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 Developmental
Neurobiology

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 To E.A.B.

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 Developmental
Neurobiology

Lynne M. Bianchi

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 Vice President: Denise Schanck
Senior Development Editor: Monica Toledo
Senior Digital Project Editor: Natasha Wolfe
Senior Production Editor: Georgina Lucas
Text Editor: Kathleen Vickers
Illustrator: Nigel Orme
Text and Cover Design: Matthew McClements, Blink Studio, Ltd.
Copyeditor: John Murdzek
Proofreader: Susan Wood
Indexer: Simon Yapp at Indexing Specialists
Permissions Coordinator: Sheri Gilbert

Lynne M. Bianchi is Professor of Neuroscience and Pre-Medical Program Director

at Oberlin College. She received her Ph.D. in Anatomy and Cell Biology from the
University at Buffalo School of Medicine and Biomedical Sciences. She joined
Oberlin College, a liberal arts college with one of the first and longest-running
undergraduate neuroscience programs in the United States, in 1998. Her research
interests focus on neuron–target interactions and the role of nerve growth factors in
the developing auditory system.

Cover image shows a light micrograph of a mouse embryo, approximately 10.5

days post-fertilisation. The specimen was stained with a fluorescent marker that
highlights the presence of precursor cells to nerve tissue then chemically treated to
make it optically transparent. Image courtesy of RPS/Jim Swoger/BNPS.

© 2018 by Garland Science, Taylor & Francis Group, LLC

This book contains information obtained from authentic and highly regarded
sources. Every effort has been made to trace copyright holders and to obtain their
permission for the use of copyright material. Reprinted material is quoted with
permission, and sources are indicated. A wide variety of references are listed.
Reasonable efforts have been made to publish reliable data and information, but
the author and the publisher cannot assume responsibility for the validity of all
materials or for the consequences of their use.

All rights reserved. No part of this publication may be reproduced, stored in a

retrieval system or transmitted in any form or by any means—graphic, electronic, or
mechanical, including photocopying, recording, taping, or information storage and
retrieval systems—without permission of the copyright holder.

ISBN 9780815344827

Library of Congress Cataloging-in-Publication Data

Names: Bianchi, Lynne, author.
Title: Developmental neurobiology / Lynne M. Bianchi.
Description: New York, NY: Garland Science, Taylor & Francis Group, LLC,
2018.
Identifiers: LCCN 2017034851 | ISBN 9780815344827
Subjects: LCSH: Developmental neurobiology.
Classification: LCC QP363.5 .B563 2018 | DDC 612.6/4018--dc23
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Published by Garland Science, Taylor & Francis Group, LLC, an informa business,
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 Preface

No one goes into science because they love to memorize facts; they go
into science because they love the process of discovery and problem
solving. The field of developmental neurobiology is filled with numerous
examples of creativity and insight that highlight the exciting process of
scientific discovery. As an instructor, it is a pleasure to be able to discuss
the motivation and experimental methods behind such studies. Whether
studies were done 125 years ago or 5 weeks ago, there is always something
intriguing to discuss—from the very first stages of neural induction in early
embryogenesis to the refinement of synaptic connections during postnatal
development.
One goal of this book is to provide historical background on topics to
help students gain a perspective on how ideas have evolved over time. As
instructors, it is sometimes tempting to focus only on the latest material.
However, somewhere along the way, I noticed that students did not always
fully grasp why a new discovery was so remarkable and realized that many
had not yet heard about the earlier work that suggested another outcome,
and were therefore unable to appreciate the excitement generated by
the newer findings. Thus, I have found providing such background to
be beneficial to students. As one reviews earlier studies, one comes to
appreciate how the experiments were done, what information influenced
how certain hypotheses were formed, and how unexpected findings have
shifted the focus of research efforts over time. While students will have to
memorize some detailed facts for a course, I hope that reading how the
facts were generated will lead to an appreciation of why those details are
so important for understanding how the nervous system develops.
A challenge often encountered by instructors teaching developmental
neurobiology is that, at many institutions, the course is an elective course
for undergraduate or first-year graduate students. Therefore, it is not
unusual for students to enter the class with different academic backgrounds.
Instructors need to balance providing enough information so students
without much advanced biology and neuroscience can keep up, without
also losing the students who have had the more advanced coursework.
In writing this book, I kept those differing levels of student experience
in mind. My goal was to provide sufficient background information in
each chapter so that all students will be able to follow the more detailed
and specific concepts as they are covered. This organization also gives
advanced students a review of material and allows instructors to skim over
background information when appropriate for a given class.
The opportunity to teach developmental neurobiology is always
a welcome experience because there are so many topics to discuss that
an instructor never runs out of material. However, when organizing the
course or planning a single lecture, an instructor is required to select
specific content to cover in the available time, knowing that other material
must be set aside. It is never an easy task. I’ve chosen to particularly
highlight experiments that had a major impact on the field or changed how
investigators approached a particular question. These examples are not the
only experiments that have shaped the field of developmental neurobiology,
but they are provided to illustrate the types of work that have been done.
To start, Chapter 1 provides an overview of concepts that will be
important for material covered in subsequent chapters. The chapter begins

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 vi PREFACe

with a review of basic cell biology and anatomy of major structures in

the nervous system, and then describes the embryonic development and
staging criteria used for common vertebrate and invertebrate animal
models, as well as for humans. Images from atlases of the different species
are provided so that students have a reference for understanding studies
discussed in later chapters. Chapter 1 concludes with a discussion of
experimental methods commonly used by investigators and frequently
discussed in subsequent chapters.
Chapters 2–10 focus on selected stages of neural development. As
with any subtopic in developmental neurobiology, it is difficult to provide
a comprehensive overview of every neural population and so examples
that highlight major developmental mechanisms were selected, though
there are certainly other equally important examples that could have been
used. Chapter 2 describes the process of neural induction beginning with
the discovery of the organizer through current discoveries identifying
subtle differences in induction mechanism across different vertebrate
and invertebrate animal models. Chapters 3 and 4 cover segmentation
and patterning along the anterior–posterior and dorsal–ventral axes,
respectively. The topics have been separated into two chapters because the
volume of information on each has advanced to the point where covering
all the material in a single chapter can become overwhelming to both the
instructor and the student. Chapter 5 discusses how cells migrate to their
proper location in the developing central and peripheral nervous systems,
while Chapter 6 covers the cellular determination of selected neural and
sensory cells. Chapter 7 explains mechanisms that guide axons to their
proper target cells, and Chapter 8 discusses how target cells influence
neuronal survival and the various signaling pathways that intersect to
mediate neuronal survival and death. Chapters 9 and 10 cover synapse
formation and reorganization at the neuromuscular junction and central
nervous system, respectively. Both chapters discuss how synapses are
formed at each region and how synapses are later eliminated or reorganized
in early postnatal development. Rather than separate chapters based on
synapse formation and synapse elimination/reorganization, the chapters
are separated by the type of synapse to provide a sense of what happens
at particular synapses over time in a given region of the nervous system.
For many experimental examples discussed in the book, the names of
lead investigators are indicated so that students can refer to the literature and
read the original papers. In several instances an investigator’s name is listed
with the very broad label “and colleagues.” In some cases, the colleagues
were a few other individuals working on the project in a single lab. In many
cases, however, “and colleagues” represents the contributions of several, if
not dozens, of researchers over the course of many years or, in some cases,
decades. While not specifically named in the text, the contributions of the
colleagues cannot be underestimated. The research of current investigators
is also highlighted in boxes to provide examples of how careers in develop-
mental neurobiology begin and evolve. Many of these boxes were written
by recent graduates of Oberlin College who are now pursuing careers in
scientific research or medicine, and illustrate some of the many career paths
available.
Writing a book takes a remarkably long time, particularly because it
has to be done in the moments that can be found outside of time dedicated
to other academic responsibilities. I greatly appreciate the support and
encouragement of my colleagues and friends throughout this process. I
also thank the many colleagues who provided background information
on various studies described in the text. The staff at the Oberlin College
Archives and Science Library were extremely helpful in providing the many
materials needed for preparing this book, and they were very patient when

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 PREFACE vii

I kept books out for extended periods of time. It is the students at Oberlin
College who motivated me to begin and continue this project, and I am
thankful for the many great conversations I have had with so many of them
over the years.
I thank Janet Foltin for initially contacting me and assuring me that
writing such a book was possible. The staff at Garland Science has made
writing a textbook a very smooth process. I greatly appreciate the careful
and thoughtful editing of Kathleen Vickers during the early stages of the
project. I am especially grateful to Monica Toledo for her commitment
to this book. She kept me on track, reviewed the text and illustrations to
make sure everything fit together, and recruited reviewers and compiled
their reviews for me. She also taught me a lot about the publishing process
along the way. I also thank Nigel Orme for his hard work and ability to turn
my sketches into clear illustrations that convey the ideas I was trying to get
across and Matthew McClements for his cover and text designs. I greatly
appreciate the time and effort of the many reviewers who read early drafts
of the chapters. The thoughtful and detailed reviews they provided were
extremely helpful and have certainly enhanced the content of the book.
And, finally, I want to acknowledge and thank my husband and children
for all of their support, good humor, and incredible patience during the
processes of completing this book. I hope they enjoy reading it as much as
I have enjoyed writing it.

ACKNOWLEDGMENTS
The author and publisher of Developmental Neurobiology gratefully
acknowledge the contributions of the following scientists and instructors
for their advice and critique in the development of this book: Coleen Atkins
(University of Miami); Karen Atkinson-Leadbeater (Mount Royal University);
Eric Birgbauer (Winthrop University); Jennifer Bonner (Skidmore College);
Martha Bosma (University of Washington); Sara Marie Clark (Tulane
University); Elizabeth Debski (University of Kentucky); Mirella Dottori
(University of Melbourne); Mark Emerson (The City College of New York);
Erika Fanselow (University of Pittsburgh); Deni S. Galileo (University
of Delaware); Suzanna Lesko Gribble (University of Pittsburgh); Jenny
Gunnersen (University of Melbourne); Elizabeth Hogan (Canisius College);
Alexander Jaworski (Brown University); John Chua Jia En (National University
of Singapore); Raj Ladher (National Centre for Biological Sciences); Stephen
D. Meriney (University of Pittsburgh); Mary Wines-Samuelson (University of
Rochester); and Richard E. Zigmond (Case Western Reserve University).

RESOURCES FOR INSTRUCTORS

The figures from Developmental Neurobiology are available in two
convenient formats: PowerPoint® and JPEG, which have been optimized
for display. Please email [email protected] to access the resources.

9780815344827_FM.indd 7 13/10/17 2:58 pm

 Contents

Chapter 1 New tissue culture methods and cell-specific

markers advanced the search for neural
An Introduction to the Field of inducers 36
Developmental Neurobiology 1
NEURAL INDUCTION: THE NEXT PHASE
CELLULAR STRUCTURES AND
OF DISCOVERIES 37
ANATOMICAL REGIONS OF THE
The search for mesoderm inducers revealed that
NERVOUS SYSTEM 4 neural induction might involve removal of animal
The central and peripheral nervous systems are cap-derived signals 37
comprised of neurons and glia 4
Mutation of the activin receptor prevents the
The nervous system is organized around three axes 7 formation of ectoderm and mesoderm but results
in the formation of neural tissue 38
ORIGINS OF CNS AND PNS REGIONS 8
Modern molecular methods led to the identification
The vertebrate neural tube is the origin of many of three novel neural inducers 39
neural structures 9
Future vertebrate CNS regions are identified NOGGIN, FOLLISTATIN, AND CHORDIN
at early stages of neural development 10 PREVENT EPIDERMAL INDUCTION 42
Timing of developmental events in various Studies of epidermal induction revealed the
vertebrates 11 mechanism for neural induction 42
Anatomical regions and the timing of The discovery of neural inducers in the fruit fly
developmental events are mapped in invertebrate Drosophila contributed to a new model for
nervous systems 15 epidermal and neural induction 42
The Drosophila CNS and PNS arise from distinct BMP signaling pathways are regulated
areas of ectoderm 15 by SMADs 46
Cell lineages can be mapped in C. elegans 18 Additional signaling pathways may influence
neural induction in some contexts 46
GENE REGULATION IN THE DEVELOPING Species differences may determine which
NERVOUS SYSTEM 20 additional pathways are needed for neural
Experimental techniques are used to label genes induction 47
and proteins in the developingnervous system 22
Altering development as a way to understand normal Summary 48
processes 22
Further Reading 49
Summary 26
Further Reading 26
Chapter 3
Chapter 2 Segmentation of the
Neural Induction 29 Anterior–Posterior Axis 51
THE ESTABLISHMENT OF NEURAL NEURAL TUBE FORMATION 52
Early segmentation in the neural tube helps
TISSUE DURING EMBRYOGENESIS 29
establish subsequent neural anatomical
Gastrulation creates new cell and tissue organization 54
interactions that influence neural induction 30
Temporal–spatial changes in the signals required
EARLY DISCOVERIES IN THE STUDY OF to induce head and tail structures 56
NEURAL INDUCTION 33 Activating, transforming, and inhibitory signals
Amphibian models were used in early interact to pattern the A/P axis 56
neuroembryology research and remain
popular today 34 SPECIFICATION OF FOREBRAIN REGIONS 57
Signals from extraembryonic tissues pattern
A region of the dorsal blastopore lip organizes the
forebrain areas 57
amphibian body axis and induces the formation
of neural tissue 34 Forebrain segments are characterized by different
patterns of gene expression 58
The search for the organizer’s neural inducer
took decades of research 35 Signals prevent Wnt activity in forebrain regions 58

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 CONTENTS ix

REGIONALIZATION OF THE MESENCEPHALON Sonic hedgehog (Shh) is necessary for floor plate
AND METENCEPHALON REGIONS 60 and motor neuron induction 86
Intrinsic signals pattern the midbrain–anterior Shh concentration differences regulate induction
hindbrain 61 of ventral neuron subtypes 89
Multiple signals interact to pattern structures Genes are activated or repressed by the Shh
anterior and posterior to the isthmus 62 gradient 90
FGF is required for development of the cerebellum 63 Shh binds to and regulates patched receptor
FGF isoforms and intracellular signaling pathways expression 91
influence cerebellar and midbrain development 63 RA and FGF signals are also used in ventral
FGF and Wnt interact to pattern the A/P axis 64 patterning 95

