broad Complex (BR-C) of zinc-finger transcription factors, possibly via induction of pointed (Deng and Bownes, 1997; see Section IVB3). Another Ets domain transcription factor, D-elg, also has mutant

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phenotypes consistent with a positive role in Egfr signaling (Schultz et al., 1993; Gajewski et al., 1995). In contrast, levels of the zinc-finger transcription factor CF2 are negatively regulated by Egfr activity (Hsu et al., 1996; see Section IVB4). Two additional targets of Egfr signaling were initially identified as P-element enhancer trap insertions (Bellen et al., 1989; Bier et al., 1989; Wilson eral., 1989) whose expression pattern within the follicular epithelium was sensitive to changes in Gurken-Egfr signaling (Musacchio and Perrimon, 1996; Dobens et al., 1997). Molecular analysis of adjacent DNA revealed that these elements had inserted into the kekkon-1 and bunched genes. Like the enhancer traps, these genes are expressed in dorsal anterior and posterior follicle cells and their expression is modulated by changes in Gurken-Egfr activity. kekkon-1 encodes a predicted transmembrane protein with an immunoglobulin loop and multiple leucine-rich repeats (Musacchio and Perrimon, 1996). A deletion of kekkon-1 does not yield a detectable mutant phenotype; therefore a requirement for kekkon-1 in DV patterning has not yet been established. It is possible that a related gene, kekkon-2, can compensate for the loss of kekkon-1. bunched, previously identified as the shortsighted gene (Treisman et al., 1995), encodes a leucine zipper protein. Follicle cell clones homozygous for a mutant bunched allele are associated with dorsal appendage defects, suggesting a possible role for bunched as an Egfr effector in cell fate determination within the follicular epithelium (Dobens er al., 1997). Since the Egfr functions in multiple developmental processes and appears to require common downstream effectors in each, an outstanding question is how activation of the same receptor in these different contexts leads to distinct cellular responses. Even within oogenesis, Egfr activation instructs at least two different cell fate decisions: the determination of posterior cell fates early in oogenesis, and the determination of dorsal cell fates in midoogenesis. A possible explanation for these two distinct responses is that the signal itself is modulated over time. For example, in the embryo different levels of Ras pathway activation can mediate the transcription of different genes and the determination of different structures (Greenwood and Struhl, 1997). Different levels of ligand could also affect the duration of signaling pathway activation and yield different responses, as observed in a vertebrate cultured cell system (for review, see Marshall 1995). Alternatively, a temporally regulated germline cofactor could be present or active at only a certain stage (Lehmann, 1995). The response to Egfr activation probably also depends on the previous history of the follicle cell receiving the signal. For example, early in oogenesis the terminal follicle cells, at either end of the developing egg chamber, can be distinguished by the expression of certain genes (Grossniklaus et al., 1989; Ruohola et al., 1991), suggesting that they have adopted a distinct fate. The position of these cells corresponds to those that receive the Egfr signal early in oogenesis to determine posterior cell fate. Therefore, the prior specification of these cells may confer competence to differentiate as posterior follicle cells in response to the Gurken-Egfr signal. Indeed, it has been recently demonstrated

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that early in oogenesis distinct follicle cell populations at either end of the egg chamber acquire the competence to respond to Gurken-Egfr signaling (GonzhlezReyes and St. Johnston, 1998).

B. Determination of Multiple Cell Fates within the Dorsal Follicular Epithelium As described earlier, Egfr activation in the follicle cells by a localized germline ligand induces dorsal follicle cell fates, thereby establishing dorsoventral polarity within the follicular epithelium. Moreover, the morphology of the eggshell indicates that this signal induces at least two distinct cell fates within the dorsal region of the follicular epithelium. The dorsalmost follicle cells secrete the dorsal midline region of the chorion, which lacks dorsal appendage material but includes the operculum, while dorsolateral follicle cells give rise to the dorsal appendages. These fates are dependent upon Gurken-Egfr signaling, but the mechanism by which Egfr activation leads to multiple cell fates within the dorsal follicular epithelium is not fully understood.

1. Does a Gradient of Egfr Activity Determine Distinct Dorsal Follicle Cell Fates? Two general models can be proposed. In one, gurken is distributed as a gradient and induces a corresponding gradient of Egfr activation within the follicular epithelium. In this model, different levels of Egfr activation determine distinct cell fates, with the highest levels inducing the midline follicle cell fates and lower levels inducing dorsolateral fates. Alternatively, activation of the Egfr by Gurken could elicit a single uniform response within a broad region of the dorsal follicular epithelium. In such a model subsequent interactions between follicle cells at the boundary between the activated region and the bordering, uninduced cells generate an additional distinct fate. Increased levels of gurken expression, achieved by generating females with extra copies of the gurken gene, result in eggs in which the dorsal appendages are often separated by a wider region of dorsal midline follicle cells and the width of the appendages themselves is increased (Neuman-Silberberg and Schupbach, 1994; see Figure 2D). This dorsalized phenotype reflects an expansion of midline follicle cell fates and a corresponding lateral shift of dorsolateral appendage fates in response to an increase in Gurken-Egfr signaling. This observation does not distinguish between the two possible models, however, since gurken overexpression could result in either an overall increase of signal and therefore an augmented gradient or simply in an increase in the size of the region of Egfr activation, thus shifting the boundaries where interactions occur toward the ventral side. This issue has also been addressed using a related approach, in which the profile of Gurken-Egfr signaling is modulated by receptor activation rather than ligand

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overexpression. When a constitutively activated form of the Egfr is expressed in midoogenesis in all follicle cells at high and relatively uniform levels, the resulting eggs lack dorsal appendages and along the entire DV axis the eggshell phenotype most closely resembles that produced by dorsal midline follicle cells (Queenan et al., 1997). This phenotype can also be detected in the ovary as a similar expansion of dorsal anterior follicle cell markers. Expression of lower levels of this activated receptor results in the production of dorsal appendage material around the entire anterior circumference of the egg. These results are more easily explained by the gradient model, in which inital high levels of receptor activation result in dorsal midline follicle cell fate and initial lower levels of activation specify the dorsal appendage cell fate. However, these results do not entirely rule out the cell-cell interaction model, and it seems likely that a combination of both processes occurs, in which an initial graded profile of Egfr activation is refined by secondary processes involving cell-cell interactions to yield the final pattern of the dorsal region.

2. Regulation of Egfr Activity in Other Tissues Spatially restricted Egfr activation by a localized ligand seems to be a common paradigm utilized in various developmental processes, where graded Egfr activation has been proposed to lead to distinct cell fates. This type of patterning process appears to involve feedback modulation of Egfr activity by some of its target genes. One of these targets is argos, which is induced by Egfr activation and encodes an inhibitory Egfr ligand, suggesting a negative feedback mechanism of Egfr regulation (Freeman et al., 1992b; Schweitzer et al., 1995a, Golembo et al., 1996b). rhomboid encodes an integral membrane protein that plays a positive role in Egfr activation, and, in the ovary, rhomboid expression is induced by Egfr activation (Bier et al., 1990; Ruohola-Baker et al., 1993; Queenan et al., 1997). The function of these feedback mechanisms in other tissues may provide a framework for analysis of Egfr-dependent pattern formation in oogenesis. In the embryo, the Egfr is required for cell fate determination along the DV axis of the ventral ectoderm (Raz and Shilo, 1992, 1993). This process is thought to involve Egfr activation by a graded distribution of the active form of its ligand, Spitz (Schweitzer et al., 1995b; Golembo et af., 1996a). spirz is expressed broadly throughout the ventral ectoderm, but spitz expression in the ventral midline cells is sufficient to induce ventral cell fates in the neighboring ectodermal cells (Golembo et af., 1996a). This nonautonomous effect, and the observation that expression of a secreted form of Spitz can also determine ventral cell fates nonautonomously, led to the proposal that processing of Spitz from a membraneassociated form to an active secreted form occurs only at the ventral midline and that subsequent diffusion from this source generates a gradient of active ligand (Schweitzer et al., 1995b; Golembo et al., 1996a). Expression of rhomboid and Star in the ventral midline cells is sufficient for ventral fate determination, and it has been proposed that these proteins function in the production or processing

