J. A. Campos-Ortega· V. Hartenstein

The Embryonic Development of Drosophila melanogaster

Springer-Verlag Berlin Heidelberg GmbH

J.A. Campos-Ortega v. Hartenstein

The Embryonic Development of Drosophila melanogaster Second Edition

With 139 Illustrations, 37 in Colour, Consisting of over 600 Separate Figures

Springer

IV

Prof. Dr. Jose A. Campos-Ortega lnstitut fur Entwicklungsbiologie der Universitat zu Koln Gyrhofstrasse 17

D-50923 Koln Germany

Prof. Dr. Volker Hartenstein Department of Molecular Cell and Developmental Biology University of California Los Angeles 405 Hildgard Avenue Los Angeles, CA 90024 USA

ISBN 978-3-662-22491-5

Library of Congress Cataloging-in-Publication Data Campos-Ortega, Jose A. (Jose Antonio), 1940-The embryonic development of Drosophila melanogaster/Jose A. Campos­Ortega, Volker Hartenstein. -- 2nd ed. p. em. Includes bibliographical references and index. ISBN 978-3-662-22491-5 ISBN 978-3-662-22489-2 (eBook)DOI 10.1007/978-3-662-22489-21. Drosophila melanogaster--Development. 2. Embryology--Insects. I. Hartenstein, Volker, 1957- . II. Title. QL958.C36 1997 571.8' 615774--dc21

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© Springer-Verlag Berlin Heidelberg 1997 Originally published by Springer-Verlag in 1997 Softcover reprint of the hardcover 2nd edition 1997

The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover Design: D & P GmbH, Heidelberg Cover Illustration: Dr. Volker Hartenstein Typesetting: Typo-Press Service GmbH Heidelberg SPIN 10054085 39/3137 5 4 3 2 1 o - Printed on acid free paper -

v

Dedicated to Donald F. Poulson

Preface

This is a monograph on embryogenesis of the fruit fly Drosophila

melanogaster, conceived as a reference book on the morphology of embryonic development. A monograph of this extent and content is not yet available in the literature on Drosophila embryology, and we believe that there is a real need for it. Thanks to the pro­gress achieved during the last ten years in the fields of develop­mental and molecular genetics, work on Drosophila development has expanQ.ed considerably, creating an even greater need for the information that we present here. Our own interest in embryonic development of the wild type arose several years ago, when we began to study the development of mutants. In the course of these studies we repeatedly had occasion to discover gaps and inade­quacies in the existing literature on the embryology of the wild type, so that we undertook to investigate many of these problems ourselves. Convinced that many of our colleagues will have encountered similar difficulties, we decided to publish the present monograph.

Although not explicitly recorded, Thomas Aquinas was proba­bly referring to the domestic fly and not to the fruit fly. Irrespec­tive of which fly he meant, however, we know that Thomas was right. The situation today is not very different from that in the thirteenth century insofar as nobody would seriously claim to have completely investigated the "nature" of the fly. Consequently, in our work we did not intend to cover all aspects of embryology; in particular, we have purposely avoided dealing with genetic aspects of development, restricting ourselves to morphology. We repeat: our intention was only to provide basic information about the anatomy of normal embryogenesis, illustrating our descripti­on with figures of appropriate technical quality.

In this book we have used a rather conventional distribution of topics, similar to that in other embryological monographs. A few of these topics, i.e. neurogenesis, the pattern of mitotic divisions of embryonic cells, some aspects of the morphogenetic move­ments and the blastoderm fate map, are based on studies by the

" ... but our knowledge is so weak that no philosopher will ever be able to completely explore the nature of even a fly ... " * Thomas Aquinas "In Symbolum Apostolorum" 079

RSV p/96

VII

VIII Preface

,. The Latin quotation is: " ... sed cognitio nostra est adeo debilis quod nullus philosophus potuit unquam perfecte investigare naturam unius muscae ...

authors and their colleagues, the results of which have already appeared in different form in the scientific literature. The third chapter deals chiefly with organogenesis, although we have also considered in some detail several aspects of the anatomical orga­nization of the larva which, in fact, do not strictly pertain to embryogenesis; for example, we have invested some effort in inve­stigating the pattern of distribution of sensory organs, peripheral nerves, muscles, etc. in the fully developed embryo.