RHOMBOMERES: SEGMENTS OF THE DORSAL PATTERNING IN THE POSTERIOR

HINDBRAIN 65 NEURAL TUBE 95
Cells usually do not migrate between adjacent TGFβ-related molecules help pattern the dorsal
rhombomeres 65 neural tube 96
Some of the signals responsible for establishing Roof plate signals pattern a subset of dorsal
and maintaining hindbrain segments have been interneurons 97
identified 67 BMP-related signals pattern class A interneurons 97
BMP-like signaling pathways are regulated by
GENES THAT REGULATE HINDBRAIN SMADS 99
SEGMENTATION 68 Wnt signaling through the β-catenin pathway
The body plan of Drosophila is a good model influences development in the dorsal neural tube 100
for studying the roles specific genes play in
Gradients of BMP and Shh antagonize each other
segmentation 68
to form D/V regions of the neural tube 102
The homeotic genes that are active in
establishing segment identity are conserved D/V PATTERNING IN THE ANTERIOR
across species 69 NEURAL TUBE 104
A unique set of expressed Hox genes defines the Roof plate signals pattern the anterior D/V axis
patterning and cell development in each by interacting with the Shh signaling pathway 105
rhombomere 71 Zic mediates D/V axis specification by integrating
Retinoic acid regulates Hox gene expression 73 dorsal and ventral signaling pathways 106
The RA-degrading enzyme Cyp26 helps regulate The location of cells along the A/P axis influences
Hox gene activity in the hindbrain 75 their response to ventral Shh signals 107
RA and FGF signaling interactions differentially Analysis of birth defects reveals roles that D/V
pattern posterior rhombomeres and spinal cord 76 patterning molecules play in normal
Cdx transcription factors are needed to regulate development 108
Hox gene expression in the spinal cord 76
Summary 109
Summary 78
Further Reading 109
Further Reading 79

Chapter 4 Chapter 5
Patterning along the Proliferation and Migration
Dorsal–Ventral Axis 81 of Neurons 111
ANATOMICAL LANDMARKS AND NEUROGENESIS AND GLIOGENESIS 111
SIGNALING CENTERS IN THE POSTERIOR Scientists debated whether neurons and glia
arise from two separate cell populations 112
VERTEBRATE NEURAL TUBE 82
Precursor cell nuclei travel between the apical
The sulcus limitans is an anatomical landmark
and basal surfaces 113
that separates sensory and motor regions 83
Interkinetic movements are linked to stages of
The roof plate and floor plate influence gene
the cell cycle 114
expression patterns to delineate cell groupings
in the dorsal and ventral neural tube 83 The plane of cell division and patterns of protein
distribution determine whether a cell proliferates
VENTRAL SIGNALS AND MOTOR NEURON or migrates 115
PATTERNING IN THE POSTERIOR NEURAL Distinct proteins are concentrated at the apical
TUBE 85 and basal poles of progenitor cells 116
The notochord is required to specify ventral The rate of proliferation and the length of the
structures 85 cell cycle change over time 118

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 x CONTENTS

CELLULAR MIGRATION IN THE CENTRAL Cells of the Drosophila PNS arise along epidermal
NERVOUS SYSTEM 122 regions and develop in response to differing
In the neocortex, newly generated neurons levels of Notch signaling activity 151
form transient layers 123 Ganglion mother cells give rise to Drosophila
Most neurons travel along radial glial cells to CNS neurons 153
reach the cortical plate 124 Apical and basal polarity proteins are differentially
Cells in the cortical plate are layered in an segregated in GMCs 153
inside-out pattern 126 Cell location and the temporal expression
Changes in cortical migration patterns lead of transcription factors influence cellular
to clinical syndromes in humans 127 determination 154
The Reeler mutation displays an inverted MECHANISMS UNDERLYING FATE
cell migration pattern 128 DETERMINATION IN VERTEBRATE
Cajal–Retzius cells release the protein Reelin, CNS NEURONS 155
a stop signal for migrating neurons 128 Changes in transcription factor expression
Cortical interneurons reach target areas by mediate the progressive development of
tangential migration 130 cerebellar granule cells 156
Cell migration patterns in the cerebellum reflect Temporal cues help mediate the fate of cerebral
its distinctive organization 131 cortical neurons 157
Cerebellar neurons arise from two zones Epigenetic factors influence determination and
of proliferation 132 differentiation in vertebrate neurons 159
Granule cell migration from external to internal
layers of the cerebellar cortex is facilitated by DETERMINATION AND DIFFERENTIATION
astrotactin and neuregulin 134 OF NEURAL-CREST-DERIVED NEURONS 161
Mutant mice provide clues to the process of Environmental cues influence the fate of
neuronal migration in the cerebellum 136 parasympathetic and sympathetic neurons 161
Sympathetic neurons can change neurotransmitter
MIGRATION IN THE PERIPHERAL production later in development 163
NERVOUS SYSTEM: EXAMPLES FROM
NEURAL CREST CELLS 136 DETERMINATION OF MYELINATING GLIA
Neural crest cells emerge from the neural plate IN THE PERIPHERAL AND CENTRAL
border 137 NERVOUS SYSTEM 164
Neural crest cells from different axial levels Neuregulin influences determination of
contribute to specific cell populations 138 myelinating Schwann cells in the PNS 166
Cranial neural crest forms structures in the head 139 Precursor cells in the optic nerve are used to
study oligodendrocyte development 167
Multiple mechanisms are used to direct neural
crest migration 140 Internal clocks establish when oligodendrocytes
will start to form 168
Trunk neural crest cells are directed by permissive
and inhibitory cues 141 DEVELOPMENT OF SPECIALIZED
Melanocytes take a different migratory route SENSORY CELLS 170
than other neural crest cells 143 Cell–cell contact regulates cell fate in the
compound eye of Drosophila 170
Summary 144
Cell–cell contacts and gene expression patterns
Further Reading 144 establish R1–R7 photoreceptor cell types 173
Cells of the vertebrate inner ear arise from the
otic vesicle 175
Chapter 6
Notch signaling specifies hair cells in the organ
Cell Determination and Early of Corti 176
Differentiation 147 Cells of the vertebrate retina are derived from
LATERAL INHIBITION AND NOTCH the optic cup 178
RECEPTOR SIGNALING 148 The vertebrate retina cells are generated in a
Lateral inhibition designates future neurons in specific order and are organized in a precise
Drosophila neurogenic regions 148 pattern 180
Lateral inhibition designates stripes of neural Temporal identity factors play a role in vertebrate
precursors in the vertebrate spinal cord 150 retinal development 181

CELLULAR DETERMINATION IN THE Summary 181

INVERTEBRATE NERVOUS SYSTEM 151 Further Reading 182

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 CONTENTS xi

Chapter 7 Amphibian retinal ganglion cell axons regenerate

to reestablish neural connections 212
Neurite Outgrowth, Axonal
Retinotectal maps are found in normal and
Path-finding, and Initial experimental conditions 214
Target Selection 185 Some experimental evidence contradicts the
GROWTH CONE MOTILITY AND chemoaffinity hypothesis 215
PATHFINDING 185 A “stripe assay” reveals growth preferences
Early neurobiologists identify the growth cone for temporal retinal axons 215
as the motile end of a nerve fiber 186 Retinotectal chemoaffinity cues are finally
In vitro and in vivo experiments confirm neurite identified in the 1990s 218
outgrowth from neuronal cell bodies 186 Eph/ephrin signaling proves to be more complex
Substrate binding influences cytoskeletal than originally thought 220
structures to promote growth cone motility 187 Axonal self-avoidance as a mechanism for
Actin-binding proteins regulate actin chemoaffinity 222
polymerization and depolymerization 189
Summary 223
Rho family GTPases influence cytoskeletal
dynamics 190 Further Reading 224

GROWTH CONE SUBSTRATE

PREFERENCES IN VITRO AND IN VIVO 191 Chapter 8
In vitro studies confirm that growth cones actively Neuronal Survival and
select a favorable substrate for extension 191
Programmed Cell Death 227
Extracellular matrix molecules and growth cone
receptors interact to direct neurite extension 192 GROWTH FACTORS REGULATE
Roles of pioneer axons and axonal fasciculation NEURONAL SURVIVAL 227
in target selection 193 The death of nerve cells was not initially
Research in invertebrate models leads to the recognized as a normal developmental event 228
labeled pathway hypothesis 196 Studies reveal that target tissue size affects the
Fasciclins are expressed on axonal surfaces 197 number of neurons that survive 228
Vertebrate motor neurons rely on local Some tumor tissues mimic the effect of extra
guidance cues 197 limb buds on nerve fiber growth 229
Several molecules help direct motor axons In vitro studies led to a bioassay method
to muscles 200 to study nerve growth factors 231
The factor released by sarcoma 180 is found
INTERMEDIATE, MIDLINE TARGETS FOR to be a protein 231
SPINAL COMMISSURAL AXONS 202 Nerve growth factor is identified in salivary glands 233
In vertebrate embryos, the axons of commissural Recognition that not all neuronal populations
interneurons are attracted to the floor plate 203 respond to NGF leads to the discovery of
Laminin-like midline guidance cues are found in brain-derived neurotrophic factor 234
invertebrate and vertebrate animal models 204 Discoveries of other NGF-related growth factors
Homologous receptors mediate midline attractive rapidly followed 236
and repulsive guidance cues 205
NGF SIGNALING MECHANISMS AND
Slit proteins provide additional guidance cues
to axons at the midline 206 NEUROTROPHIN RECEPTORS 237
NGF undergoes retrograde transport from
Slit proteins repel commissural axons away from
the nerve terminal to the cell body 237
the midline by activating Robo receptors 207
NGF receptors are first identified in the PC12
Robo signaling is regulated by additional proteins
cell line 238
expressed on commissural axons 208
Activation of Trk receptors stimulates multiple
Shh phosphorylates zip code binding proteins
intracellular signaling pathways 240
to increase local translation of actin and direct
growth of vertebrate commissural axons at the Interaction of full-length Trk receptors with
midline 209 truncated Trk receptors or p75NTR further
influences cell survival 243
THE RETINOTECTAL SYSTEM AND Other growth factors also regulate neuronal
THE CHEMOAFFINITY HYPOTHESIS 211 survival and outgrowth 244
Early scientists focus on studies of physical cues Ciliary neurotrophic factor is isolated based
and neural activity in regulating axon-target on an assay for developing ciliary ganglion
recognition 211 neurons 245

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 xii CONTENTS

The CNTF receptor requires multiple components needed for presynaptic development and
to function 246 alignment with postjunctional folds 280
Growth factors unrelated to CNTF promote
survival of developing CG and motor neurons 247 MODELS OF SYNAPTIC ELIMINATION
IN THE NMJ 282
PROGRAMMED CELL DEATH DURING The relative levels of neuromuscular activity
NEURAL DEVELOPMENT 248 determine which terminal branches remain
Studies reveal cell death is an active process at the endplate 283
dependent on protein synthesis 250 BDNF and pro-BDNF are candidates for the
Cell death genes are identified in C. elegans 251 protective and punishment signals 283
Homologs of the C. elegans ced and egl genes Perisynaptic Schwann cells influence the
contribute to the mammalian apoptotic pathway 252 stability of synaptic connections 285
p75NTR and precursor forms of neurotrophins Summary 285
help mediate neuronal death during
development 254 Further Reading 287
Summary 255
Further Reading 256 Chapter 10
Synaptic Formation and
Reorganization Part II: Synapses
Chapter 9 in the Central Nervous System 289
Synaptic Formation and Reorganization
EXCITATORY AND INHIBITORY NEURONS
Part I: The Neuromuscular Junction 259
IN THE CENTRAL NERVOUS SYSTEM 290
CHEMICAL SYNAPSE DEVELOPMENT Many presynaptic and postsynaptic elements are
IN THE PERIPHERAL AND CENTRAL similar in excitatory and inhibitory synapses 292
NERVOUS SYSTEMS 260 The postsynaptic density is an organelle found
Reciprocal signaling by presynaptic and in excitatory, but not inhibitory, neurons 293
postsynaptic cells results in the development Cell adhesion molecules mediate the initial
of unique synaptic elements 261 stabilization of synaptic contacts 294
THE VERTEBRATE NEUROMUSCULAR Neurexins and neuroligins also induce formation
of synaptic elements and stabilize synaptic
JUNCTION AS A MODEL FOR SYNAPSE contacts 295
FORMATION 262
Reciprocal signals regulate pre- and postsynaptic
At the NMJ, the presynaptic motor axon releases development 296
acetylcholine to depolarize the postsynaptic
muscle cell 263 Dendritic spines are highly motile and actively
seek presynaptic partners 297
The distribution of AChRs has been mapped in
developing muscle fibers 264 BDNF influences dendritic spine motility and
synaptogenesis 298
The density of innervation to muscle fibers
changes during vertebrate development 266 Eph/ephrin bidirectional signaling mediates
presynaptic development 299
The synaptic basal lamina is a site of NMJ
organizing signals 267 Eph/ephrin signaling initiates multiple intracellular
pathways to regulate the formation of
AChRs cluster opposite presynaptic nerve postsynaptic spine and shaft synapses 301
terminals in response to agrin released by motor
neurons 269 Wnt proteins influence pre- and postsynaptic
specializations in the CNS 304
The agrin hypothesis is revised based on
additional observations 271 Different Wnts regulate postsynaptic development
at excitatory and inhibitory synapses 305
The receptor components MuSK and Lrp4
mediate agrin signaling 272 Glial cells contribute to CNS synaptogenesis 306
Rapsyn links AChRs to the cytoskeleton 276 SYNAPSE ELIMINATION AND
AChR subunits are synthesized in nuclei adjacent REORGANIZATION IN THE CNS 307
to the nerve terminal 276 The vertebrate visual system is a popular model
Perisynaptic Schwann cells play roles in to study synapse elimination and reorganization 307
NMJ synapse formation and maintenance 279 Spontaneous waves of retinal activity stabilize
The synaptic basal lamina concentrates laminins selected synapses in LGN layers 308

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 CONTENTS xiii

Competition between neurons determines which Summary 316

synaptic connections are stabilized 309
Neural activity resulting from early visual
Further Reading 317
experience establishes ocular dominance
columns in the primary visual cortex 310 Glossary 319
Homeostatic plasticity contributes to synaptic Index 330
activity 312
Intrinsic and environmental cues continue
to influence synapse organization at all ages 313

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 An Introduction to the Field of
Developmental Neurobiology 1
D
evelopmental neurobiology is an area of study that seeks to
understand the formation of one of the most complex biological
systems—the nervous system. Fundamental questions about how
the nerve cells of the human nervous system initially form and extend
cellular processes to make the billions of necessary connections with
such precision have intrigued scientists and non-scientists for centuries.
Experimentally, it is one of the most exciting fields to work in, as the
questions are addressed using a variety of methods that range from the
classical approaches of tissue manipulations to the most sophisticated
molecular, genetic, and imaging techniques available today. It is no wonder
that developmental neurobiology is a field populated by researchers
with backgrounds in fields as diverse as anatomy, biochemistry, cellular
and molecular biology, computational sciences, embryology, genetics,
medicine, physics, physiology, and psychology (Box 1.1).
Identifying the paths that nerve fibers take as they extend from the
brain and spinal cord to target areas throughout the body has been of
interest to scientists for centuries (Figure 1.1), but significant advances
in understanding how such pathways form during development did not
occur until the mid- to late nineteenth century. During this period, detailed
descriptions of the microscopic anatomy of neural tissue were described for
the first time. These new findings were made possible by several technical
advances arising during that period. One such technological development
was the introduction of the microtome, an instrument that provides a means
to cut tissues into very thin slices. Another was the increasing availability
of microscopes with improved optics that allowed for better visualization
of these thinner tissue slices. Additionally, scientists continued to test and
refine techniques for fixing (preserving) and staining tissues, so that by the
end of the nineteenth century, several improved methods for visualizing
the cellular composition of tissues were available. These innovations led to
discoveries that were part of the “great age of cellular biology,” laying the
foundation for many fundamental concepts that we now take for granted.
Several of the first explanations of how the nervous system formed and
extended nerve fibers were based on these early microscopic observations.