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of Spitz (Schweitzer et af., 1995b; Golembo et af., 1996a). Similarly, in the eye imaginal disk diffusion of Spitz from certain photoreceptor cells induces the differentiation of neighboring cells and their recruitment to the developing ommatidium (Freeman, 1996, 1997; Tio and Moses, 1997). As in the ventral ectoderm, rhomboid and Star are expressed in the same cells where spitz is first required (Freeman et af., 1992a; Heberlein et af., 1993). A similar process may also function in the specification of vein and intervein regions in the wing imaginal disk (Sturtevant et af., 1993). Proper cell fate determination in these processes also requires argos activity. Genetic data indicate that urgos antagonizes Egfr function (Schweitzer et al., 1995a; Golembo et af., 1996b), and Argos can inhibit Egfr activation by Spitz in a cultured cell system (Schweitzer et af.,1995a), suggesting that that Argos functions in negative feedback regulation of Egfr activity. In the ventral ectoderm, argos is expressed in ectodermal cells just adjacent to the midline, presumably induced by high levels Egfr activation (Freeman et af., 1992b; Golembo et af., 1996b). Expression of argos in these cells requires induction of the pointedf I transcription factor, and inhibition of the negative transcription factor Yan, by Egfr activation (Gabay et al., 1996). In the eye imaginal disk, diffusion of Argos within the developing ommatidium has been proposed to limit the range of Spitz action (Freeman, 1997). In the embryo, it has been proposed that Argos diffuses across the ventral ectoderm, where it competes with Spitz and therefore functions as a sink to maintain the gradient of Spitz-mediated Egfr activation and compensate for any fluctuations in Spitz levels (Golembo et af., 1996b).

3. Interactions between Egfr Targets in the Ovary The foregoing results describe some of the factors involved in Egfr signaling in other tissues, and some of these have been shown to play a role in DV patterning in oogenesis. For example, pointed appears to be involved in distinguishing dorsal midline from dorsolateral cell fates (Morimoto et af., 1996). In midoogenesis, around the stage of DV polarity determination, pointed is expressed in the dorsalmost anterior follicle cells. This pattern is subsequently refined, with expression restricted in later stages to dorsolateral anterior populations that flank the dorsal midline. This expression pattern is similar for both the pointedPI and pointedf2 transcripts, although pointedPl is also detected in two posterior dorsolateral patches and is dependent upon Gurken-Egfr signaling. In mosaic egg chambers, loss of eitherpointedf I orpointedP2 activity, or both, from dorsal anterior follicle cells results in eggs with a single broad dorsal appendage. This phenotype differs from the ventralized eggs produced by weak gurken or Egfr mutants, which also have a single dorsal appendage, but of a narrow shape. The broad appendage phenotype suggests that loss of pointed activity does not result in a reduction of dorsal fates, but rather in a conversion of dorsal midline fates to appendage-producing fates. These results suggest that pointed is required for induction of dorsalmost

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Fig. 5 Model for regulation of ER-C expression by Egfr and dpp signaling. Dorsal view of egg chamber. Gurken (grk), localized adjacent to the oocyte nucleus (circle), induces Egfr activation in the dorsal follicle cells, which in turn induces BR-C expression. In the dorsalmost follicle cells, Egfr activation induces pointed (pnt) expression, which represses BR-C expression. Along the AP axis, expression of dpp by the follicle cells overlying the anterior margin of the oocyte represses ER-C expression. Thus restriction of ER-C expression to dorsolateral follicle cell populations results from a combination of induction by Gurken-Egfr signaling and repression by pointed and dpp. Also depicted is a putative role for argos (aos) in the follicle cells. argos is expressed in the dorsalmost follicle cells and is an inhibitory Egfr ligand. In other tissues, argos expression requirespointed. Although the role of nrgos in the ovary is not known, induction of pointed in dorsalmost follicle cells by high levels of Egfr activation may induce nrgos expression, which may in turn negatively regulate Egfr activity in this region. Adapted from Deng and Bownes (1997, Fig. 6, p. 4644).with permission from the Company of Biologists Ltd.

cell fates by Egfr activity. However, ectopic expression of pointed is not sufficient to cause expression of a dorsal follicle cell marker in more ventral cells, suggesting that pointed is probably not the only effector of dorsal midline follicle cell fates. Given the requirement forpoinredP1 in argos induction in the ventral ectoderm, it is tempting to speculate that argos may be a downstream target ofpointed in the ovary as well. Little is known about the role of argos in Egfr signaling in oogenesis, but its induction by Egfr activity and its expression in the dorsal midline follicle cells suggest a possible role for argos in patterning of the dorsal follicular epithelium (Queenan et al., 1997). The expression of argos in dorsal follicle cells is detected later in oogenesis than the expression of pointed in this region. Induction of pointed expression by high initial levels of Egfr activity could induce argos expression, which in turn might result in downregulation of Egfr activity in the dorsal midline follicle cells, such that at a later stage, Egfr activity in the dorsal midline follicle cells may in fact be low (Fig. 5). In such a scenario, feedback inhibition of Egfr activity would be required for determination of the dorsal midline follicle cell fates. However, a role for argos in patterning of the dorsal follicular epithelium, or induction of argos by pointed in the ovary, has yet to be demonstrated.

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A member of the BR-C group of transcription factors is also a potential target of pointed (Deng and Bownes, 1997). The BR-C is initially expressed in all follicle cells beginning early in midoogenesis, independent of Gurken-Egfr signaling. Later, just after the stage where DV polarity is established, BR-C expression is lost from the dorsalmost anterior follicle cells and then from the ventral and posterior follicle cells, so that expression remains only in two patches of dorsolateral follicle cells. Mutations in the Gurken-Egfr signaling pathway alter this later expression pattern, such that the two patches of expression are shifted dorsally in Egfr loss-of-function mutants and shifted laterally and expanded in Kf 0 mutants. Females mutant for a viable combination of BR-C alleles lay eggs with reduced dorsal appendages, consistent with a role for BR-C in dorsal appendage formation. Regulation of BR-C expression by Gurken-Egfr signaling may be mediated by pointed. Ectopic expression of pointedP2 in all anterior columnar follicle cells results in a reduction in the number of BR-C-expressing cells (Deng and Bownes, 1997). In addition, BR-C and pointed display complementary expression patterns, with pointed expressed in the dorsalmost follicle cells at the stage when BR-C is excluded from these cells. Taken together, these observations suggest that BR-C expression is induced by Egfr activation but that in the dorsalmost follicle cells, high levels of Egfr activity also induce pointed, which in turn represses BR-C (Fig. 5). Thus the BR-C expression pattern probably reflects the induction of a repressor in the dorsal midline follicle cells by high levels of Egfr signaling, rather than differential induction of BR-C expression by different levels of Egfr activation. This regulatory relationship is not entirely straightforward, however, and seems to involve a temporal aspect, since later in oogenesis BR-C and pointed expression are both detected in dorsolateral follicle cells.

