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





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 Development—Effects 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


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 provides—you may be the one who sees what others have missed.

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 gametes—eggs 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.
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


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Developmental Biology
Published by Elsevier Science under Auspices of Society for Developmental Biology
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