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 2 Chapter 1 An Introduction to the Field of Developmental Neurobiology

Box 1.1 Pathways to developmental neurobiology

Individual investigators have come to the field of

developmental neurobiology by following many
different paths. Some, such as Hans Spemann and
Rita Levi-Montalcini, began their careers studying
medicine, but ultimately decided to focus on research
instead. Both began research careers in the general
area of zoology and gradually, as they undertook one
project and then another, began to focus on questions
pertaining to neurodevelopment. Some investigators,
such as the biochemist Stanley Cohen, were recruited
to help address a particular question in the developing
nervous system, and later focused research efforts
on topics beyond the nervous system. Spemann’s
work provided pivotal insights into how neural tissue
is first formed in the early stages of embryogenesis
(Chapter 2) and Levi-Montalcini and Cohen identified
the first protein to promote the survival of developing
neurons (Chapter 8).
Figure 1 Roger Sperry as the captain of the basketball
Researchers also come from a variety of backgrounds.
team. Like many college students, Roger Sperry was unsure of
Some who study developmental neurobiology were the what career he would pursue. Prior to entering Oberlin College,
first in their families to attend college, whereas others dn Box
he expressed interest 1.01and
in science Figure
athletic1coaching. As an
descend from families comprised of several scientists undergraduate, Sperry majored in English and was captain of
the basketball team. After graduating in 1935, he remained at
and physicians. Some completed their undergraduate
Oberlin to complete a master’s degree in Psychology (1937),
studies at large universities, whereas others began then took additional courses in zoology to prepare for his
their studies at small colleges. Some came to college doctoral studies at the University of Chicago. (Courtesy of
expecting to study science, while others began with Oberlin College Archives.)
different majors and uncertain career goals. Roger
Sperry, for example, whose early work addressed how successful career could have easily taken a different
neurons are able to extend nerve fibers to the correct path. On his college application, when asked about
target cell, graduated from Oberlin College in 1935 with his future career plans, one of his suggestions was
a major in English. He later earned a master’s degree college athletic coach due to his interests and talents
in psychology and studied zoology at Oberlin College in various sports (Figure 1). It is certain that none of
prior to beginning his doctoral studies. In addition the scientists whose work is featured throughout this
to his influential contributions to developmental book had any idea as undergraduate students where
neurobiology, Sperry won the Nobel Prize for Physiology their careers would take them, what questions they
or Medicine in 1981 for his work on split-brain might address in the future, how long their careers in
patients that revealed how the two hemispheres of the science would last, or how many other scientists they
brain communicate with one another. Yet, this very would influence.

Among the most influential scientists of that period was Santiago Ramón y
Cajal, whose work is described in subsequent chapters. What is remarkable
about the work of Cajal and his contemporaries is that their descriptions
of how neurons grew and behaved in an embryonic environment were
all formulated based on images of fixed tissues. By careful observation at
different stages of development, the researchers were able to formulate
reasonable hypotheses about how cell growth and movement would take
place. While not every hypothesis put forth in the late nineteenth century
was found to be accurate, a surprisingly large number of the ideas were
later found to be correct or very nearly so.
By the early twentieth century scientists had developed a variety of
surgical, histological, electrophysiological, and tissue culture techniques
that advanced studies in the area of developmental neurobiology. Major
scientific milestones in the field often paralleled advances in other areas.

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 An Introduction to the Field of Developmental Neurobiology 3

Figure 1.1 Early illustration of the nervous system. Scientists have long been
interested in understanding the paths nerve fibers take from the brain and spinal cord
to target regions throughout the body. This illustration was completed by the physician
Amé Bourdon in 1678. (Image courtesy of U.S. National Library of Medicine, Historical
Anatomies.)
dn 1.01

For example, in the mid-twentieth century, the electron microscope made it

possible to view cellular organelles and led to the conclusive identification
of the synapse as the site of connection between two nerve cells. Advances
in electronics were similarly influential. As recording equipment became
more precise, researchers were able to detect the tiny electrical impulses
produced by nerve cells. Additionally, instrumentation was developed that
provided a means for making precision microelectrodes to record activity
outside of cells, inside of cells, and even on a restricted patch of cell
membrane. Because of these technical advances, researchers can measure
isolated ionic currents and neural activity and monitor changes that occur
as the nervous system develops. Also in the mid-twentieth century, the
discovery of DNA (deoxyribonucleic acid), the various forms of RNA
(ribonucleic acid), amino acid structures, and the genetic code ushered
in the entirely new field of molecular biology, which provided insight into
the importance of regulated gene and protein expression during neural
development. Technological advances continue to be made in imaging,
electronics, molecular biology, and genetics. As in the past, researchers
commonly combine the available techniques to get a fuller picture of what
happens as neurons progress through various developmental stages.
Discoveries regarding development of the nervous system are made
using a variety of animal models, including flies, worms, frogs, fish, chicks,
and mice, to track normal developmental events as well as manipulate

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 4 Chapter 1 An Introduction to the Field of Developmental Neurobiology

developing systems and evaluate the impact of such changes on neural

development. It is now recognized that many developmental mechanisms
are highly conserved among species, and scientists working with the fruit
fly (Drosophila manangaster), the nematode worm (Caenorhabditis elegans),
and other invertebrate species often are the first to discover genes and
signaling molecules that regulate a particular aspect of nervous system
formation in multiple species.
This book describes many of the primary mechanisms by which the
nervous system develops, from the initial specification of neural tissue to
the refinement of neural connections during early postnatal periods. Each
chapter highlights some of the experiments that were key to advancing
understanding of a particular stage of neural development. These many
experiments highlight the remarkable creativity and insight of the early
neurobiologists who made so many major contributions, even without
benefit of the more sophisticated techniques available today. One also
quickly appreciates how some questions simply could not be answered
until suitable technical approaches became available. It is likely that many
of the techniques that are considered advanced today will appear crude to
scientists in the future. Yet as in the past, the discoveries made today will
add to the foundation of knowledge that will be used by future scientists,
and together these discoveries will elucidate the mechanisms that govern
the formation of the nervous system.

CELLULAR STRUCTURES AND ANATOMICAL

REGIONS OF THE NERVOUS SYSTEM

In order to help orient readers to topics discussed in subsequent

chapters, the following sections provide a brief overview of some major
cellular, anatomical, and developmental features found in vertebrate and
invertebrate animal models discussed in this text. The cellular composition
and anatomical organization of key neural structures are described first,
followed by information on specific developmental stages documented in
the chick, mouse, human, fish, frog, fly, and worm nervous systems. These
descriptions focus on the timing of shared developmental milestones,
including gastrulation, neural plate and neural tube formation, and early
brain segmentation. For more detailed explanations of these topics, refer
to the references at the end of the chapter.

The central and peripheral nervous systems are

comprised of neurons and glia

The vertebrate nervous system is divided into two main regions, the
central nervous system (CNS) and the peripheral nervous system
(PNS). The vertebrate CNS is comprised of the brain and spinal cord,
while the PNS consists of collections of neurons called ganglia that lie
outside of the CNS. The vertebrate PNS includes the neurons of the spinal
sensory (dorsal root) ganglia, cranial nerve ganglia, the enteric ganglia,
and ganglia of the autonomic nervous system (ANS). The invertebrate
nervous system is also divided into CNS and PNS regions; however,
different terminology is used for the various CNS and PNS structures, as
described in this chapter.
The cells of the CNS and PNS are the neurons and glia. A vertebrate
neuron consists of a cell body and cellular processes called axons and
dendrites (Figure 1.2A). Each neuron has only one axon but may
have several dendrites. The axon is typically longer than other neural
processes, has a uniform diameter, and ends in specialized regions called

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 CELLULAR STRUCTURES AND ANATOMICAL REGIONS OF THE NERVOUS SYSTEM 5

(A) presynaptic (B)

neuron
neuron
cell body
dendrites axon

presynaptic
synaptic axon terminal
vesicles
axon
action neurotransmitters
potential

axon
terminals
neurotransmitter postsynaptic
axon receptors neuron
dendrites

postsynaptic
neuron

Figure 1.2 Neurons release neurotransmitters to communicate with other cells.

(A) Vertebrate neurons consist of a cell body and cellular processes called axons and
dendrites. The majority of neuronsdn in the vertebrate nervous system signal to other cells
1.02
by conducting the electrical activity of an action potential down the axon to stimulate
the release of a neurotransmitter from the axon terminals and, in this example, to the
dendrites and cell body of the postsynaptic neuron. (B) The neurotransmitter is released
from synaptic vesicles then diffuses across the synaptic cleft to bind to specific receptors
on the postsynaptic cell. Depending on the neurotransmitter and receptor pair, the
binding will either increase or decrease the likelihood that an action potential will occur in
the postsynaptic cell.

axon terminals. In contrast, the dendrites tend to be shorter, branch

extensively, and have tapered ends. In some circumstances, the term
neurite is used to refer to either axons or dendrites. For example, when
viewing neuronal processes at early stages of neural development or in
tissue culture preparations, it is often difficult to conclusively identify a
process as an axon or a dendrite and therefore the term neurite is used.
Neurons primarily communicate with one another through electrical
signals (action potentials) that are conducted along the length of the
axon to initiate the release of chemical signals (neurotransmitters)
from synaptic vesicles that accumulate in the axon terminals. The
release of neurotransmitter occurs at the synapse, a small gap or cleft
between the axon terminal of one neuron (the presynaptic cell) and
the cell body or processes of another (the postsynaptic cell). The
neurotransmitter diffuses across the synaptic cleft to bind to receptors
on the postsynaptic cell, which may be another neuron or a muscle
cell (Figure 1.2B). Neurotransmitter–receptor pairs that increase the
likelihood that an action potential will occur are found at excitatory
synapses. In contrast, neurotransmitter–receptor pairs that reduce the
likelihood of an action potential firing are found at inhibitory synapses.
A small percentage of vertebrate neurons and some invertebrate neurons
communicate through gap junctions—channels that are formed between
two cells that are in direct contact with each other. In vertebrates, the
chemical synapses and gap junction synapses can work together to
enhance neural transmission.
The nervous system is also comprised of a number of distinct cell
types called glia. Originally called neuroglia in the mid 1800s, these cells
were thought to be connective tissue—the “glue”—needed to support

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 6 Chapter 1 An Introduction to the Field of Developmental Neurobiology

the structures of the nervous system. For over a century and half, glia
were thought to be limited to this role. However, it is now clear that glia
serve a number of important functions in the nervous system and in
some cases participate in cell signaling. The glia in the vertebrate CNS
are the oligodendrocytes, astrocytes, microglia, and ependymal cells.
Oligodendrocytes extend cellular processes that form the myelin around
axons in the CNS. Each oligodendrocyte extends processes to wrap around
several nearby axons. Myelin provides a type of insulation that speeds
the propagation of action potentials. Thus, action potentials are conducted
faster along myelinated axons than along unmyelinated axons. Astrocytes
are star-shaped cells that perform many functions in the central nervous
system, such as maintaining the balance of ions in the extracellular fluid
surrounding neurons, interacting with cells that form the blood–brain
barrier, and communicating with neurons. Microglia are the smallest of
the glia cell types and generally function as the immune cells of the brain
to remove debris and pathogens in the CNS. Microglia may also interact
with signals from the immune system to modify the stability of synaptic
connections during development and in neurodegenerative conditions
(Figure 1.3A). Ependymal cells line the ventricles of the CNS, where they
produce cerebral spinal fluid (CSF).
In the vertebrate PNS, glial cells consist of the Schwann cells and
satellite cells. Most Schwann cells function similarly to oligodendrocytes.
However, each Schwann cell wraps around only one axon and does not
extend processes to nearby axons. The satellite cells surround neuronal

(A) CNS microglial cell

axon
neuron

axon
terminals
dendrites

oligodendrocytes astrocyte

(B) PNS
satellite cells

Schwann cells axon

axon
terminals
neuron

dendrites

Figure 1.3 The vertebrate nervous system is comprised of neurons and glia.
dn 1.03
Neurons in the vertebrate nervous system are characterized by a single axon and many
dendrites. Axons are generally longer and of uniform diameter, while the dendrites tend
to be shorter, with tapered ends. Glial cells surround the neurons and perform diverse
functions. (A) Neurons in the central nervous system (CNS) are surrounded by numerous
glia, including astrocytes, microglia, and the myelinating oligodendrocytes that wrap
around the axons. (B) In the peripheral nervous system (PNS), the cell bodies of neurons
are surrounded by glial satellite cells, whereas the axons are wrapped by myelinating
Schwann cells.

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 CELLULAR STRUCTURES AND ANATOMICAL REGIONS OF THE NERVOUS SYSTEM 7

cell bodies and appear to have functions similar to astrocytes (Figure 1.3B).
In recent years, glia in the vertebrate CNS and PNS have also been found
to release signals that regulate various aspects of neural development and
roles for specific types of invertebrate glial have begun to be determined.