Several persons and institutions have contributed to this book. Chapter 2 presents a classification of embryogenetic stages which is based on similar classifications by other authors. The author­ship of this chapter is actually shared by Christiane Nusslein-Vol­hard and Eric Wieschaus, with whom J. A. Campos-Ortega defi­ned the stages almost identically several years ago. Work on this manuscript was initiated at the Institut fur Biologie III of the Uni­versity of Freiburg i. Br., FRG, of which both authors were staff members. Many friends and colleagues helped us while working in Freiburg. The most important contribution was by Sigrid Krien, our collaborator of over ten years, who patiently prepared most of the material for this work. Important, too, was the "moral" support of Rainer Hertel. Many of the ideas presented in this book originated in discussions with Ursula Dietrich, Fernan­do Jimenez, Gerd Jurgens, Ruth Lehmann, Christiane Nusslein­Volhard, Gerd Technau and Eric Wieschaus. We would particular­ly like to thank Klaus Sander, who repeatedly contributed enligh­tening discussions on the embryogenesis of insects, insisted on the importance of using appropriate terminology, and critically read the entire manuscript, correcting several mistak.es. Finally we want to thank Gerd Technau for permission to quote his unpu­blished results, and Alfonso Martinez-Arias for a discussion on tracheal pits. Financial support was provided by the Deutsche Forschungsgemeinschaft (DFG, grants SFB 46, Ca 60/6-1, Ca 60/7-1 and SFB 74).

Kaln, September 1985 Jose A. Campos-Ortega Volker Hartenstein

Preface to the 2nd edition

The first edition of this book appeared in 1985, over 10 years ago. Prior to its publication, Drosophila embryogenesis was the field of study of only a handful of scientists. Consequently, at that time this topic was poorly understood since we all knew very little indeed. Nevertheless, many important questions related to Drosophila embryonic development had already been asked by our predeces­sors, and we added a few more. Unfortunately, most of these que­stions rem!lined unanswered because of the inadequacies of the available techniques and other adverse factors.

Since then, work on Drosophila embryogenesis has made con­siderable progress, and most of the questions posed by earlier workers have already found satisfactory solutions. Today, the Dro­sophila embryo is in fact one of the best understood developmen­tal systems. Accordingly, the task of preparing this second edition has taken much longer, since much more had to be reviewed; paradoxically, it has also been a much easier job, as the informati­on can now be more convincingly integrated in the larger context. The mass of information available, very much increased as com­pared to the situation 10 years ago, has forced us to increase the number of chapters from five to eighteen. To accommodate this new material, the previous third chapter (Organogenesis) has been expanded and now comprises ten different chapters, in which the development of each different organ system is treated separately. However, the organization of the book has remained in principle the same. Most of the data discussed in the first edition were derived from our own work; this time, many data are derived from the work by other authors.

We would like to thank Detlev Buttgereit, Christian Klambt, Renate Renkawitz-Pohl, Thomas Menne, Evelin Sadlowski, Ger­hard Technau, and Andrea Wolk, who provided slides and/or unpublished photographs for inclusion in this book. We would also like to thank Paul Hardy, Elisabeth Knust and Christian Klambt (Koln), Torsten Bossing, Gerald Udolph and Gerhard M. Technau (Mainz), Corey Goodman (Berkeley), Amelia Younossi-

Preface IX

X Preface 2nd edition

Hartenstein, Judith Lengyel and Ulrich Tepass (Los Angeles), for many discussions, communication of unpublished results and rea­ding of the manuscript. Jose A. Campos-Ortega would like to thank Dr. Kazumoto Imahozi, past president of the Mitsubishi­Kasei Institute of Life Sciences, Machida (Tokyo), and Dr. Daisuke Yamamoto, of the same Institute, for the hospitality they provided in February 1992 when part of the text of this book was written. Work in our laboratories was supported by grants of the Deutsche Forschungsgemeinschaft (SFB 243), the "Fonds der Chemischen Industrie", the NSF (IBN 9221407) and the NIH (Grant NS29367).

K6ln and Los Angeles, June 1996

Jose A. Campos-Ortega Volker Hartenstein

Introductory Remarks

The importance of Drosophila melanogaster for biological re­search does not need to be expressly emphasized; it has been suf­ficiently demonstrated and documented on a multitude of occasi­ons during the 90 years in which the fruitfly has been used in experimental work (Allen 1975, 1978). Due to its very elaborate genetics, the fruitfly is now one of the preferred models for tack­ling most problems in developmental biology.

