Mary S. Tyler
Developmental Biology, A Guide for Experimental Study, Second Edition, 2000
Sinauer Associates, Inc. Publishers, Sunderland, MA
ISBN 0-87893-843-5

• Contents
• Preface

Preface xiii

1. Getting Started 1

Things you will need 1

Your laboratory notebook 1

A format for your notebook 3

Formal laboratory reports 3

Anatomy of a scientific paper 3

Some hints about writing 4

Using the library 6

2. Embryological Tools 9

Microknives 9

Microneedles 10

Hairloops 10

Pipettes 11

Embryo spoons 12

Instrument tray 12

Retooling metal instruments 13

Sterile technique 14

The workbench 14

Dissecting tools 14

Glassware 15

Fluids 15

Rules for the road 16

3. Using the Compound Microscope 19

The microscope 19

Safety first 20

Stage 20

Magnifications 20

Oculars 20

Objectives 21

Koehler illumination 22

The condenser 22

Iris diaphragm 23

Other settings when maximum resolution is not desired 23

Oil immersion 24

Make your $1000 microscope into a $10,000 instrument for pennies 25

Color filters 25

Polarizing filters 25

Dark-field optics 26

Measurement under the microscope 27

Care and maintenance of your microscope: "Twelve good rules" 27

4. Cellular Slime Molds 31

Life cycle 31

The vegetative stage 31

Aggregation 31

Pseudoplasmodium 33

Culmination 34

Sexual reproduction: A rare event 34

Preparing for your laboratory studies 35

Culture procedures 35

Experiments using Dictyostelium 36

Labeling with vital dyes 36

Transecting, grafting, and disaggregating pseudoplasmodia 37

Disruption of cyclic AMP levels 38

Behavioral studies 39

Completing your experiments 40

5. Gametogenesis 45

Meiosis: An outline 45

Mammalian spermatogenesis 47

Stereology 48

Tunica albuginea 48

Seminiferous tubules 48

Spermiogenesis 50

Interstitial cells 51

Comparative sperm morphology 51

Mammalian oogenesis 52

Follicles 52

Theca 54

Oocyte 54

Corpus luteum 55

Histological Hitchcocking 55

6. Echinoid Fertilization and Development 59

Collecting 59

Fertilization 61

Instructions for normal fertilization 62

Watching fertilization 63

Interfering with fertilization 65

Parthenogenesis 65

Cleavage, gastrulation, and larval stages 66

7. Sea Urchin DevelopmentÑEffects of Ultraviolet Radiation 73

Ultraviolet radiation in Earth's atmosphere 73

Biological effects of UV radiation 74

Mechanisms that protect against damage from UV 75

Using sea urchins as a model organism for UV studies 75

Setting up your experiments 76

Special lightbulbs that can be used 77

Filters that can be used to exclude UV-A or UV-B 78

Filters that block UV-B 78

Filters that block both UV-A and UV-B (UV opaque) 79

Filters that do not block either UV-A or UV-B (UV transparent) 79

Safety first 79

Monitoring your cultures 80

Keeping up with the issues 80

8. Development of the Fruit Fly 85

Life cycle 85

Culturing Drosophila melanogaster 86

Collecting eggs 87

Collecting chamber 87

Mating behavior of adult flies 88

Observations of the egg 88

The chorion 89

Dechorionating an egg 90

Embryogenesis 90

Cleavage 90

Gastrulation 91

Later development 91

Embryonic staging series 91

Larval development 94

Anatomy of the larva 95

External anatomy 96

Internal anatomy 96

Development of imaginal discs 96

Dissection of imaginal discs 98

Eversion of imaginal discs 100

Whole-mount preparations of imaginal discs 100

Chromosome squash from salivary glands 101

Pupation 101

9. Early Development of the Chick 107

The chick egg 107

The female chick reproductive tract 111

Cleavage 112

Gastrulation 113

24-Hour chick whole mount 116

10. 33-Hour Chick Embryo 121

Central nervous system 121

Circulatory system 122

33-Hour whole mount 122

Folds and tucks: Morphogenesis proceeds 122

Ectodermal structures 123

A category unto themselves: Color them green 125

Endodermal structures 125

Mesodermal structures 125

33-Hour serial sections 128

Experiment with evolution 134

Create a puppet 134

11. The Living Embryo and Making of Whole Mounts 137

Incubation of eggs 137

Preparing whole mounts 138

Fixation 1 41

Washing 142

Staining 143

Dehydration and clearing 143

Mounting 144

12. Histological Techniques 147