The nervous system is organized around three axes

When describing the location of different anatomical structures in the

nervous system, scientists often refer to structures relative to other
structures along one of three axes. The dorsal–ventral axis (also called
the dorsoventral axis) runs from the back (from the Latin dorsum) to
the belly (venter) side of the animal, and can easily be envisioned in any
number of vertebrate species such as mice or humans (Figure 1.4A).
However, other terms are more easily envisioned in embryos and four-
legged animals than in humans. The main body axis of a mouse, for
example, is the rostral–caudal (or rostrocaudal) axis. Rostral comes
from the Latin word rostrum, meaning beak or stiff snout, and caudal from
the word cauda, meaning tail. In many species, as well as in the early
embryonic nervous system, this axis is often called the anterior–posterior
(anteroposterior) axis, where the terms anterior and posterior substitute
for rostral and caudal, respectively. These terms apply to the main body
axis as well as the neuraxis established by the brain and spinal cord
(Figure 1.4B). However, this axis is not as readily envisioned in the adult
human nervous system, because the brain and spinal cord (neuraxis) are at
a nearly 90-degree angle. For example, along the neuraxis, the cerebellum
is caudal (posterior) to the cerebrum. Because of the angle, however, it
may at first mistakenly appear that the cerebellum is “dorsal” to portions
of the cerebrum (Figure 1.4B). In the adult human, the terms anterior and
posterior are often used differently, and when used to describe locations
along the torso, these terms correspond to dorsal and ventral. Throughout
this book, anterior and posterior refer to the locations along the neuraxis,
as shown in Figure 1.4B. The medial–lateral axis is the third axis used
to described structures relative to one another. Structures that are located
closer to the midline are said to be medial, while those located further from
the midline are called lateral (Figure 1.4C).

(A) dorsal (B) dorsal/superior (C)

lateral medial lateral

rostral caudal rostral cerebrum
(anterior) (posterior) (anterior) dorsal

cerebellum
spinal cord ventral/
cerebrum inferior ventral
cerebellum dorsal
ventral dorsal
spinal cord
ventral
ventral
caudal lateral medial lateral
(posterior)
Figure 1.4 The nervous system is organized around three axes. The positions of different neural structures are described relative to one
another along three axes. (A) In four-legged animals such as mice, the rostral–caudal or anterior–posterior axis of the nervous system is easily
seen as it extends from the region of the snout toward the tail. The dorsal–ventral axis extends from the back to the belly side of the animal.
(B) These same axes are present in the adult human nervous system, but the curvature of the brain and spinal cord lead to a corresponding
bending of the rostral–caudal (anterior–posterior) axis. (C) In the medial–lateral axis, those structures closest to the midline are called medial,
while those further from the midline are designated lateral as shown in these sections from the brain (pink) and spinal cord (yellow).

dn 1.04

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 8 Chapter 1 An Introduction to the Field of Developmental Neurobiology

ORIGINS OF CNS AND PNS REGIONS

A wide variety of invertebrate and vertebrate animal models have been

used to study neural development, each with its own advantages and
disadvantages. Common animal models include fruit flies, worms, frogs,
chicks, and mice. Many investigators focus on only one animal model,
while some use two or more for comparative studies. Few researchers are
fully versed in all of the developmental events of every animal model used,
yet having a general idea of how the nervous system forms in different
model systems can be extremely useful when reading the literature or when
formulating questions to test in another model system. Aspects of neural
development in some of the commonly used animal models discussed in
later chapters are described in the following sections. These descriptions
highlight common developmental events and the general timing of these
events in different model systems. Further details of each species can be
found in the references at the end of the chapter.
Among the early structures formed during vertebrate neural development
are the blastula, gastrula, neural plate, neural tube, and primary and
secondary brain vesicles. Similar structures are also found in many
invertebrate models. Each of these structures forms at a specific time
during embryogenesis in a given animal model. Because formation of these
structures is common across many species, these developmental milestones
are often used as a general means for comparing developmental progress
in different animal models. Specific details on the induction of neural tissue
and origins of blastula, gastrula, neural plate, neural tube, and primary and
secondary brain vesicles are provided in Chapters 2, 3, and 4.
The egg cell (zygote) begins to divide following fertilization, creating
a group of cells called the blastoderm. The blastoderm lies above a hollow
cavity and together the blastoderm and hollow cavity form a structure that
is often called the blastula. While the term blastula is often used for all
embryos at this stage, more specific terms are used for a given species
based on its morphological appearance. For example, blastula is the term
used for amphibians, blastocyst is used for many mammals, blastodisc
is used for birds, fish, and some mammals (Figure 1.5). The blastula-stage
embryo is organized around the animal and vegetal poles, with the animal
pole being the region that gives rise to the nervous system and epidermis
(skin) and the vegetal pole being the site of origin for tissues associated
with the gut. Blastula-stage embryos are used in a number of experimental
preparations from numerous vertebrate models and therefore is a key
structure identified in many studies of developmental neurobiology.

(A) BLASTULA (B) BLASTODISC

Figure 1.5 The blastula-stage embryo is ANIMAL POLE ANIMAL POLE

used in numerous studies of early neural blastoderm
development. Soon after fertilization, the blastoderm
egg cell divides, creating a group of cells that
lies above a hollow cavity. The cells are called blastomere
the blastoderm, while the cells and the hollow
cavity together comprise a structure that is
blastocoel
often referred to as the blastula. However, in
different animal models the morphology of
these regions varies and more specific terms yolk
are applied. For example, in amphibians
the ball-shaped structure is called a blastula
(A), whereas in birds, fish, and humans, the yolk cells
structure is more flattened and is called a VEGETAL POLE VEGETAL POLE
blastodisc (B).

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 ORIGINS OF CNS AND PNS REGIONS 9

As described in Chapter 2, gastrulation is the process that begins

as the cells of the blastula start to migrate through an indentation that
forms on the outer surface of the blastula. As cells migrate though this
indentation, the three primary germ layers are formed. The innermost
layer becomes endoderm, the middle layer forms mesoderm, and the
outermost layer forms the ectoderm. The ectoderm gives rise to both
the neural tissue and epidermal (skin) tissue. The vertebrate CNS derives
from neural ectoderm along the dorsal surface of the embryo, whereas the
invertebrate CNS arises from the ventral ectoderm.

The vertebrate neural tube is the origin of many neural

structures

The early-stage vertebrate CNS is formed from the neural tube, an

ectoderm-derived region that forms along the dorsal region of the embryo.
The neural ectoderm begins as a flattened sheet of cells called the neural
plate. The neural plate extends along the anterior–posterior (rostral–
caudal) body axis and is wider at the cephalic (head) end. Along the length
of the neural plate, a central indentation forms called the neural groove.
The lateral (outermost) edges of the neural plate then begin to curl upward
to form the neural folds. The neural folds continue to curve over and
eventually contact one another, thereby forming the neural tube. The
former lateral regions of the neural plate thus become the dorsal surface of
the neural tube, while the medial section becomes the ventral region of the
neural tube. The neural tube lies below overlying epidermal ectoderm. The
central lumen of the neural tube will later expand to form the ventricles
of the brain and the narrow central canal of the spinal cord, all of which
contain cerebral spinal fluid (Figure 1.6).
In the zebrafish (Brachydanio renio) which has become another popular
animal model for developmental studies in recent years, the hollow center
of the neural tube does not form as a result of the edges of the neural plate
curling over. Instead, the neural plate first bends to form the neural keel
and then the neural rod, both of which are solid structures lacking a central
lumen. The cells at the center of the rod then migrate, leaving the hollow
center of the neural tube.
Many of the neurons and glia of the vertebrate PNS originate from
a group of cells that is unique to vertebrates. These cells are called the
neural crest cells because they originate in the crest of the neural folds.
Neural crest cells migrate out of the dorsal neural tube to form many of
the ganglia of the PNS (Chapter 4). Other neurons and glia of the PNS form
from thickened patches of ectoderm called placodes that arise in specified
regions of the developing embryo (Chapter 5).

(A) neural plate future neural crest (B) (C)

ANTERIOR
future
epidermal neural grooves
ectoderm

neural folds

L–M–L POSTERIOR

Figure 1.6 The nervous system arises from neural plate ectoderm. (A) The neural plate ectoderm, located on the dorsal surface of the
embryo, is wider at the cephalic (head) region. (B) The lateral edges of the neural plate begin to curve upward, leading to the identification
of neural folds and a central indentation called the neural groove. Neural crest cells form at the crest of the neural folds. (C) The neural folds
eventually curl over and contact one another, thus forming the neural tube (blue). Epidermal ectoderm (yellow) surrounds the neural plate
ectoderm. M, medial; L, lateral.

dn 3.01/1.06
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 10 Chapter 1 An Introduction to the Field of Developmental Neurobiology

Future vertebrate CNS regions are identified at early

stages of neural development

Soon after the neural tube closes, the anterior region of the neural tube
expands and constricts at specific locations to form three primary brain
vesicles. These vesicles are called the prosencephalon (forebrain),
which is located at the most anterior (rostral) region of the neural
tube, the mesencephalon (midbrain), and the rhombencephalon
(hindbrain), which is located just anterior to the developing spinal cord
(Figure 1.7A). As development continues, five secondary brain vesicles
are formed. The prosencephalon forms two vesicles, the telencephalon
and diencephalon, the mesencephalon remains as a single vesicle,
and the rhombencephalon is divided into the metencephalon and
myelencephalon (Figure 1.7B).
The five secondary vesicles correspond to the sites of origin for
adult CNS structures. The telencephalon gives rise to cerebral cortex,
hippocampus, basal ganglia, basal forebrain nuclei, olfactory bulb, and
lateral ventricles. The diencephalon gives rise to structures that include
the thalamus, hypothalamus, and the optic cup—the precursor of the retina
that contains the sensory cells of the visual system. The mesencephalon
gives rise to the midbrain tegmentum, or central gray matter, of the
brainstem as well as the tectal regions (the superior and inferior colliculi)
that are important relay centers for visual and auditory information,
respectively. The metencephalon will ultimately form the cerebellum and
pons, while the myelencephalon will form the medulla. The signals that
coordinate to regulate the formation of these different CNS regions along
the anterior–posterior and dorsal–ventral axes are described in Chapters 3
and 4, respectively.

(A) (B)

ANTERIOR

telencephalon

diencephalon

prosencephalon mesencephalon

mesencephalon metencephalon

rhombencephalon myelencephalon

Figure 1.7 The neural tube forms primary presumptive

and secondary brain vesicles. (A) The early- spinal cord spinal
stage neural tube forms three primary brain
cord
vesicles designated the prosencephalon,
mesencephalon, and rhombencephalon.
(B) The primary vesicles further divide
into the five secondary brain vesicles
designated the telencephalon, diencephalon,
mesencephalon, metencephalon, and
myelencephalon. Each of the vesicles is the POSTERIOR
site of origin for different brain structures.

dn n3.100/1.07

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 ORIGINS OF CNS AND PNS REGIONS 11

Timing of developmental events in various vertebrates

The formation of the blastula, gastrula, neural plate, neural tube, and brain
vesicles occurs at specific times in embryonic development in each of
the animal models studied. While the sequence of developmental events
is consistent across all vertebrate species, the actual time that these
structures arise varies. Developmental age is reported as the number of
hours, days, or weeks post fertilization or by staging criteria established
for each species. The stages are based on various morphological criteria,
including embryo length and the presence of key developmental features,
such as the number of somites (the blocks of mesoderm that line either
side of the neuraxis). Such staging corrects for any variations that might
arise from genetic or environmental influences.
Human development is often referred to in terms of weeks of
gestation or Carnegie stages—stages first defined in the early twentieth
century by Franklin Mall and George Streeter, both of whom worked at
the Carnegie Institute in Washington, DC. Mice are described in terms of
embryonic (E) days or days post coital (d.p.c.) and are often staged using
criteria established by Karl Theiler. Development of chick embryos is
reported based on the hours or days post fertilization or by the duration
of incubation. Chick embryos are staged using criteria published by Viktor
Hamburger and Howard Hamilton.
The development of the chick embryo begins in utero and continues
after the egg is laid (about 20 hours after fertilization). In utero development
involves the formation of the blastoderm at 10–11 hours after fertilization.
The onset of gastrulation begins about the time the egg is laid and
subsequent developmental events are easily monitored by cutting a small
hole, or window, in the egg. Thus, the chick embryo is an extremely useful
model for viewing neural development. Another useful feature of the chick
egg is that development of a fertilized egg can be halted for several days if
the eggs are maintained at room temperature. Development resumes when
the eggs are placed in an incubator at 37.5°C. Figure 1.8 shows several
of the stages first published by Hamburger and Hamilton in 1951. At these
stages of development, hours refer to the number of hours the eggs were
incubated, rather than the hours post fertilization. The first somites and
the head neural folds are visible at Hamburger and Hamilton (HH) stage 7
(23–26 hours of incubation). In the chick, the three primary brain vesicles
are detected at HH10 (33–38 hours) and the five secondary vesicles are
observed after 40–45 hours of incubation (HH11). The embryo turns to the
side beginning at HH13 (48–52 hours) and the enlargement and refinement
of the brain vesicles is easily viewed in the translucent embryo from HH
14–21 (about 2–3 days after incubation).
Figure 1.9 compares the development in humans and mice using
Carnegie and Theiler criteria, respectively. In humans, the blastula is
detected in the uterus at four days after fertilization (Carnegie stage 3, CS3)
and gastrulation begins at day 16 (CS7). The neural plate and neural folds
become evident at day 18 (CS8). The three primary vesicles form during
the third and fourth week of gestation (days 20–24; CS8–11) and the five
secondary vesicles become visible during the fifth week of gestation (CS14).
In mice, the blastula stage embryo is formed 3 to 4 days post coitus
(d.p.c.), corresponding to Theiler stages 4–5 (TS04–05). Gastrulation begins
at 6.5–7.5 d.p.c. (TS09) and the neural plate forms at 7.5–8 d.p.c. (TS11). The
three primary vesicles are visible at 8–8.5 days of embryogenesis (TS12–13)
and the five secondary vesicles are detected at days 9–10 (TS15–16). The
development of a newborn mouse (TS27; 19–20 d.p.c.) is similar to that of a
9-week human (CS23). Human development continues for about 38 weeks
(9 months).