The suit?-bility of Drosophila for embryogenetic studies, and, in particular, the fact that it allows genetic dissection of embryonic development, was recognized almost 6o years ago by Poulson (1937a, 1940, 1943, 1945), who studied the effects of a few chromo­somal deficiencies on embryonic development. In fact, more important than his description of the effects of the lack of certain genes on development was Poulson's contribution to establishing the genetic approach to embryogenesis on a firm basis. Poulson was among the first to claim explicitly that genes are involved in directing developmental processes, on the same lines as Gold­schmidt's Physiological Genetics (1927, 1938), and he called the attention of experimenters to deficiencies and other chromosomal mutations with lethal effects on the homozygotes as tools for inve­stigating the contribution of genes to embryonic development (Poulson 1943, 1945). The genetic approach to embryonic develop­ment has recently received a tremendous boost. It suffices to com­pare Ted Wright's (1970) review on the genetics of embryogenesis in Drosophila with some of the pertinent, up-to-date papers on aspects of the same topic (e.g. the contributions to the book "Development of Drosophila melanogaster" edited by C.M. Bate and A. Martinez-Arias, 1993), to realize how profoundly this field has been transformed, both from the conceptual and the material point of view.

Published work on normal embryogenesis of the fruitfly was very scarce until the mid-eighties of this century. The very promi­sing foundation of a modern embryology of dipteran insects represented by Weismann's contribution (1863; see Sander 1985a,

XI

XII Introductory Remarks

for a critical appraisal of Weismann's studies on insect develop­ment) was unfortunately followed by only a few further exten­sions (e.g. Poulson 1950, Sonnenblick 1950, Schoeller 1964, Ander­son 1962, 1972). The standard works on organogenesis, and some other aspects of Drosophila embryogenesis, for many years were those of Sonnenblick (1941, 1950) and Poulson (1937b, 1950). These authors enunciated several of the embryogenetic questions with which we are still concerned, although the technical quality of their histological material was unsatisfactory. Subsequent accounts of organogenesis in the fruit fly (e.g. Fullilove and Jacob­son 1978) follow Poulson's more or less literally; our account has also been heavily influenced by his.

The small size of the Drosophila egg, and the technical difficul­ties associated with its investigation (for example, the lack for many years of reliable fixation methods for histological analyses, other than those requiring pricking, or otherwise damaging the integrity of the egg), were certainly two of the reasons for the pau­city of publications on normal embryogenesis of the fruitfly for a very long time. However, most of these technical difficulties were eliminated by the elaboration of reliable techniques for egg per­meabilization (Zalokar 1970, 1976; Zalokar and Erk 1976, 1977). The significance of these techniques for the embryology of the fruitfly is inestimable; they permitted considerable improvement in the fixation of specimens for histological investigation, which then became an easy undertaking, and initiated a series of studies on cell biological aspects of early embryogenesis that is still in progress. For example, the excellent preparation of the material used in Turner and Mahowald's (1976, 1977, 1979) scapning elec­tron microscope studies on morphogenetic movements was made possible by Zalokar's permeabilization techniques.

However, the most important contribution to Drosophila embryology has come from the combination of phenotypic and molecular analyses of morphogenetic mutants and the correspon­ding genes (e.g. Niisslein-Volhard et al. 1982) that has been exploi­ted during the last 15 years. A key element in this process was the development of a reliable and easy technique for germ line trans­formation (Rubin and Spradling 1982; Spradling and Rubin 1982), which opened wholly unexpected opportunities for the analysis of the genetic bases of development. Wholemount in situ hybridiza­tion (Tautz und Pfeifle 1989), antibodies and enhancer trap lines (O'Kane and Gehring 1987) contributed to facilitate the cellular analysis of the embryogenetic mutants and their phenotypes. All these elements have made the embryo of Drosophila one of the best understood developmental systems.

A few terminological clarifications are necessary for following our text. The egg of Drosophila melanogaster is a regular ovoid, slightly flattened dorsoventrally. It has become customary in embryogenetic studies to use the percentage of egg length (% EL, o % at the posterior pole, Krause 1939) and to the percentage of ventrodorsal circumference (% VD, o o/o at the ventral midline) as a reference system that permits developing structures to be loca­ted; this system has been used throughout this book, together with the terms dorsal and ventral, medial and lateral, cephalic and caudal or anterior and posterior. The term anlage is used for the progenitor cells of a given organ in the blastoderm; the term pri­mordium is reserved for the progenitor cells once the organ is recognizable, prior to completion of proliferation and cytodiffe­rentiation. The terms stomodeum and proctodeum are used to designate the primordia of foregut and hindgut, respectively, before regional differentiations have appeared; foregut or hindgut is used on~e regionalization appears, i.e. when pharynx and oeso­phagus, Malpighian tubules and hindgut have differentiated. Due to germ band elongation, abdominal levels of the Drosophila embryo are located in mirror-image relative to thoracic and gnat­hal levels during a substantial fraction of embryogenesis; this peculiarity poses some problems in orienting structures. Unless expressly stated, orientation is indicated with respect to the pola­rity of the embryo. The term germ band refers to the overtly meta­meric region of the embryo, as distinct from the procephalon.

