Methods and Protocols

Edited by Paul T. Sharpe and Ivor Mason

METHODS IN MOLECULAR BIOLOGY ™

Molecular Embryology Methods and Protocols

Edited by Paul T. Sharpe and Ivor Mason

Dental and Medical Schools of Guy's, King's, and St. Thomas's Hospitals, King's College, London, UK

Humana press ^ Totowa, New Jersey

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Library of Congress Cataloging in Publication Data Main entry under title:

Methods in molecular biology ^ .

Molecular embryology: methods and protocols / edited by Paul T. Sharpe and Ivor Mason.

p. cm. ~ (Methods in molecular biology ^ ; 97) Includes index.

ISBN 0-89603-387-2 (alk. paper)

1. Embryology--Vertebrates--Methodology. 2. Chemical embryology—Methodology I. Sharpe, Paul T. II. Mason, I. III. Series: Methods in molecular biology (Totowa, NJ); 97. QL959.M65 1998 571.8'616—dc21 98-23234

PREFACE

Most people have some interest in embryos; this probably results, in part, from their interest in understanding the biological origins of themselves and their offspring and, increasingly, concerns about how environmental change such as pollution might affect human development. Obviously, ethical considerations preclude experimental studies of human embryos and, consequently, the developmental biologist has turned to other species to examine this process. Fortunately, the most significant conclusion to be drawn from the experimental embryology of the last two decades is the manner in which orthologous or closely related molecules are deployed to mediate similar developmental processes in both vertebrates and invertebrates. The molecular mechanisms regulating processes fundamental to most animals, such as axial patterning or axon guidance, are frequently conserved during evolution. (It is now widely believed that the differences between phyla and classes are the result of new genes, arising mostly by duplication and divergence of extant sequences, regulating the appearance of derived characters.)

Other vertebrates are obviously most likely to use the same developmental mechanisms as humans and, within the vertebrate subphylum, the apparent degree of conservation of developmental mechanism is considerable. It has long been recognized that particular vertebrate species offer either distinct advantages in investigating particular stages of development or are especially amenable to particular manipulations. No single animal can provide all the answers because not all types of experiments can be carried out on a single species. Traditionally, developmental biologists have worked on their particular experimental favorite, working, for example, solely on Drosophila, orXenopus, or the mouse. In the last few years, this has started to change and there are now increasing numbers of laboratories that have acquired the expertise to work on several different animals and are thus able to harness the experimental advantages of different developmental systems to address specific developmental questions. Alternatively, Developmental Biology Departments are becoming organized such that they have expertise in several model organisms. It is the increasing necessity to be able to move between embryos of different vertebrate classes as a project progresses that prompted us to as-

semble Molecular Embryology: Methods and Protocols. We hope that it will allow researchers to familiarize themselves with the various commonly studied vertebrate embryos, to make informed choices about which might be best suited to their investigations, and to understand the techniques by which they might be manipulated.

Sadly, while this book was going to press, Nigel Holder, one of its contributors, died. Nigel was an excellent developmental biologist, a founder of the Developmental Biology Research Group at King's College, and had recently been appointed to the Chair of Anatomy and Human Biology at University College London. He was both a colleague and friend to ourselves and to many of the other contributors to this volume. He is greatly missed.

PAUL T. SHARPE IVOR MASON

LIST OF COLOR PLATES

Color plates 1-12 appear as an insert following p. 368.

Plate 1 (Fig. 1 from Chapter 37). Schematic view of the steps in constructing a subtracted cDNA library or subtracted probe. Plate 2 (Fig. 1 from Chapter 23). Cells labeled with Dil and DiA in the chick embryo neural plate.

Plate 3 (Fig. 1 from Chapter 48). Apoptosis in the rhombencephalic neural crest of the chick embryo as revealed by acridine orange staining and TUNEL.

Plate 4 (Fig. 4 from Chapter 51). (1) Color temperature: effects of a blue filter on a specimen under tungsten illumination. (2) Pseudocolor: three examples of the use of pseudocolor with black-and-white digital images.

Plate 5 (Fig. 1 from Chapter 45). Nonradioactive in situ hybridization on wax-embedded tissues in combination with immunohistochemical staining.

Plate 6 (Fig. 1 from Chapter 29). The reticulospinal complex of the zebrafish.

Plate 7 (Fig. 1 from Chapter 46). Regrowing axons from a transfected peripheral nerve.

Plate 8 (Fig. 2 from Chapter 46). Schwann cells of a transected peripheral nerve.