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 12 Chapter 1 An Introduction to the Field of Developmental Neurobiology

Figure 1.8 Chick development is easily 4 5 6 7

viewed throughout embryogenesis.
Images of developing chick embryos
reveal some of the key events in neural
development. Numbers in the corners of the
images indicate the stage of development
as determined by Hamburger and Hamilton
(HH). The neural folds are first identified at
23–26 hours (HH stage 7, arrow). The primary
brain vesicles are visible at HH 10 (arrow)
and the secondary vesicles at HH 11 (arrow).
The embryo begins to turn to the side at
HH 13 and further development of the brain 8– 8 9
regions is observed through the embryo’s
10
translucent body until HH 21 (embryonic day
4). (From Hamburger V & Hamilton HL [1992]
Dev Dyn 195:231–272.)

11 12 13 14

15 16 17

18

19 20
21

dn 1.08

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 ORIGINS OF CNS AND PNS REGIONS 13

1 cm

(× 35) (× 1.5)

oocyte TS01 TS02–08 TS09 TS11 TS13 TS16 TS19 TS21 TS22 TS23 TS25 TS27

CS1–10 CS08–11 CS11–14 CS15–16 CS17–18 CS19–22 CS23

(week 4) (week 5) (week 6) (week 7) (week 8) (week 9)

Figure 1.9 Comparison of stages of mouse and human embryonic development. Morphological criteria are used to identify the stages
of embryonic development. Mouse development is staged by the criteria of Theiler, whereas human development is marked by Carnegie
stages. As shown in the diagram, Theiler stage 13 (TS13) is equivalent to Carnegie stage 11 (CS11). At these stages, the three primary vesicles
are observed. TS16 is equivalent to CS11–14, the stages when the five secondary brain vesicles are formed. TS27 is a newborn mouse, which is
at a similar stage of development as a nine-week human (CS23). Arrows indicate times at which human and mouse development are at a similar
stage. (From Xue L, Cai J-Y, Ma J et al. [2013] BMC Genomics 14:568.)
dn 1.09
Fish and amphibians have also been very popular vertebrate animal
models in studies of neural development. The stages of development in the
zebrafish have been documented by Monte Westerfield, Charles Kimmel,
and colleagues. The cells of the zygote begin to divide about 40 minutes
after fertilization and are easily viewed above the yolk. The zebrafish
blastula forms at a little over two hours post fertilization, when 128 cells,
the blastomeres, are present. The blastula-stage embryo continues to
develop through multiple stages during the first five hours after fertilization
and the blastoderm is identifiable a little over 4.5 hours post fertilization
(h.p.f.; Figure 1.10). After the blastula-stage is complete, epiboly and
gastrulation, the movement and thinning of cell layers, begins just over
5 h.p.f. At these stages, the embryo begins to curl around the central
yolk. Development is measured in terms of the percentage of epiboly,
indicating the percent of the yolk that is surrounded by the blastoderm. At
50% epiboly, gastrulation begins. At just over 6 hours, the embryonic shield,
a key structure in the process of neural induction (Chapter 2), is present.
At 90% epiboly, 9 h.p.f., the neural plate is visible. At the completion of
epiboly and gastrulation, somites are detected (10 h.p.f.), and by 16 hours,
there are 14 somites and the three primary brain vesicles are observed. At
24 hours, the five secondary brain vesicles are present. At the same time,

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 14 Chapter 1 An Introduction to the Field of Developmental Neurobiology

eight cells sixty four 256 high stage dome 30% epiboly 50% epiboly shield
zygote

70% epiboly 90% epiboly 2-somite

15-somites

25-somites

24 hr
prim 5
33 hr
prim 20

48 hr

Figure 1.10 Examples of zebrafish development from zygote to hatching. Cells of the zebrafish zygote begin to divide about 40 minutes
after fertilization. The resulting blastomeres continue to divide as the blastodisc forms above the yolk, as seen in the eight-cell stage shown
in this figure. The blastula stage of development begins at the 128-256 cell stage (2.25-2.5 hours post fertilization, h.p.f.) then progresses
through multiple stages. The high stage, for example, indicates the period that the blastodisc is located “high” on the yolk. The blastula stage
continues until a little over 4 h.p.f. (dome stage). By about 4.5 hours, epiboly can be measured, indicating the percentage of the yolk surface
that is surrounded by the embryo. At 50% epiboly gastrulation begins and at 90% epiboly, the neural plate is present. Somites are first visible
by 10 h.p.f., and the divisions of the brain vesicles are first observed at the 14-16 somite stage. The embryo begins to straighten away from the
yolk at 24 h.p.f. (prim 5), when development is measured by the myotome number that the tip of the primoridum (prim) of the lateral line organ
reaches. By 48 h.p.f., the embryo hatches. (From Westerfield M [1993] The Zebrafish Handbook, 2nd ed, University of Oregon Press.)

the embryo begins to straighten away from the yolk sac. The embryos are
now measured by indicating which myotome (the segment of the somite
dn 1.10
that later gives rise to muscle) that the tip of primordium (prim) of the lateral
line organ (a sensory organ found in aquatic vertebrates) reaches. Thus,
prim 5 indicates that the tip of the primordium of the lateral line reaches the
fifth myotome. The embryo hatches at about 48 hours and enters the larval
stages by 72 hours post fertilization. The larval stages last up to 29 days.
Juvenile zebrafish form at day 30 and become adults by day 90.
Frogs are another frequently used vertebrate animal model in studies
of neural development. Among the most commonly used frogs are Xenopus
laevis. These frogs provide many advantages for researchers, including
the ability to induce frogs to produce eggs year round and the one-year
cycle needed to complete development from a fertilized egg to an adult
frog. The description that follows refers to the timing of developmental
events in Xenopus based on the time post fertilization and the staging
criteria of Pieter Nieuwkoop and Jacob Faber. The timing of these events
may be slightly different in other frogs. In Xenopus, blastula-stage embryos
are noted by four hours after fertilization (stage 7). Gastrulation begins
approximately 7–10 hours post fertilization (stages 10–12) and leads to the
formation of the neurula-stage embryo, the stage when the neural tissue
begins to form (12 and 13.5 hours post fertilization; Figure 1.11). The
neurula stage (stages 13–21) continues until the early tailbud-stage embryo
forms 24–32 hours post fertilization (stages 22–28). Primary brain vesicles
appear around 24 hours after fertilization (stage 22) and the five secondary
vesicles about 32 h.p.f (stage 28). The embryo develops into a tadpole by
96 hours (stages 45–50), before undergoing metamorphosis (stages 51–65)
and reaching the adult stage approximately 12 months later (stage 66).

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 ORIGINS OF CNS AND PNS REGIONS 15

Figure 1.11 Development of the frog from

egg through adult stages. Examples from
the criteria established by Nieuwkoop and
Faber in 1967 identify some of the key stages
of development in the frog Xenopus laevis.
Images show embryos from posterior-dorsal,
stage 7 stage 10 stage 12 stage 13 stage 17 stage 21 lateral, and dorsal views. By stage 7 (4 hours
(dorsal) (vegetal) (vegetal) (posterior/ (dorsal) (dorsal) after fertilization), the blastula-stage embryo
dorsal) is present. Gastrulation begins 7–10 hours
after fertilization (stages 10–12). The neurula-
stage embryo, when the neural structures first
form, continues from 12 to 22.5 hours after
fertilization (stages 13–21). The embryo then
enters the tailbud stage at 24–36 hours post
fertilization (stages 22–44). The brain vesicles
stage 22 stage 25 stage 26 stage 33–34 are visible beginning 24 hours after fertilization
(lateral) (lateral) (lateral) (lateral) (stage 22). The embryo develops into a tadpole
by 96 hours (stages 45–50), before undergoing
metamorphosis (stages 51-65). An adult frog is
formed approximately 12 months later (stage 66).

stage 40 stage 43 stage 50

(lateral) (lateral) (lateral)

stage 63 stage 66
(dorsal) (dorsal)

Anatomical regions and the timing of developmental

events are mapped in invertebrate nervous systems

Several invertebrate animal models, particularly the fruit fly Drosophila

melanogaster and the round worm dn 1.11
Caenorhabditis elegans (C. elegans),
are also utilized in a number of pivotal studies described in subsequent
chapters. Drosophila became a popular animal model for research in areas
of genetics and developmental biology beginning with the pioneering
work of Thomas Hunt Morgan in the early twentieth century. The work
of Seymour Benzer and colleagues in the 1960s helped make Drosophila
an animal model of ongoing interest to developmental neurobiologists.
C. elegans also became a popular model beginning in the 1960s, largely
through the work of Sydney Brenner’s lab. These animal models are easily
bred, have a short life cycle from the time of fertilization to the adult form,
and exhibit naturally occurring and experimentally induced mutations that
provide a means to test how specific genes regulate development of the
various cells within the nervous system. Like the vertebrate nervous system,
the nervous system of invertebrates arises from the ectoderm. However,
there are significant differences in how and where neural structures arise
in these animal models.

The Drosophila CNS and PNS arise from distinct areas of

ectoderm

When Drosophila are maintained at 25°C, embryogenesis occurs over a

period of approximately 22 hours and adult flies are formed within 9–12 days
(Figure 1.12). This allows for the generation of large numbers of animals

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 16 Chapter 1 An Introduction to the Field of Developmental Neurobiology

fertilized egg syncytial embryo

blastoderm
cleavage gastrulation
hatching

(stage 1) (stage 2) (stage 4) (stage 6–7) (stage 12–17)

0 1–2 2–3 3 7–22

approximate hours
after fertilization

larval stages pupa adult fly

1st instar 2nd instar 3rd instar

metamorphosis

1 2 3–4 5–8
approximate days
after fertilization

9–12

Figure 1.12 Development of the fruit fly Drosophila melanogaster. The fertilized Drosophila egg (stage 1) undergoes cleavage (stage
2) and forms a syncytial blastoderm 2–3 hours after fertilization (stage 4) followed by a cellular blastoderm (stage 5, not shown). The embryo
then begins gastrulation (stages 6-7) and forms the late stage embryo 7–22 hours after fertilization (stages 12-17). The embryo enters the first
larval stage by 24 hours after fertilization, and continues through three larval instar stages before forming a pupa at 5–8 days post fertilization.
Metamorphosis takes place and the adult fly emerges 9–12 days after fertilization.

to evaluate in a short period of time. Staging of Drosophila embryos is often

dn 1.12
noted using the criteria of Volker Hartenstein and Jose Campos-Ortega.
In Drosophila, the cytoplasm of the fertilized egg contains many nuclei
that divide rapidly (stages 1–2) prior to migrating to the outer cortex of the
cell to form a syncytial blastoderm (stage 4). Each nucleus is then surrounded
by a cell membrane to form the cellular blastoderm (stage 5). Gastrulation
(stages 6–7) occurs within 3 hours of fertilization, and by the end of the
first day (stages 16–17), the embryo hatches to enter the first larval stage
called the first instar. Drosophila larvae progress through three instar stages,
with each stage lasting one to two days (Figure 1.12). An epidermal-derived
hardened shell called a cuticle surrounds each instar stage larva. At the end
of each instar stage, the cuticle sheds to accommodate the growth of the
larva. A new cuticle is then produced for the larger larva. Following the third
instar stage, the cuticle contributes to extracellular case that surrounds the
prepupa. During the pupal stage, metamorphosis occurs. Adult tissues that
are derived from ectoderm, such as the nervous system, arise from pockets
of epithelium formed during the larval stages. These pockets of tissue, called
imaginal discs, attach to the inside of the larval epidermis and later evert
during metamorphosis to form adult structures of the head, thorax, legs, and
wings. Unlike most other larval-stage organs, the components of the gut and
nervous system persist in the adult fly. Cells of the nervous system proliferate
during the larval stages and begin to differentiate in the pupal stage.
In Drosophila, the nervous system arises from ventral ectoderm
(the ventral neurogenic ectoderm), rather than the dorsal ectoderm as
in vertebrates. This ectoderm gives rise to the neuroblasts (beginning at
stage 9, about 4 hours after fertilization) that form the adult brain and
ventral nerve cord, a structure with functions similar to the vertebrate
spinal cord (Figure 1.13). During development, neuroblasts segregate
from the surrounding ectoderm, then move inside the embryo along

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 ORIGINS OF CNS AND PNS REGIONS 17

embryo Figure 1.13 The Drosophila central nervous

system (CNS) forms in the embryo and
continues to develop during the larval
stages. The outline of the Drosophila
embryo (A) and third instar larval stage CNS
(C) are shown in blue. (B) CNS structures of
the embryo are identified in red. The arrow
indicates the brain region and the arrowhead
indicates the ventral nerve cord. (D) The brain
(A) (B) (arrow) and ventral nerve cord (arrowhead)
continue to develop and enlarge during the
third instar larvae (L3) third instar larval stage. In this panel, the areas
of red reveal sites of synaptic connections.
(From Diaper DC & Hirth F [2014] In Brain
Development Methods and Protocols [SG
Sprecher ed], pp 3–17. Humana Press.)

(C) (D)

the anteroposterior axis. As development progresses through the larval

stages, the number of neurons increases and definitive CNS regions
form. In the adult, the anterior-most region of the CNS is divided further,
reminiscent of subdivisions found in the mammalian brain (Figure 1.14).
These anterior brain regions arednformed
1.13 from three pairs of ganglia, with
each pair controlling specific functions: the protocerebrum (forebrain,
largely associated with visual regions), the deutocerebrum (midbrain,
largely associated with sensory information from the antennae), and
the tritocerebrum (hindbrain, primarily integrates information from the
protocerebrum and deutocerebrum; linked to the ventral nerve cord).
The more posterior ganglia of the CNS are part of the ventral nerve cord
(Figure 1.14). These include the subesophageal ganglia (associated with
head and neck regions), the thoracic ganglia (associated with legs and
wings structures), and the abdominal ganglia (associated with abdominal
structures).
Many of the neurons of the PNS arise from sensory organ
progenitors (SOPs) located in the surface ectoderm. The SOPs ultimately
develop into the mechanosensory, chemosensory, and chordotonal organs
of the fly. PNS neurons generally develop later than the cells of the CNS.
Mechanisms regulating the formation of various CNS and PNS structures
in Drosophila are described in Chapters 2, 3, 4, and 6.
There are four types of glial cells in Drosophila that are designated
cortex, surface, neuropil, and peripheral glia. Many of the functions of these

protocerebrum
Figure 1.14 The Drosophila central
deutocerebrum circulatory system
nervous system is comprised of pairs of
tritocerebrum digestive system ganglia. The three pairs of ganglia that make
up the Drosophila brain are divided into
protocerebrum (forebrain), deutocerebrum
(midbrain), and tritocerebrum (hindbrain).
These ganglia connect to the ventral nerve
cord comprised of subesophageal, thoracic,
and abdominal ganglia. The nervous system
(blue) is shown relative to the digestive (green)
subesophageal thoracic abdominal and circulatory (yellow) systems. (Adapted
ganglia ganglia ganglia from Agricultural and Life Sciences, General
Entomology, North Carolina State University.)