4. Role of rhomboid in Follicle Cell Patterning

rhomboid is also involved in patterning the follicular epithelium and seems to actively promote dorsal appendage fates. rhomboid exhibits a dynamic expression pattern in midoogenesis, with an initially broad expression pattern in the dorsal anterior follicle cells that is later refined to two dorsolateral stripes that correspond to the position of the future dorsal appendages. Ectopic expression of rhomboid results in an expansion of dorsal follicle cell fates and dorsalization of the eggshell and embryo, whereas expression of an antisense rhomboid construct results in ventralization (Ruohola-Baker et al., 1993). These phenotypes suggest that rhomboid is a positive regulator of Egfr activity. Interestingly, expression of rhomboid is dependent upon Gurken-Egfr signaling, suggesting that rhomboid functions in a positive feedback loop to regulate Egfr activity, possibly acting to amplify the initial DV asymmetry in Egfr signaling. In such a model, the dynamic rhomboid expression pattern could be interpreted as a broad enhancement of Egfr activity by the initial activation of rhomboid in the entire dorsal region. Subsequent refinement of rhomboid expression into two separate dorsolateral stripes, possibly also

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requiring the activity of repressors in the dorsal midline cells, would result in highest levels of Egfr activity in these lateral follicle cells which will subsequently reorganize themselves into the rows of migrating follicle cells that form the dorsal appendages. A candidate negative regulator of rhomboid expression is the transcription factor CF2 (Hsu et al., 1996; Mantrova and Hsu, 1998) In midoogenesis, CF2 is detected throughout the follicular epithelium, with the exception of the dorsal anterior follicle cells. The region of cells lacking CF2 is expanded in dorsalizing mutants and decreased in ventralizing mutants, indicating that CF2 expression is inhibited by Gurken-Egfr signaling. This inhibition is post-transcriptional and appears to result from cytoplasmic retention and degradation of CF2, probably in response to phosphorylation by MAPK (Mantrova and Hsu, 1998). Downregulation of CF2 levels appears to be required for induction of dorsal follicle cell fates by Egfr signaling, because ectopic CF2 expression results in a weakly ventralized eggshell phenotype. Ectopic CF2 expression also represses rhomboid expression, suggesting that rhomboid is negatively regulated by CF2. Conversely, rhomboid expression is expanded in response to loss of CF2 throughout the follicular epithelium (Hsu et al., 1996). These results suggest that Gurken-Egfr signaling in the dorsal anterior follicle cells negatively regulates CF2 levels, which in turn induces the initial broad pattern of rhomboid expression in the dorsal anterior follicle cells (Hsu et al., 1996; Mantrova and Hsu, 1998). Taken together, these results indicate that rhomboid expression is induced by Gurken-Egfr signaling, possibly through inhibition of CF2, and that rhomboid is required for induction of dorsal follicle cell fates. However, the mechanism of rhomboid function and its effect on Egfr activity remain unclear. As described earlier, rhomboid can function nonautonomously and is expressed in the same cells that act as a source of active Spitz, leading to the proposal that the role of rhomboid in Egfr activation is in local processing of the Spitz precursor to an active form. Although spitz expression in follicle cells has not been demonstrated, recent rhomboid misexpression experiments have demonstrated nonautonomous effects of rhomboid within the follicular epithelium (Sapir et al., 1998).This observation has led to the suggestion that rhomboid may also function to activate the Egfr by promoting the production or processing of a second, unidentified, Egfr ligand in the follicle cells. Alternatively, rhomboid may activate the Egfr in the follicle cells by another, yet unknown, mechanism.

C. Dorsal Patterning along the AP Axis

The specification of midline and appendage-producing follicle cell fates, discussed earlier, represents a patterning process occurring along the DV axis of the developing egg chamber. However, the final pattern of the columnar follicular epithelium also depends on the pattern of cell fates along the AP axis. The dorsal

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side of the egg chamber is not uniform along the AP axis, as is evident in the positioning of the dorsal appendages at the anterior of the eggshell and the restricted expression pattern of certain genes along the AP axis. Furthermore, although expression of the constitutively activated Egfr at appropriate levels throughout the follicular epithelium can yield dorsal appendage material around the entire dorsoventral axis, this ectopic material is confined to the anterior of the chorion (Queenan el al., 1997). Similarly, the expansion of rhomboid expression along the DV axis in these egg chambers was restricted to the anterior follicle cells. Activation of the Egfr therefore does not induce a uniform response at all positions within the follicular epithelium. These asymmetries further indicate that spatial information along the AP axis is required to specify the position within the follicular epithelium of the cells that will give rise to the dorsal appendages. Accumulating evidence suggests that expression of decupentaplegic (dpp) within the anterior follicular epithelium provides this spatial information and that induction of dorsal anterior follicle cell fates may involve an interaction between the Egfr and dpp signaling pathways. dpp encodes a member of the transforming growth factor-p superfamily and has been shown to act as a diffusible signaling molecule in other Drosophila tissues (for review, see Kingsley, 1994). dpp is expressed in the anterior follicular epithelium beginning in midoogenesis, in the follicle cells that overlie the nurse cells and in a subset of cells at the leading edge of the centripetally migrating follicle cells, which are located at the anterior margin of the oocyte in midoogenesis and later migrate between the oocyte and nurse cells (Twombly et ul., 1996). Consistent with this expression pattern, genetic evidence demonstrates a role for dpp activity in determination of anterior cell fates on the dorsal side of the follicular epithelium. Decreased dpp activity during oogenesis results in eggs with reduced anterior eggshell structures. Defects in the length or appearance of the dorsal appendages are observed, but the most striking defect is a reduction in the size of the operculum in most eggshells. Conversely, overexpression of dpp causes a dramatic increase in the size of the operculum. This phenotype is also accompanied by a range of dorsal appendage defects. Taken together, these results suggest a requirement for dpp in the determination of anterior eggshell structures. Evidence that the Egfr and dpp signaling pathways coordinately specify cell fates within the dorsal anterior follicular epithelium is provided by analysis of BR-C expression. As described earlier, BR-C expression depends on the profile of Egfr activation along the DV axis, and is expressed in two dorsolateral domains that presumably correspond to the cells that will secrete the dorsal appendages (Deng and Bownes, 1997). Along the AP axis, BR-C expression is absent from the anteriormost follicle cells, overlying the oocyte, so that the two patches of BR-C expression are located about 2-3 cells posterior to the centripetally migrating follicle cells. This positioning of BR-C expression along the AP axis is controlled by dpp expression. When dpp activity is reduced during oogenesis, the

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BR-C expression is shifted anteriorly. Conversely, when dpp is ectopically expressed thoughout the follicular epithelium in midoogenesis, the two dorsolateral BR-C expression domains extend further posterior. However, this ectopic dpp expression does not induce BR-C expression in the dorsalmost or ventral follicle cells, reflecting the dependence of BR-C expression on Egfr signaling along the DV axis. These results indicate that BR-C expression is dependent on input from two different signaling pathways, with expression regulated along the DV axis by Egfr activity and along the AP axis by dpp (Fig. 5 ) . Thus the specification of the dorsolateral follicle cell subpopulations is determined by the intersection of the appropriate levels of input from these two signaling pathways. An interaction between the dpp and Egfr signaling pathways has also been demonstrated in the process of cell fate determination within the tracheal placode (Wappner et al., 1997).

D. Model: Initial Signal from Germline and Refinement within the Follicular Epithelium

Taken together, these results suggest a model for patterning of the dorsal follicular epithelium in wild-type egg chambers. Initially, induction of follicle cell gene expression is determined by the level of Egfr activation, which is in turn determined by the distribution of its ligand, gurken, in the germline. The observation that some of the genes induced by Gurken-Egfr signaling are only expressed in a narrow region on the dorsal midline suggests that different genes may have different thresholds for induction by Egfr activation. The modulation of the expression patterns of some of these genes over time suggests that the initial response to Gurken-Egfr signaling is then refined, at least in part by interactions between some of these Egfr targets. For example, pointed and CF2 appear to be primary targets of Egfr activity, and these factors in turn regulate the expression of other genes, such as BR-C and rhomboid, respectively, although these genes may be themselves directly regulated by Gurken-Egfr signaling as well. Thus interactions occurring downstream of the Egfr appear to play a role in determination of patterns of gene expression and, therefore, follicle cell fates. In addition to interactions between downstream Egfr targets, determination of dorsal follicle cell fates seems to involve feedback regulation of the Egfr itself. For example, urgos is expressed in the dorsalmost follicle cells and, by analogy to other tissues, may function to inhibit Egfr activity in these and/or adjacent follicle cells. In contrast, rhomboid is expressed in dorsolateral follicle cells and is proposed to enhance Egfr activity, and thus may function to maintain or enhance the expression of Egfr target genes in these dorsolateral regions. The interaction between these putative positive and negative feedback mechanisms could therefore regulate Egfr activity over time, resulting in a later profile in which Egfr

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activity is low in the dorsalmost follicle cells, where it was initially the highest, and highest in the dorsolateral follicle cells. Thus the opposing actions of rhomboid and argos could serve to sharpen over time the initial boundaries between adjacent cell fates. This patterning process is also modulated by spatial cues along the AP axis. Expression of dpp in the anterior follicle cells appears to control the development of anterior follicle cell fates and anterior eggshell structures. Although the temporal and mechanistic details of the interaction of the dpp and Egfr signaling pathways are not understood, current evidence suggests that their intersection determines the coordinates of the dorsolateral follicle cell populations that will later migrate to form the dorsal appendages. In this way, spatial information along both the AP and DV axes is integrated to achieve the final pattern of the follicular epithelium.