Plate 9 (Fig. 1 from Chapter 10). Dynamic progression of myogenin gene expression during the development of the skeletal musculature.

Plate 10 (Fig. 9 from Chapter 15). Stage 8 (HH) (28-34 h).

Plate 11 (Fig. 3 from Chapter 15). Stage 7 (HH) (26-30 h) shows the segmentation of the first pair of somites from thepara-notochordal mesoderm.

Plate 12 (Fig. 1 from Chapter 42). In situ hybridization of Fgf-8 to a stage 20 chicken embryo using a mouse cDNA sequence as probe.

CONTENTS

Preface v

List of Color Plates vii

Contributors xiii

Part I. The Mouse Embryo 1

The Mouse As a Developmental Model Paul T. Sharpe

Culture of Postimplantation Mouse Embryos Paul Martin and David L. Cockroft

3 23

Organ Culture in the Analysis of Tissue Interactions Irma Thesleff and Carin Sahlberg

4 33

Treatment of Mice with Retinoids in Vivo and in Vitro: Skeletal Staining Gillian M. Morriss-Kay

5 41

Cell Grafting and Labeling in Postimplantation Mouse Embryos Gabriel A. Quinlan, Paul A. Trainor, and Patrick P. L. Tam

6 61

Production of Transgenic Rodents by the Microinjection of Cloned DNA into Fertilized One -Celled

Eggs

San Ling Si-Hoe and David Murphy

7 101

Cre Recombinase Mediated Alterations of the Mouse Genome Using Embryonic Stem Cells Anna-Katerina Hadjantonakis, Melinda Pirity, and Andras Nagy

8 123

Gene Trapping in Mouse Embryonic Stem Cells Jane Brennan and William C. Skarnes

9 139

Production and Growth of Conditionally Immortal Cell Lines from the H-2KbtsA58 Transgenic Mouse Mark Noble

10 159

Reporter Genes for the Study of Transcriptional Regulation in Transgenic Mouse Embryos Jonathan D. Gilthorpe and Peter W. J. Rigby

11 183

Application of lacZ Transgenic Mice to Cell Lineage Studies

Paul A. Trainor, Sheila X. Zhou, Maala Parameswaran, Gabriel A. Quinlan, Monica Gordon, Karin Sturm, and Patrick P. L. Tam

Mouse Primordial Germ Cells: Isolation and in Vitro Culture Patricia A. Labosky and Brigid L. M. Hogan

Part II. Chicken Embryo 213

13 215

The Avian Embryo: An Overview Ivor Mason

14 221

Chick Embryos: Incubation and Isolation Ivor Mason

15 225

New Culture Amata Hornbruch

16 245

Grafting Hensen's Node Claudio D. Stern

17 255

Grafting of Somites Claudio D. Stern

18 265

Notochord Grafts

Andrew Lumsden and Susanne Dietrich

19 273

Transplantation of Avian Neural Tissue Sarah Guthrie

20 281 Grafting of Apical Ridge and Polarizing Region Cheryl Tickle

21 293

Tissue Recombinations in Collagen Gels Marysia Placzek and Kim Dale

22 305

Quail—Chick Chimeras

Marie-Aimée Teillet, Catherine Ziller, and Nicole M. Le Douarin

23 319

Using Fluorescent Dyes for Fate Mapping, Lineage Analysis, and Axon Tracing in the Chick

Embryo

Jonathan D. W. Clarke

Part III. Amphibian Embryo 329

24 331

An Overview of Xenopus Development C. Michael Jones and James C. Smith

25 341

Mesoderm Induction Assays C. Michael Jones and James C. Smith

26 351

Experimental Embryological Methods for Analysis of Neural Induction in the Amphibian

Ray Keller, Ann Poznanski, and Tamira Elul

27 393

A Method for Generating Transgenic Frog Embryos Enrique Amaya and Kristen L. Kroll

Axolotl/newt Malcolm Maden art IV. Zebrafish 429

29 431

The Zebrafish: An Overview of Its Early Development Nigel Holder and Qiling Xu

30 441 Small-Scale Marker-Based Screening for Mutations in Zebrafish Development

Peter D. Currie, Thomas F. Schilling, and Philip W. Ingham

31 461 Transgenic Zebrafish

Trevor Jowett

32 487 Microinjection of DNA, RNA, and Protein into the Fertilized Zebrafish Egg for Analysis of Gene

