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Principles of Development
 Principles of Development
Sixth Edition

Lewis Wolpert | Cheryll Tickle | Alfonso Martinez Arias

Peter Lawrence

James Locke

1
 1
Great Clarendon Street, Oxford, OX2 6DP,
United Kingdom
Oxford University Press is a department of the University of Oxford.
It furthers the University’s objective of excellence in research, scholarship,
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© Oxford University Press 2019

The moral rights of the authors have been asserted
Third edition 2006
Fourth edition 2011
Fifth edition 2015
Impression: 1
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a retrieval system, or transmitted, in any form or by any means, without the
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contained in any third party website referenced in this work.
Preface

The fundamental questions about how an embryo develops into a new individual
were posed long ago by the ancient Greek scientists and philosophers. Over the cen-
turies, advances in experimental embryology and, more recently, the application
of genetics, cell biology, and advances in imaging have revealed many similarities
between the development of different organisms, particularly during the early stages,
suggesting the existence of general principles underlying the process. Developmental
biology is now a mature discipline, and this is the foundation on which this book,
focused on principles, was built. We have not included in this sixth edition every new
detail that has emerged since the previous edition, which are many and wonderful,
but instead, where appropriate, have provided up-to-date examples to illustrate the
general principles.
Principles of Development is designed for undergraduates, and we have tried to
make these principles as clear as possible and to provide numerous summaries, in
both words and pictures. As we understand the principles better, the book should
become shorter, but we have not yet achieved this!
We concentrate on the development of vertebrates and Drosophila but include other
organisms, such as the nematode, the sea urchin, Hydra, planarians, and crustaceans,
where they best illustrate a concept. Chapter 1 provides a brief history of embryology
and an introduction to some of the main general principles and processes involved.
Chapter 2 considers the process of pattern formation in laying down the body plan in
Drosophila (Chapter 2). This small fly has played, and still plays, a central role in elu-
cidating developmental mechanisms. Chapter 3 describes the embryology and genetics
of our vertebrate model organisms—Xenopus, zebrafish, chick, and mouse—together
with some of the main methods used to study them, with coverage in this edition of
new methods of gene editing such as CRISPR-Cas9. Although the fundamental prin-
ciples of development are still largely illuminated by studies on the embryos of model
organisms, there is an emerging focus on human embryonic development, and we have
expanded our coverage of the topic to include advances in studying early stages, an
outline of the development of the human placenta, and a box on human twinning. The
mechanisms involved in pattern formation in the early development of our vertebrate
model organisms are considered in Chapters 4 and 5. These chapters are organized as
in the previous edition, with the process of laying down the early body plan being first
described in its entirety in Xenopus (Chapter 4), the vertebrate in which the general
principles were discovered. This is followed by comparisons with the process in the
zebrafish (Chapter 4), and in chick and mouse (Chapter 5). Chapter 5 also considers
how the body plan is completed, which mainly rests on studies in chick and mouse
embryos. Chapter 6 focuses on pattern formation in two invertebrate model organisms,
the nematode and the sea urchin, with an online section on ascidian development.
Chapters 7 and 8 focus on the fundamental processes of morphogenesis and differ-
entiation, respectively, and have been extensively revised, with particular reference to
the role of planar cell polarity and convergent extension in Chapter 7, and the impact
of single-cell transcriptomics in dissecting cell-fate decisions, with the blood cell lin-
eages as an example, in Chapter 8. Chapter 8 also includes more extensive coverage of
the role of epigenetics in development and cell differentiation, and some new boxes,
including one describing organoids—three-dimensional organ cultures that mimic tis-
sues and organs—and their potential clinical uses. Chapter 9 deals with germ cells and
vi Preface

fertilization. Organogenesis (Chapter 10) and the development of the nervous system
(Chapter 11) are huge topics, so we have had to be very selective in our coverage, but
have included a new box in Chapter 10 on mammary gland development and its rel-
evance to understanding breast cancer. Sections of the chapter on the development of
the Drosophila tracheal system and the mammalian vascular system have been moved
to Chapter 7, and other sections from previous editions have been placed online.
Growth and regeneration are considered together in the same chapter (Chapter 12).
Chapter 13 deals with plant development. This chapter has been updated and includes
a new section on vernalization. The last chapter (Chapter 14) deals with development
in relation to evolution and has been reorganized and updated throughout, highlight-
ing the impact of genomics and the many insights it has brought to this aspect of
developmental biology. Evolution and development are inextricably linked in ways
we are only now beginning to understand, with the convergence of genomics and cell
biology on embryos. Together with the new technologies for gene editing, genomics is
rapidly expanding the range of species accessible to evolutionary studies.
We have assumed that students have some familiarity with basic cell and molecular
biology and genetics, but all key concepts, such as the control of gene activity, are
explained in the text. There is an extensive Glossary, which means that the book is
self-contained. The illustrations are a special feature and have been carefully designed
and chosen to illuminate both experiments and mechanisms. New diagrams and pho-
tographs are included throughout the book, together with information about their
sources. In providing further reading, our prime concern has been to guide the student
to particularly helpful papers and reviews, rather than to give credit to all the scientists
who have made major contributions: to those whom we have neglected, we apologize.
The main authors for this new edition are, as for the last edition, Lewis Wolpert,
Cheryll Tickle, and Alfonso Martinez Arias. We have bid farewell to co-authors of previ-
ous editions, Jim Smith, Elliot Meyerowitz, Elizabeth Robertson, and Andrew Lumsden,
and thank them for their pivotal contributions over several editions. The remaining
long-standing co-author, Peter Lawrence, has been joined by James Locke for this edi-
tion, who has revised the chapter on plants. Each chapter has also been reviewed by a
number of experts (see page xxv), to whom we are very grateful. The authors made the
initial revisions, which were then deciphered, edited, and incorporated by our editor,
Eleanor Lawrence, who has been helped for this edition by Amanda Tromans. We thank
them both. Eleanor’s involvement has been absolutely essential in the preparation of
this edition and her expertise and influence pervades the book. Her input has also been
invaluable in ensuring that the information in the book is readily accessible to students.
The new illustrations were brilliantly drawn or adapted by Matthew McClements, who
created the illustrations for the first and subsequent editions.
We are indebted to Roseanna Levermore and Jonathan Crowe at Oxford University
Press for their help and suggestions throughout the preparation of this new edition.

L. W.

London
October 2018

C. T.

Bath
October 2018

A. M. A.

Cambridge
October 2018
Learning from this book

Principles of Development includes a number of features to help make it easy to use, and to make your learning as effective as possible.

Experimental boxes discuss both classic and current experimental research,

EXPERIMENTAL BOX 8C Single-cell analysis of cell-fa
demonstrating ‘how we know what we know.’
During development, differentiating cells progress from a multi
potent state towards terminally differentiated states, in which
they express a specific set of genes that are associated with
the specialized structure and function of the fully differentiated
cells. This progression is generally envisaged as a sequence o

Cell Biology boxes equip you with a robust conceptual framework on which to
add further detail from the vast amount of scientific information available to us CELL BIOLOGY BOX 4B The Wnt/β-catenin signaling pat

today.
The important developmental
Wnt abs
signaling pathway initiated by
the Wnt family of intercellular
signaling proteins (pronounced LRP5/6
‘Wints’) is named after the proteins

Medical boxes illustrate the direct relevance of developmental biology to medi-

cine and health-related issues. MEDICAL BOX 11D Autism: a developmental disorder

The brain works through the release of neurotransmitter mol

ecules at synapses, which interconnect neurons in highly com
plex networks. Correct synapse formation and refinement during
development is therefore of crucial importance to cognitive func
tion. Autism is a common (1 in around 120 live births) and per

Special interest boxes highlight topics of interest such as ‘The development of

the neural circuit for the knee-jerk reflex’ in Chapter 11 and the ‘Origins of mor- BOX 11C The development of the neural circuit for the

phological diversity in dogs’ in Chapter 14.

Neural circuit that underlies the knee-jerk reflex

dorsal root sensory muscle quadriceps

neuron spindle

spinal

Summary boxes provide a brief overview of each main section, which we hope
SUMMARY
you will find particularly useful when revising for examinations. Summary boxes
are augmented by a bullet-point review of the chapter’s major concepts at the Transcriptional control is a key feature of cell differentiation. Tis
sion of a eukaryotic gene depends on control elements located
end of each chapter.
regions flanking the gene. These elements comprise both the
adjacent to the start site of transcription, which bind RNA po
distant elements such as enhancers that control tissue-specifi
stage specific gene expression The combination of different
viii Learning from this book

Online resources

The online resources that accompany this text contain additional teaching and learning
resources for both lecturers and students.
Visit the site at www.oup.com/uk/wolpert6e

For students

Flashcard glossary

Flashcards, which can be downloaded your mobile phone, help you to test your recall
of key terminology.

Multiple-choice questions

Use the extensive bank of multiple-choice questions to check your understanding of

concepts introduced in the book, and get instant feedback on your progress.

Answer guidance

The authors have written answer guidance to the long-answer questions found at the
end of each chapter, so you can check that you have considered all the appropriate
points when responding to each question.

Web links and web activities

Links to websites, with notes to explain how each site relates to concepts featured in
the book, are provided to help you explore topics in the book in more detail. Complete
the associated activities to get to grips with the material in a hands-on way.

In silico practicals

In the textbook, we set out the current understanding of developmental processes

and provide some examples of ‘how we know what we know’ in experimental boxes.
However, it is impossible to present the raw data that provide the evidence on which
our knowledge is based. The purpose of the practicals is to give you the opportunity
to examine raw data and appreciate the way in which results are interpreted and lead
to advances in understanding. The in silico practicals include questions to help you
think more deeply about the material you have learned.

Movies from real research

The movies show key developmental processes occurring in real embryos, to help you
visualize the processes of developmental biology as they unfold in three dimensions.

Signaling pathway animations

Custom-made animations of key signaling pathways break down these complex pro-
cesses into stages, making them easier to understand and remember.
 Learning from this book ix

Online extracts

Online extracts provide additional information on a range of extra topics, including:

● P-element-mediated transformation
● Genetic mosaics and mitotic recombination
● Kidney development
● Development of the Drosophila compound eye
● Reaction–diffusion mechanisms (extended version)
● How the bird wing evolved
● Ascidians
● Directed dilation
● Chromosome capture techniques

For registered adopters

Electronic artwork

Figures from the book are available to download, for use in lecture slides.

Journal clubs

Journal clubs consist of discussion questions focused around primary literature arti-
cles that relate to topics featured in the book. Use these as an additional learning tool
to help your students become more adept at assimilating knowledge from the research
literature.

Test bank

A test bank of questions is available for you to use when assessing your students.
About the authors
Lewis Wolpert is Emeritus Professor of Biology as Applied to Medicine, in the
Department of Anatomy and Developmental Biology, University College London,
London, UK. He is the author of The Triumph of the Embryo, A Passion for Science,
The Unnatural Nature of Science, and Six Impossible Things Before Breakfast.

Cheryll Tickle is Emeritus Professor in the Department of Biology and Biochemistry,

University of Bath, Bath, UK.

Alfonso Martinez Arias is Professor of Developmental Mechanick at the University

of Cambridge, UK.

Peter Lawrence is in the Department of Zoology, University of Cambridge, UK, and

Emeritus member of the Medical Research Council Laboratory of Molecular Biology,
Cambridge, UK. He is the author of The Making of a Fly.

James Locke is Royal Society University Research Fellow and Research Group Leader
in the Sainsbury Laboratory, University of Cambridge, UK, where he examines noise
in gene regulation.

Eleanor Lawrence is a freelance science writer and editor.

Matthew McClements is an illustrator who specializes in design for scientific, techni-

cal, and medical communication.
Summary of contents
Preface  v

Learning from this book  vii

About the authors  x

List of boxes  xxiii

Reviewer acknowledgments  xxv

Chapter 1 History and basic concepts 1

Chapter 2 Development of the Drosophila body plan 37

Chapter 3 Vertebrate development I: life cycles and experimental

techniques94

Chapter 4 Vertebrate development II: Xenopus and zebrafish 142

Chapter 5 Vertebrate development III: chick and mouse—completing

the body plan 183

Chapter 6 Development of nematodes and sea urchins 235

Chapter 7 Morphogenesis: change in form in the early embryo 271

Chapter 8 Cell differentiation and stem cells 333

Chapter 9 Germ cells, fertilization, and sex determination 397

Chapter 10 Organogenesis435

Chapter 11 Development of the nervous system 505

Chapter 12 Growth, post-embryonic development, and regeneration 553

Chapter 13 Plant development 609

Chapter 14 Evolution and development 651

Glossary 702

Index 725
Contents

Preface  v 1.13 Inductive interactions make cells different from

each other 24
Learning from this book  vii
■■ Cell Biology Box 1E Signal transduction and intracellular
About the authors  x signaling pathways 26
List of boxes  xxiii 1.14 The response to inductive signals depends on the
state of the cell 26
Reviewer acknowledgments  xxv
1.15 Patterning can involve the interpretation of positional
information27
Chapter 1 History and basic concepts 1 ■■ Medical Box 1F When development goes awry 28
The origins of developmental biology3 1.16 Lateral inhibition can generate spacing patterns 30
1.1 Aristotle first defined the problem of epigenesis versus 1.17 Localization of cytoplasmic determinants and asymmetric
preformation3 cell division can make daughter cells different from each other 30
■■ Box 1A Basic stages of Xenopus laevis development 4 1.18 The embryo contains a generative rather than
1.2 Cell theory changed how people thought about a descriptive program 32
embryonic development and heredity 4 1.19 The reliability of development is achieved by various
1.3 Two main types of development were originally proposed 6 means32

■■ Cell Biology Box 1B The mitotic cell cycle 7 1.20 The complexity of embryonic development is due
to the complexity of cells themselves 33
1.4 The discovery of induction showed that one group of
cells could determine the development of neighboring cells 8 1.21 Development is a central element in evolution 33

1.5 Developmental biology emerged from the coming together Summary34

of genetics and embryology 8 Summary to Chapter 1 35
1.6 Development is studied mainly through selected model
organisms9 Chapter 2 Development of the Drosophila
1.7 The first developmental genes were identified as body plan 37
spontaneous mutations 11 Drosophila life cycle and overall development38
Summary 13 2.1 The early Drosophila embryo is a multinucleate
A conceptual tool kit13 syncytium38
1.8 Development involves the emergence of pattern, change in 2.2 Cellularization is followed by gastrulation and
form, cell differentiation, and growth 14 segmentation40
■■ Cell Biology Box 1C Germ layers 15 2.3 After hatching, the Drosophila larva develops through
1.9 Cell behavior provides the link between gene action and several larval stages, pupates, and then undergoes
developmental processes 17 metamorphosis to become an adult 41

1.10 Genes control cell behavior by specifying which 2.4 Many developmental genes were identified in Drosophila
proteins are made 17 through large-scale genetic screening for induced mutations 42

1.11 The expression of developmental genes is under ■■ Experimental Box 2A Mutagenesis and genetic screening

tight control 19 strategy for identifying developmental mutants in Drosophila 43

■■ Experimental Box 1D Visualizing gene expression Summary44

in embryos 20 Setting up the body axes44
1.12 Development is progressive and the fates of cells 2.5 The body axes are set up while the Drosophila embryo
become determined at different times 22 is still a syncytium 45
xiv Contents