V. Determination of Embryonic DV Polarity by local Egfr Activation In addition to its role in eggshell patterning, the positional information generated within the follicular epithelium during oogenesis by Gurken-Egfr signaling is also required for determination of the dorsoventral pattern of the future embryo. As described earler, Gurken-Egfr signaling is required for dorsal follicle cell fate determination, and females mutant for genes involved in this process lay ventralized eggs. The embryos that develop within these eggs also display a ventralized phenotype, whereas mutations that result in dorsalization of the eggshell give rise to dorsalized embryos. These phenotypes indicate that Gurken-Egfr signaling is required for establishment of the DV axis of the embryo. This effect on the embryo implies that patterning information is conveyed from the follicular epithelium to the future embryo and that therefore the communication between the germline and soma must be bidirectional. That is, in midoogenesis, localized gurken in the oocyte conveys spatial information to the dorsal follicle cells, inducing them to adopt a distinct cell fate. This signaling event distinguishes dorsal from ventral follicle cells, generating a DV asymmetry. This asymmetry provides spatial information that is later conveyed back to the embryo to define its dorsoventral axis (for review, see Ray and Schiipbach, 1996). A. Role of the Ras Pathway in Embryonic DV Polarity Determination

Comparison of the eggshell and embryonic mutant phenotypes from certain mutant alleles has suggested a possible differential requirement for Egfr signaling in patterning of the eggshell and the embryo. As described earler, gurken or Egfr mutations result in ventralization of both the eggshell and the embryo. In general,

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the severity of the embryonic defect correlates well with that of the eggshell, although the correlation is not absolute (Clifford and Schupbach, 1989). Interestingly, some hypomorphic alleles of the Egfr effectors Rasl, RaJ; and D-mek exhibit differential effects on eggshell and embryonic dorsoventral patterning (Brand and Perrimon, 1994; Hsu and Perrimon, 1994; Schnorr and Berg, 1996). These mutant females lay ventralized eggs but, although a similar degree of ventralization derived from a gurken or Egfr mutant female yields ventralization of the embryo, many of these embryos develop with a normal dorsoventral pattern and hatch normally. The Ras pathway is clearly required downstream of the Egfr for embryonic dorsoventral patterning, however, because mutant alleles of Rasl and D-mek can suppress the dorsalized phenotype of embryos from KIO mutant females (Hsu and Perrimon, 1994; Schnorr and Berg, 1996). Therefore, the differential effects of certain mutant alleles likely reflect a greater sensitivity of the eggshell to a reduction in Ras pathway activity. Consistent with this interpretation, higher levels of an activated form of 0-raf are required to dorsalize the embryo than the eggshell (Brand and Perrimon, 1994). These results suggest that Rasl functions downstream of the Egfr to determine the DV pattern of both the eggshell and embryo but imply a greater requirement for Ras pathway activity in eggshell patterning than in embryonic patterning. Alternatively, the involvement of an unidentified Ras-independent pathway in embryonic DV patterning has been proposed as a possible explanation for this difference (Schnorr and Berg, 1996).

B. Negative Regulation of Ventral Embryonic Cell Fates by Egfr Activity

The dorsoventral pattern of the early embryo is specified by a gradient of nuclear localization of the Dorsal protein. Dorsal is a member of the NF-KB/rel family of transcription factors (Steward, 1987) and regulates the expression of zygotic genes in a concentration-dependent manner, resulting in the determination of different cell fates along the embryonic DV axis (Steward er al., 1988; Roth et al., 1989; Rushlow et al., 1989; Steward, 1989). The Dorsal gradient forms early in embryogenesis when, in response to an extraembryonic signal, the Dorsal protein moves from the cytoplasm into the nuclei of the syncitial embryo. This process requires the function of 12 maternally required genes, known as the dorsal group (for review, see Chasan and Anderson, 1993; Morisato and Anderson, 1995). One of these genes, Toll, encodes a transmembrane protein that is present uniformly throughout the embryonic plasma membrane (Hashimoto et al., 1988). Activation of Toll by its presumptive ligand, the product of the spurzle gene, occurs only on the ventral side of the embryo and leads to import of Dorsal into ventral nuclei (Anderson et al., 1985; Stein et al., 1991; Morisato and Anderson, 1994; Schneider et al., 1994). Genetic experiments have ordered the other dorsal group genes into those that function downstream of Toll to transduce the signal leading to nuclear translocation of Dorsal and those that act upstream to produce ventrally

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restricted Toll ligand. Most of the dorsal group genes are required in the germline, but three, windbeurel, pipe, and nudel, are required in the soma, presumably in the follicular epithelium (Schupbach et al., 1991; Stein et al., 1991). This somatic requirement is consistent with the model that spatial information from the follicle cells is required to determine the dorsoventral axis of the embryo. The production of the extraembryonic ventral cue leading to nuclear Dorsal import is negatively regulated by Egfr activation in the dorsal follicle cells. In the ventralized embryos derived from females with defects in Gurken-Egfr signaling, the Dorsal gradient is expanded, resulting in an expansion of the ventralmost embryonic cell fates and a shift of ventrolateral fates toward the dorsal side (Schiipbach, 1987; Roth and Schiipbach, 1994). This phenotype suggests that in these mutants the extraembryonic signal leading to nuclear import of Dorsal functions in a wider ventral domain. This expansion of the Dorsal gradient suggests that Gurken-Egfr signaling on the dorsal side of the egg acts to negatively regulate the production of the ventral signal, confining this process to a restricted ventral region. The existence of such a functionally distinct ventral region has been demonstrated in recent studies of the spatial requirements within the follicular epithelium for the somatically-required dorsal group genes windbeurel, pipe, and nudel (Nilson and Schupbach, 1998). In these experiments, the spatial requirements for these genes were determined through analysis of mosaic egg chambers, in which clones of follicle cells homozygous for mutant alleles of these genes were generated within a heterozygous follicular epithelium. Incorporation of a follicle cell marker visible in the mature eggshell allowed the position of a mutant clone to be compared directly with the resulting embryonic phenotype. Analysis of the positions of clones that were associated with embryonic DV patterning defects revealed no localized requirement for nudel but demonstrated that windbeutel and pipe activity is required only in the ventralmost 20-30% of the follicular epithelium. This ventral region presumably corresponds to the source of active Toll ligand, which governs Toll activation and nuclear import of Dorsal and is predicted to be expanded in gurken or Egfr mutants (Fig. 6). Furthermore, this ventral follicle cell can determine lateral cell fates within the embryo. That is, lateral embryonic cell fates are generated both at positions corresponding to the normal lateral boundaries of this region and at the ectopic boundaries created at the borders of clones mutant for windbeurel or pipe. Although inductive interactions between follicle cells at these boundaries have not been ruled out, this “nonautonomy” suggests that diffusion of some activity from this ventral region ultimately generates the Dorsal gradient. The diffusible factor could be the Toll ligand or any upstream component of the pathway, and diffusion could occur either upstream or downstream of Toll. The establishment of this functionally distinct ventral subpopulation of follicle cells appears to involve some sort of secondary patterning process acting downstream of the Egfr within the follicular epithelium. The existence of such a process is suggested by the observation that the expansion of the Dorsal gradient in