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 18 Chapter 1 An Introduction to the Field of Developmental Neurobiology

glia appear similar to vertebrate glia. The cortex glia are most like astro-
cytes, the neuropil glia are similar to oligodendrocytes, and the peripheral
glia function similar to Schwann cells. The cortex, surface, and neuropil
glia can also function like the microglia found in the vertebrate CNS.

Cell lineages can be mapped in C. elegans

Most adult C. elegans are hermaphrodites, with a smaller percentage of the

adults being male. The adult hermaphrodite C. elegans has a total of 959 cells
of which 302 are neurons and 56 are glial. The lineage of each cell has been
documented through serial electron micrographs and by following the progeny
and fate of individual cells through the translucent body of the tiny worm.
The timing of developmental events established for C. elegans
maintained at 22°Celsius are shown in Figure 1.15. The egg cell is fertilized
inside the worm and cells begin to divide in a specific sequence beginning
about 40 minutes after fertilization. The eggs are laid at the gastrulation
26-cell; beginning of gastrulation
eggs laid outside

synthesis of larval cuticle starts

pharyngeal pumping starts

visible sexual dimorphism
1.5-fold (tadpole) stage

3-fold (pretzel) stage

ventral cleft closed,

hatching (558 cells)

end of gastrulation

2-fold (plum) stage

excretory cell born

twitching starts
comma stage
bean stage
fertilization

174-cell
190-cell
44-cell

87-cell
99-cell
2-cell

4-cell

8-cell

310 330 360 430 460 490 510 690

470

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 840 min

Ea, Ep P4, MS elongation

cell
migrations D C

AB

gastrulation

Figure 1.15 An adult C. elegans can form within three days of fertilization. A timeline of developmental events through hatching for
C.elegans maintained at 22 degrees Celsius. The egg is fertilized and begins to divide in the worm. Gastrulation begins when the egg is laid,
about 150 minutes after fertilization, when there are 26 cells present. Gastrulation continues until 330 minutes (5.5 hours) after fertilization, when
421 cells are present. The timing of the migration of founder cells during gastrulation is indicated below the timeline. As the worm continues to
elongate and become thinner, the worm folds over itself 1.5 times (tadpole stage), then two times (plum stage) and finally three times (pretzel
stage). Worms hatch 14 hours after fertilization, when
dnthere
1.15are 558 cells. The resulting larvae then progresses through four larval stages (L1–L4)
before forming an adult worm. Under favorable environmental conditions, the adult worm emerges about 56 hours after fertilization. (Adapted
from The Worm Atlas.)

9780815344827_Ch01.indd 18 13/10/17 2:05 pm

 ORIGINS OF CNS AND PNS REGIONS 19

stage, approximately 150 minutes after fertilization, when there are about
26 cells present. Gastrulation is initiated as cells move inward to form the
gut and muscle tissues, while the hypodermis, the equivalent of ectoderm
in vertebrates, remains as the outermost layer. Cells of the hypodermis that
subsequently move to the inside of the embryo give rise to the majority
of the neurons. The remaining cells of the hypodermis migrate over the
surface of the embryo to form the epidermis. Gastrulation continues until
the number of cells increase to 421 (about 5.5 hours after the first cleavage).
During the final stages of embryogenesis, the shape of the embryo
changes from a spherical structure to the elongated shape of the adult. As
elongation continues, the worm begins to fold over first 1.5 times (tadpole
stage), then 2 times (plum stage), and eventually 3 times (pretzel stage).
Hatching occurs about 14 hours after fertilization, when there are 558 cells.
The resulting larva then progresses through the four larval stages (L1–L4)
before forming an adult worm. During the larval stages, additional neurons
are produced, with the majority born during the late L1 stage. The length
of time in each larval stage depends, in part, on environmental conditions
such as temperature, food supply, and population density. Under favorable
conditions, it takes less than 2.5 days for a C. elegans to complete the life
cycle from fertilized egg to adult worm.
The cell fate options available to a particular cell in C. elegans are
established early in development with the asymmetric division of the
zygote into the AB and P founder cells. A series of cell divisions then results
in a total of six founder cells that are designated AB, P, E, MS, C, and D
(Figure 1.15). Each founder cell gives rise to progeny in a specific pattern.
The initial division of the zygote yields an AB cell located at the anterior pole
and the P1 cell at the posterior pole of the embryo (Figure 1.16A). Next,
the AB cell divides to produce two daughter cells, the anterior AB.a and the

P2 P3 D
P1 EMS
POSTERIOR P4 (germ line)
AB.p E
AB.pr

egg
AB.pl
AB.ar
ANTERIOR
AB.al AB P1
fertilized egg
AB.a MS
AB (epidermis,
(A) neurons, pharynx)
AB.a AB.p EMS P2

head ganglia (brain) dorsal cord MS E C P3

D P4

(B) ventral cord tail ganglia

Figure 1.16 Cells in C. elegans divide in a precise order. (A) The asymmetric division of the fertilized egg leads to formation of a larger AB
founder cell at the anterior pole of the developing embryo and a smaller P1 founder cell at the posterior pole. The cells continue to divide until
a total of six founder cells are produced (AB, P, E, MS, C, and D; bold letters, inset). As shown in the diagram, each dividing cell produces cells in
a specific location. The AB cell (green) gives rise to the AB.a cell at a more anterior site and the AB.p at a more posterior site. These cells divide
to produce additional daughter cells designated as AB.al and AB.ar, the left- and right-handed daughter cells of AB.a. Similarly AB.p divides to
produce AB.pl and AB.pr. The P1 cell (red) establishes the germ line (red cells) and various somatic cells (yellow, orange, purple, and blue). The
AB founder cell gives rise to most neurons in C. elegans. Descendants of MS and C give rise to a small number of the neurons in C. elegans. (B)
Regions of the worm nervous system are stained with green dn 6.03/1.16
fluorescent protein to outline regions of the brain (head ganglia), the tail ganglia,
and the dorsal and ventral nerve cords. (A, adapted from Alberts B, Johnson A, Lewis J et al. [2008] Molecular Biology of the Cell, 5th ed.
Garland Science. B, courtesy of Harold Hutter.)

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 20 Chapter 1 An Introduction to the Field of Developmental Neurobiology

posterior AB.p. Shortly after the AB cell divides, the P1 cell divides, produc-
ing the more posterior P2 cell and the more anterior EMS cell (Figure 1.16A).
As more AB cells are generated, the location of a cell relative to its
sister cell is specified along the anterior–posterior axis. Thus, the AB.a1 cell
is the “left-handed” daughter cell of an anterior AB cell, whereas AB.pr is the
“right-handed” daughter of the posterior AB daughter cell (Figure 1.16A).
The EMS cell divides to produce E and MS cells, whereas P2 produces C and
P3. P3 then divides, producing D and P4, the cell that establishes the germ
line. Thus, the P1 founder cell functions like a stem cell in that it gives rise
to both somatic and germ cells.
Each founder cell produces progeny that go on to contribute to
specific tissues. However, not all cells of a given body system arise from a
single founder cell type. For example, most of the 302 neurons arise from
descendants of the AB founder cell, but some arise from the MS and C cells
that descend from the P1 founder cell. In all cases, however, the individual
neuronal types always arise from the same precursor and are always found
in the same location in the body.
C. elegans has a somatic and pharyngeal nervous system. The somatic
nervous system contains 282 neurons found in the head and tail ganglia as
well as in the ventral and dorsal nerve cords (Figure 1.16B). These regions
contain sensory, motor, and interneurons. The head region also contains
numerous sense organs called sensilla that are comprised of free nerve
endings and glial sheath and socket cells. The pharyngeal nervous system
contains 20 neurons. The pharynx in C. elegans is segregated from the rest
of the tissues of the body by a unique basement membrane and functions
largely independent of the other parts of the worm.
There are 56 glial cells in C. elegans that are designated into three
categories: sheath, socket, and glial-like nerve ring (GLR). The 24 sheath
and 26 socket glia are derived from ectoderm, whereas the six GLR cells
are derived from mesoderm. While the functions of glia in C. elegans are
not as well characterized as in other animal models, they appear to assist
in synaptic signaling and play roles in the development, maintenance, and
activity of their associated synapses. The GLR cells seem to be specifically
associated with signaling that regulates motor movement. Unlike verte-
brates and Drosophila, axons in C. elegans are not myelinated, so glia do
not wrap around axons to speed neural conduction.
Despite the variations in anatomy and cellular organization among
the different animal models, many of the genes and signaling pathways
are conserved across species, allowing discoveries in one animal model to
impact discoveries in another. This is particularly helpful when a technique
is more readily applied to a simpler invertebrate animal model than a more
complex vertebrate model.

GENE REGULATION IN THE DEVELOPING

NERVOUS SYSTEM

In all animals, each neuron found in the nervous system must selectively
express specific cellular components, such as neurotransmitters, ion
channels, cell surface receptors, cytoskeletal elements, and other proteins.
The regulated production of these specialized proteins gives individual
neurons their unique characteristics and allows them to perform specific
functions in the nervous system. Neurons, like other cells, produce only
the proteins required at a particular stage of development. In order to
selectively produce these proteins, individual genes must be turned on
(expressed) or turned off (repressed) at the correct stage of development.
The process of turning genes on or off is called gene regulation.

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 GENE REGULATION IN THE DEVELOPING NERVOUS SYSTEM 21

A gene is a segment of DNA, the double-stranded, helical molecule DNA

synthesized in the nucleus of all cells. While DNA is the same in every cell of
the body, the genes that are expressed in an individual cell at a particular stage
of development will determine which messenger RNA (mRNA) nucleotides
are transcribed from the DNA template and therefore which amino acids DNA synthesis
REPLICATION DNA
are translated into proteins (Figure 1.17). Among the numerous proteins a
cell produces are transcription factors—proteins that bind to specific DNA
sequences to enhance or suppress expression of a gene.
In many instances, gene expression and protein production are RNA synthesis
influenced by extracellular signals. The extracellular signal is typically a nucleotides TRANSCRIPTION
ligand that binds to a cell surface receptor protein to initiate intracellular
RNA
signal transduction pathways. Cell signaling or signal transduction
is the process by which signals originating outside a cell are conveyed
to cytoplasmic components or the nucleus to influence cell behavior. protein synthesis
Because the ligand is often thought of as the first messenger in a signal TRANSLATION
transduction pathway, the subsequent intracellular events are often called PROTEIN
second messenger pathways. The activation of various signal transduction
pathways regulates cellular events such as survival, death, growth, amino acids
differentiation, movement, and intracellular communication.
Figure 1.18 outlines an example of a signal transduction pathway, Figure 1.17 Gene regulation determines
or cascade, where an extracellular signal (ligand) binds to a cell surface which proteins are produced in a cell.
DNA synthesis (replication) takes place in the
receptor. Once the ligand binds to the receptor, subsequent signaling
nucleus of the cell, where the template strand
molecules are activated inside the cell. There are often several sequential of DNA synthesizes a complementary strand of
signaling molecules influenced before the final cellular response is achieved. DNA. A DNA template strand can also initiate
A signal is said to activate a target downstream when it influences the synthesis (transcription) of a complementary
dn During
strand of RNA. m1.04/1.17
protein synthesis
next molecule in the signal transduction pathway. The signal transduction
(translation) the sequence of nucleotides
pathway eventually regulates effector proteins that serve a variety of in a strand of messenger RNA determines
different cellular functions. Common effector proteins are ion channels, the order of amino acids and therefore the
metabolic enzymes, cytoskeletal proteins, and gene regulatory proteins resulting protein structure. (Adapted from
(Figure 1.18). Several examples of specific signal transduction pathways Alberts B, Johnson A, Lewis J et al. [2015]
Molecular Biology of the Cell, 6th ed. Garland
utilized during neural development are detailed in subsequent chapters. Science.)
The structure of each ligand and receptor is unique so that a given ligand
only binds to corresponding receptors. This allows for binding specificity.

EXTRACELLULAR
LIGAND

CELL-SURFACE
RECEPTOR

plasma membrane
of target cell

Figure 1.18 Signal transduction pathways

transfer extracellular information to the
INTRACELLULAR
cell. When an extracellular signal (ligand)
SIGNALING MOLECULES
binds to its corresponding receptor located on
the surface of a cell, one of many intracellular
signal transduction cascades can be initiated.
Each step in the cascade stimulates the next
molecule in the pathway until an effector
protein is influenced. Examples of effector
proteins include ion channels, such as those
EFFECTORS
that alter a neuron’s membrane potential,
ion metabolic cytoskeletal gene metabolic enzymes that impact cellular
channel enzyme protein regulatory metabolism, cytoskeletal proteins that
proteins influence cell shape and movement, and gene
regulatory proteins, such as transcription
altered cell factors, that influence whether a gene is
metabolism gene
membrane shape or expressed or repressed. (Adapted from Luo
expression
potential movement L [2016] Principles of Neurobiology. Garland
Science.)

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 22 Chapter 1 An Introduction to the Field of Developmental Neurobiology

Binding specificity combined with the ability of cells to regulate the expression
of the multitude of ligands and receptors ensure that specific signaling
pathways are only available to a cell when needed. Thus, cells become
specialized so they only respond to required signals at each developmental
stage and ignore other signals that may also be present at that time.