2.6 Maternal factors set up the body axes and direct the Summary75
early stage of Drosophila development 46 Segmentation genes and segment patterning75
2.7 Three classes of maternal genes specify the 2.23 Expression of the engrailed gene defines the boundary
antero-posterior axis 47 of a parasegment, which is also a boundary of cell lineage
2.8 Bicoid protein provides an antero-posterior gradient of a restriction76
morphogen48 2.24 Segmentation genes stabilize parasegment boundaries 77
2.9 The posterior pattern is controlled by the gradients 2.25 Signals generated at the parasegment boundary delimit
of Nanos and Caudal proteins 50 and pattern the future segments 78
2.10 The anterior and posterior extremities of the embryo ■■ Cell Biology Box 2E The Hedgehog signaling pathway 80
are specified by activation of a cell-surface receptor 51
■■ Experimental Box 2F Mutants in denticle pattern provided
2.11 The dorso-ventral polarity of the embryo is specified by clues to the logic of segment patterning 81
localization of maternal proteins in the egg vitelline envelope 52
Summary83
2.12 Positional information along the dorso-ventral axis
Specification of segment identity83
is provided by the Dorsal protein 53
2.26 Segment identity in Drosophila is specified by Hox genes 84
■■ Cell Biology Box 2B The Toll signaling pathway: a
multifunctional pathway 54 2.27 Homeotic selector genes of the bithorax complex are
responsible for diversification of the posterior segments 85
Summary54
2.28 The Antennapedia complex controls specification
Localization of maternal determinants during oogenesis55
of anterior regions 86
2.13 The antero-posterior axis of the Drosophila egg is
2.29 The order of Hox gene expression corresponds to the
specified by signals from the preceding egg chamber and by
order of genes along the chromosome 87
interactions of the oocyte with follicle cells 56
2.30 The Drosophila head region is specified by genes other
■■ Cell Biology Box 2C The JAK–STAT signaling pathway 58
than the Hox genes 87
2.14 Localization of maternal mRNAs to either end of the egg
Summary88
depends on the reorganization of the oocyte cytoskeleton 58
Summary to Chapter 289
2.15 The dorso-ventral axis of the egg is specified by
movement of the oocyte nucleus followed by signaling
Chapter 3 Vertebrate development I: life cycles
between oocyte and follicle cells 60
and experimental techniques 94
Summary61
Vertebrate life cycles and outlines of development95
Patterning the early embryo62
3.1 The frog Xenopus laevis is the model amphibian for
2.16 The expression of zygotic genes along the dorso-ventral studying development of the body plan 98
axis is controlled by Dorsal protein 62
3.2 The zebrafish embryo develops around a large mass
2.17 The Decapentaplegic protein acts as a morphogen to of yolk 102
pattern the dorsal region 64
3.3 Birds and mammals resemble each other and differ from
2.18 The antero-posterior axis is divided up into broad Xenopus in some important features of early development 105
regions by gap gene expression 66
3.4 The early chicken embryo develops as a flat disc of cells
2.19 Bicoid protein provides a positional signal for the overlying a massive yolk 106
anterior expression of zygotic hunchback67
3.5 The mouse egg has no yolk and early development
2.20 The gradient in Hunchback protein activates and involves the allocation of cells to form the placenta and
represses other gap genes 68 extra-embryonic membranes 110
■■ Experimental Box 2D Targeted gene expression and Experimental approaches to studying vertebrate
misexpression screening 69 development115
Summary70 3.6 Gene expression in embryos can be mapped by in situ
Activation of the pair-rule genes and the establishment nucleic acid hybridization 116
of parasegments71 ■■ Experimental Box 3A Gene-expression profiling by DNA
2.21 Parasegments are delimited by expression of pair-rule microarrays and RNA seq 117
genes in a periodic pattern 71 3.7 Fate mapping and lineage tracing reveal which cells
2.22 Gap gene activity positions stripes of pair-rule gene in which parts of the early embryo give rise to particular
expression73 adult structures 118
 Contents xv

3.8 Not all techniques are equally applicable to all vertebrates 120 4.7 Mesoderm induction occurs during a limited period in the
3.9 Developmental genes can be identified by spontaneous blastula stage 155
mutation and by large-scale mutagenesis screens 121 4.8 Zygotic gene expression is turned on at the mid-blastula
■■ Experimental Box 3B Large-scale mutagenesis screens transition156
for recessive mutations in zebrafish 123 4.9 Mesoderm-inducing and patterning signals are produced
3.10 Transgenic techniques enable animals to be produced by the vegetal region, the organizer, and the ventral mesoderm 157
with mutations in specific genes 124 4.10 Members of the TGF-β family have been identified as
■■ Experimental Box 3C The Cre/loxP system: a strategy mesoderm inducers 158
for making gene knock-outs in mice 127 ■■ Experimental Box 4D Investigating receptor function using

■■ Experimental Box 3D The CRISPR-Cas9 genome-editing dominant-negative proteins 159

system128 4.11 The zygotic expression of mesoderm-inducing and
3.11 Gene function can also be tested by transient patterning signals is activated by the combined actions
transgenesis and gene silencing 130 of maternal VegT and Wnt signaling 159

Human embryonic development131 4.12 Threshold responses to gradients of signaling proteins

are likely to pattern the mesoderm 161
3.12 The early development of a human embryo is similar
to that of the mouse 131 Summary162

■■ Medical Box 3E Preimplantation genetic diagnosis 134 The Spemann organizer and neural induction163

3.13 The timing of formation and the anatomy of the ■■ Cell Biology Box 4E The fibroblast growth factor

human placenta differs from that in the mouse 135 signaling pathway 163

3.14 Some studies of human development are possible 4.13 Signals from the organizer pattern the mesoderm
but are subject to strict laws 136 dorso-ventrally by antagonizing the effects of ventral signals 164

■■ Box 3F Identical twins 137 4.14 The antero-posterior axis of the embryo emerges during
gastrulation165
Summary to Chapter 3 138
4.15 The neural plate is induced in the ectoderm 168
Chapter 4 Vertebrate development II: Xenopus 4.16 The nervous system is patterned along the
and zebrafish 142 antero-posterior axis by signals from the mesoderm 170
Setting up the body axes143 4.17 The final body plan emerges by the end of gastrulation
4.1 The animal–vegetal axis is maternally determined and neurulation 171
in Xenopus143 Summary172
■■ Cell Biology Box 4A Intercellular protein signals Development of the body plan in zebrafish172
in vertebrate development 145 4.18 The body axes in zebrafish are established by maternal
■■ Cell Biology Box 4B The Wnt/β-catenin signaling pathway 146 determinants173
4.2 Local activation of Wnt/β-catenin signaling specifies 4.19 The germ layers are specified in the zebrafish blastoderm
the future dorsal side of the embryo 147 by similar signals to those in Xenopus173
4.3 Signaling centers develop on the dorsal side of the 4.20 The shield in zebrafish is the embryonic organizer 176
blastula149 ■■ Box 4F A zebrafish gene regulatory network 176
Summary150 Summary to Chapter 4 178
The origin and specification of the germ layers150
4.4 The fate map of the Xenopus blastula makes clear Chapter 5 Vertebrate development III: chick and
the function of gastrulation 151 mouse—completing the body plan 183
4.5 Cells of the early Xenopus embryo do not yet have their Development of the body plan in chick and mouse and
fates determined and regulation is possible 152 ­generation of the spinal cord184
4.6 Endoderm and ectoderm are specified by maternal 5.1 The antero-posterior polarity of the chick blastoderm is
factors, whereas mesoderm is induced from ectoderm by related to the primitive streak 184
signals from the vegetal region 152
5.2 Early stages in mouse development establish separate
■■ Cell Biology Box 4C Signaling by members of the TGF-β cell lineages for the embryo and the extra-embryonic
family of growth factors 155 structures186
xvi Contents

5.3 Movement of the anterior visceral endoderm indicates Chapter 6 Development of nematodes and
the definitive antero-posterior axis in the mouse embryo 190 sea urchins 235
5.4 The fate maps of vertebrate embryos are variations on Nematodes236
a basic plan 192 ■■ Cell Biology Box 6A Apoptotic pathways in nematodes,
■■ Cell Biology Box 5A Fine-tuning Nodal signaling 193 Drosophila, and mammals 238
5.5 Mesoderm induction and patterning in the chick 6.1 The cell lineage of Caenorhabditis elegans is
and mouse occurs during primitive streak formation 195 largely invariant 239
5.6 The node that develops at the anterior end of the streak 6.2 The antero-posterior axis in Caenorhabditis elegans is
in chick and mouse embryos is equivalent to the Spemann determined by asymmetric cell division 239
organizer in Xenopus196 ■■ Experimental Box 6B Gene silencing by antisense RNA
5.7 Neural induction in chick and mouse is initiated by FGF and RNA interference 241
signaling with inhibition of BMP signaling being required in 6.3 The dorso-ventral axis in Caenorhabditis elegans is
a later step 198 determined by cell–cell interactions 242
■■ Cell Biology Box 5B Chromatin-remodeling complexes 201 6.4 Both asymmetric divisions and cell–cell interactions
5.8 Axial structures in chick and mouse are generated from specify cell fate in the early nematode embryo 245
self-renewing cell populations 202 6.5 Cell differentiation in the nematode is closely linked
Summary204 to the pattern of cell division 246
Somite formation and antero-posterior patterning205 6.6 Hox genes specify positional identity along the
■■ Cell Biology Box 5C Retinoic acid: a small-molecule antero-posterior axis in Caenorhabditis elegans 247
intercellular signal 206 6.7 The timing of events in nematode development is under
5.9 Somites are formed in a well-defined order along genetic control that involves microRNAs 248
the antero-posterior axis 206 ■■ Box 6C Gene silencing by microRNAs 250
■■ Cell Biology Box 5D The Notch signaling pathway 211 6.8 Vulval development is initiated through the induction of
5.10 Identity of somites along the antero-posterior axis is a small number of cells by short-range signals from a single
specified by Hox gene expression 213 inducing cell 251

■■ Box 5E The Hox genes 214 Summary253

5.11 Deletion or overexpression of Hox genes causes Echinoderms254

changes in axial patterning 217 6.9 The sea urchin embryo develops into a free-swimming
5.12 Hox gene expression is activated in an anterior larva255
to posterior pattern 219 6.10 The sea urchin egg is polarized along the
5.13 The fate of somite cells is determined by signals from animal–vegetal axis 256
the adjacent tissues 221 6.11 The sea urchin fate map is finely specified, yet
Summary223 considerable regulation is possible 257

The origin and patterning of neural crest223 6.12 The vegetal region of the sea urchin embryo acts
as an organizer 258
5.14 Neural crest cells arise from the borders of the neural plate
and migrate to give rise to a wide range of different cell types 223 6.13 The sea urchin vegetal region is demarcated by the
nuclear accumulation of β-catenin260
5.15 Neural crest cells migrate from the hindbrain to populate
the branchial arches 225 6.14 The animal–vegetal axis and the oral–aboral
axis can be considered to correspond to the
Summary226
antero-posterior and dorso-ventral axes of other
Determination of left–right asymmetry227 deuterostomes261
5.16 The bilateral symmetry of the early embryo is broken 6.15 The pluteus skeleton develops from the primary
to produce left–right asymmetry of internal organs 227 mesenchyme262
5.17 Left–right symmetry breaking may be initiated within 6.16 The oral–aboral axis in sea urchins is related to the
cells of the early embryo 229 plane of the first cleavage 263
Summary230 6.17 The oral ectoderm acts as an organizing region for
Summary to Chapter 5 230 the oral–aboral axis 264
 Contents xvii

■■ Experimental Box 6D The gene regulatory network for 7.15 Gastrulation in chick and mouse embryos involves the
sea urchin endomesoderm specification 265 separation of individual cells from the epiblast and their
Summary266 ingression through the primitive streak 306

Summary to Chapter 6 267 Summary309

Neural tube formation311
Chapter 7 Morphogenesis: change in form in 7.16 Neural tube formation is driven by changes in cell
the early embryo 271 shape and convergent extension 311
Cell adhesion273 ■■ Cell Biology Box 7D Eph receptors and their ephrin ligands 313
■■ Cell Biology Box 7A Cell-adhesion molecules and cell ■■ Medical Box 7E Neural tube defects 314
junctions274
Summary315
7.1 Sorting out of dissociated cells demonstrates differences
Formation of tubes and branching morphogenesis316
in cell adhesiveness in different tissues 275
7.17 The Drosophila tracheal system is a prime example of
7.2 Cadherins can provide adhesive specificity 276
branching morphogenesis 316
7.3 The activity of the cytoskeleton regulates the mechanical
7.18 The vertebrate vascular system develops by
properties of cells and their interactions with each other 277
vasculogenesis followed by sprouting angiogenesis 318
■■ Cell Biology Box 7B The cytoskeleton, cell-shape change,
7.19 New blood vessels are formed from pre-existing
and cell movement 278
vessels in angiogenesis 319
7.4 Transitions of tissues from an epithelial to a mesenchymal
Summary320
state, and vice versa, involve changes in adhesive junctions 279
Cell migration320
Summary280
7.20 Embryonic neural crest gives rise to a wide range
Cleavage and formation of the blastula280
of different cell types 321
7.5 The orientation of the mitotic spindle determines the
7.21 Neural crest migration is controlled by
plane of cleavage at cell division 281
environmental cues 321
7.6 The positioning of the spindle within the cell also
7.22 The formation of the lateral-line primordium in
determines whether daughter cells will be the same or different
fishes is an example of collective cell migration 323
sizes283
7.23 Body wall closure occurs in Drosophila, Caenorhabditis,
7.7 Cells become polarized in the sea urchin blastula and
mammals, and chick 324
the mouse morula 285
Summary325
7.8 Fluid accumulation as a result of tight-junction
formation and ion transport forms the blastocoel of Summary to Chapter 7 326
the mammalian blastocyst 287
Summary288 Chapter 8 Cell differentiation and stem cells 333
Gastrulation movements289 ■■ Box 8A Conrad Waddington’s ‘epigenetic landscape’
provides a framework for thinking about how cells differentiate 335
7.9 Gastrulation in the sea urchin involves an epithelial-to-
mesenchymal transition, cell migration, and invagination of the The control of gene expression337
blastula wall 289 8.1 Control of transcription involves both general and
7.10 Mesoderm invagination in Drosophila is due to changes tissue-specific transcriptional regulators 338
in cell shape controlled by genes that pattern the 8.2 Gene expression is also controlled by epigenetic chemical
dorso-ventral axis 293 modifications to DNA and histone proteins that alter chromatin
7.11 Germ-band extension in Drosophila involves myosin- structure341
dependent remodeling of cell junctions and cell intercalation 295 ■■ Cell Biology Box 8B Epigenetic control of gene expression

7.12 Planar cell polarity confers directionality on a tissue 296 by chromatin modification 344

7.13 Gastrulation in amphibians and fish involves involution, 8.3 Patterns of gene activity can be inherited by persistence
epiboly, and convergent extension 299 of gene-regulatory proteins or by maintenance of chromatin
modifications347
■■ Box 7C Convergent extension 302
8.4 Changes in patterns of gene activity during differentiation can
7.14 Xenopus notochord development illustrates the
be triggered by extracellular signals 348
dependence of medio-lateral cell elongation and cell
intercalation on a pre-existing antero-posterior polarity 305 Summary349
xviii Contents

Cell differentiation and stem cells350 Chapter 9 Germ cells, fertilization,

8.5 Muscle differentiation is determined by the MyoD family and sex determination 397
of transcription factors 350 The development of germ cells398
8.6 The differentiation of muscle cells involves withdrawal 9.1 Germ cell fate is specified in some embryos by a distinct
from the cell cycle, but is reversible 352 germplasm in the egg 399
8.7 All blood cells are derived from multipotent stem cells 354 9.2 In mammals germ cells are induced by cell–cell interactions
8.8 Intrinsic and extrinsic changes control differentiation of during development 401
the hematopoietic lineages 357 9.3 Germ cells migrate from their site of origin to the gonad 402
■■ Experimental Box 8C Single-cell analysis of cell-fate 9.4 Germ cells are guided to their destination by
decisions358 chemical signals 403
8.9 Developmentally regulated globin gene expression is 9.5 Germ cell differentiation involves a halving of
controlled by control regions far distant from the coding chromosome number by meiosis 404
regions361 ■■ Box 9A Polar bodies 405
8.10 The epidermis of adult mammalian skin is continually 9.6 Oocyte development can involve gene amplification
being replaced by derivatives of stem cells 363 and contributions from other cells 408
■■ Medical Box 8D Treatment of junctional epidermolysis 9.7 Factors in the cytoplasm maintain the totipotency of the egg 408
bullosa with skin grown from genetically corrected
9.8 In mammals some genes controlling embryonic growth
stem cells 366
are ‘imprinted’ 409
8.11 Stem cells use different modes of division to maintain
Summary412
tissues367
Fertilization412
8.12 The lining of the gut is another epithelial tissue
that requires continuous renewal 368 9.9 Fertilization involves cell-surface interactions between
egg and sperm 413
8.13 Skeletal muscle and neural cells can be renewed
from stem cells in adults 370 9.10 Changes in the egg plasma membrane and enveloping
layers at fertilization block polyspermy 415
8.14 Embryonic stem cells can proliferate and differentiate
into many cell types in culture and contribute to normal 9.11 Sperm–egg fusion causes a calcium wave that results
development in vivo 372 in egg activation 416

■■ Experimental Box 8E The derivation and culture of mouse Summary418

embryonic stem cells 374 Determination of the sexual phenotype419
Summary375 9.12 The primary sex-determining gene in mammals is on
The plasticity of the differentiated state376 the Y chromosome 419