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Fig. 6 Model for embryonic DV patterning. Gurken-Egfr signaling at the dorsal side of the egg chamber determines dorsal follicle cell fates and establishes a functionally distinct population of follicle cells on the ventral side (upper left, dark gray). This ventral region is presumably the source of active Toll ligand and determines the Dorsal gradient (lower left, shaded circles), which defines the DV pattern of the embryo. In mutants in which Gurken-Egfr signaling is decreased, this ventral region is presumably expanded (upper right, dark gray). Interestingly, the increased ventral region of nuclear Dorsal localization in these embryos exhibits two ventrolateral peaks (lower right, shaded circles). This “splitting” of the pattern indicates that some patterning process, the nature of which is unknown,

embryos derived from females mutant for strong gurken or Egfr alleles is often accompanied by ventral pattern duplications (Schupbach, 1987; Roth and Schupbach, 1994). At the beginning of gastrulation the cells on the ventral side of the embryo organize to form two ventral furrows, instead of the single furrow formed in wild-type embryos. As demonstrated by the analysis of molecular markers, such embryos exhibit two peaks of nuclear Dorsal protein, instead of a single ventral high point, and a new ventrolateral domain separates the duplicated “ventral” domains (Fig. 6).

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The observation that reduction of Egfr activity yields a pattern duplication, rather than a uniform expansion of ventral cell fates, indicates that some patterning process intervenes downstream of the initial Gurken-Egfr signal to determine the profile of the Dorsal gradient. These results suggest a model in which GurkenEgfr signaling at the dorsal side of the egg chamber acts to define the initial DV asymmetry within the follicular epithelium. Once this initial distinction between dorsal and ventral is established, a second patterning process within the ventral follicular epithelium refines the initial DV asymmetry to yield a ventral region of activity. If the ventral domain is expanded, this process resolves this region into two separate ventral high points. The nature of such a pattern refinement process, however, is unknown.

VI. Summary The spatial regulation of Egfr activity in the follicular epithelium of the ovary is achieved by the localization of its ligand, Gurken, within the germline. The final distribution of Gurken within the oocyte appears to be specified both by the localization of the gurken RNA and by regulation of Gurken protein accumulation, possibly at the level of translation. Localized activation of the Egfr distinguishes certain subpopulations of follicle cells, thereby generating asymmetry within the follicular epithelium. In early oogenesis, Egfr activation in posterior follicle cells defines the AP polarity of the egg chamber, and in midoogenesis restriction of Egfr activity to dorsal follicle cells determines DV polarity. A number of factors required downstream of the Egfr have been identified, but the mechanism by which the observed patterning of the follicular epithelium is achieved remains unclear. The dynamic expression patterns of some of these targets suggest that the initial Gurken-Egfr signal at the dorsal side of the follicular epithelium mediates an initial distinction between dorsal and ventral follicle cells and also initiates subsequent refinement processes that determine the final pattern of cell fates. In the dorsal follicle cells, this refinement appears to involve interactions between Egfr targets and may also involve feedback regulation of Egfr activity such that the profile of Egfr activity is modulated over time. In addition, the initial Gurken-Egfr signal negatively regulates the functional domain of another patterning process that governs the establishment of the DV axis of the developing embryo.

Acknowledgments We thank B. Shilo, M.Bownes, C. Van Buskirk, and T. Gupta for help with the figures and A. Ghabrial, A. Norvell, L.-M. Pei, A. M.Queenan, G. Thio, and C. Van Buskirk for helpful comments on the manuscript.

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Index

A Actin, zygote symmetry breaking in fucoid algae, photopolarization role, 114-1 15 Algae, see Fucus; Pelvetia a-Catenin, pronephric tubule induction in Xenopus, 82 Angiosperms, see also specific species apical dominance main shoot zonation, 132-133 mechanisms, 133-135 meristem potential, 129-131 overview, 127-129, 160-162 bud outgrowth genetic study approaches, 143-160 altered hormone response mutants, 145- I46 Arabidopsis, 144-147 auxin studies, 157-158, 162 branching phenotype mutants, 146-147 cytokinin studies, 158 grafting studies, 155-157 growth habit genes, 151, 154 Petunia, 147-1 5 I photoperiod response genes, 152-154 Pisum sarivum, 151-160 Rmsgenes, 156-158, 160 shoot-root signals, 158-160 induced mutation studies, 135-137 outgrowth potential, 131-133 overview, 127-129, 160-162 recent gene isolation, 137-140 Is gene, 139-140 Tbf gene, 138-139 transgenic hormonal modulation a p proaches, 140-143 auxin level modulation, 141-143 cytokinin level increases, 141-143 homeostasis, 142-143 mRNA stability regulation, 174-175 Apical dominance bud outgrowth

genetic study approaches, 143-160 altered hormone response mutants, 145-146 Arabidopsis, 144-147 auxin studies, 157-158 branching phenotype mutants, 146-147 cytokinin studies, 158 grafting studies, 155-157 growth habit genes, 151, 154 Petunia, 147-15 1 photoperiod response genes, 152-154 Pisum sativum, 151-160 Rms genes, 156-158, 160 shoot-root signals, 158-160 induced mutation studies, 135-137 outgrowth potential, 131-133 overview, 127-129, 160-162 recent gene isolation, 137-140 Is gene, 139-140 T b f gene, 138-139 transgenic hormonal modulation approaches, 140- I43 auxin level modulation, 141-143 cytokinin level increases, 141-143 homeostasis, 142-143 main shoot zonation, 132-133 mechanisms, 133-135 meristem potential, 129-131 overview, 127-129.160-162 Arabidopsis bud outgrowth, genetic study approaches, 144-147 branching phenotypes, 146-147 mutants with altered hormone response, 145-146 mRNA stability regulation, 175 Auxin, bud outgrowth in angiosperms genetic study approaches, 157-158 transgenic hormonal modulation approaches, 141-143

24.5

246 Aves, mRNA stability regulation, 186-189 Axillary buds, see Bud outgrowth

B P-Galactosyltransferase, green fluorescent protein compared, I , 14 hicoid gene, mRNA stability regulation in Drosophila, 178-184 Biochemical markers, see Markers Bone morphogenetic proteins, pronephric tubule induction in Xenupus, 81-82 Bud outgrowth, in angiosperms genetic study approaches, 143-160 altered hormone response mutants, 145 - I46 Arabidopsis, 144-147 auxin studies, 157-158 branching phenotype mutants, 146-147 cytokinin studies, 158 grafting studies, 155-157 growth habit genes, 151, 154 Petunia, 147-15 I photoperiod response genes, 152-1 54 Pisum sativum, I5 I - I60 Rms genes, 156-158, 160 shoot-root signals, 158-160 induced mutation studies, 135-137 outgrowth potential, 131-133 overview, 127-129, 160-162 recent gene isolation, 137-140 Is gene, 139-140 Tbl gene, 138-139 transgenic hormonal modulation approaches, 140-143 auxin level modulation, 141-143 cytokinin level increases, 141-143 homeostasis, 142-143

C Caenorhahditis elegans, mRNA stability regulation, 176-178 Calcium ion, zygote symmetry breaking in fucoid algae, photopolarization and rhizoidal growth, 108-112 Calmodulin, zygote symmetry breaking in fucoid algae, photopolarization and rhizoidal growth, 112-1 13 caudal gene, mRNA stability regulation in Drusophila, 178-184 Cell adhesion molecules, pronephric tubule induction in Xenopus, 82