Experimental techniques are used to label genes and

proteins in the developing nervous system

Because each cell subtype in the nervous system expresses a unique set
of genes and proteins, researchers have developed several techniques to
identify where and when these molecules are expressed during development
and in adulthood. Among the techniques are those that use microscopy to
identify the distribution of genes and proteins in tissues or individual cells.
These approaches lead not only to understanding the cellular distribution of
genes and proteins, but also provide a way to label or mark particular cells
and track them over the course of development. This has been especially
helpful, because the outward morphological appearance of embryonic
neurons is often homogeneous, making it difficult, or impossible, to identify
a cell with any certainty following any sort of experimental manipulation.
To visualize gene expression in neural tissues, scientist use in situ
hybridization. With this technique, mRNA is visualized by incubating
whole embryos, tissue sections, or cultured cells with probes made up of
a DNA nucleotide sequence of interest. These probes are labeled with a
radioactive or fluorescent marker so that when the DNA probe binds to
the corresponding mRNA sequence, the cells expressing that mRNA can
be visualized. Identifying gene expression patterns often provides insight
into the putative function of that gene in a given cell, while also providing
a labelling method to track changes in gene expression under normal
and experimental conditions. Examples of experiments using in situ
hybridization are found in Figures 3.24 and 4.20.
Scientists use immunohistochemistry or immunocytochemistry
to label proteins in tissues or cells, respectively. These methods take
advantage of the immune response in which an animal develops antibodies
to a foreign substance. For example, one method of generating antibodies
is to inject rabbits with a protein of interest. The animals develop
antibodies to the protein that are then isolated from the blood serum. The
resulting antibodies, called primary antibodies, are then added to tissues
or cell cultures, where they bind to the target protein. These antibodies
are visualized by either adding an enzymatic reporter molecule, such as
horseradish peroxidase, or a fluorescent probe directly to the primary
antibody, or by adding one of the labels to a secondary antibody that
recognizes and binds the primary antibody. These methods often provide fine
details on the distribution of a protein within a cellular region. Examples of
such methods are shown in Figure 1.13, in which a neuron-specific protein
is used to visualize the entire nervous system (Figure 1.13B), and another is
used to identify a single presynaptic element known as the active zone protein
(see Chapters 9 and 10), which is localized to presynaptic nerve terminals
(Figure 1.13D). Another example of immunolabelling to identify the
distribution of synaptic contacts on a single neuron is shown in Figure 10.1.

Altering development as a way to understand normal

processes

One of the most common ways to assess normal developmental events

is to alter some aspect of development and see what happens. This allows
researchers to test whether a given tissue, cell, or protein is necessary for normal
development to occur. Over the past century and a half, a number of methods

9780815344827_Ch01.indd 22 13/10/17 2:05 pm

 GENE REGULATION IN THE DEVELOPING NERVOUS SYSTEM 23

have been used to alter development. Among the common approaches used
today are techniques to manipulate tissues in vivo and in vitro and methods to
evaluate naturally occurring and experimentally induced genetic mutations.
Tissue manipulations have been used since the earliest studies of
developmental neurobiology. These methods typically involve surgically
removing or rotating a particular region of the developing embryo or grafting
extra tissue onto a region of the embryo. Several examples of these types of
studies are highlighted in Chapters 2, 4, 7, and 8. Scientists can also observe
effects of tissue manipulations in cell culture preparations. In these assays,
tissues of interest are surgically dissected from an embryo at a given stage of
development and placed into a cell culture dish. The dishes are often coated
with substrate molecules that support the attachment and growth of the cells
under investigation. The tissues are then covered in a nutrient-containing
fluid (cell culture medium). To identify sources of signals that promote the
survival, growth, or differentiation of a neural population, the tissues may be
grown in the presence of other tissues. In some experiments, specific proteins
may be added to test whether they have a direct effect on the developing
cells. Examples using these approaches are discussed in Chapters 4, 7, and 8.
Cell culture techniques to study neural development were introduced in the
1920s and remain a very popular method for analyzing the development of
neural cells. An advantage of cell culture is the ability to test single reagents
on a select population of cells. A limitation to the method is that the artificial
environment removes other tissue-derived cues that may interact with and
alter the effects of the reagent under investigation.
Scientists also observe the effects of additional or missing genes. Such
genetic manipulations have been instrumental in understanding neural
development in both invertebrate and vertebrate animal models. Studies of
naturally occurring gene mutations in Drosophila, C. elegans, and mice have
been documented for nearly a century. A number of these spontaneously
occurring mutations have provided an extensive body of data on the
development of the nervous system. As detailed in Chapter 6, scientists
investigating Drosophila initially relied on naturally occurring mutations,
but soon developed methods to experimentally mutate genes of interest.
Methods for blocking, reducing, or increasing gene expression were also
developed for many vertebrate animal models. Examples of mutations
induced in frogs are found in Figures 2.10, 2.11, and 6.2, while examples from
Drosophila are shown in Figures 7.20 and 7.22. A method to experimentally
delete, or knock out, individual genes in mice was introduced in the 1980s.
The development of a technique for generating gene knockout mice
greatly advanced studies of mammalian neural development (Box 1.2).
Other methods to selectively interfere with gene expression use short
interfering RNAs (siRNAs). Segments of RNA consisting of 20–25 base pairs
that are complementary to a gene sequence of interest are introduced to cells
by electroporation, a method in which an electrical current is used to make
cell membranes more permeable. siRNAs can be electroporated into specific
regions of an embryo, where they degrade the target mRNA and prevent
translation of the protein, thereby providing insight into the normal function
of the protein in vivo. Examples of this approach are shown in Figure 4.10.
Researchers continue to refine techniques to selectively alter gene and
protein expression in cells at specific stages of development, providing finer
resolution of the molecular pathways involved in neural development. There
are limits to these approaches, however, and researchers are aware that
induced changes represent an artificial environment and that complementary
studies are needed to test the role of the molecules during normal
development. Despite the inherent limitations of these approaches, to date
such tissue and genetic manipulation studies have provided considerable
insight into mechanisms underlying normal neural development.

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 24 Chapter 1 An Introduction to the Field of Developmental Neurobiology

Box 1.2 Knockout mice

The term “knockout mouse” is now commonly used this first litter contain cells that arise from both mice.
throughout the scientific literature. The technique has These chimeras, made up of genetic contributions from
become so widely used and discussed that it may be the blastocyst and surrogate mice, can be identified by a
difficult to imagine what a surprising and significant coat color that differs from that of the surrogate mother.
impact it had when it first emerged in the early The male chimeras are then mated with females of
1980s. In fact, when Mario Cappecchi first proposed another coat color, such as white (step 4). The resulting
the technique to a funding agency, the proposal was pups are again selected by coat color to identify those
turned down because reviewers believed the process that carry genes from the ES cells (for example, mice
could not work effectively. The technique relies on the that are black). The DNA from these mice is then
process of homologous recombination—the ability sequenced and those mice that are heterozygous—that
of an inserted DNA sequence to line up in the correct is, the mice that contain one copy of the disrupted gene
orientation and location and replace a specific gene. and one copy of the normal gene—are then mated with
Homologous recombination takes place naturally and littermates that are also heterozygous for the mutation.
frequently in bacteria, yeast, and viruses, but under One quarter of the resulting litter will contain mice
normal conditions is rare in mammalian cells, except that are homozygous for the mutation. These are the
in germ-line and embryonic stem (ES) cells—that knockout mice that contain two copies of the mutated
is, the cells that have the ability to give rise to all the gene and are the ones to be examined for anatomical,
cells in an organism. Mammalian cells are also capable physiological, or behavioral deficits. Other mice in the
of the process when foreign genes are intentionally litter will be normal, or wild-type mice, which carry
inserted, such as occurs in the process of generating two copies of the normal gene, and the others will be
knockout mice. heterozygous.
Figure 1 outlines the steps used to generate mice Depending on the particular gene disrupted, the
lacking a gene of interest. In the first step, the target resulting changes—the phenotype of the mice—can be
gene is removed from a segment of DNA and selector mild, suggesting that another gene compensates for
genes are inserted to create a targeting vector. Two the loss of the targeted gene, or severe, sometimes
commonly used selector genes are the neomycin even resulting in death of the embryo prior to birth. As
resistance (neoR) gene—a positive selector gene—and scientists have found over the years, the heterozygous
the herpes thymidine kinase (tk) gene—a negative mice often have a milder phenotype than the
selector gene. The neor gene is flanked by DNA present homozygous mice, displaying a gene dosage effect—
in the target gene, while the tk gene is located outside that is, those with one copy disrupted are impacted
the targeted sequence. less than those with two copies disrupted. Embryonic
In the second step, the target vector is introduced into lethal mutations can sometimes provide information
mouse embryonic stem (ES) cells. Electroporation on the role of the gene, depending on the stage when
provides a small electrical charge that opens the the embryos die. Such severe mutations are often
cell membranes and permits entry of the DNA. The of limited value, however, particularly if the embryo
cells are grown in a culture medium that contains dies prior to the onset of gene function in the cell
the drugs neomycin and glanciclivor. Cells that have population of interest.
inserted the neoR gene in place of the targeted gene A refinement to the gene knockout technique was
will survive in the medium containing the antibiotic introduced in the late 1980s that allows for researchers
neomycin. Glanciclivor will kill any cells that retain the to delete a gene in selected tissues or at specific
tk gene; thus, the cells that have randomly inserted the stages of development and therefore overcome the
targeting vector outside the gene sequence of interest limitations of deleting a gene in every cell of the body.
will be eliminated. By using both positive and negative The basic method used to create such conditional
selector genes, all or nearly all of the cells surviving in knockout mice involves inserting loxP (locus of
the culture medium will be those with the targeted gene X-over P1) in noncoding regions of the DNA sequence
disrupted. of interest using homologous recombination. These
The remaining ES cells are then injected into blastocyst- segments are said to be “floxed” (flanked by loxP).
stage mouse embryos from mice of a particular coat color The floxed sites of the DNA are recognized by Cre
(Figure 1, step 3). The blastocysts are implanted into a recombinase that mediates the exchange of DNA.
surrogate, or foster, female mouse of a different coat The conditional expression of the gene is regulated
color to develop to term. The tissues of the pups from in one of two ways—namely, Cre recombinase can

9780815344827_Ch01.indd 24 13/10/17 2:05 pm

 GENE REGULATION IN THE DEVELOPING NERVOUS SYSTEM 25

step 1 designing the target vector

target genes and
flanking sequences
homologous target homologous
DNA 1 gene DNA 2

neoR tk

neoR tk
targeting
vector
homologous homologous
DNA 1 DNA 2

step 2 inserting the targeting vector into ES cells

in some cells, the targeting vector in other cells, the targeting vector
recombines with the target gene and recombines in the wrong place, a
knocks out one copy of the target gene random section of the chromosome

knocked-out neoR neoR tk random

gene sequence chromosome
result: cells with knocked-out gene are result: cells with random recombination are
– neomycin-resistant – neomycin-resistant
– ganciclovir-resistant (no TK) – ganciclovir-sensitive (TK present)

step 3 step 4
injecting cells into breeding
a new embryo

Cells that have successfully The resulting chimeric (spotted) mouse Among their offspring are
incorporated the target vector contains a mix of its own cells and the mice that are capable of
are then selected and injected heterozygous knockout cells. This passing the knockout gene to
into a normal developing mouse is bred with a normal (white) their own offspring.
mouse embryo. mouse.

Figure 1 The creation of a knockout mouse. An outline of the steps used to generate mice with the targeted gene disruption.

be linked to tissue-specific promoters or to inducible population or at a specific time in development. A

proteins. Thus, depending on the method used, number of modifications to these initial knockout and
the gene of interest will only be altered in selected conditional knockout methods have been made since
tissues or only at the developmental stages in which they were first introduced over 25 years ago so that
it is experimentally induced, thus allowing researchers researchers now have the ability to track, delete, or
to investigate the role of a gene in a selected cell overexpress multiple genes in a single animal.

dn Box 1.02 Fig 1

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 26 Chapter 1 An Introduction to the Field of Developmental Neurobiology

Summary

In the past 125 years, scientists have made remarkable progress in

identifying the many mechanisms that lead the human nervous system to
develop from a simple region of ectoderm into a complex nervous system
comprised of hundreds of billions of neurons and supporting cells. These
insights have come from several vertebrate and invertebrate animal models
and ongoing studies continue to provide more details regarding the types
of signals used to form the human nervous system.
The development of the nervous system as a whole, as well as of
specific subtypes of neural cells, often occurs through processes that
intentionally designate or actively restrict tissues or cells to a particular fate
option. Chapter 2 begins by explaining how a specific region of ectoderm
is designated to form neural tissue rather than epidermal tissue. Chapters
3 and 4 describe mechanisms that regulate patterning along the anterior–
posterior and dorsal–ventral axes of the neural tube. Such patterning
establishes early anatomical boundaries and establishes restricted regions
where signals that promote or inhibit subsequent developmental events
originate. Mechanisms that influence cellular migration (Chapter 5) and
cellular fate options (Chapter 6) are then described, thus demonstrating
how further specialization of neuronal subtypes are established throughout
the nervous system.
Nervous system development depends not only on the formation
of highly specialized cell types, but also on the formation of precise
connections between individual neurons. Chapter 7 explains how axons
extend through the developing embryo to establish initial connections.
Because the nervous system of vertebrates often over-produces the
number of neurons necessary, additional signals are needed to determine
which cells and connections persist and which are eliminated. Chapter 8
describes how neurons are selected for survival or death, while Chapters 9
and 10 discuss how synapses are formed and select connections are lost at
the neuromuscular and CNS synapses, respectively.
It is thought that some of the same signals that underlie development
of the nervous system may also influence repair or regeneration of
neural tissues in the adult. Regeneration of many nerves is common in
invertebrates; and peripheral nerves regenerate, at least to some extent,
in vertebrates. Thus, knowledge gained from studies of developmental
neurobiology has the potential to lead to clinical treatments to enhance
nerve regeneration and possibly treat neurodegenerative diseases such as
multiple sclerosis, spinal cord injuries, amyotrophic lateral sclerosis (ALS),
Parkinson’s disease, Alzheimer’s disease, and others.

Further Reading
Alberts B, Johnson A, Lewis J et al. (2015) Molecular Biology of Gilbert SF (2014) Developmental Biology, 10th ed. Sinauer
the Cell, 6th ed. Garland Science. Associates.
Capecchi M (1994) Targeted gene replacement. Sci Amer Hamburger V & Hamilton HL (1951) A series of normal stages
270(3)52–59. in the development of the chick embryo. J Morph 88(1):49–92.
Diaper DC and Hirth, F (2014) Immunostaining of the Haretenstein V (1993) Atlas of Drosophila Development. Cold
developing embryonic and larval Drosophila brain. In Brain Spring Harbor Laboratory Press.
Development Methods and Protocols (Sprecher SG ed), pp
Kimmel CB, Ballard WW, Kimmel SR et al. (1995) Stages of
3–17. Humans Press.
embryonic development of the zebrafish. Dev Dyn 203:253–310.
Friedel RH, Wurst W, Wefers B and Kuhn, R. (2012) Generating
Luo L (2016) Principles of Neurobiology. Garland Science.
conditional knockout mice. In Transgenic Mouse Methods and
Protocols, Methods in Molecular Biology. (Hofker MH & van Nieuwkoop PD & Faber J (1967) Normal Table of Xenopus
Deursen JM eds), pp 205–231. Humana Press. laevis. Elsevier.