8.15 Nuclei of differentiated cells can support development 376 9.13 Mammalian sexual phenotype is regulated by gonadal
hormones420
8.16 Patterns of gene activity in differentiated cells can be
changed by cell fusion 378 9.14 The primary sex-determining factor in Drosophila is the
number of X chromosomes and is cell autonomous 422
8.17 The differentiated state of a cell can change by
transdifferentiation379 9.15 Somatic sexual development in Caenorhabditis is
determined by the number of X chromosomes 424
8.18 Adult differentiated cells can be reprogrammed to form
pluripotent stem cells 381 9.16 Determination of germ cell sex depends on both genetic
constitution and intercellular signals 425
■■ Experimental Box 8F Induced pluripotent stem cells 382
9.17 Various strategies are used for dosage compensation of
8.19 Stem cells could be a key to regenerative medicine 382
X-linked genes 427
■■ Experimental Box 8G Stem cells can be cultured in vitro
Summary429
to produce ‘organoids’—structures that mimic tissues and
organs386 Summary to Chapter 9 431

8.20 Various approaches can be used to generate

Chapter 10 Organogenesis435
differentiated cells for cell-replacement therapies 388
The insect wing and leg436
Summary391
10.1 Imaginal discs arise from the ectoderm in the
Summary to Chapter 8 391
early Drosophila embryo 437
 Contents xix

10.2 Imaginal discs arise across parasegment boundaries 10.20 Self-organization may be involved in the
and are patterned by signaling at compartment boundaries 438 development of the limb 475
10.3 The adult wing emerges at metamorphosis after folding ■■ Box 10E Reaction–diffusion mechanisms 476
and evagination of the wing imaginal disc 439 10.21 Limb muscle is patterned by the connective tissue 477
10.4 A signaling center at the boundary between anterior 10.22 The initial development of cartilage, muscles, and
and posterior compartments patterns the Drosophila wing tendons is autonomous 478
disc along the antero-posterior axis 440
10.23 Joint formation involves secreted signals and
■■ Box 10A Positional information and morphogen gradients 443 mechanical stimuli 478
10.5 A signaling center at the boundary between dorsal 10.24 Separation of the digits is the result of programmed
and ventral compartments patterns the Drosophila wing cell death 479
along the dorso-ventral axis 445
Summary480
10.6 Vestigial is a key regulator of wing development that
Teeth481
acts to specify wing identity and control wing growth 445
10.25 Tooth development involves epithelial–mesenchymal
10.7 The Drosophila wing disc is also patterned along
interactions and a homeobox gene code specifies tooth identity 482
the proximo-distal axis 447
Summary484
10.8 The leg disc is patterned in a similar manner to the
wing disc, except for the proximo-distal axis 448 Vertebrate lungs484

10.9 Different imaginal discs can have the same 10.26 The vertebrate lung develops from a bud of endoderm 484
positional values 450 ■■ Medical Box 10F What developmental biology can teach

Summary450 us about breast cancer 486

The vertebrate limb452 10.27 Morphogenesis of the lung involves three modes of
branching488
10.10 The vertebrate limb develops from a limb bud and its
development illustrates general principles 452 Summary489

10.11 Genes expressed in the lateral plate mesoderm The vertebrate heart489
are involved in specifying limb position, polarity, and identity 454 10.28 The development of the vertebrate heart involves
10.12 The apical ectodermal ridge is required for limb-bud morphogenesis and patterning of a mesodermal tube 489
outgrowth and the formation of structures along the proximo- The vertebrate eye492
distal axis of the limb 457 10.29 Development of the vertebrate eye involves
10.13 Formation and outgrowth of the limb bud involves interactions between an extension of the forebrain and
oriented cell behavior 458 the ectoderm of the head 493
10.14 Positional value along the proximo-distal axis of Summary497
the limb bud is specified by a combination of graded signaling Summary to Chapter 10 497
and a timing mechanism 460
10.15 The polarizing region specifies position along the limb’s Chapter 11 Development of the nervous system 505
antero-posterior axis 462 Specification of cell identity in the nervous system507
10.16 Sonic hedgehog is the polarizing region morphogen 464 11.1 Initial regionalization of the vertebrate brain involves
■■ Medical Box 10B Too many fingers: mutations that affect signals from local organizers 507
antero-posterior patterning can cause polydactyly 465 11.2 Local signaling centers pattern the brain along the
■■ Cell Biology Box 10C Sonic hedgehog signaling and the antero-posterior axis 508
primary cilium 466 11.3 The cerebral cortex is patterned by signals from the
10.17 The dorso-ventral axis of the limb is controlled by the anterior neural ridge 509
ectoderm468 11.4 The hindbrain is segmented into rhombomeres by
■■ Medical Box 10D Teratogens and the consequences of boundaries of cell-lineage restriction 509
damage to the developing embryo 470 11.5 Hox genes provide positional information in the
10.18 Development of the limb is integrated by interactions developing hindbrain 512
between signaling centers 470 11.6 The pattern of differentiation of cells along the
10.19 Hox genes have multiple inputs into the patterning dorso-ventral axis of the spinal cord depends on ventral
of the limbs 472 and dorsal signals 513
xx Contents

11.7 Neuronal subtypes in the ventral spinal cord are Chapter 12 Growth, post-embryonic development,
specified by the ventral to dorsal gradient of Shh 515 and regeneration 553
11.8 Spinal cord motor neurons at different dorso-ventral Growth554
positions project to different trunk and limb muscles 516 12.1 Tissues can grow by cell proliferation, cell enlargement,
11.9 Antero-posterior pattern in the spinal cord is or accretion 555
determined in response to secreted signals from the node 12.2 Cell proliferation is controlled by regulating entry into
and adjacent mesoderm 517 the cell cycle 556
Summary518 12.3 Cell division in early development can be controlled
The formation and migration of neurons518 by an intrinsic developmental program 557
11.10 Neurons in Drosophila arise from proneural clusters 519 12.4 Extrinsic signals coordinate cell division, cell growth,
11.11 The development of neurons in Drosophila involves and cell death in the developing Drosophila wing 558
asymmetric cell divisions and timed changes in gene ■■ Cell Biology Box 12A The core Hippo signaling pathways in
expression521 Drosophila and mammals 559
11.12 The production of vertebrate neurons involves lateral 12.5 Cancer can result from mutations in genes that control
inhibition, as in Drosophila522 cell proliferation 560
■■ Box 11A Specification of the sensory organs of 12.6 The relative contributions of intrinsic and extrinsic
adult Drosophila523 factors in controlling size differ in different mammalian organs 562
11.13 Neurons are formed in the proliferative zone of the 12.7 Overall body size depends on the extent and the
vertebrate neural tube and migrate outwards 524 duration of growth 564
■■ Experimental Box 11B Timing the birth of cortical 12.8 Hormones and growth factors coordinate the growth of
neurons526 different tissues and organs and contribute to determining
11.14 Many cortical interneurons migrate tangentially 528 overall body size 565

Summary528 12.9 Elongation of the long bones illustrates how growth

can be determined by a combination of an intrinsic growth
Axon navigation529
program and extracellular factors 566
11.15 The growth cone controls the path taken by a
■■ Box 12B Digit length ratio is determined in the embryo 568
growing axon 530
12.10 The amount of nourishment an embryo receives can
■■ Box 11C The development of the neural circuit for
have profound effects in later life 570
the knee-jerk reflex 532
Summary571
11.16 Motor neuron axons in the chick limb are guided
by ephrin–Eph interactions 533 Molting and metamorphosis572

11.17 Axons crossing the midline are both attracted 12.11 Arthropods have to molt in order to grow 572
and repelled 534 12.12 Insect body size is determined by the rate and
11.18 Neurons from the retina make ordered connections duration of larval growth 573
with visual centers in the brain 535 12.13 Metamorphosis in amphibians is under hormonal
Summary538 control575

Synapse formation and refinement539 Summary576

11.19 Synapse formation involves reciprocal interactions 539 Regeneration577

11.20 Many motor neurons die during normal development 542 12.14 Regeneration involves repatterning of existing
tissues and/or growth of new tissues 578
■■ Medical Box 11D Autism: a developmental disorder that
involves synapse dysfunction 543 12.15 Amphibian limb regeneration involves cell
dedifferentiation and new growth 578
11.21 Neuronal cell death and survival involve both intrinsic
and extrinsic factors 544 ■■ Box 12C Regeneration in Hydra580

11.22 The map from eye to brain is refined by neural ■■ Box 12D Planarian regeneration 582
activity545 12.16 Limb regeneration in amphibians depends on the
Summary546 presence of nerves 586

Summary to Chapter 11 547 12.17 The limb blastema gives rise to structures with
positional values distal to the site of amputation 587
 Contents xxi

12.18 Retinoic acid can change proximo-distal positional 13.12 The regular arrangement of leaves on a stem is
values in regenerating limbs 589 generated by regulated auxin transport 629
12.19 Mammals can regenerate the tips of the digits 590 13.13 The outgrowth of secondary shoots is under
12.20 Insect limbs intercalate positional values by both hormonal control 630
proximo-distal and circumferential growth 591 13.14 Root tissues are produced from Arabidopsis root apical
■■ Box 12E Why can’t we regenerate our limbs? 592 meristems by a highly stereotyped pattern of cell divisions 633

12.21 Heart regeneration in zebrafish involves the 13.15 Root hairs are specified by a combination
resumption of cell division by cardiomyocytes 594 of positional information and lateral inhibition 635

Summary596 Summary636

Aging and senescence597 Flower development and control of flowering636

12.22 Genes can alter the timing of senescence 598 13.16 Homeotic genes control organ identity in the flower 637

12.23 Cell senescence blocks cell proliferation 600 ■■ Box 13C The basic model for the patterning of the
Arabidopsis flower 639
12.24 Elimination of senescent cells in adult salamanders
explains why regenerative ability does not diminish with age 601 13.17 The Antirrhinum flower is patterned dorso-ventrally,
as well as radially 640
Summary602
13.18 The internal meristem layer can specify floral
Summary to Chapter 12 602
meristem patterning 641

Chapter 13 Plant development 609 13.19 The transition of a shoot meristem to a floral
meristem is under environmental and genetic control 642
13.1 The model plant Arabidopsis thaliana has a short life
cycle and a small diploid genome 611 ■■ Box 13D The circadian clock coordinates plant growth and
development643
Embryonic development612
13.20 Vernalization reflects the epigenetic memory of winter 643
13.2 Plant embryos develop through several distinct stages 612
13.21 Most flowering plants are hermaphrodites, but
■■ Box 13A Angiosperm embryogenesis 614
some produce unisexual flowers 645
13.3 Gradients of the signal molecule auxin establish the
Summary646
embryonic apical–basal axis 616
Summary to Chapter 13 647
13.4 Plant somatic cells can give rise to embryos
and seedlings 617
Chapter 14 Evolution and development 651
13.5 Cell enlargement is a major process in plant growth
■■ Box 14A Darwin’s finches 654
and morphogenesis 619
The evolution of development655
■■ Experimental Box 13B Plant transformation and
genome editing 620 14.1 Multicellular organisms evolved from single-celled
ancestors655
Summary621
14.2 Genomic evidence is throwing light on the evolution
Meristems622
of animals 657
13.6 A meristem contains a small, central zone of
■■ Box 14B The metazoan family tree 658
self-renewing stem cells 623
14.3 How gastrulation evolved is not known 659
13.7 The size of the stem cell area in the meristem is kept
constant by a feedback loop to the organizing center 623 14.4 More general characteristics of the body plan develop
earlier than specializations 660
13.8 The fate of cells from different meristem layers can be
changed by changing their position 624 14.5 Embryonic structures have acquired new functions
during evolution 661
13.9 A fate map for the embryonic shoot meristem can
be deduced using clonal analysis 626 14.6 Evolution of different types of eyes in different animal
groups is an example of parallel evolution 663
13.10 Meristem development is dependent on signals
from other parts of the plant 627 Summary664

13.11 Gene activity patterns the proximo-distal and The diversification of body plans665
adaxial–abaxial axes of leaves developing from the 14.7 Hox gene complexes have evolved through gene
shoot meristem 628 duplication665
xxii Contents

14.8 Differences in Hox gene expression determine the 14.15 Adaptive evolution within the same species provides
variation in position and type of paired appendages a way of studying the developmental basis for evolutionary
in arthropods 667 change684
14.9 Changes in Hox gene expression and their target ■■ Experimental Box 14D Pelvic reduction in sticklebacks
genes contributed to the evolution of the vertebrate is based on mutations in a gene control region 686
axial skeleton 671 Summary687
14.10 The basic body plan of arthropods and vertebrates Changes in the timing of developmental processes687
is similar, but the dorso-ventral axis is inverted 672
14.16 Changes in growth can modify the basic body plan 687
Summary673
■■ Box 14E Origins of morphological diversity in dogs 689
The evolutionary modification of specialized characters674
14.17 Evolution can be due to changes in the timing of
14.11 Limbs evolved from fins 674 developmental events 690
14.12 Limbs have evolved to fulfill different specialized functions 14.18 The evolution of life histories has implications for
678 development692
14.13 The evolution of limblessness in snakes is associated ■■ Box 14F Long- and short-germ development in insects 693
with changes in axial gene expression and mutations in a
Summary695
limb-specific enhancer 679
Summary to Chapter 14 696
14.14 Butterfly wing markings have evolved by
redeployment of genes previously used for other functions 680
■■ Experimental Box 14C Using CRISPR-Cas9 genome-editing Glossary  702
techniques to test the functioning of the snake ZRS 681 Index  725
List of Boxes
Special Interest Boxes Box 7B The cytoskeleton, cell-shape change, and cell
Box 1A Basic stages of Xenopus laevis development 4 movement278
Box 3F Identical twins 137 Box 7D Eph receptors and their ephrin ligands 313
Box 4F A zebrafish gene regulatory network 176 Box 8B Epigenetic control of gene expression by chromatin
Box 5E The Hox genes 214 modification344
Box 6C Gene silencing by microRNAs 250 Box 10C Sonic hedgehog signaling and the
Box 7C Convergent extension 302 primary cilium 466
Box 8A Conrad Waddington’s ‘epigenetic landscape’ provides a Box 12A The core Hippo signaling pathways in Drosophila and
framework for thinking about how cells differentiate 335 mammals559
Box 9A Polar bodies 405
Box 10A Positional information and morphogen gradients 443 Experimental Boxes
Box 10E Reaction–diffusion mechanisms 476 Box 1D Visualizing gene expression in embryos 20
Box 11A Specification of the sensory organs of adult Drosophila 523 Box 2A Mutagenesis and genetic screening strategy for
Box 11C The development of the neural circuit for the identifying developmental mutants in Drosophila 43
knee-jerk reflex 532 Box 2D Targeted gene expression and misexpression screening 69
Box 12B Digit length ratio is determined in the embryo 568 Box 2F Mutants in denticle pattern provided
Box 12C Regeneration in Hydra580 clues to the logic of segment patterning 81
Box 12D Planarian regeneration 582 Box 3A Gene-expression profiling by DNA microarrays and
Box 12E Why can’t we regenerate our limbs? 592 RNA seq 117
Box 13A Angiosperm embryogenesis 614 Box 3B Large-scale mutagenesis screens for recessive mutations
Box 13C The basic model for the patterning of the in zebrafish 123
Arabidopsis flower 639 Box 3C The Cre/loxP system: a strategy for making gene
Box 13D The circadian clock coordinates plant growth knock-outs in mice 127
and development 643 Box 3D The CRISPR-Cas9 genome-editing system 128
Box 14A Darwin’s finches 654 Box 4D Investigating receptor function using dominant-negative
Box 14B The metazoan family tree 658 proteins159
Box 14E Origins of morphological diversity in dogs 689 Box 6B Gene silencing by antisense RNA and RNA interference 241
Box 14F Long- and short-germ development in insects 693 Box 6D The gene regulatory network for sea urchin
endomesoderm specification 265
Cell Biology Boxes Box 8C Single-cell analysis of cell-fate decisions 358
Box 1B The mitotic cell cycle 7 Box 8E The derivation and culture of mouse embryonic
Box 1C Germ layers 15 stem cells 374
Box 1E Signal transduction and intracellular signaling pathways 26 Box 8F Induced pluripotent stem cells 382
Box 2B The Toll signaling pathway: a multifunctional pathway 54 Box 8G Stem cells can be cultured in vitro to produce
Box 2C The JAK–STAT signaling pathway 58 ‘organoids’—structures that mimic tissues and organs 386
Box 2E The Hedgehog signaling pathway 80 Box 11B Timing the birth of cortical neurons 526
Box 4A Intercellular protein signals in vertebrate development 145 Box 13B Plant transformation and genome editing 620
Box 4B The Wnt/β-catenin signaling pathway 146 Box 14C Using CRISPR-Cas9 genome-editing techniques to
Box 4C Signaling by members of the TGF-β family of test the functioning of the snake ZRS 681
growth factors 155 Box 14D Pelvic reduction in sticklebacks is based on mutations
Box 4E The fibroblast growth factor signaling pathway 163 in a gene control region 686
Box 5A Fine-tuning Nodal signaling 193
Box 5B Chromatin-remodeling complexes 201 Medical Boxes
Box 5C Retinoic acid: a small-molecule intercellular signal 206 Box 1F When development goes awry 28
Box 5D The Notch signaling pathway 211 Box 3E Preimplantation genetic diagnosis 134
Box 6A Apoptotic pathways in nematodes, Drosophila, Box 7E Neural tube defects 314
and mammals 238 Box 8D Treatment of junctional epidermolysis bullosa
Box 7A Cell-adhesion molecules and cell junctions 274 with skin grown from genetically corrected stem cells 366
xxiv Contents

Box 10B Too many fingers: mutations that affect Box 10F What developmental biology can teach
antero-posterior patterning can cause polydactyly 465 us about breast cancer 486
Box 10D Teratogens and the consequences of Box 11D Autism: a developmental disorder that
damage to the developing embryo 470 involves synapse dysfunction 543
Reviewer acknowledgments

We would like to reiterate our thanks to the people who contributed to the first
five editions. Many people gave their advice based on the fifth edition, and others
reviewed the draft chapters for the sixth edition as they emerged. We wish to express
our gratitude to the following colleagues.