Index Cell lineages, retrospective relationship analysis, mouse chimeras, 34-35 Chimeras, in mouse developmental genetics studies, 46-53 cytogenetic studies, 46-47 genomic imprinting, 49-50 knockout analysis, 50-53 Mash2 gene analysis, 51-53 mutant gene phenotype analysis, 50-53 nodal gene analysis, 53 Pax6 gene analysis, 50-5 I sex determination in chimeras, 47-49 development studies, 30-40 cell distribution analysis, 37-40 founder cell number estimates, 35-37 lineage relationship analysis, 34-35 prospective studies, 30-33 qualitative spatial analysis, 37-38 quantitative spatial analysis, 39-40 retrospective studies, 33-40 experimental properties embryonic stem cell chimera production, 28-29 genetic effects on composition, 29-30 mosaics compared, 30 production, 22-28 genetic studies, 40-46 dr gene, 46 muscle degeneration analysis, 42-43 quantitative genetic traits, 40-41 retinal degeneration analysis, 43-46 rl gene, 46 single mutant phenotypic analysis, 41 -46 green fluorescent protein marker, 12-14 overview, 21-22.53-55 Chloramphenicol acetyltransferase, green fluorescent protein compared, I , 14 Collagen, mRNA stability regulation in aves, 186-189 c-rer gene, pronephric duct induction in XenoP U S , 84-85 Cytokinin, bud outgrowth in angiosperms genetic study approaches, 158 transgenic hormonal modulation approaches, 14 1- I43

D dickkopf-1 gene, pronephric mesoderm patterning role, 72 Drosophila epidermal growth factor receptor-signaling in oogenesis, 203-236

Index activation gurken gene translational regulation, 2 17-22 1 gurken RNA localization, 213-217 post-translational regulation, 221 spatial regulation, 212-221 axial polarity, 203 -206 dorsoventral polarity, 232-236 Ras pathway role, 232-233 ventral embryonic cell fate regulation, 233-236 epidermal growth factor receptor role, 206 -2 12 follicle cell response, 221-232 dorsal follicular epithelium cell fate determination, 224 -232 downstream targets, 222-224 epidermal growth factor receptor gradient effects, 224-225 germline signal initiation, 231 ovary target interactions, 226-228 pattern formation, 228-231 rhomboid gene role, 225,228 overview, 203-204.236 mRNA stability regulation, 178-184 d f gene, mouse chimera phenotypic analysis, 46

E Egg cells, see also Oogenesis in vivo gene expression studies, green fluorescent protein marker, 10-1 1 E g / gene, mRNA stability regulation in Xenopus, 184-186 emb-1 gene, mRNA stability regulation in angiosperms, 174-175 Embryonic stem cells, chimera production in mouse, 28-29 E m - 2 gene, pronephric duct induction in Xenopus, 86 Epidermal growth factor receptor, signaling in Drosophilu oogenesis, 203 -236 activation gurken gene translational regulation, 217-221 gurken RNA localization, 213-217 post-translational regulation, 22 1 spatial regulation, 212-221 axial polarity, 203-206 dorsoventral polarity, 232-236 Ras pathway role, 232-233 ventral embryonic cell fate regulation, 233-236

247 epidermal growth factor receptor role, 206-212 follicle cell response, 221-232 dorsal follicular epithelium cell fate determination, 224-232 downstream targets, 222-224 epidermal growth factor receptor gradient effects, 224-225 germline signal initiation, 23 1 ovary target interactions, 226 -228 pattern formation, 228-23 I rhomboid gene role, 225.228 overview, 203-204.236

F fern-3 gene, mRNA stability regulation in Caenorhabdiris eleguns, 176 -I 78 Fertilization, fucoid algae physiology, 103105 Fibroblast growth factors mRNA stability regulation in aves, 186-1 89 pronephric tubule induction in Xenopus, 81-82 Flow cytometry, green fluorescent protein analysis in mammals, 16 Fluorescence microscopic analysis, green fluorescent protein analysis in mammals, 15 Fluorometry, green fluorescent protein analysis in mammals, 15 Follicle cells, epidermal growth factor receptorsignaling in Drosophilu oogenesis, 22 I 232 dorsal follicular epithelium cell fate determination, 224-232 downstream targets, 222-224 epidermal growth factor receptor gradient effects, 224-225 germline signal initiation, 231 ovary target interactions, 226-228 pattern formation, 228-231 rhomboid gene role, 225,228 Founder cells, mouse chimera number estimates, 35-37 frizzled gene, pronephric tubule induction, 78-80 frzb gene, pronephric mesoderm patterning role, 72 frt gene, mRNA stability regulation in Drosophila, 178, 182 Fucus mRNA stability regulation, 174-175 zygote symmetry breaking

248 Fucus (continued) axis formation axis fixation, 115-1 17 cortical pH gradients, 113-1 14 fertilization physiology, 103-105 light response, 105-108 overview, 101-103, 121-122 photopolarization actin microfilament role, 114-1 15 calcium role, 108-1 12 calmodulin role, 112-1 13 signal transduction process, 117-1 18 speculative model, 119-121 rhizoidal growth, 108-1 14 calcium role, 108-1 12 calmodulin role, 112-1 13 cortical pH gradients, 113-1 14

Index Growth factor, see speci’fic types gurken gene, epidermal growth factor receptorsignaling in Drosophilu oogenesis dorsal follicle cell fates, 224-22.5 mechanisms, 206 -2 I2 overview, 203,236 RNA localization, 2 13-2 I7 translational regulation, 217-221

H Hedgehog genes, pronephric tubule induction in Xenopus, 82 Helix transcription factors, pronephric tubule induction in Xenopus, 77-78 hunchback gene, mRNA stability regulation in Drosophilu, 178-184

G Genetic markers, see Markers Genomic imprinting. mouse chimeras, 49-50 Glial cell line-derived neurotrophic factor, pronephric duct induction in Xenopus, 84-85 Glial cell line-derived neurotrophic factor receptor-a, pronephric duct induction in XenoPUS, 87-88 Glycophosphatidylinositol-linkedproteins, pronephric duct induction in Xenopus, 86-87 Green fluorescent protein, gene expression marker in mammals, 1-16 characteristics, 2 - 4 future research directions, 14 in virro expression, 4-7 cell marking, 4-6 intracellular biosensor function, 6-7 tag attachment, 6 in vivo expression, 7-14 chimeras, 12-14 egg study, 10-1 1 mouse study, 7-10 SPMI study, 11-12 observation methods, 14-16 fixation, 14-15 flow cytometry, 16 fluorescence microscopic analysis, 15 fluorometry, 15 immunostaining, 16 Western blot, 16 overview, 1-2 variants, 2-4 gremlin gene, pronephric duct induction in Xenopus. 84

I Immunostaining, green fluorescent protein analysis in mammals, 16 Imprinting, mouse chimeras, 49-50 Integrin 016, pronephric tubule induction in Xenopus. 82

K Kidneys, pronephric development, 67-92 early development, 70-71 glomus, 88-90 background, 88 platelet-derived growth factor-p. 90 vascular endothelial growth factor, 90 Wilms’ tumor-suppressor gene, 88-90 molecular mechanisms, 7 1-73 embryonic patterning, 71 laterally expressed genes, 72-73 molecular data, 7 1-73 organizer-specific genes, 72-73 pan-mesodermal genes, 7 1-72 pronephros-inducing genes, 73 pronephros induction, 71 ventralizing pathway, 72 ventrally expressed genes, 72 overview, 67-69.90-92 pronephric duct, 83-88 background, 83-84 c-ret, 84-85 Em-2 gene, 86 glial cell line-derived neurotrophic factor, 84 - 85

Index glial cell line-derived neurotrophic factor receptor-a, 87-88 glycophosphatidylinositol-linkedproteins, 86-87 gremlin, 84 Id gene, 85-86 pronephric tubules, 74-83 background, 74 bone morphogenetic protein, 8 1-82 cell adhesion molecules, 82 fibroblast growth factor, 81-82 Frz, 78-80 hedgehog, 82 helix transcription factors, 77-78 Lim-I. 75-76 Pax-2,80-81 Pax-8.76-77 3G8,82 Wnt, 78-80 structure and function, 69-70 Knockout genes, chimera analysis in mouse, 50-53