Fixation 147

Washing 149

Dehydration and clearing 149

Special microwaving techniques that save time 150

Paraffin infiltration 151

Paraffin molds 151

Paraffin embedding 152

Mounting and trimming paraffin blocks 152

Sectioning 153

Mounting sections 155

Staining 156

Using the hematoxylin, eosin, and alcian blue staining series 158

Hematoxylin 159

Mounting coverslips 159

13. Planarian Regeneration 163

Collecting and maintaining planarians 164

Observations of normal anatomy and behavior 164

Anatomy 165

Behavior 166

The regeneration process: Patterns and theories of regeneration 167

The regeneration process 167

Pattern formation and positional information 168

Diffusion gradient model 168

Polar coordinate model 169

Patterns of regeneration 171

Experimental procedures 173

Planning your experiments 173

Procedures 173

Record keeping and analysis 175

14. Amphibian Development 179

Breeding 179

Environmental hazards affecting amphibian development 180

Preparing for the field trip 181

Learn about your local amphibians first 181

Equipment needed and recording of data 181

Equipment for pH measurements 187

Come dressed for the field 188

Know government regulations 188

The field trip 188

Rules for the road 190

Night sounds 191

Back at the laboratory 191

Species identification 191

Normal development 191

Environmental effects on amphibian development 197

Adopt an egg mass 199

Stay in touch 199

Index 204

Preface

Development is a magnificent and mysterious journey that we all participate in until the cold ground claims us. It is a journey so complex that we travel mostly unaware of the means by which we are speeding along. But the means of travel are well worth our attention. This book invites you to look through the windows that science providesÑyou may be the one who sees what others have missed.

THE BOOK AT A GLANCE

This guidebook begins with a discussion of the tools you need for studying developing organisms and moves on through the events of development, first in simple and then in more complex organisms.

Chapters 1-3 & 12: Tools of the trade

Much of the study of developmental biology involves craft. The first three chapters and chapter 12 will school you in the methods of this craft, teaching you how to record and report data, how to create your own tools, how to be master of your microscope, and how to prepare tissue for microscopic study.

Chapter 4: A simple organism

Not all organisms use sexual reproduction as the preferred method for passing on their genes. Sex is expensive, burdened as it is with the costs of meiosis. The the frugal cellular slime mold, consisting of only a few cell types, seldom indulges in sex, instead undergoing repeated cycles of asexual reproduction. Chapter 4 shows you how to design experiments that use this organism to explore principles of development operating in the realm of simple organization.

Chapter 5: Gametogenesis

In organisms that do use sexual reproduction, gametogenesis marks the beginning of development. Chapter 5 shows you how to sleuth your way through the events that lead to the formation of dimorphic gametesÑeggs and sperm, the cells that each carry half a load of genetic material for the new organism.

Chapter 6: Fertilization

Mature gametes have their cellular triggers cocked; fertilization is the explosion that is set off when egg and sperm meet, fusing the two gametes and propelling the new individual off along the road of development. Chapter 6 invites you to explore questions surrounding the mechanisms of this event using sea urchin gametes.

Chapters 6-11: Cleavage, gastrulation, and organogenesis

The journey started at fertilization continues at breakneck speed through cleavage. Cleavage provides the embryo with a large number of cells for continuing the trip. Its product, a beautiful blastula, may be the hollow ball of cells you will see among the sea urchin embryos spiraling across your dish in the experiments in Chapters 6 and 7; a ring of cells surrounding a yolk mass, as you will see in fruit fly embryos in Chapter 8; or a plate of cells balanced on a yolky sphere, as you will see in chick embryos in Chapter 9.