Katie Abley, University of Cambridge

Mike Akam, University of Cambridge
Willy M. Baarends, University Medical Centre, Rotterdam
Hegias Mira Bontenbal, University Medical Centre, Rotterdam
Thomas Butts, University of Liverpool
Sinéad Drea, University of Leicester
Sander van den Driesche, University of Edinburgh
Anna-Pavlina Haramis, Leiden University
Brigitte Galliot, University of Geneva
Erik Griffin, Dartmouth College
Jane Kenney-Hunt, Westminster College, Fulton, Missouri
Jane Langdale, University of Oxford
Tony Perry, University of Bath
Mary Elizabeth Pownall, University of York
Aida Rajic, University of Suffolk
Joanna Richardson, University of Sussex
Guojun Sheng, Kumamoto University
Kate Storey, University of Dundee
Shahragim Tajbakhsh, Institut Pasteur, Paris
Michael Taylor, Cardiff University
Adri Thomas, Utrecht University
Fiona Wardle, King’s College London
Deneen Wellik, University of Michigan Medical School,
Ann Arbor
Larissa Williams, Bates College, Lewiston, Maine

We would also like to thank our colleagues who wrote the online Journal Clubs to
accompany this edition.

Katie Abley, University of Cambridge

Zoe Burke, University of Bath
Andrew Economou, The Francis Crick Institute
Caitlin McQueen, University of Sheffield
Adriana Amorim Torres, AAT Assessoria Cientìfica
 1
History and basic concepts
● The origins of ● A conceptual tool kit
developmental biology

The aim of this chapter is to provide a conceptual framework for the study of develop-
ment. We start with a brief history of the study of embryonic development, which illus-
trates how some of the key questions in developmental biology were first formulated,
and continue with some of the essential principles of development. The big question
is: How does a single cell—the fertilized egg—give rise to a multicellular organism, in
which a multiplicity of different cell types are organized into tissues and organs to
make up a three-dimensional body? This question can be studied from many different
viewpoints, all of which have to be fitted together to obtain a complete picture of de-
velopment: which genes are expressed, and when and where; how cells communicate
with each other; how a cell’s developmental fate is determined; how cells proliferate
and differentiate into specialized cell types; and how major changes in body shape
are produced. All the information for embryonic development is contained within the
fertilized egg. We shall see that an organism’s development is ultimately driven by
the regulated expression of its genes in space and time, determining which proteins
are present in which cells and when. In turn, proteins largely determine how a cell
behaves. The genes provide a generative program for development, not a blueprint,
as their actions are translated into developmental outcomes through cellular behavior
such as intercellular signaling, cell proliferation, cell differentiation, and cell movement.

The development of a multicellular organism from a single cell—the fertilized egg—is

a brilliant triumph of evolution. The fertilized egg divides to give rise to many mil-
lions of cells, which form structures as complex and varied as eyes, arms, heart, and
brain. This amazing achievement raises a multitude of questions. How do the cells
arising from division of the fertilized egg become different from each other? How do
they become organized into structures such as limbs and brains? What controls the
behavior of individual cells so that such highly organized patterns emerge? How are
the organizing principles of development embedded within the egg, and, in particular,
within the genetic material, DNA? Much of the excitement in developmental biology
today comes from our growing understanding of how genes direct these developmen-
tal processes, and genetic control is one of the main themes of this book. Thousands
of genes are involved in controlling development, but we will focus only on those that
have key roles, and illustrate general principles.
2 Chapter 1 History and basic concepts

a Understanding how embryos develop is a huge intellectual challenge, and one of the
ultimate aims of the science of developmental biology is to understand how we humans
develop (Fig. 1.1). We need to understand human development for several reasons. We
need to properly understand why it sometimes goes wrong and why a fetus may fail to
be born or a baby be born with congenital abnormalities. The link here with genetic con-
trol of development is very close, as mutations in genes can lead to abnormal develop-
ment; environmental factors, such as drugs and infections, can affect it too. Another area
of medical research related to developmental biology is regenerative medicine—finding
out how to use cells to repair damaged tissues and organs. The focus of regenerative
medicine is currently on stem cells. Stem cells that can proliferate and give rise to all
the different tissues of the body are present in embryos. These, and the stem cells with
more limited developmental potential that are found in adult tissues, are discussed in
Chapter 8. Cancer cells also display some properties of embryonic cells, such as the abil-
b ity to divide indefinitely, and so the study of embryonic cells and their behaviour could
lead to new and better treatments for cancer, as many of the same genes are involved.
The development of an embryo from the fertilized egg is known as embryogenesis.
One of the first tasks that cells have in an embryo is to lay down the overall body plan
of the organism, and we shall see that different organisms solve this fundamental
problem in several ways. The focus of this book is mainly on animal development, in
particular that of vertebrates—frogs, birds, fish, and mammals—whose early develop-
ment is discussed in Chapters 3–5. We also look at selected invertebrates, particularly
the fruit fly and the nematode worm, and also the sea urchin. Our understanding of
the genetic control of development is founded on work with fruit flies and nematodes,
where it is also most advanced, and the main features of their early development are
Fig. 1.1 Human fertilized egg and embryo.
considered in Chapters 2 and 6, respectively. The fruit fly is also used throughout the
(a) Human fertilized egg. The nuclear
book to illustrate particular aspects of development. In Chapter 13 we look briefly at
membranes of the sperm and egg nuclei
(pronuclei) have not yet broken down to allow some aspects of plant development, which differs in many respects from that of ani-
the parental chromosomes to mingle. (b) Human mals but involves similar basic principles.
embryo at around 51 days’ gestation (Carnegie Morphogenesis, or the development of form, is discussed in Chapter 7. In Chapter
stage 20), which is equivalent to a mouse 9 we look at how sex is determined and how germ cells develop. The differentiation of
embryo at 13.5 days post-fertilization. A human unspecialized cells into cells that carry out particular functions, such as muscle cells
embryo at this stage is about 21–23 mm long. and blood cells, is considered in Chapter 8. Structures such as the vertebrate limb, and
(a) Courtesy of A. Doshi, CRGH, London. organs such as insect and vertebrate eyes, the heart and the nervous system, illustrate
(b) Reproduced courtesy of the MRC/ the problems of multicellular organization and tissue differentiation in embryogen-
Wellcome-funded Human Developmental esis, and we consider some of these systems in detail in Chapters 10 and 11. The study
Biology Resource. of developmental biology, however, goes well beyond the development of the embryo.
Post-embryonic growth and aging, how some animals undergo metamorphosis, and
how animals can regenerate lost organs is discussed in Chapter 12. Taking a longer
view, we shall consider in Chapter 14 how developmental mechanisms have evolved
and how they constrain the very process of evolution itself.
One might ask whether it is necessary to cover so many different organisms in order
to understand the basic features of development. The answer is yes. Developmental
biologists do, indeed, believe that there are general principles of development that
apply to all animals, but life is too wonderfully diverse to find all the answers in a sin-
gle organism. As it is, developmental biologists have tended to focus their efforts on a
relatively small number of animals, chosen because they were convenient to study and
amenable to experimental manipulation or genetic analysis. This is why some crea-
tures, such as the frog Xenopus laevis (Fig. 1.2) and the fruit fly Drosophila melano-
gaster, have such a dominant place in developmental biology. Similarly, work with the
thale cress, Arabidopsis thaliana, has uncovered many features of plant development.
One of the most exciting and satisfying aspects of developmental biology is that
understanding a developmental process in one organism can help to illuminate sim-
Fig. 1.2 The South African claw-toed frog, ilar processes elsewhere—for example, giving insights into how humans develop.
Xenopus laevis. Scale bar = 1 cm. Nothing illustrates this more dramatically than the influence that our understanding
Photograph courtesy of J. Smith. of Drosophila development, and especially of its genetic basis, has had throughout
 The origins of developmental biology 3

developmental biology. The identification of genes controlling early embryogenesis

in Drosophila has led to the discovery of related genes being used in similar ways in
the development of mammals and other vertebrates. Such discoveries encourage us
to believe in the existence of general developmental principles.
Amphibians have long been favorite organisms for studying early development
because their eggs are large and their embryos are easy to grow in a simple culture
medium and relatively easy to experiment on. Embryogenesis in the South African frog
Xenopus (Box 1A) illustrates some of the basic stages of development in all animals.
In the rest of this chapter we first look briefly at the history of embryology—as the
study of developmental biology used to be called. The term developmental biology itself is
of much more recent origin and reflects the appreciation that development is not restricted
to the embryo alone. Traditionally, embryology described experimental results in terms
of morphology and cell fate, but we now understand development in terms of molecular
genetics and cell biology as well. In the second part of the chapter we will introduce some
key concepts that are used over and over again in studying and understanding development.

The origins of developmental biology

Many questions in embryology were first posed hundreds, and in some cases thou-
sands, of years ago. Appreciating the history of these ideas helps us to understand
Fig. 1.3 Malpighi’s description of the
why we approach developmental problems in the way that we do today.
chick embryo. The figure shows Malpighi’s
drawings, made in 1673, depicting the
1.1 Aristotle first defined the problem of epigenesis versus preformation early embryo (top) and at 2 days’ incubation
(bottom). His drawings accurately illustrate
A scientific approach to explaining development started with Hippocrates in Greece in the shape and blood supply of the embryo.
the fifth century bc. Using the ideas current at the time, he tried to explain development Reprinted by permission of the President and
in terms of the principles of heat, wetness, and solidification. About a century later the Council of the Royal Society.
study of embryology advanced when the Greek philosopher Aristotle formulated a ques-
tion that was to dominate much thinking about development until the end of the nine-
teenth century. Aristotle addressed the problem of how the different parts of the embryo
were formed. He considered two possibilities: one was that everything in the embryo was
preformed from the very beginning and simply got bigger during development; the other
was that new structures arose progressively, a process he termed epigenesis (which
means ‘upon formation’) and that he likened metaphorically to the ‘knitting of a net’.
Aristotle favored epigenesis and his conjecture was correct. Aristotle’s influence on Euro-
pean thought was enormous and his ideas remained dominant well into the seventeenth
century. The contrary view to epigenesis, namely that the embryo was preformed from
the beginning, was championed anew in the late seventeenth century. Many could not
believe that physical or chemical forces could mold a living entity such as the embryo.
Along with the contemporaneous background of belief in the divine creation of the world
and all living things, was the belief that all embryos had existed from the beginning of the
world, and that the first embryo of a species must contain all future embryos.
Even the brilliant seventeenth-century Italian embryologist Marcello Malpighi could
not free himself from preformationist ideas. While he provided a remarkably accurate
description of the development of the chick embryo, he remained convinced, against
the evidence of his own observations, that the fully formed embryo was present from
the beginning (Fig. 1.3). He argued that at very early stages the parts were so small
that they could not be seen, even with his best microscope. Other preformationists
believed that the sperm contained the embryo, and some even claimed to see a tiny
human—an homunculus—in the head of each human sperm (Fig. 1.4). Fig. 1.4 Some preformationists believed
The preformation/epigenesis issue was vigorously debated throughout the eigh- that an homunculus was curled up in the
teenth century. But the problem could not be resolved until one of the great advances head of each sperm.
in biology had taken place—the recognition that living things, including embryos, An imaginative drawing, after N. Harspeler
were composed of cells. (1694).
4 Chapter 1 History and basic concepts

BOX 1A Basic stages of Xenopus laevis development

Blastula

Egg
Sperm Animal pole

Gastrula (section)
blastocoel
Cleavage
Vegetal pole

Fertilization blastopore

Adult Gastrulation
mesoderm

Metamorphosis
Free-swimming
tadpole endoderm
Dorsal
Neurulation
ectoderm future gut
(section)
Ventral Organogenesis
Neurula
Anterior
Anterior neural
folds
brain
spinal cord
notochord
Ventral Dorsal
somites
Posterior
(dorsal view)
yolk mass

Posterior
Tailbud-stage embryo Tailbud-stage
(dorsal view with embryo
surface removed) (lateral view)

Fig. 1

1.2 Cell theory changed how people thought about

embryonic development and heredity

The invention of the microscope around 1600 was essential for the discovery of
cells, but the ‘cell theory’ of life was only developed between 1820 and 1880 by,
among others, the German botanist, Matthias Schleiden, and the physiologist, The-
odor Schwann. It recognized that all living organisms consist of cells, that these are
the basic units of life, and that new cells can only be formed by the division of pre-
existing cells. The cell theory was one of the most illuminating advances in biology,
and had an enormous impact. Multicellular organisms, such as animals and plants,
could now be viewed as communities of cells. Development could not, therefore, be
based on preformation, but must be by epigenesis, because during development many
 The origins of developmental biology 5

Although vertebrate development is varied, there are a number stage, these cells are still on the surface of the embryo. During
of basic stages that can be illustrated by following the develop- the next stage—gastrulation—there is a dramatic rearrangement
ment of the frog Xenopus laevis (Fig. 1). The unfertilized egg is a of cells; the endoderm and mesoderm move inside, and the basic
large cell. It has a pigmented upper surface (the animal pole) and body plan of the tadpole is established. Internally, the mesoderm
a lower region (the vegetal pole) characterized by an accumula- gives rise to a rod-like structure (the notochord), which runs from
tion of yolk granules. the head to the tail, and lies centrally beneath the future nervous
After fertilization of the egg by a sperm, and the fusion of male system. On either side of the notochord are segmented blocks of
and female pronuclei, cleavage begins. Cleavages are mitotic mesoderm called somites, which will give rise to the muscles and
divisions in which cells do not grow between each division, and so vertebral column, as well as the dermis of the skin (somites can
with successive cleavages the cells become smaller. After about be seen in the cutaway view of the later tailbud-stage embryo).
12 division cycles, the embryo, now known as a blastula, consists Shortly after gastrulation, the ectoderm above the notochord
of many small cells surrounding a fluid-filled cavity (the blasto- folds to form a tube (the neural tube), which gives rise to the
coel) above the larger yolky cells. Already, changes have occurred brain and spinal cord—a process known as neurulation. By this
within the cells and they have interacted with each other so that time, other organs, such as limbs, eyes, and gills, are specified
the three germ layers—mesoderm, endoderm, and ectoderm— at their future locations, but only develop a little later, during
are specified (see Box 1C). The animal region gives rise to ecto- organogenesis. During organogenesis, specialized cells such as
derm, which forms both the epidermis of the skin and the nervous muscle, cartilage, and neurons differentiate. By four days after
system. The vegetal region gives rise to the future endoderm and fertilization, the embryo has become a free-swimming tadpole
mesoderm, which are destined to form internal organs. At this with typical vertebrate features.

new cells are generated by division from the egg, and new types of cells are formed. A
crucial step forward in understanding development was the recognition, in the 1840s,
that the egg itself is but a single, albeit specialized, cell.
An important advance in embryology was the proposal by the nineteenth-century
German biologist, August Weismann, that an offspring does not inherit its charac-
teristics from the body (the soma) of the parent but only from the germ cells—egg
and sperm. Weismann drew a fundamental distinction between germ cells and the
body cells or somatic cells (Fig. 1.5). Characteristics acquired by the body during an
animal’s life cannot be transmitted to the germline. As far as heredity is concerned,
the body is merely a carrier of germ cells. As the English novelist and essayist Samuel
Butler put it: ‘A hen is only an egg’s way of making another egg.’
Work on sea urchin eggs showed that after fertilization the egg contains two nuclei,
which eventually fuse; one of these nuclei belongs to the egg, whereas the other comes
from the sperm. Fertilization therefore results in a single cell—the zygote—carrying a
nucleus with contributions from both parents, and it was concluded that the cell nucleus
must contain the physical basis of heredity. The climax of this line of research was the
Fig. 1.5 The distinction between germ
cells and somatic cells. In each generation
First generation Second generation Third generation a germ cell contributes to the zygote, which
mutations in mutation in gives rise to both somatic cells and germ
somatic cells do germ cell cells, but inheritance is through the germ
not affect germline affects somatic cells only (first panel). Changes that occur
cells and
somatic cells

somatic cells

somatic cells

germline due to a mutation (red) in a somatic cell can

be passed on to its daughter cells but do not
affect the germline, as shown in the second
panel. In contrast, a mutation in the germline
(green) in the second generation will be
germline present in every cell in the body of the new
mutation organism to which that cell contributes, and
will also be passed on to the third and future
zygote zygote zygote generations through the germline, as shown
germ cells germ cells germ cells
in the third panel.
6 Chapter 1 History and basic concepts

First cleavage Second cleavage

Fig. 1.6 Weismann’s theory of nuclear

determination. Weismann assumed that
there were factors in the nucleus that were
distributed asymmetrically to daughter cells
during cleavage and directed their future
Weismann’s nuclear determinants
development.

demonstration, towards the end of the nineteenth century, that the chromosomes within
the nucleus of the zygote are derived in equal numbers from the two parental nuclei,
and the recognition that this provided a physical basis for the transmission of genetic
characters according to the laws developed by the Austrian botanist and monk, Gregor
Mendel. The number of chromosomes is kept constant from generation to generation by
a specialized type of cell division that produces the germ cells, called meiosis, which
halves the chromosome number; the full complement of chromosomes is then restored
at fertilization. The zygote and the somatic cells that arise from it divide by the process
of mitosis, which maintains chromosome number (Box 1B). Germ cells contain a single
copy of each chromosome and are called haploid, whereas germ-cell precursor cells and
the other somatic cells of the body contain two copies and are called diploid.