L IacZ, green Huorescent protein compared, I , 14 Id gene, pronephric duct induction in Xenopus. 85 -86 Light response, see Photopolarization Lim-lgene. pronephric tubule induction in Xenopus, 75-76 Lineages, retrospective relationship analysis, mouse chimeras, 34-35 Is gene, bud outgrowth in angiosperms, 139140

M Mammals, see nlso .specific species characteristics. 2-4 future research directions, 14 green Huorescent protein marker, 1-16 in vitro expression, 4-7 cell marking, 4 - 6 intracellular biosensor function. 6-7 tag attachment, 6 in vivo expression, 7-14 chimeras. 12-14 egg study, 10-1 1 mouse study, 7-10 sperm study, 11-1 2 mRNA stability regulation, 189-194

249 observation methods, 14 - I6 fixation, 14-15 flow cytometry, 16 fluorescence microscopic analysis, 15 fluorometry, 15 immunostaining, 16 Western blot, 16 overview, 1-2 variants, 2-4 Markers, green fluorescent protein in mammals, 1-16 characteristics, 2 - 4 future research directions, 14 in v i m expression, 4-7 cell marking, 4 - 6 intracellular biosensor function, 6-7 tag attachment, 6 in vivo expression, 7-14 chimeras, 12-14 egg study, 10-1 I mouse study, 7- 10 sperm study, 11-12 observation methods, 14-16 fixation, 14-15 flow cytometry, 16 fluorescence microscopic analysis, 15 Huorometry, 15 immunostaining, 16 Western blot, 16 overview, 1-2 variants, 2-4 Marsilea. mRNA stability regulation, 174-175 Mash2 gene, chimera analysis in mouse, 5 1-53 Meristems, apical dominance in angiosperms, 129-1 3 I Mosaics, chimeras compared, 30 Mouse chimeras developmental genetics studies, 46-53 cytogenetic studies, 46-47 genomic imprinting, 49-50 knockout analysis, 50-53 Mash2 gene analysis, 5 1-53 mutant gene phenotype analysis, 50-53 nudul gene analysis, 53 Pax6 gene analysis, 50-5 I sex determination in chimeras, 47-49 development studies, 30-40 cell distribution analysis, 37-40 founder cell number estimates, 35-37 lineage relationship analysis, 34-35 prospective studies, 30-33 qualitative spatial analysis, 37-38

250 Mouse (continued) quantitative spatial analysis, 39-40 retrospective studies, 33-40 experimental properties embryonic stem cell chimera production, 28-29 genetic effects on composition, 29-30 mosaics compared, 30 production, 22-28 genetic studies. 40-46 dt gene, 46 muscle degeneration analysis, 42-43 quantitative genetic traits, 40-41 retinal degeneration analysis, 43-46 rl gene, 46 single mutant phenotypic analysis, 41-46 green fluorescent protein marker, 12-14 overview, 21-22.53-55 green fluorescent protein marker chimeras, 12-14 in vivo gene expression studies, 7-10 Muscle cells, degeneration, mouse chimera phenotypic analysis, 42-43 myf-5 gene, pronephric mesoderm patterning role, 73 myoD gene, pronephric mesoderm patterning role, 73

N nunos gene, mRNA stability regulation in Drosophilu, 180-184 N-cadherin, pronephric tubule induction in Xenopus, 82 Nodal gene, chimera analysis in mouse, 53 nr-3 gene, pronephric mesoderm patterning role, 72

0 Oogenesis, epidermal growth factor receptorsignaling in Drosophilu, 203 -236 activation gurken gene translational regulation, 217-221 gurken RNA localization, 213-217 post-translational regulation, 22 1 spatial regulation, 212-221 axial polarity, 203-206 dorsoventral polarity, 232-236 Ras pathway role, 232-233

Index ventral embryonic cell fate regulation, 233 -236 epidermal growth factor receptor role, 206-212 follicle cell response, 22 1-232 dorsal follicular epithelium cell fate determination, 224-232 downstream targets, 222-224 epidermal growth factor receptor gradient effects, 224-225 germline signal initiation, 23 1 ovary target interactions, 226-228 pattern formation, 228-23 I rhomboid gene role, 225,228 overview, 203-204.236 Opsin-like photoreceptors, Pelvetia zygote symmetry breaking, 1 18-1 19 Organizer, pronephric mesoderm patterning genes, 72-73

P pal-I gene, mRNA stability regulation in Cuenorhabditis eleguns, 176-178 Pan-mesodermal genes, pronephric development role, 7 1-72 Pattern formation dorsoventral axis polarity in Drosophila oogenesis epidermal growth factor receptor activation, 232-236 Ras pathway role, 232-233 ventral embryonic cell fate regulation, 233-236 epidermal growth factor receptor role, 206 -2 12 follicle cell response, 221-232 anterior-posterior axis, 229-23 I dorsal follicle cell fates, 224-225 downstream targets, 222-224 epidermal growth factor receptor gradient effects, 224-225 germline signal initiation, 23 1-232 rhomboid gene role, 225, 228 target interactions, 226 -228 overview, 203-206, 236 spatial regulation, 212-221 gurken RNA localization, 2 I3 -2 I7 post-translational regulation, 221 translational regulation, 2 17-22 1 pronephros induction, 7 1-73 zygote symmetry breaking in fucoid algae

Index axis formation axis fixation, 115-1 17 cortical pH gradients, 113-1 14 fertilization physiology, 103-105 light response, 105 - I08 opsin-like photoreceptor, I 18- I 19 overview, 101-1 03, 12 1- 122 photopolarization actin microfilament role, 114-1 15 calcium role, 108-1 12 calmodulin role, 112-1 13 signal transduction process, 117-1 18 speculative model, 119-121 rhizoidal growth, 108-1 14 calcium role, 108-1 12 calmodulin role, 112-1 13 cortical pH gradients, 113-1 14 Pax-2 gene, pronephric tubule induction in Xenopus, 80 -8 I Pax-6 gene, chimera analysis in mouse, 50-5 I Pax-8 gene, pronephric tubule induction in Xenopus, 76-77 Pelveria, zygote symmetry breaking axis formation axis fixation, 115-I 17 cortical pH gradients, 113-1 14 fertilization physiology, 103-105 light response, 105-108 opsin-like photoreceptor, 118-1 19 overview, 101- 103, 121- I22 photopolarization actin microfilament role, 114-1 15 calcium role, 108-1 12 calmodulin role, 112-1 13 signal transduction process, 117-1 18 speculative model, 119-121 rhizoidal growth, 108-1 14 calcium role, 108-112 calmodulin role, 112-1 13 cortical pH gradients, 113-1 14 Petunia, bud outgrowth studies, 147-15 1 pH, zygote symmetry breaking in fucoid algae, axis formation and rhizoidal growth role, 113-1 14 Photoperiod response genes, bud outgrowth in angiosperms, 152- 154 Photopolarization, zygote symmetry breaking in fucoid algae actin microfilament role, 114-1 15 calcium role, 108-1 12 calmodulin role, 112-1 13 opsin-like photoreceptor, 118-1 19

251 overview, 105-108 signal transduction process, I 17-1 18 speculative model, 119-121 Pisum sativum, bud outgrowth studies genetic approaches, 151- 160 auxin studies, 157-158 branching, 152-1 54 cytokinin studies, 158 grafting studies, 155-157 growth habit, 151 photoperiod response genes, 152-154 Rms gene studies, 156-158, 160 shoot-root signals, 158-160 outgrowth potential, 131-132 Plants, see specific aspects; specific species Platelet-derived growth factor-p, glomus development role, 90 Pronephric development, 67-92 early development, 70-71 glomus, 88-90 background, 88 molecules, 88-90 platelet-derived growth factor-p, 90 vascular endothelial growth factor, 90 Wilms’ tumor-suppressor gene, 88-90 molecular mechanisms, 71-73 embryonic patterning, 7 1 molecular data, 7 1-73 laterally expressed genes, 72-73 organizer-specific genes, 72-73 pan-mesodermal genes, 7 1-72 pronephros-inducing genes, 73 ventralizing pathway, 72 ventrally expressed genes, 72 pronephros induction, 71 overview, 67-69,90-92 pronephric duct, 83-88 background, 83- 84 molecules, 84-88 c-ret, 84-85 E m - 2 gene, 86 glial cell line-derived neurotrophic factor, 84-85 glial cell line-derived neurotrophic factor receptor-a, 87-88 glycophosphatidylinositol-linkedproteins, 86-87 gremlin, 84 Id gene, 85-86 pronephric tubules, 74-83 background, 74 molecules, 75-82