Following cleavage, cells start using a new set of instructions, newly transcribed messenger RNAs (mRNAs), for their developmental journey. Some of these messages instruct the cells to don new clothes, in the form of new cell-surface molecules. These molecules identify the cells according to germ layer: ectoderm, mesoderm, or endoderm. Once they put on their new clothes, the cells can't sit still. They begin moving to new positions in the process of gastrulation, which brings the germ layers into positions appropriate for their specific fates. You will see how these movements are choreographed in the sea urchin, fruit fly, and chick in Chapters 6 through 10.

With the germ layers in position, the trip enters a phase of dynamic interactions known as organogenesis. Cells in the embryo relay information back and forth, and these interactions lead to organ differentiation. The embryo is now complete; the embryonic portion of the trip is over. In Chapters 6 through 11 you will glimpse this final leg of the embryonic journey as you raise sea urchin larvae, dissect and experiment with imaginal discs of fruit fly larvae, and test the responses of one of the earliest products of organogenesis in the chick, the embryonic heart.

Chapter 13: Regeneration and pattern formation

Development is a long journey that extends well beyond the event of hatching or birth. One of the fascinating side roads is that of regeneration. Few organisms are capable of such impressive feats of regeneration as those seen in planaria. Forming complete replicas of the intact worm from fragments as small as 1/279th the size of the original organism, the planarian, as you will see in Chapter 11, allows you to venture into the mysteries of regeneration.

Chapters 7 and 14: Development and the environment

Development is always influenced by the environment in which it takes place, and often environmental hazards affecting the delicate embryonic stages of an organism threaten its survival. Organisms such as sea urchins and amphibians, which must entrust their poorly protected embryos to the open wilds, can be particularly vulnerable, and therefore make sensitive barometers of environmental hazards. In Chapter 7, the hazards of ultraviolet radiation, an ever increasing threat, are examined using the delicate sea urchin embryo. In Chapter 14 we end our study of the developmental journey by exploring the woods and streams for evidence of threats to embryos of our cool and thin-skinned amphibian relatives. .

One goal of this book is to help students be independent scientists. All the exercises are described in sufficient detail that any student should be able to perform them on their own. No secrets have been sequestered to a separate instructor's manual. In addition to instructions and background information, the book provides recipes for solutions, bibliographies for further study, and lists of scientific suppliers.

The illustrations in the book are line drawings designed to clearly delineate components and to draw attention to general principles of structure. Photographs and videos for all chapters can be found on the supplementary CD-ROM, Vade Mecum.

VADE MECUM - A Supplementary CD-ROM

Vade Mecum, an Interactive Guide to Developmental Biology, by this author along with R. N. Kozlowski, and published by Sinauer Associates, was developed primarily to augment this laboratory manual. The goal of the combined manual and CD is to provide students with all that they need to be truly independent learners. Each chapter of the laboratory manual is represented by a chapter on the CD, including over 130 videos and 300 labeled photographs that explain the development of the organisms and give step-by-step explanations of techniques. For example, where useful, color-coding is superimposed on living embryos to illustrate positioning of different germ layers. A complete set of cross-sections of a 33-hour chick embryo and wholemounts with definitions of terms are included. A "virtual microscope" section shows how to achieve Koehler and dark-field illumination and how to use polarizing filters. For each chapter, there are study questions and websites listed. A section on laboratory safety is also included. Vade Mecum can be purchased with the laboratory manual at reduced cost and also comes packaged with Scott Gilbert's Developmental Biology textbook.

ACKNOWLEDGMENTS

Many people have contributed ideas, inspiration, and hard work to these pages, and to them I am very grateful. I thank all the reviewers who added their wisdom and careful comments:

Nikki Adams, John Dearborn, Brian Hall, Malcolm Hunter, Richard Kessin, John Lucchesi, Robert Mead, David McClay, Drew Noden, John Ringo, Brian Sullivan, Seth Tyler, and Bonnie Wood. To those at Sinauer Associates, publisher Andrew Sinauer, production specialist Chris Small, and editors Kathaleen Emerson, Chelsea Holabird, and Carol Wigg, I extend a special warm thanks for bringing the book in its second edition to reality. Their creativity, attention to detail, and generous patience and support were invaluable. To Ryan Genz I am indebted for his creative cover design. And to my always-curious students, I am beholden for their attentiveness, which has taught me to see more than I would alone.

Mary S. Tyler

Orono, Maine