1.3 Two main types of development were originally proposed

The next big question was how cells became different from one another during
embryonic development. With the increasing emphasis on the role of the nucleus, in
the 1880s Weismann put forward a model of development in which the nucleus of
the zygote contained a number of special factors, or determinants (Fig. 1.6). He pro-
Fig. 1.7 Roux’s experiment to posed that while the fertilized egg underwent the rapid cycles of cell division known
investigate Weismann’s theory of mosaic as cleavage (see Box 1A), these nuclear determinants would be distributed unequally
development. After the first cleavage of to the daughter cells and so would control the cells’ future development. The fate of
a frog embryo, one of the two cells is killed each cell was therefore predetermined in the egg by the factors it would receive during
by pricking it with a hot needle; the other cleavage. This type of model was termed ‘mosaic’, as the egg could be considered to
remains undamaged. At the blastula stage the be a mosaic of discrete localized determinants. Central to Weismann’s theory was the
undamaged cell can be seen to have divided
assumption that early cell divisions must make the daughter cells quite different from
as normal into many cells that fill half of the
each other as a result of unequal distribution of nuclear components.
embryo. The development of the blastocoel,
In the late 1880s, initial support for Weismann’s ideas came from experiments
a small fluid-filled space in the center of the
blastula, is also restricted to the undamaged
carried out independently by the German embryologist, Wilhelm Roux, who experi-
half. In the damaged half of the embryo, no mented with frog embryos. Having allowed the first cleavage of a fertilized frog egg,
cells appear to have formed. At the neurula Roux destroyed one of the two cells with a hot needle and found that the remain-
stage, the undamaged cell has developed into ing cell developed into a well-formed half-larva (Fig. 1.7). He concluded that the
something resembling half a normal embryo.

Fertilized frog egg Two-cell stage Blastula stage (section) Neurula stage

hot needle blastocoel remains half

of embryo
killed
cell

neural
tube
 The origins of developmental biology 7

CELL BIOLOGY BOX 1B The mitotic cell cycle

When a eukaryotic cell duplicates itself it goes through a fixed

sequence of events called the cell cycle. The cell grows in size,
the DNA is replicated, and the replicated chromosomes then
undergo mitosis and become segregated into two daughter Resting
nuclei. Only then can the cell divide to form two daughter cells, M
G0
which can go through the whole sequence again.
G2 Division
The standard eukaryotic mitotic cell cycle is divided into well-
marked phases (Fig. 1). At the M phase, mitosis and cell cleavage
give rise to two new cells. The rest of the cell cycle, between one Growth
factors
M phase and the next, is called interphase. Replication of DNA G1
occurs during a defined period in interphase, the S phase (the S I nte r p h a s e
S stands for synthesis of DNA). Preceding S phase is a period
known as G1 (the G stands for gap), and after it another interval
known as G2, after which the cells enter mitosis (see Fig. 1). G1,
S phase, and G2 collectively make up interphase, the part of the
cell cycle during which cells synthesize proteins and grow, as well
Fig. 1
as replicate their DNA. When somatic cells are not proliferating
they are usually in a state known as G0, into which they withdraw
after mitosis. The decision to enter G0 or to proceed through G1 Particular phases of the cell cycle are absent in some cells: dur-
may be controlled by both intracellular state and extracellular ing cleavage of the fertilized Xenopus egg G1 and G2 are virtually
signals such as growth factors. Growth factors enable the cell to absent, and cells get smaller at each division. In Drosophila sali-
proceed out of G0 and progress through the cell cycle. Cells such vary glands there is no M phase, as the DNA replicates repeatedly
as neurons and skeletal muscle cells, which do not divide after without mitosis or cell division, leading to the formation of giant
differentiation, are permanently in G0. polytene chromosomes.

‘development of the frog is based on a mosaic mechanism, the cells having their
character and fate determined at each cleavage’.
But when Roux’s fellow countryman, Hans Driesch, repeated the experiment on sea
urchin eggs, he obtained quite a different result (Fig. 1.8). He wrote later: ‘But things
turned out as they were bound to do and not as I expected; there was, typically, a

Normal development of sea urchin larva from two-cell stage

Driesch’s separation of cells at two-cell stage resulted in the death of one cell.
The surviving cell developed into a small but otherwise normal larva Fig. 1.8 The outcome of Driesch’s
experiment on sea urchin embryos, which
first demonstrated the phenomenon of
regulation. After separation of cells at the
two-cell stage, the remaining cell developed
into a small, but whole, normal larva. This
is the opposite of Roux’s earlier finding
one of the
separated cells that when one of the cells of a two-cell
usually died frog embryo is damaged, the remaining cell
develops into a half-embryo only (see Fig. 1.7).
8 Chapter 1 History and basic concepts

Dorsal lip of blastopore grafted from an whole gastrula on my dish the next morning, differing only by its small size from a nor-
unpigmented species of newt to the mal one; and this small but whole gastrula developed into a whole and typical larva.’
blastocoel roof of a pigmented species
Driesch had completely separated the cells at the two-cell stage and obtained a
Triton cristatus normal but small larva. That was just the opposite of Roux’s result, and was the first
gastrula clear demonstration of the developmental process known as regulation. The experi-
ment of Roux on frogs was later repeated by the American T.H. Morgan, who sepa-
dorsal lip rated the two blastomeres instead of killing one of them and leaving it attached, and
of blastopore
he obtained the same result as Driesch did with sea urchins. This showed the general
blastocoel ability of vertebrate embryos to regulate, that is, to restore normal development, even
if some portions are removed or rearranged very early in development. The basis
for this phenomenon is explained later in the chapter. The extent to which embryos
can regulate differs in different species and we shall see many examples of regula-
tion throughout the book. The existence of regulation does not mean, however, that
the unequal distribution of determinants that make two daughter cells different from
Triton taeniatus
gastrula each other is not important during development. But Weismann was wrong in one
crucial respect, in that such determinants are not nuclear, but are located in the cell
cytoplasm. We shall see many examples of developmentally important proteins and
A secondary embryo is induced RNAs that act in this way as cytoplasmic determinants.

1.4 The discovery of induction showed that one group of cells could
determine the development of neighboring cells

The fact that embryos can regulate implies that cells must communicate and interact with
each other, but the central importance of cell–cell interactions in embryonic develop-
ment was not really established until the discovery of the phenomenon of induction. This
secondary (induced) embryo
is where one cell, or tissue, directs the development of another, neighboring, cell or tissue.
X Y The importance of induction and other cell–cell interactions in development was proved
Primary Secondary dramatically in 1924 when Hans Spemann and his assistant, Hilde Mangold, carried out a
structures (induced)
structures now famous transplantation experiment in amphibian embryos. They showed that a par-
neural tube
notochord tial second embryo could be induced by grafting one small region of an early newt embryo
notochord
neural tube
onto another at the same stage (Fig. 1.9). The grafted tissue was taken from the dorsal lip
of the blastopore—the slit-like invagination that forms where gastrulation begins on the
dorsal surface of the amphibian embryo (see Box 1A). This small region they called the
organizer, as it seemed to be ultimately responsible for controlling the organization of a
complete embryonic body; it is now known as the Spemann–Mangold organizer, or just
Fig. 1.9 The dramatic demonstration by
the Spemann organizer. For their discovery, Spemann received the Nobel Prize for Physi-
Spemann and Mangold of the induction
of a new main body axis by the organizer ology or Medicine in 1935, the first Nobel Prize ever given for embryological research.
region in the early amphibian gastrula. Sadly, Hilde Mangold had died earlier, in an accident, and so could not be honored.
A piece of tissue (yellow) from the dorsal lip
of the blastopore of a newt (Triton cristatus)
1.5 Developmental biology emerged from the coming together of
gastrula is grafted to the opposite side of
genetics and embryology
a gastrula of another, pigmented, newt
species (Triton taeniatus, pink). The grafted When Mendel’s laws were rediscovered in 1900 there was a great surge of interest in
tissue induces a new body axis containing mechanisms of inheritance, particularly in relation to evolution, but less so in relation to
neural tube and somites. The unpigmented
embryology. Genetics was seen as the study of the transmission of hereditary elements
graft tissue forms a notochord at its new
from generation to generation, whereas embryology was the study of how an individual
site (see section in lower panel), but most
organism develops and, in particular, how cells in the early embryo became different
of the neural tube and the other structures
of the new axis have been induced from the from each other. Genetics seemed, in this respect, to be irrelevant to development.
pigmented host tissue. The organizer region The fledgling science of genetics was put on a firm conceptual and experimental
discovered by Spemann and Mangold is footing in the first quarter of the twentieth century by T.H. Morgan. Morgan chose the
known as the Spemann organizer. fruit fly D. melanogaster as his experimental organism. He noticed a fly with white
eyes rather than the usual red eyes, and by careful cross-breeding he showed that
inheritance of this mutant trait was linked to the sex of the fly. He found three other
sex-linked traits and worked out that they were each determined by three distinct
‘genetic loci’, which occupied different positions on the same chromosome, the fly’s
 The origins of developmental biology 9

X chromosome. The rather abstract hereditary ‘factors’ of Mendel had been given real-
ity. But even though Morgan was originally an embryologist, he made little headway
in explaining development in terms of genetics. That had to wait until the nature of
the gene was better understood.
An important concept in understanding how genes influence physical and physi-
ological traits is the distinction between genotype and phenotype. This was first put
forward by the Danish botanist, Wilhelm Johannsen, in 1909. The genetic endowment
of an organism—the genetic information it inherits from its parents—is the genotype.
The organism’s visible appearance, internal structure, and biochemistry comprise the
phenotype. While the genotype certainly controls development, environmental fac-
tors interacting with the genotype influence the phenotype. Despite having identical
genotypes, identical twins can develop differences in their phenotypes as they grow
up (Fig. 1.10), and these tend to become more evident with age.
Following Morgan’s discoveries in genetics, the problem of development could now
be posed in terms of the relationship between genotype and phenotype: how the Fig. 1.10 The difference between
genetic endowment becomes ‘translated’ or ‘expressed’ during development to give genotype and phenotype. These identical
rise to a functioning organism. But the coming together of genetics and embryology twins have the same genotype because
was slow and tortuous. The discovery in the 1940s that genes are made of DNA and one fertilized egg split into two during
encode proteins was a major turning point. It was already clear that the properties development. Their slight difference in
appearance is due to non-genetic factors,
of a cell are determined by the proteins it contains, and so the fundamental role of
such as environmental influences.
genes in development could at last be appreciated. By controlling which proteins were
Photograph courtesy of Robert and Lewis
made in a cell, genes could control the changes in cell properties and behaviour that
McCaffrey.
occurred during development. A further major advance in the 1960s was the discov-
ery that some genes encode proteins that control the activity of other genes.

1.6 Development is studied mainly through selected model organisms

Although the embryology of many different species has been studied at one time or
another, a relatively small number of organisms provide most of our knowledge about
developmental mechanisms. We can thus regard them as ‘models’ for understanding the
processes involved, and they are often called model organisms. Sea urchins and amphib-
ians were the main animals used for the first experimental investigations because their
developing embryos are easy to obtain and, in the case of amphibians, relatively easy to
manipulate experimentally, even at quite late stages. Among vertebrates, the frog Xeno-
pus laevis, the mouse (Mus musculus), the chicken (Gallus gallus), and the zebrafish
(Danio rerio) are the main model organisms now studied. Among invertebrates, the fruit
fly Drosophila melanogaster and the nematode Caenorhabditis elegans have been the
focus of most attention, because a great deal is known about their developmental genet-
ics and they can be easily genetically modified. Two Nobel prizes have been awarded for
discoveries about development in Drosophila and Caenorhabditis, respectively. With the
advent of modern methods of genetic analysis, there has also been a resurgence of inter-
est in the sea urchin Strongylocentrotus purpuratus. For plant developmental biology,
Arabidopsis thaliana serves as the main model organism. The life cycles and background
details for these model organisms are given in the relevant chapters later in the book.
The evolutionary relationships of these organisms are shown in Figure 1.11.
The reasons for these choices are partly historical—once a certain amount of
research has been done on one animal it is more efficient to continue to study it
rather than start at the beginning again with another species—and partly a question
of ease of study and biological interest. Each species has its advantages and disad-
vantages as a developmental model. The chick embryo, for example, has long been
studied as a model for vertebrate development because fertile eggs are easily available
and the embryo withstands experimental microsurgical manipulation very well. A
disadvantage, however, was that until very recently little was known about the chick’s
developmental genetics. In contrast, we know a great deal about the genetics of the
mouse, although the mouse is more difficult to study in some ways, as development
10 Chapter 1 History and basic concepts

Plants (Arabidopsis)
Nematodes (Caenorhabditis)
Insects (Drosophila)
Arthropods
Crustaceans
Annelids
Mollusks
Platyhelminthes (planarians)
Single-
celled Echinoderms (sea urchin)
ancestors
Mammals (mouse, human)
Birds (chick)
Reptiles
Chordates
Amphibians (Xenopus)
Fig. 1.11 Phylogenetic tree showing the
placement of the main developmental Fish (zebrafish)
model organisms. The organisms discussed
Ascidians (Ciona)*
in this book are highlighted in blue. *Ascidian
development is described in Section S5 Cnidarians (Hydra)
online (http://bit.ly/wolpert6oes5a).

normally takes place entirely within the mother. It is, however, possible to fertilize
mouse eggs in culture, let them develop for a short time outside the uterus, when they
can be observed, and then re-implant the early embryos for them to complete their
development. Many developmental mutations have been identified in the mouse, and
it is also amenable to genetic modification by transgenic techniques, by which genes
can be introduced, deleted, or modified in living organisms. It is also the best experi-
mental model we have for studying mammalian development. The zebrafish is a more
recent addition to the select list of vertebrate model organisms; it is easy to breed in
large numbers, the embryos are transparent and so cell divisions and tissue move-
ments can be followed visually, and it has great potential for genetic investigations.
A major goal of developmental biology is to understand how genes control embry-
onic development, and to do this one must first identify which genes, out of the
many thousands in the organism, are critically and specifically involved in control-
ling development. This task can be approached in various ways, depending on the
organism involved, but the general starting point is to identify gene mutations that
alter development in some specific and informative way, as described in the following
section. Techniques for identifying such developmental genes and for detecting and
manipulating their expression in the organism are described throughout the book,
along with techniques for manipulating the genes themselves.
As we have just discussed, some of our model organisms are more amenable to conven-
tional genetic analysis than others. Despite its importance in developmental biology, little
conventional genetics has been done on X. laevis, which has the disadvantage of being
tetraploid (it has four sets of chromosomes in its somatic cells, as opposed to the two
sets carried by the cells of diploid organisms, such as humans and mice) and a relatively
long period of 1–2 years to reach sexual maturity and breed. Using modern genetic and
bioinformatics techniques, however, many developmental genes have been identified in
X. laevis by direct DNA sequence comparison with known genes in Drosophila and mice.
The closely related frog Xenopus tropicalis is a more attractive organism for genetic analy-
sis; it is diploid and can also be genetically manipulated to produce transgenic organisms.
The hereditary information of a particular organism, comprising its complete
set of genes and non-coding DNA sequences, is known as its genome. In sexually
reproducing diploid species, two complete copies of the genome are carried by the
 The origins of developmental biology 11

chromosomes in each somatic cell—one copy inherited from the father and one from
the mother. Genome sequences are available for all our model organisms highlighted
in Figure 1.11, even for the tetraploid X. laevis. In the zebrafish, a genome-duplication
event in its evolutionary history means that there are duplicates of at least 2900 out of
the estimated 20,000 protein-coding genes in its genome. Thus, the zebrafish genome
contains over 26,000 protein-coding genes, whereas the mouse genome has only
22,000. Having the complete DNA sequences of the genomes of our model organ-
isms helps enormously in identifying developmental genes and other developmentally
important DNA sequences. These developmentally important sequences include non-
coding control regions that regulate the expression of the protein-coding genes.
In general, when an important developmental gene has been identified in one ani-
mal, it has proved very rewarding to see whether a corresponding gene is present and
is acting in a developmental capacity in other animals. Such genes are often identified
by a sufficient degree of nucleotide sequence similarity to indicate descent from a com-
mon ancestral gene. Genes that meet this criterion are known as homologous genes.
As we shall see in Chapter 5, this approach identified a hitherto unsuspected class of
vertebrate genes that control the regular segmented pattern from head to tail, which is
represented by the different types of vertebrae at different positions. These genes were
identified by their homology with genes that determine the identities of the different
body segments in Drosophila (described in Chapter 2). The DNA sequences that act
as gene control regions can also often be identified by homology.