Introductory Remarks XIII

XIV

CONTENTS

Chapter 1

A Summary of Drosophila Embryogenesis 1

Chapter 2

Stages of Drosophila Embryogenesis 9

Chapter 3 Mesoderm Development 101

3.1 Gastrulation 101 3.2 Splanchnopleura 102 3.3 The procephalic mesoderm 104

Chapter 4 Musculature 107

4.1 Visceral musculature 107 4.2 Somatic musculature III

Chapter 5 Circulatory System , 123

5.1 Structure of the dorsal vessel in the mature embryo 123 5.2 Development of the dorsal vessel 127 5.3 The fat body 129

Chapter 6 Macrophages 133 6.1. The pattern of embryonic cell death 134 6.2. Origin and dispersal of macrophages 134

Chapter 7 The Gut and its Annexes 139 7.1. The foregut 139 7.2 The salivary glands 147 7.3 The midgut 148 7.4 The hindgut and the Malpighian tubules 148

Chapter 8 Epidermis 161

8.1 The. pattern of early ectodermal mitoses 164 8.2 Morphogenesis of the epidermal primordium 164 8.3 Cell differentiation in the epidermis 167 8.4 The oenocytes 168 8.5 The imaginal discs and histoblasts 171

Chapter 9 Peripheral Nervous System 175 Sensory Organs and Peripheral Nerves 175

9.1 Sensory organs 175 9.2 Peripheral nerves 208

Chapter 10 Peripheral Nervous System 219 Stomatogastric Nervous System 219 10.1 Organization of the stomatogastric nervous

system in the fully developed embryo 219 10.2 The ring gland 223 10.3 Development of the stomatogastric nervous

system and ring gland 225

Chapter 11 Central Nervous System 231 11.1 Early neurogenesis in the segmented germ band 232 11.2 Early neurogenesis in the procephalon 248 11.3 Neuronal differentiation 255 11.4 The glial cells 263 11.5 Origin of the imaginal nervous system 266

Chapter 12 Tracheal Tree 269

Chapter 13

Gonads 277 13.1 Formation of the pole cells 13.2 Migration of pole cells 13.3 Formation of the gonads

277 279 282

Contents XV

XVI Contents

Chapter 14 The Pattern of Embryonic Cell Divisions 285 14.1 Overview 285 14.2 The pattern of the first postblastoderm mitosis 287 14.3 The pattern of the second postblastoderm mitosis 297 14.4 The pattern of the third and fourth postblastoderm

mitoses 301 14.5 The orientation of mitotic spindles 306

Chapter 15 Morphogenetic Movements 307 15.1 Gastrulation 308 15.2 The cephalic and the transverse furrows 311 15.3 Germ band elongation 313 15.4 The behaviour of blastoderm cells during early

morphogenetic movements 313 15.5 Morphogenetic movements associated with

organogenesis 15.6 Germ band shortening 15.7 Dorsal closure

Chapter 16 Cephalogenesis 16.1 Early events 16.2 Late events: larval structure of the atrium,

cephalopharyngeal skeleton and dorsal pouch 16.3 Atrium formation 16.4 Cell death during cephalogenesis 16.5 Retraction of the labrum 16.6 The embryonic origin of head imaginal discs

Chapter 17 Some Aspects of Segmentation 17.1 Introduction

315 316

317

319 319

32 7 331 335 335 336

339 339

17.2 Metamerical pattern elements of the germ band 340 17.3 Homologies and differences between segments 347 17.4 Embryology of segmentation 349 17.5 Segmentation in the head 353 17.6 The tail region 354

Chapter 18

A Fate Map of the Blastoderm 18.1 Th~ fate map 18.2 The rationale of fate map construction 18.3 The size of the blastoderm anlagen References

357 359 359 361

367

Contents XVII