Function

Nigel Holder and Qiling Xu

33 491 Retinoids in Nonmammalian Embryos

Malcolm Maden

Part V. Nonvertebrate Chordates 511

34 513 Protochordates

Peter W. H. Holland and Hiroshi Wada

Part VI. Retroviruses 517

35 519 Gene Transfer to the Rodent Embryo by Retroviral Vectors

Grace K. Pavlath and Marla B. Luskin

36 539 Gene Transfer in Avian Embryos Using Replication-Competent Retroviruses

Cairine Logan and Philippa Francis-West

Part VII. Molecular Techniques 553

37 555 Subtractive Hybridization and Construction of cDNA Libraries

Bruce Blumberg and Juan Carlos Izpisúa Belmonte

38 575 Differential Display of Eukaryotic mRNA

Antonio Tugores and Juan Carlos Izpísua Belmonte

39 591 RT-PCR on Embryos Using Degenerate Oligonucleotide Primers

Anthony Graham

40 601 Single-Cell RT-PCR cDNA Subtraction

Damian L. Weaver, César Núñez, Clare Brunet, Victoria Bostock, and Gerard Brad

41 611 In Situ Hybridization of Radioactive Riboprobes to RNA in Tissue Sections

Radma Mahmood and Ivor Mason

In Situ Hybridization to RNA in Whole Embryos Huma Shamim, Radma Mahmood, and Ivor Mason

Wholemount in Situ Hybridization to Xenopus Embryos C. Michael Jones and James C. Smith

44 641

Whole-Mount in Situ Hybridization to Amphioxus Embryos Peter W. H. Holland

45 645

In Situ Hybridization to Sections (Nonradioactive) Maria Rex and Paul J. Scotting

46 655

Immunohistochemistry Using Polyester Wax Andrew Kent

47 663

Immunohistochemistry on Whole Embryos Ivor Mason

48 667

Whole Embryo Assays for Programmed Cell Death Anthony Graham

49 673

Gene Interference Using Antisense Oligodeoxynucleotides on Whole Chick Embryos: Optimal Ring and Roller-Bottle Culture Technique Jonathan Cooke and Alison Isaac

50 699

Protein Techniques: Immunoprecipitation, in Vitro Kinase Assays, and Western Blotting David I. Jackson and Clive Dickson

Part VIII. Microscopy and Photography 709

51 711

Microscopy and Photomicrography Techniques

Richard J. T. Wingate

Index

CONTRIBUTORS

Enrique Amaya • Wellcome, CRC Institute, Cambridge, UK

Juan Carlos Izpísua Belmonte • Gene Expression Laboratory, The Salk Institute for Biological Sciences, La Jolla, CA

Bruce Blumberg • Gene Expression Laboratory, The Salk Institute for Biological Sciences, La Jolla, CA

Victoria Bostock • School of Biological Sciences, University of Manchester, UK

Gerard Brady • School of Biological Sciences, University of Manchester, UK

Jane Brennan • BBSRC Centre for Genome Research, University of Edinburgh, Scotland, UK

Clare Brunet • School of Biological Sciences, University of Manchester, UK

Jonathan D. W. Clarke • Department of Anatomy and Developmental Biology, University College, London

David L. Cockroft • Imperial Cancer Research Fund, Department of Zoology, University of Oxford, UK

Jonathan Cooke • National Institute for Medical Research, London, UK

Peter D. Currie • Molecular Embryology Unit, Imperial Cancer Research Fund, London, UK

Kim Dale • Developmental Genetics Programme, The Krebs Institute, University of Sheffield, UK

Clive Dickson • Imperial Cancer Research Fund, London, UK

Susanne Dietrich • Department of Craniofacial Development, Guy's Campus, King's College, London, UK Tamira Elul • Graduate Group in Biophysics, University of California, Berkeley, CA

Philippa Francis-West • Department of Craniofacial Development, Guy's Campus, King's College, London, UK

Jonathan D. Gilthorpe • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Monica Gordon • Embryology Unit, Children's Medical Research Institute, Wentworthville, Australia

Anthony Graham • Department of Experimental Pathology, Guy's Campus, King's College, London, UK

Sarah Guthrie • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Anna-Katerina Hadjantonakis • Samuel LunenfeldResearch Institute, Mount Sinai Hospital, Toronto, Canada

Brigid L. M. Hogan • Department of Cell Biology, Howard Hughes Medical Institute, Vanderbilt University Medical School, Nashville, TN

Nigel Holder • Department of Anatomy and Developmental Biology, University College London, UK (deceased)