1.7 The first developmental genes were identified as spontaneous mutations

Most of the organisms dealt with in this book are sexually reproducing diploid organ-
isms: their somatic cells contain two copies of each gene, with the exception of those
on the sex chromosomes. In a diploid species, one copy, or allele, of each gene is
contributed by the male parent and the other by the female. For many genes there
are several different alleles present in the population, which leads to the variation in
phenotype one normally sees within any sexually reproducing species. Occasionally,
however, a mutation will occur spontaneously in a gene and there will be marked
change, usually deleterious, in the phenotype of the organism.
Many of the genes that affect development have been identified by spontaneous or
induced mutations that disrupt their function and produce an abnormal phenotype. Muta-
tions are classified broadly according to whether they are dominant or recessive (Fig. 1.12).

Recessive mutation (e.g. vestigial–) Semi-dominant mutation (e.g. brachyury)

Genotype Phenotype Genotype Phenotype

wild type normal wild type normal

Fig. 1.12 Types of mutations. Left: a
vg+ + mutation is recessive when it only has an
effect in the homozygous state; that is, when
+ both copies of the gene carry the mutation.
vg+
The vestigial mutation in Drosophila leads to
the absence of wings when homozygous. A
heterozygous mutation normal heterozygous mutation deformed tail plus sign (+) denotes the ‘normal’ allele (wild
vg+ +
type), and a minus sign (−) the recessive
allele. Right: by contrast, a dominant or semi-
dominant mutation produces an effect on the
vg– T
phenotype in the heterozygous state; that
is, when just one copy of the gene carries
homozygous mutation vestigial wings homozygous mutation embryonic lethal
the mutation. The Brachyury mutation in the
vg– T
mouse has an obvious but non-lethal effect
in the heterozygous state, but is lethal in the
vg– T
homozygous state. T denotes the dominant
mutant allele of the brachyury gene.
12 Chapter 1 History and basic concepts

Dominant and semi-dominant mutations are those that produce a distinctive phenotype
when the mutation is present in just one allele of a pair; that is, it exerts an effect in the
heterozygous state. By contrast, recessive mutations alter the phenotype only when both
alleles are of the mutant type; this is known as the homozygous state. In recessive muta-
tions, the function of the one wild-type gene is sufficient to confer a normal phenotype in
heterozygotes, whereas in dominant or semi-dominant mutations, the effect of the mutant
allele completely or partly overrides that of the wild-type gene in the heterozygote or one
copy of the normal gene is not enough to provide function.
In general, dominant mutations are more easily recognized, particularly if the phenotype
involves alterations in gross anatomy or coloration, provided that they do not cause the early
death of the embryo in the heterozygous state. True dominant mutations are rare. However,
if one functional allele is not enough to provide the full function of a gene, a heterozygous
mutant will have an abnormal phenotype. An example of this is seen in mice that have one
copy of the mutant allele of the brachyury gene, denoted by T. These heterozygous mice
(T/+) have short tails. Because of this phenotype, the gene affected in the mutants was
called brachyury (from the Greek brachy, short, and oura, tail). When the mutation is homo-
zygous (T/T), it has a much greater effect, and embryos die at an early stage, with very short
bodies, indicating that the brachyury gene is required for normal embryonic development
(Fig. 1.13). Once breeding studies had confirmed that a single gene was involved, the muta-
tion could be mapped to a location on a particular chromosome by classical gene-mapping
techniques, and it was found that the T allele represents a deletion of the brachyury gene.
Identifying genes affected by recessive mutations is more laborious, as the het-
erozygote has a phenotype identical to a normal wild-type animal, and a carefully
worked-out breeding program is needed to obtain homozygotes. In mammals, iden-
tifying potentially lethal recessive developmental mutations requires careful observa-
tion and analysis, as the homozygotes may die unnoticed inside the mother.
Many mutations in invertebrates have been identified as conditional mutations.
The effects of these mutations only show up when the animal is kept in a particular
condition—most commonly at a higher temperature, in which case the mutation is
known as a temperature-sensitive mutation. At the usual ambient temperature, the
animal appears normal. Temperature sensitivity is usually due to the encoded mutant
protein being able to fold into a functional structure at the normal temperature, but
being less stable at the higher temperature.
Very rigorous criteria must be applied to identify mutations that are affecting a genuine
developmental process and not just affecting some vital but routine ‘housekeeping’ func-
tion without which the animal cannot survive. One simple criterion for a developmental

T +
+ +

Short tail

Fig. 1.13 Genetics of the semi-dominant

T mutation, which deletes the brachyury
T T + +
gene in the mouse. A male heterozygote + + + +
carrying the T mutation merely has a short
tail. When mated with a female homozygous
for the normal, or wild-type, brachyury gene
(+/+), some of the offspring will also be
heterozygotes and have short tails. Mating
two heterozygotes together will result in homozygous
T T + T embryonic lethal
some of the offspring being homozygous (T/T) + + +
T (abnormal posterior
for the mutation, resulting in a severe and mesoderm)
lethal developmental abnormality in which the
posterior mesoderm does not develop.
 A conceptual tool kit 13

mutation is that it leads to the death of the embryo, but mutations in genes involved in
vital housekeeping functions can also have this effect. Mutations that produce observable
abnormal embryonic development are more promising candidates for true developmental
mutations. In later chapters we shall see how large-scale screening for mutations after
mutagenesis by chemicals or X-rays has identified many more developmental genes than
would ever have been picked up just by looking for rare spontaneous mutations.
In recent years, new techniques for identifying developmental genes have appeared.
These depend on knowing the existence and sequence of a gene (e.g. from genomic
sequences) and working backwards to determine its function in the animal, usu-
ally by removing the gene or blocking its function. Such methods are known as
reverse genetics, as opposed to the traditional methods of forward genetics outlined
above which starts with a mutant phenotype and then identifies the gene responsible.
Examples of reverse genetics include gene knock-out, in which the gene is effectively
deleted from the animal’s genome by transgenic techniques (discussed in Section 3.10),
and gene knockdown or gene silencing, in which gene expression is prevented by
techniques such as RNA interference or antisense RNA (discussed in Box 6B). Over the
past few years a new DNA-editing technology, CRISPR-Cas9, has revolutionized genetic
approaches to development. This technique allows an efficient and clean introduction
of mutations in any organism in which the sequence of the gene to be targeted is known
and the editing components can be effectively introduced into cells. Mutations can be
introduced in both protein-coding and regulatory DNA sequences (discussed in Box 3D).

SUMMARY

The study of embryonic development started with the Greeks more than 2000 years ago.
Aristotle put forward the idea that embryos were not contained completely preformed in
miniature within the egg, but that form and structure emerged gradually, as the embryo
developed. This idea was challenged in the seventeenth and eighteenth centuries by those
who believed in preformation, the idea that all embryos that had been, or ever would be, had
existed from the beginning of the world. The emergence of the cell theory in the nineteenth
century finally settled the issue in favor of epigenesis, and it was realized that the sperm
and egg are single, albeit highly specialized, cells. Some of the earliest experiments showed
that very early sea urchin embryos are able to regulate—that is, to develop normally even if
cells are removed or killed. This established the important principle that development must
depend, at least in part, on communication between the cells of the embryo. Direct evidence
for the importance of cell–cell interactions came from the organizer graft experiment carried
out by Spemann and Mangold in 1924, showing that the cells of the amphibian organizer
region could induce a new partial embryo from host tissue when transplanted into another
embryo. The role of genes in controlling development, by determining which proteins are
made, has only been fully appreciated in the last 50 years and the study of the genetic basis
of development has been made much easier in recent times by the techniques of molecular
biology and the availability of the DNA sequences of whole genomes.

A conceptual tool kit

Development into a multicellular organism is the most complicated fate a single liv-
ing cell can undergo; in this lies both the fascination and the challenge of develop-
mental biology. Yet only a few basic principles are needed to start to make sense of
developmental processes. The rest of this chapter is devoted to introducing these key
concepts. These principles are encountered repeatedly throughout the book, as we
look at different organisms and developmental systems, and should be regarded as a
conceptual tool kit, essential for embarking on a study of development.
Genes control development by determining where and when proteins are synthesized.
Gene activity sets up intracellular networks of interactions between proteins and genes,
14 Chapter 1 History and basic concepts

and between proteins and proteins, that give cells their particular properties. One of these
properties is the ability to communicate with, and respond to, other cells. It is these cell–
cell interactions that determine how the embryo develops; no developmental process can
therefore be attributed to the function of a single gene or single protein. The amount of
genetic and molecular information on developmental processes is now enormous. In this
book, we will be highly selective and describe only those molecular details that give an
insight into the mechanisms of development and illustrate general principles.

1.8 Development involves the emergence of pattern, change in form, cell

differentiation, and growth

In most embryos, fertilization is immediately followed by cell division. This stage

is known as cleavage, and divides the fertilized egg into a number of smaller cells
(Fig. 1.14). Unlike the cell divisions that take place during growth of a tissue, there
is no increase in cell size between each cleavage division; the cleavage cell cycles
consist simply of phases of DNA replication, mitosis, and cell division (see Box 1B).
Early embryos are therefore no bigger than the zygote, and there is little increase in
size during embryogenesis unless the embryo has access to a substantial source of
material it can use to grow—for example, the large yolk available to the chick embryo
and the nutrients provided to the mammalian embryo through the placenta.
Development is, in essence, the emergence of organized structures from an initially very
simple group of cells. It is convenient to distinguish four main developmental processes,
which occur in roughly sequential order in development, although, in reality, they overlap
with, and influence, each other considerably. They are pattern formation, morphogenesis,
cell differentiation, and growth. Pattern formation is the process by which cellular activity
is organized in space and time so that a well-ordered structure develops within the embryo.
It is of fundamental importance in the early embryo and also later in the formation of
organs. In the developing arm, for example, pattern formation enables cells to ‘know’
whether to make an upper arm or fingers, and where the muscles should form. There is
no universal strategy of patterning; rather, it is achieved by various cellular and molecular
mechanisms in different organisms and at different stages of development.
Pattern formation initially involves laying down the overall body plan—defining the
main body axes of the embryo that run from head (anterior) to tail (posterior), and
from back (dorsal) to underside (ventral). Most of the animals in this book have a head
at one end and a tail at the other, with the left and right sides of the body being out-
wardly bilaterally symmetrical—that is, a mirror image of each other. In such animals,
the main body axis is the antero-posterior axis, which runs from the head to the tail.
Bilaterally symmetrical animals also have a dorso-ventral axis, running from the back
to the belly. A striking feature of these axes is that they are almost always at right angles
to one another and so can be thought of as making up a system of coordinates on which
any position in the body could be specified (Fig. 1.15). Internally, animals have distinct
differences between the left and right sides in the disposition of their internal organs—
the human heart, for example, is on the left side. In plants, the main body axis runs from
the growing tip (the apex) to the roots and is known as the apical–basal axis. Plants also
Fig. 1.14 Light micrographs of cleaving have radial symmetry, with a radial axis running from the center of the stem outwards.
Xenopus eggs. Even before the body axes become clear, eggs and embryos often show a distinct
Images courtesy of Dr. H. Williams. polarity, which in this context means that they have an intrinsic orientation, with
 A conceptual tool kit 15

Xenopus laevis tailbud-stage embryo

Dorsal (back)

Right

Anterior Posterior
(head) (tail)

Fig. 1.15 The main axes of a developing

embryo. The antero-posterior axis and the
Left
Ventral (front) dorso-ventral axis are at right angles to one
another, as in a coordinate system.

one end different from the other. Polarity can be specified by a gradient in a protein
or other molecule, as the slope of the gradient provides direction. Individual cells
within the embryo can also have their own polarity. One type of polarity is called
apico-basal polarity, and is seen in simple epithelia, single-layered sheets of cells in
which the two sides of the sheet (the apical and basal sides) are different, and which
are building blocks of tissues and organs (discussed in detail in Chapter 7). A second
axis of polarity in cells can be discerned along the plane of the tissue and this provides
a larger-scale coordinate system for the tissue. This type of individual cell polarity is
known as planar cell polarity and is discussed further in Chapter 7. Planar cell polar-
ity is sometimes easily visible in structures—the hairs produced by epidermal cells on
the edge of the Drosophila wing all point in the same direction.
At the same time that the axes of the embryo are being formed, cells are being allo-
cated to the different germ layers—ectoderm, mesoderm, and endoderm (Box 1C).

CELL BIOLOGY BOX 1C Germ layers

The concept of germ layers is useful to distin-

Germ layers
guish between regions of the early embryo that
give rise to quite distinct types of tissues. It Dorsal Dorsal
Vertebrates Insects
applies to both vertebrates and invertebrates.
All the animals considered in this book, except
for the cnidarian Hydra, are triploblasts, with
three germ layers: the ectoderm, which gives
rise to the epidermis of the skin and nervous
system; the mesoderm, which gives rise to the
skeleto-muscular system, connective tissues,
and other internal organs, such as the kidney
and heart; and the endoderm, which gives
Ventral Ventral
rise to the gut and its derivatives, such as the
liver and lungs in vertebrates (Fig. 1). These Germ layers Organs
are specified early in development. There are,
however, some exceptions to the types of Endoderm gut, liver, lungs gut
tissue that arise from a particular germ layer.
Mesoderm skeleton, muscle, kidney, heart, blood muscle, heart, blood
The neural crest in vertebrates, for example,
is ectodermal in origin, but gives rise not only Ectoderm epidermis of skin, nervous system cuticle, nervous system
to neural tissue, but also to some skeletal ele-
Fig. 1
ments that generally arise from mesoderm.
16 Chapter 1 History and basic concepts

mouth

Fig. 1.16 Gastrulation in the sea

urchin. Gastrulation transforms the spherical
blastula into a structure with a tube—the
gut—through the middle. The left-hand side
of the embryo has been removed. gut anus