252 Pronephric development (continued) bone morphogenetic protein, 81-82 cell adhesion molecules, 82 fibroblast growth factor, 8 1-82 Frz, 78-80 hedgehog, 82 helix transcription factors, 77-78 Lim- I , 75-76 Pax-2.80-81 Pax-8.76-77 3G8,82 Wnt, 78-80 structure and function, 69-70

R Ras pathway, dorsoventral axis polarity determination in Drosophila oogenesis, 232-233 Reporter genes, see specific types Retina, degeneration, mouse chimera phenotypic analysis, 43-46 rhomboid gene, epidermal growth factor receptor-signaling in Drosophila oogenesis activation regulation, 225 follicle cell patterning role, 228 rl gene, chimera phenotypic analysis in mouse, 46 Rms genes, bud outgrowth studies, in angiosperms auxin role, 157-158, 162 branching control, 160 cytokinin role, 158 genetic approaches. 158, 160 grafting, 155-157 growth habit, 154 shoot-root signals, 158-160 RNA localization in gurken gene, 213-217 mRNA stability regulation, 171-195 aves, 186-1 89 Caenorhabditis elegans, 176 - I78 Drosophila, 178-184 mammals, 189-194 overview, I7 I - 173 plants, 174-175 Xenopus. I84 - I86 S Sex determination. mouse chimeras, 47-49 Shoots, see Apical dominance; Bud outgrowth Signal transduction, zygote symmetry breaking

Index in fucoid algae, photopolarization role, 117-118 sizzled gene, pronephric mesoderm patterning role, 73 Spermatozoa, in vivo gene expression studies, green fluorescent protein marker, I I - I2 Stem cells, chimera production in mouse, 28-29 string gene, mRNA stability regulation in Drosophilu, 183-184

T Tbl gene, bud outgrowth in angiosperms, 138-1 39 3G8 gene, pronephric tubule induction, 82 torpedo gene, epidermal growth factor receptorsignaling in Drosophilu oogenesis, 206, 208 Transcription factors, pronephric tubule induction in Xenopus. 77-78 Transgenics, bud outgrowth in angiosperms, hormone modulation, 140-143 auxin level modulation, 141-143 cytokinin level increases, 141-143 homeostasis, 142-143

V Vascular endothelial growth factor, glomus development role, 90 Vent-1 gene, pronephric mesoderm patterning role. 72

W Western blot, green fluorescent protein analysis in mammals, 16 Wilms’ tumor-suppressor gene, glomus development role, 88-90 Wnf genes pronephric mesoderm patterning role, 72-73 pronephric tubule induction, 78-80

X Xenopus mRNA stability regulation, 184-186 pronephric development, 67-92 early development, 70-7 1 glomus, 88-90 background, 88 platelet-derived growth factor-p, 90

Index vascular endothelial growth factor, 90 Wilms’ tumor-suppressor gene, 88-90 molecular mechanisms, 7 1-73 embryonic patterning, 71 laterally expressed genes, 72-73 molecular data, 7 1-73 organizer-specific genes, 72-73 pan-mesodermal genes, 71-72 pronephros-inducing genes, 73 pronephros induction, 71 ventralizing pathway, 72 ventrally expressed genes, 72 overview, 67-69,90-92 pronephric duct, 83-88 background, 83-84 c-ret, 84-85 Em-2 gene, 86 glial cell line-derived neurotrophic factor, 84-85 glial cell line-derived neurotrophic factor receptor-a, 87-88 glycophosphatidylinositol-linkedpro. teins, 86-87 gremlin, 84 Idgene, 85-86 pronephric tubules, 74-83 background, 74 bone morphogenetic protein, 8 1-82 cell adhesion molecules, 82 fibroblast growth factor, 81-82 Frz, 78-80

253 hedgehog, 82 helix transcription factors, 77-78 Lim-I, 75-76 Pax-2.80-81 Pax-8.76-77 3G8.82 Wnt, 78-80 structure and function, 69-70 Xlist gene, mRNA stability regulation in mammals, 189-194 Z

Zygotes, symmetry breaking in fucoid algae axis formation axis fixation, 115-1 17 cortical pHgradients, I 13-1 14 fertilization physiology, 103- I05 light response, 105-108 opsin-like photoreceptor, 118-1 19 overview, 101-103, 12 1- 122 photopolarization actin microfilament role, 114-1 15 calcium role, 108-1 12 calmodulin role, 112-1 13 signal transduction process, 1 17-1 18 speculative model, 119-121 rhizoidal growth, 108-1 14 calcium role, 108-1 12 calmodulin role, 112-1 13 cortical pH gradients, 113-1 14

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Contents of Previous Volumes

1 The Murine Allantois Karen M . Downs

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

3 Maternal Control of Pattern Formation in Early Caenorhabditis elegans Embryos Bruce Bowerman

4 Eye Development in Drosophila: Formation of the Eye Field and

Control of Differentiation Jessica E. Treisrnan and Ulrike Heberlein

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

6 Molecular Regulationof Neuronal Apoptosis Santosh R. D’Mello

7 A Novel Protein for Ca2+Signaling at Fertilization J. Parrington, F. A. Lai, and K. Swann

8 The Development of the Kidney Jonathan Bard

1 Homeobox Genes in Cardiovascular Development Kristin D. Patterson, Ondine Cleaver, Wendy V. Gerber, Matthew W. Grow, Craig S. Newrnan, and Paul A. Krieg 255

256

Contents of Previous Volumes

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

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

4 Retinoids and Related Signals in Early Development of the Vertebrate Central Nervous System A. J. Durston, J. van der Wees, W. W. M. Pijnappel, andS. 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 NonbilaterianAnimals: Implicationsfor the Evolution of the Hox Cluster and the Zootype John R. Finnerty

7 The Conflict Theory of Genomic Imprinting: How Much Can Be Explained? Yoh lwasa

1 Pattern Formation in Zebrafish-Fruitful liaisons between Embryology and Genetics Lilianna Solnica-Krezel

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

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

4 DevelopmentalGenetics of Caenorhabditiseregans Sex Determination Patricia E. Kuwabara

5 Petal and Stamen Development Vivian F. Irish

Contents of Previous Volumes

257

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

Cumulative Subject Index, Volumes 20 through 41

1 Epigenetic Modification and Imprinting of the Mammalian Genome during Development Keith E. Latham 2 A Comparison of Hair Bundle Mechanoreceptorsin Sea Anemones and Vertebrate Systems Glen M. Watson and Patricia Mire

3 Development of Neural Crest in Xenopus Roberto Mayor, Rodrigo Young, and Alexander Vargas

4 Cell Determination and Transdeterminationin Drosophila Imaginal Discs Lisa Maves and Gerold Schuhiger

5 Cellular Mechanisms of Wingless/ Wnt Signal Transduction Herman Dierick and Amy Bejsovec

6 Seeking Muscle Stem Cells Jeffrey Boone Miller, Laura Schaefer, and JaniceA. Dominov

7 Neural Crest Diversification Andrew K. Groves and Marianne Bronner-Fraser

8 Genetic, Molecular, and MorphologicalAnalysis of Compound leaf Development Tom Goliber, Sharon Kessler, Ju-JiunChen, Geeta Bharathan, and Neelima Sinha

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