Peter W. H. Holland • School of Animal and Microbial Sciences, The University of Reading, Whitenights, UK

Amata Hornbruch • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Philip W. Ingham • Developmental Genetics Programme, The Krebs Institute, University of Sheffield, UK Alison Isaac • National Institute for Medical Research, London, UK David I. Jackson • Imperial Cancer Research Fund, London, UK C. Michael Jones • Chester Beatty Laboratories, London, UK

Trevor Jowett • Department of Biochemistry and Genetics, University of Newcastle Upon Tyne Medical School, Newcastle Upon Tyne, UK

Ray Keller • Department of Biology, University of Virginia, Charlottsville, VA

Andrew Kent • Division of Anatomy, Cell, and Human Biology, Guy's Campus, King's College, London, UK Kristen L. Kroll • Wellcome, CRC Institute, Cambridge, UK

Patricia A. Labosky • Department of Cell Biology, Howard Hughes Medical Institute, Vanderbilt University Medical School, Nashville, TN

Nicole M. Le Douarin • Centre National de la Recherche Scientifique, Instituit d'Embryologie Cellulaire et Moleculaire, College de France, Nogent-sur-Marne, France

Cairine Logan • Neuroscience Research Group, Department of Anatomy and Cell Biology, University of Calgary, Canada

Andrew Lumsden • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Marla B. Luskin • Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA

Malcolm Maden • Developmental Biology Research Centre, King's College London, UK

Radma Mahmood • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Paul Martin • Department of Anatomy and Developmental Biology, University College London, UK

Ivor Mason • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Gillian M. Morriss-Kay • Department of Human Anatomy, Oxford, UK David Murphy • Department of Medicine, University of Bristol, UK

András Nagy • Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada

Mark Noble • Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT

César Núñez • School of Biological Sciences, University of Manchester, UK

Maala Parameswaran • Embryology Unit, Children's Medical Research Institute, Wentworthville, Australia

Grace K. Pavlath • Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA

Melinda Pirity • Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada

Marysia Placzek • Developmental Genetics Programme, The Krebs Institute, University of Sheffield, UK

Ann Poznanski • Department of Biology, University of Virginia, Charlottsville, VA

Gabriel A. Quinlan • Embryology Unit, Children's Medical Research Institute, Wentworthville, Australia

Maria Rex • Department of Genetics, University of Notingham, UK

Peter W. J. Rigby • Chester Beatty Laboratories, London, UK

Carin Sahlberg • Biocentre, University of Helsinki, Finland

Thomas F. Schilling • Department of Anatomy and Developmental Biology, University College, London, UK Paul J. Scotting • Department of Genetics, University of Notingham, UK

Huma Shamim • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Paul T. Sharpe • Department of Craniofacial Development, Guy's Campus, King's College, London, UK San Ling Si-Hoe • Department of Medicine, University of Bristol, UK

William C. Skarnes • BBSRC Centre for Genome Research, University of Edinburgh, Scotland, UK

James C. Smith • Laboratory of Developmental Biology, National Institute for Medical Research, London, UK

Claudio D. Stern • Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, NY

Karin Sturm • Embryology Unit, Children's Medical Research Institute, Wentworthville, Australia Patrick P. L. Tam • Embryology Unit, Children's Medical Research Institute, Wentworthville, Australia

Marie-Aimee Teillet • Centre National de la Recherche Scientifique, Instituit d'Embryologie Cellulaire et Moleculaire, College de France, Nogent-sur-Marne, France

Irma Thesleff • Biocentre, University of Helsinki, Finland

Cheryl Tickle • The Wellcome Trust Building, University of Dundee, UK

Paul A. Trainor • National Institute for Medical Research, London, UK

Antonio Tugores • Gene Expression Laboratory, The Salk Institute for Biological Sciences, La Jolla, CA Hiroshi Wada • School of Animal and Microbial Sciences, The University of Reading, UK Damian L. Weaver • School of Biological Sciences, University of Manchester, UK

Richard J. T. Wingate • MRC Brain Development Programme, Department of Developmental Neurobiology, Guy's Campus, King's College, London, UK

Qiling Xu • National Institute for Medical Research, London, UK

Sheila X. Zhou • Embryology Unit, Children's Medical Research Institute, Wentworthville, Australia

Catherine Ziller • Centre National de la Recherche Scientifique, Instituit d'Embryologie Cellulaire et Moleculaire, College de France, Nogent-sur-Marne, France

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