During further pattern formation, cells of these germ layers acquire different identities
so that organized spatial patterns of differentiated cells finally emerge, such as the
arrangement of skin, muscle, and cartilage in developing limbs, and the arrangement
of neurons in the nervous system.
The second important developmental process is change in form, or morphogenesis
(discussed in Chapter 7). Embryos undergo remarkable changes in three-dimensional
form—you need only think about the complicated anatomy of structures such as
your hands and feet. At certain stages in development, there are characteristic and
dramatic changes in form, of which gastrulation is the most striking. Almost all
Weeks after fertilization animal embryos undergo gastrulation, during which endoderm and mesoderm move
8 12 16 Birth
inside, the gut is formed, and the main body plan emerges. During gastrulation, cells
Change in size on the outside of the embryo move inwards and, in animals such as the sea urchin,
gastrulation transforms a hollow spherical blastula into a gastrula with a tube through
the middle—the gut (Fig. 1.16). Morphogenesis in animal embryos can also involve
extensive cell migration. Most of the cells of the human face, for example, are derived
from cells that migrated from a tissue called the neural crest, which originates on
the dorsal side of the embryo. Morphogenesis can also involve developmentally pro-
grammed cell death, called apoptosis, which is responsible, for example, for the sepa-
ration of our fingers and toes from an initially solid plate of tissue.
The third developmental process we must consider here is cell differentiation, in
which cells become structurally and functionally different from each other, ending up
as distinct cell types, such as blood, muscle, or skin cells. Differentiation is a gradual
process, with cells often going through several divisions between the time at which
Change in proportions they start differentiating and the time they are fully differentiated (when many cell
types stop dividing altogether), and is discussed in Chapter 8. In humans, the fertil-
ized egg gives rise to hundreds of clearly distinguishable types of cell.
Pattern formation and cell differentiation are very closely interrelated, as we can see
by considering the difference between human arms and legs. Both contain exactly the
same types of cell—muscle, cartilage, bone, skin, and so on—yet the pattern in which
they are arranged is clearly different. It is essentially pattern formation that makes us
look different from elephants and chimpanzees.
The fourth process is growth—the increase in size. In general, there is little growth
during early embryonic development and the basic pattern and form of the embryo
is laid down on a small scale, always less than a millimeter in extent. Subsequent
growth can be brought about in various ways: cell proliferation, increase in cell size,
Fig. 1.17 The human fetus changes shape and deposition of extracellular materials, such as, for example, in bone. Growth can
as it grows. From the time the body plan
also be morphogenetic, in that differences in growth rates between organs, or between
is well established, at 8 weeks, the fetus
parts of the body, can generate changes in the overall shape of the embryo (Fig. 1.17),
increases in length some ten-fold until birth
as we shall see in more detail in Chapter 12.
(upper panel), while the relative proportion
of the head to the rest of the body decreases These four developmental processes are neither independent of each other nor
(lower panel). As a result, the proportions and strictly sequential. In very general terms, however, one can think of pattern forma-
the shape of the fetus change. Scale bar = 10 tion in early development specifying differences between cells that lead to changes
cm. After Moore, K.L.: The Developing Human, in form, cell differentiation, and growth. But in any real developing system there are
1983. Saunders. many twists and turns in this sequence of events.
 A conceptual tool kit 17

1.9 Cell behavior provides the link between gene action and developmental
processes

Gene expression within cells leads to the synthesis of proteins that are responsible
for particular cellular properties and behavior, which, in turn, determine the course
of embryonic development. The past and current patterns of gene activity confer a
certain state, or identity, on a cell at any given time, which is reflected in its molecular
organization—in particular which proteins are present. As we shall see, embryonic
cells and their progeny undergo many changes in state as development progresses.
Other categories of cell behavior that will concern us are intercellular communication,
also known as cell–cell signaling or cell–cell interactions, changes in cell shape and
cell movement, cell proliferation, and cell death.
Changing patterns of gene activity during early development are essential for pat-
tern formation. They give cells identities that determine their future behavior and lead
eventually to their final differentiation. And, as we saw in the example of induction by
the Spemann organizer, the capacity of cells to influence each other’s fate by producing
and responding to signals is crucial for development. By their response to signals for cell
localized
movement or a change in shape, for example, cells generate the physical forces that bring contraction
about morphogenesis (Fig. 1.18). The curvature of a sheet of cells into a tube, as hap-
pens in Xenopus and other vertebrates during formation of the neural tube (see Box 1A), Fig. 1.18 Localized contraction of
is the result of contractile forces generated by cells changing their shape at certain posi- particular cells in a sheet of cells can cause
tions within the cell sheet. An important feature of cell surfaces is the presence of adhe- the whole sheet to fold. Contraction of a
sive proteins known as cell-adhesion molecules, which serve various functions: they line of cells at their apices due to changes in
hold cells together in tissues; they enable cells to sense the nature of the surrounding the cells’ internal structure causes a furrow
extracellular matrix; and they serve to guide migratory cells such as the neural crest cells to form in a sheet of epithelium.
of vertebrates, which leave the neural tube to form structures elsewhere in the body.
We can therefore describe and explain developmental processes in terms of how
individual cells and groups of cells behave. Because the final structures generated by
development are themselves composed of cells, explanations and descriptions at the
cellular level can provide an account of how these adult structures are formed.
Because development can be understood at the cellular level, we can pose the
question of how genes control development in a more precise form. We can now ask
how genes are controlling cell behavior. The many possible ways in which a cell can
behave therefore provide the link between gene activity and the morphology of the
adult animal—the final outcome of development. Cell biology provides the means by
which the genotype becomes translated into the phenotype.

1.10 Genes control cell behavior by specifying which proteins are made

What a cell can do is determined very largely by the proteins it makes. The hemoglo-
bin in red blood cells enables them to transport oxygen; the cells lining the gut secrete
specialized digestive enzymes; and skeletal muscle cells are able to contract because
they contain contractile structures composed of the proteins myosin, actin and tropo-
myosin, as well as other muscle-specific proteins needed to make muscle function
correctly. All these are specialized proteins and are not involved in the ‘housekeep-
ing’ activities that are common to all cells and keep them alive and functioning.
Housekeeping activities include the production of energy and the metabolic pathways
involved in the breakdown and synthesis of molecules necessary for the life of the
cell. Although there are qualitative and quantitative variations in housekeeping pro-
teins in different cells, they are not important players in development. In development
we are concerned primarily with those proteins that make cells different from one
another and which are often known as tissue-specific proteins.
Genes control development mainly by specifying which proteins are made in which
cells and when. In this sense the genes are passive participants in development, compared
18 Chapter 1 History and basic concepts

with the proteins they encode. These are the agents that directly determine cell behavior,
which includes determining which genes are expressed. To produce a particular pro-
tein, its gene must be switched on and transcribed (transcription) into messenger RNA
(mRNA). This process is known as gene expression. The protein encoded by the mRNA is
then synthesized in the process of translation. The initiation of transcription, or switch-
ing on of a gene, involves combinations of specialized gene-regulatory proteins binding
to the control region of the gene. The control region is made up of cis-regulatory regions
in the DNA (the cis simply refers to the fact that the regulatory region is on the same DNA
molecule as the gene it controls). These cis-regulatory regions are often modular, and in
many developmental genes the time of gene expression and the tissue in which the gene
is expressed are controlled by different cis-regulatory modules.
Both transcription and translation are subject to several layers of control, and transla-
tion does not automatically follow transcription. Figure 1.19 shows the main stages in
gene expression at which the production of a protein can be controlled. Even after a
gene has been transcribed, for example, the mRNA may be degraded before it can be
Control region
exported from the nucleus. Even if an mRNA reaches the cytoplasm, its translation may
DNA be prevented or delayed. In the eggs of many animals, preformed mRNA is prevented
cis-regulatory from being translated until after fertilization. mRNAs may also be targeted by specific
regions
microRNAs (miRNAs), short non-coding RNAs that can render the mRNA inactive
transcription RNA polymerase and so block translation (see Box 6C). Some microRNAs are known to be involved
factor
in gene regulation in development. Another process that determines which proteins
are produced is RNA processing. In eukaryotic organisms—all organisms other than
promoter region bacteria and archaea—the initial RNA transcripts of most protein-coding genes are
cut and rejoined in a processing step called RNA splicing to give the functional
Coding region
mRNA. In some genes, the transcripts can be cut and spliced in different ways, called
DNA
alternative splicing, to give rise to two or more different mRNAs; in this way, a num-
ber of different proteins with different properties can be produced from a single gene.
1 transcription Even if a gene has been transcribed and the mRNA translated, the protein may still
not be able to function immediately. Many newly synthesized proteins require fur-
RNA ther post-translational modification before they acquire biological activity. One very
common permanent modification that occurs to proteins destined for the cell mem-
2 processing brane or for secretion is the addition of carbohydrate side chains, or glycosylation.
Nucleus
Reversible post-translational modifications, such as phosphorylation, can also signifi-
mRNA cantly alter protein function. Together, alternative RNA splicing and post-translational
modification mean that the number of functionally different proteins that can be
produced is considerably greater than the number of protein-coding genes would indi-
3 transport cate. For many proteins, their subsequent localization in a particular part of the cell,
Cytoplasm
nuclear for example the nucleus, is essential for them to carry out their function.
envelope
Fig. 1.19 Gene expression and protein synthesis. A protein-coding gene comprises a coding
region—a stretch of DNA that contains the instructions for making the protein—and adjacent
DNA sequences that act as a control region. The control region includes the promoter, to which
general transcription factors and the enzyme RNA polymerase bind to start transcription, and
a cis-regulatory region, consisting of one or more modules, at which other transcription factors
ribosome 4 translation bind to switch the gene on or off. This latter control region may be thousands of base pairs
away from the promoter. When the gene is switched on, the DNA sequence is transcribed to
produce RNA (1). The RNA formed by transcription is spliced to remove introns and the non-
protein coding region at the start of the gene (yellow) and processed within the nucleus (2) to produce
mRNA. This is exported from the nucleus to the cytoplasm (3) for translation into protein at the
ribosomes (4). Control of gene expression and protein synthesis occurs mainly at the level of
5 modification transcription, but can also occur at later stages. For example, mRNA may be degraded before it
can be translated, or if it is not translated immediately it may be stored in inactive form in the
cytoplasm for translation at a later stage. mRNAs may also be targeted by specific microRNAs
carbohydrate so that translation is blocked. Some proteins require a further step of post-translational
modification (5) to become biologically active. A very common post-translational modification is
the addition of carbohydrate side-chains (glycosylation), as shown here.
Another random document with
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“Acts of kindness,” replied Emma’s mother, “always produce a
feeling of pleasure. This every one may know. And it is the purest
and truest pleasure we experience in this world. Try and remember
this little incident of the flowers as long as you live, my child; and let
the thought of it remind you that every act of self-denial brings to the
one who makes it a sweet delight.”
 The Timely Aid.
“TAKE care of that wolf, my son,” said Mrs. Maylie to a boy about
twelve years old, who had come from school in a very ill humour with
a playmate, and kept saying harsh things about him, which were but
oral evidences of the unkind feelings he cherished within.
“What wolf, mother?” asked Alfred, looking up with surprise.
“The wolf in your heart. Have you already forgotten what I told you
last evening about the wild beasts within you?”
“But you told us too,” spoke up little Emily, “about the innocent
lambs. There are gentle and good animals in us, as well as fierce
and evil ones.”
“Oh yes. Good affections are the innocent animals of your hearts,
and evil affections the cruel beasts of prey that are lurking there,
ever ready, if you will permit them, to rise up and destroy your good
affections. Take care, my children, how you permit the wild beasts to
rage. In a moment that you know not, they may ravage some sweet
spot.”
“But what did you mean by saying that there was a wolf in brother
Alfred? Tell us the meaning of that, mother.”
“Yes, do, mother,” joined in Alfred, whose ill humour had already
begun to subside. “I want to know what the wolf in my heart means.”
“Do you know anything about the nature of wolves?” asked Mrs.
Maylie.
“They are very cruel, and love to seize and eat up dear little innocent
lambs,” said Emily.
“Yes, my children, their nature is cruel, and they prey upon innocent
creatures. Until now, Alfred, you have always loved to be with your
playmate, William Jarvis.”
Alfred was silent.
“Was it not so, my dear?”
“Yes, ma’am; I used to like him.”
“Frequently you would get from me a fine large apple, or a choice
flower from the garden, to present to him. But the tender and
innocent feelings that prompted you to do this have perished. Some
wolf has rushed in and destroyed them. Is it not so?”
Alfred sat in thoughtful silence.
“Think, my son,” continued Mrs. Maylie, “how innocent, like gentle
lambs, were your feelings until now. When you thought of William, it
was with kindness. When you played by his side, it was with a warm,
even tender regard. But it is not so now. Some beast of prey has
devoured these lambs—these innocent creatures that sported in
your bosom. If the angry, raging wolf has not eaten them up, where
are they? Before you permitted yourself to feel anger against
William, gentle creatures leaped about happily in your breast; but
you feel them no longer—only the wolf is there. Will you let him still
rage, and devour your lambs, or will you drive him out?”
“I will drive him out, mother, if I can. How shall I do it?” Alfred said
earnestly, and with a troubled look.
“By resisting him even unto the death. You have the power. You have
weapons that will prevail. Try to forget the fault of William; try to
excuse him; think of his good qualities; and assure yourself of what I
know to be true—that he never meant to offend you. If the angry wolf
growl in your bosom, thrust bravely at him, as you would, were you,
weapon in hand, defending a sheepfold; and he will and must retire,
or die at your feet. Then innocent lambs will again be seen, and their
sports delight your heart. Then you will feel no more anger towards
your young friend, but love instead.”
“I don’t think I am angry with William, mother,” Alfred said.
“But you were just now.”
“Yes; but the wolf is no longer in my heart,” the boy replied smiling.
“He has been driven out.”
“And innocent creatures can now sport there unharmed. I am glad of
it. Do not again, Alfred, do not any of you, my children, permit
ravenous beasts to prey upon the lambs of your flocks. Fly from
them in as much terror as you would fly from the presence of a wolf,
a tiger, or a lion, were one to meet you in a forest. They are equally
hurtful—one injures the body, the other the soul.”
“Tell us now, mother, about the wolf that had nearly killed uncle
Harper when he was a little boy no bigger than me,” spoke up
Charley, the youngest of Mrs. Maylie’s treasures.
“Oh yes, mother, tell us all about it,” said Alfred.
“I’ve told you that very often,” the mother returned.
“But we want to hear it again. Tell it to us; won’t you, mother?”
“Oh, certainly. Many years ago, when I was a little girl not bigger
than Emily, we lived at the foot of a high mountain, in a wild,
unsettled country. There were but few neighbours, and they were at
great distances from us. At that time bears, wolves, and panthers
were in the region where we lived, and often destroyed the sheep of
the settlers, and otherwise annoyed them. The men used frequently
to go out and hunt them, and kill off these their forest enemies in
great numbers.
“One day, when your uncle Harper was about five years old, our
father took us in his waggon to visit a neighbour about six miles up
among the mountains. This neighbour had a little boy just Harper’s
age, and they were together in the garden and about the house all
the morning. After dinner, they were dressed up nicely, and again
went out to play.
“‘Come,’ said Harper’s companion, ‘let us go and see brother Allen’s
bird-trap. He caught three pheasants yesterday. Maybe we’ll find one
in it to-day.’
“Harper was very willing to go. And so they started right into the
woods; for the forest came up close to the house, and went off quite
out of sight. They had not been gone long before a neighbour, who
lived about a mile off, came over to say that a very large wolf had
been seen a few hours before.
“‘Where is Harper?’ my mother asked quickly, going to the door and
looking out.
“‘I saw him a little while ago, playing about here with Johnny,’ some
one replied.
“‘But where is he now?’ and our mother went out of doors, looking all
around the house and in the garden.
“‘They’ve gone off to my bird-trap, without doubt,’ said Allen, a stout
boy about sixteen years of age. ‘Johnny has been there several
times within a day or two.’
“‘Do run and see,’ urged our mother. Allen took up his gun and
started off quickly towards the place where he had set his bird-trap.
Two or three took other directions; for, now that it was known a wolf
had been seen, all were alarmed at the absence of the children. In
about five minutes after Allen had left the house, we were startled by
the sharp crack of a rifle in the direction he had taken. For the next
five minutes we waited in dreadful suspense; then we were
gladdened by the sight of Allen, bringing home the two children. But
when we heard all that had occurred, we trembled from head to foot.
Allen had gone quickly towards the place where he expected to find
the little truants. When he came in sight of the trap, he saw them on
the ground close to it, and was just going to call out to them to take
care or they would spring it, when the dark body of a large wolf came
quickly in between him and the children. There was not a moment to
be lost; if the cruel beast reached them, destruction would be
inevitable. Quickly presenting his rifle, he took a steady aim and
fired. A fierce howl answered the report: as the smoke arose from
before his eyes, he saw the ‘gaunt gray robber’ of the wilderness
rolling upon the ground. The bullet had sped with unerring certainty.
“How thankful we were,” added Mrs. Maylie, “when, knowing how
great had been the danger, we saw the children safe from all harm!”
“Does uncle Harper remember it?” asked Charley.
“Yes; he says he can just remember something about it; but he was
a very little boy then.”
“That was a real wolf,” remarked Emily; “but the wolves, and tigers,
and lambs you have been telling us about are not real, are they?
Real animals can’t live in us.”
“If there was nothing real about them, could they hurt you, dear?”
“No.”
“But the wolves I spoke about do hurt you. Must they not be real
then?”
“Not real like the big hairy wolf I saw at the show?”
“Oh no; not real like that; not clothed in flesh; but still real, so far as
power to harm you is concerned: and surely that is reality enough.
Don’t you think so?”
“Yes, real that way. But still,” Alfred said, “I can’t understand how a
real wolf can be in me; for a wolf is much bigger than I am.”
“But I don’t mean a flesh and blood wolf, but something in you that
partakes of the wolf’s cruel nature, and, like the wolf, seeks to
destroy all in you that is good, and harmless, and innocent. There
may be in you something that corresponds to the fierce nature of the
wolf, and something that corresponds to the gentle nature of the
lamb. Both of these cannot be active at the same time. If you let the
wolf rule, your gentle lambs, as I before told you, will be destroyed.”
The children now understood their mother better, though they could
not clearly comprehend all that was meant by the wild beasts and
innocent creatures of the human heart.
 The Double Fault.
“WHY, Arthur,” exclaimed Mrs. Mason, on coming into the room
where she had left her two boys playing, and finding one of them
there with a bunch of flowers in his hand; “how came you to pull my
flowers? Haven’t I positively forbidden you to do so?”
“I did not do it, mother. I did not do it. It was John.”
“Where is John?”
“He’s in the yard.”
“Call him in,” said Mrs. Mason.
While Arthur was at the window calling to his brother, Mr. Mason, the
father, came into the room.
“John has been pulling my flowers. Isn’t it too bad that a boy as big
as he is should have so little consideration? They were coming out
into bloom beautifully.”
Just then John entered, with a bunch of flowers also in his hand.
“John, how came you to pull my flowers?” said Mrs. Mason. “You
knew it was wrong.”
“I did not think, when I pulled off a rosebud and two or three
larkspurs,” replied John.
“Two or three larkspurs and a rosebud! Why, your hand is full of
flowers.”
“Oh, but William Jones gave me all but the larkspurs and the
rosebud. Indeed, mother, I didn’t touch any more; and I am sorry I
took them; but I forgot that it was wrong when I did so.”
“But Arthur says you pulled that large bunch in his hand.”
“Arthur knows I didn’t. He knows he pulled them himself, and that I
told him he’d better not do it; but he said he’d as much right to the
flowers as I had.”
Mr. and Mrs. Mason both looked at Arthur in surprise and
displeasure. His countenance showed that he had been guilty of
wrongly accusing his brother.
“Is it true that you did pull the flowers, Arthur?” asked his mother.
But Arthur was silent.
“Speak, sir!” said the father sternly. “Did you pull the flowers?”
“Yes, sir.”
“And then falsely accused your brother of the wrong you had done.
That my boy should be guilty of an evil act like this! I could not have
believed it. It is a wicked thing to tell a lie to hide a fault, simply; but
falsely to accuse another of what we have ourselves done, is still
more wicked. Can it be possible that a son of mine has fallen so
low? It grieves me to the heart.”
Mr. Mason spoke as he felt. He was deeply grieved. Nothing had
occurred for a long time that so hurt him. He loved honesty and truth;
but how opposite to both had been the conduct of his boy!
“Go up to your chamber, and stay there until I see you or send for
you,” he said; and Arthur retired in shame from the presence of his
parents, and the brother he had so meanly attempted to injure. Of
course he felt very unhappy. How could he feel otherwise? The
rebuking words of his father fell like heavy blows upon his heart, and
the pain they occasioned was for a long time severely felt.
What punishment the parents thought it right to inflict upon Arthur we
do not know; but, no doubt, he was punished in some way, as he
deserved. And besides this, he had the still severer punishment
which always follows that meanest fault of which any one can be
guilty—that of accusing another and innocent person of what we
have ourselves done.
Bad as this fault is, it is, alas! too common. But no manly, honest-
minded, truthful boy will be betrayed into it. To the better impulses of
our young readers who have been so wicked as to fall into this sin,
either from sudden impulse or deliberate purpose, we would
earnestly appeal, and beg of them to think more wisely and act more
justly in the future. No cause is ever made better, but always worse,
by a falsehood. Even where detection does not follow, suspicion is
almost always created; for it is impossible for a boy to tell a lie
without betraying it in the face or voice, and causing a doubt to pass
through the minds of his parents, and set them to making inquiry into
the truth or falsehood of what he has stated.
Truth—the open, bold, honest truth—is always the best, always the
wisest, always the safest for every one, in any and all circumstances.
Let no boy deviate from it, even though he have been guilty of a
fault. Better—a thousand times better—is it to own to the wrong, and
keep a clear conscience.
 A Story about a Dog.
“TELL us a story, father, before we go to bed,” said a little boy, who
spoke for two brothers as well as for himself.
“What shall it be about?” asked Mr. Melville, their father.
“Oh, about a dog. I love to hear stories about dogs.”
“Oh yes! let it be about a dog.”
“Yes, papa, let it be about a dog,” ran through the circle of children.
“Wouldn’t you rather hear a story about the innocent lamb; the pure,
snow-white lamb that sports in the green meadows?” said the father.
“Dogs are evil animals.”
“Oh no, father! dogs are not evil animals. You don’t call our Carlo an
evil animal? He’s a good, kind, generous dog. Didn’t he save the life
of Mr. Graham’s little Harry, when he fell into the river? And doesn’t
he love us, and go with us everywhere? And didn’t he jump on Mr.
Parker’s Nero and beat him, when he flew out at us as we were
passing, and was going to bite us? I am sure Carlo is a good dog.
He watches our house at night, and keeps all the robbers away.”
“Carlo is one of the better class of dogs,” said Mr. Melville. “Many of
these animals have generous qualities, and can be taught by man to
perform many good acts; but I hardly think the dog can be called a
good animal, like the noble horse or the useful cow and sheep.
These serve man in a great variety of ways, and do not, even in their
wild state, prey upon other animals, or attack and injure man as the
dog will. The only use of the dog is for a protection against evil; and
he is able to do this from something in him that is cruel and
destructive. But I own that in some dogs there are to be found many
noble and generous qualities; but these they derive from long
association with man, and from being employed by him from one
generation to another in doing useful things. The dogs of St.
Bernard, of which you have so often read, are noble specimens of
this improved race. So are the Newfoundland dogs. But still they are
not good and innocent,—like sheep, for instance, or cows, or like the
gentle dove. Those are truly innocent animals, and correspond in
nature to certain good affections in our minds.”
But the children still thought that Carlo must be a good animal, and
insisted that it was so, and upon having a story about a dog instead
of a lamb.
“Very well,” said Mr. Melville: “I will tell you a story about a dog, and a
very interesting one it is too. I heard it or read about it somewhere
recently, but I cannot now tell where.”
“Tell it, father, do tell it,” urged the children.
Mr. Melville then told the following story:—
“There was a boy,—we will call his name Thomas,—whose father
bought him a fine horse, upon which he used to ride out almost
every day, accompanied by a large Newfoundland dog named
Bruno. One day Thomas had his horse brought out for a ride, and
after he had mounted the animal, he whistled for Bruno, who was
lying on a mat in front of the house. But Bruno only wagged his tail.
He did not even lift his head from between his fore paws, although
his dark bright eyes were fixed upon his young master. ‘Come,
Bruno, come!’ called Thomas. But the dog only wagged his tail more
quickly. ‘You are a lazy fellow, Bruno,’ said Thomas, in a half-chiding,
disappointed tone. ‘I shan’t half enjoy my ride unless you come.’ And
he whistled loud for Bruno, as he gave his horse the rein and trotted
off. Although he looked back and called for Bruno many times, as he
rode away, the dog evinced no disposition to follow him.
“It was near sunset, and the father and mother of Thomas were
sitting in front of their door, enjoying the cool refreshing air. Bruno
still lay upon the mat, and seemed to be sleeping.
“‘I wonder why that dog didn’t go with Thomas?’ said the father,
looking at Bruno.
“‘He’s lazy to-day,’ replied the mother. ‘Thomas called him, and tried
his best to get him off with him, as usual, but Bruno never stirred.’
“On hearing his name, the dog rose up, and came and rubbed
himself against his master, who patted him kindly upon the head.
While standing thus by his master’s side, Bruno all at once pricked
up his ears and rose, and seemed all attention. Almost at the same
instant the father of Thomas heard the distant clattering of a horse’s
hoofs, which drew nearer every moment. He arose quickly; as he did
so, Bruno gave a short, uneasy bark, and went a few steps towards
the road, holding his head very high, and looking first in one direction
and then in another. This suspense did not continue long. In less
than a minute from the time the first distant sound was heard, they
saw the horse of Thomas come dashing down the road at a fearful
speed, with his little rider clinging to his neck. The house stood
nearly a hundred yards from the road, and the horse approaching at
such a rapid rate, that, although the father sprang forward to catch
him, if possible, at the moment of passing, yet he was instantly
conscious that before he could possibly reach the road the
frightened animal would be beyond his reach. Just as his mind felt
this painful certainty, Bruno went past him like an arrow, cleared the
fence at a bound, and at the moment the horse was passing the gate
caught him by the bridle. To this he held on, checking the animal’s
speed so much that his master found it easy to come up with and
stop him.”
“Oh, what a noble dog!” cried the children. “How Thomas must have
loved him!”
“But how,” said one, “did Bruno know that the horse was going to run
away?”
“He did not know it,” said Mr. Melville.
“Then why didn’t he go with Thomas? He must have known it,
father.”
“Oh no; that doesn’t follow, my son, at all. But the Lord, in his
omnipotence and providence, knew what would take place, and
provided just the means that were needed to save Thomas from
being killed.”
“Then he made Bruno stay at home that he might be ready to save
his young master’s life?” said one of the children.
“The Lord’s protecting Spirit is everywhere,” replied Mr. Melville, “and
governs in all circumstances by which we are preserved from harm.
Without doubt, it was an influence from Heaven that produced in the
dog an indisposition to go with Thomas.”
“How good the Lord is!” said the child who had last spoken, in a
thoughtful tone.
“Yes, my dear,” returned Mr. Melville; “the Lord is good to all, and
kind even to the unthankful. He maketh his sun to shine upon the evil
and the good, and sendeth his rain upon the just and the unjust.”
 The Discontented Shepherd.
IN a quiet valley there once dwelt a shepherd, who led a peaceful,
happy life. He had large flocks, from whose fleecy backs the wool
was regularly shorn, and sold to the merchants; and the merchants
paid him money, with which he bought all things needful for health
and bodily comfort.
One day the shepherd drove his flocks to the sea-side, and as he
looked abroad upon the great expanse of water, and saw the ships
moving over its surface, he felt, for the first time, discontented with
his lot. A desire to see the world took possession of his mind.
“I will no longer shut myself up in this narrow valley,” he said. “I will
become a merchant. I will pass over the wide sea, and go among the
people of many lands.”
So the shepherd sold his flocks, and with the money bought
merchandise, which he placed in a ship, and started for a distant
country. During the first day after leaving the land, he could do little
else but admire the wonderful ocean upon whose surface he was
sailing, and think how happy he was at having escaped the dull life
of a shepherd in an unknown vale. But on the second day after
leaving the land, the motion of the ship made him very sick. He could
no longer enjoy the great expanse of ocean and sky spread out
above and around him, but had to remain in the cabin, unable even
to lift his head from his pillow. As he lay sick in the dark, narrow
cabin, filled with polluted air, he thought of the green shady places,
cool refreshing streams, and pure air of his native valley, and, for the
first time, he repented of what he had done.
It was more than a week before the shepherd could go upon deck,
and feel pleasure in the sky and ocean as he had done at first.
At last the vessel arrived at its destined place: the shepherd landed
his goods and offered them for sale. He soon found a merchant
willing to buy them. The price was agreed upon, the merchandise
delivered, and the money demanded. But it happened, as it almost
always happens when men get dissatisfied with the business or
calling with which they are perfectly familiar, and enter into one they
know nothing about, the shepherd fell into dishonest hands. The
merchant refused to pay him his money.
In order to get this wrong redressed, the shepherd called upon a
magistrate of the country, who promised to see that justice was done
to him. But the merchant knew the magistrate to be as unfitted for his
calling as he was for his, and so he offered him a bribe, which the
wicked magistrate accepted. In vain did the shepherd seek for justice
at his hands; no justice could he get. His importunities at last
became so great, that the magistrate threatened to have him put into
prison if he troubled him any more.
In his own peaceful valley there was no wrong and oppression like
this. The merchants who came for his fleece were good and true
men, and paid the prices agreed upon. The ignorant shepherd had
not dreamed that there were such wicked men in the world as this
merchant and this magistrate, into whose hands he had fallen.
In a strange land, among strange people, thousands of miles away
from his home, and all his money and property gone, the poor
shepherd was about giving up in despair. But he bethought him that
he would go to the king of the country, and ask justice at his hands.
The king, when he heard the shepherd’s story, was very angry at the
wrong that had been done in his kingdom. He sent immediately, and
had the magistrate and the merchant brought before him and
confronted with their accuser. On seeing the shepherd, their hearts
became filled with alarm, and their faces betrayed what was in their
hearts. When accused they could answer nothing. So the king
caused the merchant to pay the shepherd for his goods; and
besides, imposed upon him a heavy fine. From the magistrate he
took away his office, and had him cast into prison.
As soon as the shepherd had received his money, he returned in the
first ship that sailed for his native country, and buying more flocks,
was ever after contented to follow them in the peaceful valley where
no wrong, oppression, or dishonesty had yet come.
 The Shilling.
GEORGE HANSON’S uncle had given him a shilling; and George,
like most boys, felt very anxious to spend it. But, among his many
wants, he found it a hard matter to decide upon which to gratify. If it
had been a half-crown instead of a shilling, the difficulty would have
been lessened, for then George could have supplied at least half a
dozen wants. But it was only a shilling.
He stood at the window, looking out upon the passengers who were
going quickly by, the frosty air of December giving lightness to many
a step that, in a milder day, would have been less hurriedly taken.
While standing here, his mind half made up to gratify his love of
cakes and oranges by a whole shilling’s worth, a man went by with
some pretty little glass toys in a box, which he held up to the window,
and asked if he did not want to buy some.
George beckoned to the man to stop, and then ran to the front door.
The man was a glass-blower, and had manufactured some
handsome birds, and sheep, and deer, from white glass, which
looked, certainly, curious and beautiful.
“How much is this?” asked George, pointing to a bird of paradise.
“Eighteen-pence.”
“But I’ve only got a shilling,” returned George.
“Well, here’s a robin redbreast for a shilling; and here’s a deer, and a
sheep. All these on this side are a shilling.”
But George liked the bird of paradise best of all, and couldn’t think of
taking anything else.
While the man stood trying to persuade him to buy one of the birds
that were sold for a shilling, George looked up and saw going by a
poor old man, who was bent with age. He led a little girl by the hand,
who appeared to shrink in the cold. The old man looked sick and
feeble, and very poor.
“They shall have my shilling!” exclaimed George, speaking from a
sudden impulse; and he stepped forward, and placing the coin in the
old man’s hand, said, as he did so,—
“I was just going to spend this for a little glass toy that would be
broken in a day. But I want it put to a better use. Take it, and buy
something for your little girl.”
The poor old man stopped, and said, with a look of surprise and
pleasure as he received the coin,—
“Thank you, my young master! This will give my little Alice a nice
bowl of bread and milk for her supper and breakfast. She will think of
you with a grateful heart while she eats them.”
“Well done, my good boy!” said the glass-blower, as the old man
went on his way. “That poor little girl’s bread and milk will taste sweet
to her to-night. And as a reward for your generous self-denial, here is
the bird of paradise that has pleased you so much: take it.”
But George drew back, and said he hardly thought that would be
right.
“Why not, I wonder?” returned the man. “Am I to be outdone in
generosity by a boy? Take it, and whenever you look upon it let it
teach you this lesson—that it is more blessed to give than to receive;
for I am sure the thought of the good done to the old man and the
little girl will be more pleasant to you than the thought of possessing
this pretty toy.”
And so it was. The toy pleased for a short time only, but the thought
of the little girl who had been made happy by his shilling never
passed through his mind without giving him pleasure.
 The Wounded Bird.
“FATHER,” said Henry Thompson, a boy just eleven years old,
“won’t you buy me a gun?”
“A gun! Oh no; I can’t buy you a gun,” Mr. Thompson replied in a
decided voice.
Henry turned away disappointed, and went out of his father’s
warehouse, into which he had come specially to ask for a gun. He
was not pleased at the refusal he had met with, and felt much
inclined, as are too many children, to indulge hard thoughts against
his kind father for not gratifying his wish. As he walked along, he met
Alfred Lyon, a lad about his own age, whose father had given him a
gun, and who then had it on his shoulder.
“Come, Henry,” said Alfred, “I’m going out a-shooting. Won’t you go
with me?”
Henry at once said “Yes.” It was a holiday, and his mother had told
him that he might go out and spend the morning as he liked, only
that he must not go into danger, nor harm anything. So he did not
hesitate to go with Alfred. He had seen the little boy the day before,
and then learned that he had received from his father the present of
a gun, and this was what had made him desire to have one also.
The two little boys then took their way to the woods. It was a bright
day in early summer. The trees were all covered with tender foliage,
the fields bright and green, and the singing birds made the air thrill
with delicious melody. To mar this scene of innocence, beauty, and
peace, came these two thoughtless boys. They saw the woods
mantled in their dark, rich drapery, that moved gracefully in the light