9780815344827_Ch01.indd 26 13/10/17 2:05 pm

 Online sources for atlases of embryonic development 27

Oikonomou G & Shaham S (2011) The glia of Caenorhabditis Stix G (1999) Profile (Mario Capecchi): Of survival and science.
elegans. Glia 59:1253–1263. Sci Amer 281(2):26–27.
Sadler TW (2014) Langman’s Medical Embryology, 13th ed. Vanderah TW & Gould DJ (2016) Nolte’s The Human Brain: An
Wolters Kluwer Health. introduction to its Functional Anatomy, 7th ed. Elsevier.
Schoenwolf GC, Bleyl SB, Brauer PR & Francis-West PH (2014) Westerfield M (1993) The Zebrafish Handbook, 2nd ed.
Larsen’s Human Embryology, 5th ed. Churchill Livingstone. University of Oregon Press.
Stern CD & Holand PWH (1993) Essential Developmental
Biology. IRL Press.

Online sources for atlases of embryonic development

The Endowment for Human Development: http://www. Xenbase

ehd.org/virtual-human-embryo/stage.php http://www.xenbase.org/anatomy/alldev.do

eMouseAtlas Atlas of Drosophila Development

https://www.emouseatlas.org/emap/ema/theiler_ http://www.sdbonline.org/sites/fly/atlas/00atlas.htm
stages/StageDefinition/stagedefinition.html

C. elegans
eChickAtlas
Altun, Z. F. and Hall, D. H. 2005. Introduction to C. elegans
http://www.echickatlas.org/ecap/hamburger_hamilton_ anatomy. In WormAtlas. http://www.wormatlas.org/
stages/hamburger_hamilton_stages.html ver1/handbook/anatomyintro/anatomyintro.htm

The Zebrafish Information Network

https://www.zfin.org/zf_info/zfbook/stages/stages.
html

9780815344827_Ch01.indd 27 13/10/17 2:05 pm

 Neural Induction
2
T
he first step in neural development is neural induction, the process
by which undifferentiated embryonic tissue is specified to develop as
neural tissue. Once specified, neural regions continue to differenti-
ate and specialize, eventually generating the many distinct regions of the
nervous system. The steps involved in creating a functional nervous system
are numerous and complex, and the process of neural differentiation begins
very early in development. In fact, it is often within the egg cell itself that
cellular regions competent to become neural tissue are first designated. Seg-
regating neural regions at the earliest stages of development prepares cells
to receive the correct signals at the right time so that normal development
can occur. The following quote from Hans Spemann, a pioneer in the study
of neural induction, emphasizes the importance of embryonic organization
and segregation in the development of neural tissues:

“We are standing and walking on parts of our body which we

could have used for thinking if they had been developed in
another position in the embryo.”
Hans Spemann, 1924

Neural induction has been the focus of research efforts for well over a
century. As detailed below, these processes remain highly conserved
across vertebrate species, though certain differences are noted and will
be outlined in the examples that follow. Despite significant differences in
nervous system morphology, the signaling molecules used to designate
neural tissues are largely homologous in vertebrates and invertebrates,
demonstrating the evolutionary conservation of this fundamental, early
process of neural development.

THE ESTABLISHMENT OF NEURAL TISSUE

DURING EMBRYOGENESIS

During the earliest stages of development, specific areas of the vertebrate

embryo are designated to form neural tissues. Even prior to fertilization, an
egg cell is compartmentalized as a first step in establishing neural tissue. In

DevNeuro_Chapter02.indd 29 13/10/17 2:42 pm

 30 Chapter 2 Neural Induction

ANIMAL POLE egg cells from amphibians, for example, the cytoplasm is segregated into a
lighter pigmented vegetal pole and more densely pigmented animal pole
(Figure 2.1). The vegetal pole gives rise to gut structures, whereas the
DORSAL animal pole gives rise to the nervous system and the epidermis (the surface
layer of skin). In animals with large eggs, such as amphibians, cytoplasmic
VENTRAL differences are easily identified prior to fertilization, and subsequent
rearrangements in the cytoplasm are observed following fertilization.
In other vertebrates, including mammals and birds, the cytoplasmic
VEGETAL POLE differences are not as obvious, even after fertilization.
Following fertilization, the egg cell (zygote) divides into a number
Figure 2.1 Regions competent to become
neural tissue are established in the egg of cells called blastomeres that surround a central cavity known as a
cell. Eggs from amphibians are divided blastocoel (Figure 2.2). A group of blastomeres that aggregate above the
into animal and vegetal poles that go on to cavity is called the blastoderm. This entire structure—that is, the cells and
establish areas that will give rise to specific the hollow cavity they surround—is known by different names, depending
tissues. The animal pole appears denser due
on the species: the blastula in amphibians, the blastocyst in many mam-
to the accumulation of cytoplasmic lipids
dn 2.01/2.01 mals, and the blastodisc in birds, fish, and some mammals. The difference
and granules. This pole will later give rise
to epidermis and neural tissues. The lighter in terminology refers to the morphology of the cells. In birds, for example,
appearing vegetal pole will later give rise to the cells form a disc-like structure that is distinct from the cyst-like mor-
structures associated with the gut.
phology observed in mice. In many cases, the term “blastula” is used as a
generic term for all species.
The amphibian blastula-stage embryo (Figure 2.2A) is spherical,
and like the egg itself, the embryo at this stage is characterized by animal
and vegetal poles. In birds (Figure 2.2B), the blastodisc lies above the
yolk, where it forms two sheets of cells designated the epiblast and
hypoblast—regions that roughly correspond to the animal and vegetal
poles, respectively. The epiblast and hypoblast are also found in mammals,
however the size of the yolk, a source of nutrients for the embryo, is much
greater in birds than in mammals. In fact, in most mammals the size of
the yolk is negligible due to maternal sources of nutrients. Yet, despite
species differences in blastula shape and yolk size, the next step in early
development is comparable across vertebrate species.

Gastrulation creates new cell and tissue interactions that

influence neural induction

Gastrulation is the next crucial stage in early embryogenesis—the stage

during which cells first begin to reorganize into three germ cell layers to form
specific tissues. These layers, the ectoderm, mesoderm, and endoderm, are
called the germ cell layers or primary cell layers, because it is from
these three layers that all tissues of the body arise. Gastrulation involves
the migration, or invagination, of cells through an indentation on the outer

Figure 2.2 Comparison of blastula

formation in amphibians and birds. Following (A) ANIMAL POLE (B) ANIMAL POLE
fertilization, the egg cell divides repeatedly
and the resulting new cells are called blastoderm blastoderm epiblast
blastomeres that surround a hollow, fluid-
filled cavity, the blastocoel. The consolidated blastomere
group of cells above the blastocoel is called
the blastoderm. In frogs (A), the blastula is a
spherical structure, whereas in birds (B), the blastocoel
blastula, also called the blastodisc, appears
as a flattened sheet of cells overlying the yolk, yolk
with the blastocoel cavity formed between.
The two layers of cells in the blastodisc are hypoblast
termed the epiblast and hypoblast, regions yolk cells
that are roughly equivalent to the animal and
vegetal poles. (Adapted from Patten BM [1958] VEGETAL POLE VEGETAL POLE
Foundations of Embryology. McGraw-Hill.)

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 THE ESTABLISHMENT OF NEURAL TISSUE DURING EMBRYOGENESIS 31

surface of the developing embryo. Here again, the terminology varies

according to species. This site of indentation is called the blastopore in
amphibians, the embryonic shield in zebrafish, and the primitive streak
in birds and mammals.
As surface cells begin to migrate through the surface indentation (that
is, the blastopore, embryonic shield, or primitive streak), new arrangements
of the germ cell layers occur (Figure 2.3A, B). In all cases, as a result of
gastrulation, the endoderm forms as the innermost germ cell layer and
gives rise to the gut and organs associated with it. The mesoderm forms as
the middle layer and gives rise to muscle, bone, connective tissues, and the
cardiovascular and urogenital systems. The outermost layer, the ectoderm,
remains at the embryo’s surface (animal pole or epiblast) and gives rise to
the epidermis and nervous system. In all of these animal models, during a
limited period in the late gastrula-stage embryo, the ectodermal cells have
the potential to become either epidermal cells or neural cells.
Figure 2.3C–E shows sketches of amphibian gastrulation modified
from the original 1929 drawings by Walter Vogt. The sketches provide
Figure 2.3 Transition from the blastula- to
(A) ANIMAL POLE (B) the gastrula-stage embryo in amphibians.
(A) A surface view of an early stage amphibian
blastula outlining the general areas of
presumptive ectoderm, mesoderm, and
ectoderm endoderm. The animal pole will give rise to
ectoderm (blue), the vegetal pole will give rise
to endoderm (green), while the mesoderm
(light red) will arise from the middle segment.
mesoderm (B) Schematic fate map shown from the dorsal
surface of a blastula-stage amphibian embryo
endoderm just prior to the onset of gastrulation. The sight
of blastopore formation (white) indicates the
blastopore area through which surface cells will migrate
VEGETAL POLE (arrows). The regions fated to become the
head process/notochord three germ layers (ectoderm, mesoderm, or
endoderm) and the head process/notochord
(C) (D) (brown) are also shown. (C–E) show sagittal
nonneural presumptive sections through the center of the blastula
ectoderm neural plate to reveal the inward movements of cells and
ectoderm the subsequent arrangement of tissue layers
notochord
as gastrulation progresses. Dorsal is to the
endoderm cells right in all panels. The arrows indicate the
direction of cellular movements. As the cells
of the presumptive endoderm (green) and
mesoderm (light red) push inward through the
blastopore (C, D) the primitive gut (gastrocoel)
is established (E). At the dorsal surface (C),
chorda- the chordamesoderm (axial mesoderm) also
mesoderm migrates inward, forming the notochord
blastopore (brown, D) that extends below the forming
neural plate ectoderm (blue, C–E). The cells
of the ventral surface ectoderm will give
mesoderm mesoderm
rise to the nonneural, epidermal ectoderm
(yellow). The mesoderm-derived dorsal and
ventral blastopore lips at the margins of
(E) endoderm cells neural plate ectoderm (F) neural the blastopore are seen in panel E. Signals
ectoderm emanating from the dorsal blastopore lip
(shown in gray) are critical for formation of
epidermal gastrocoel or neural ectoderm. (F) Surface view showing
ectoderm primitive gut epidermal regions of neural (blue) and epidermal
ectoderm (yellow) ectoderm. (B, adapted from Nagy A,
notochord
Gertsenstein M, Vinterstan K & Behringer R
dorsal lip of [2003] Manipulating the Mouse Embryo, 3rd
reduced blastopore ed. Cold Spring Harbor Press; C–E, adapted
blastocoel from Voght W (1929) Gestaltungsanalyse am
ventral lip of amphibienkeim mit örtlicher vitalfarbung. II.
blastopore Teil. Gastrulation und mesodermbildung bei
yolk-laden mesoderm oroden und anuren. Wilhem Roux’ Arch 120:
endoderm cells 385–706.)

DevNeuro_Chapter02.indd 31 13/10/17 2:42 pm

 32 Chapter 2 Neural Induction

remarkable details of the cellular rearrangements as they were viewed

through the microscope at different stages. For example, the early migration
of cells through the blastopore (Figure 2.3B) leads to the creation of the
endoderm layer (Figure 2.3C, D) and the primitive gut (Figure 2.3E). In fact
the word gastrulation comes from the Greek word gaster meaning stomach.
Other cells then migrate through the blastopore and come to lie between
the endoderm and surface ectoderm, thus forming the mesoderm. At the
dorsal side, a specified group of these cells migrates inward to form a
band of chordamesoderm (also called the axial mesoderm). This band of
mesoderm gives rise to the notochord, a transient embryonic structure that
lies below the surface ectoderm and is important for subsequent aspects of
neural development (Figure 2.3D, E). At the opening of the blastopore, the
margins form the mesoderm-derived dorsal and ventral blastopore lips. As
described below, it is the dorsal blastopore lip (DBL) that is critical for
designating neural ectoderm from epidermal ectoderm, the two cell types that
remain at the outermost surface of the embryo (Figure 2.3E, F). Gastrulation
in zebrafish would be similar to amphibians, because the embryonic shield
more closely resembles the blastopore than the primitive streak.
As a comparison to amphibian gastrulation, Figure 2.4 shows
gastrulation in the chick embryo, a process that is similar in human embryos.
The outer surface, the epiblast, gives rise to all of the future cell layers (Figure
2.4A). A portion of the cells migrates through the primitive streak to form
the middle mesoderm and innermost endoderm layers. As the endoderm
Figure 2.4 Gastrulation in chick embryos.
forms, the hypoblast that lies just above the yolk is displaced (Figure 2.4B).
(A) A surface view of a chick blastodisc at
approximately 16 hours of development. The The hypoblast cells will give rise to extraembryonic tissues—tissues that
surface cells (epiblast) give rise to all of the
future germ layers. The primitive streak, which
(A) CEPHALIC END (B)
forms the longitudinal axis of the embryo, primitive
establishes the site where cells migrate inward prechordal plate streak epiblast
during gastrulation. Hensen’s node, located at
the anterior end of the primitive streak, is the epiblast
equivalent of the amphibian dorsal blastopore
Hensen’s node
lip. The area of the prechordal plate, where
ectoderm and endoderm adhere, is outlined
anterior to Hensen’s node. (B) A cross section
of a chick embryo demonstrates how the primitive
surface cells of the epiblast (gray) migrate streak
medially into the opening of the primitive hypoblast
migrating
streak, then migrate laterally to establish cells
the mesoderm and endoderm layers. The CAUDAL END mesoderm endoderm
hypoblast, lying above the yolk, is displaced displacing
as the endoderm forms. The hypoblast later hypoblast
gives rise to extraembryonic tissues. (C) Cells
migrating inward along the midline give rise
to the notochord, which lies below the future (C) prechordal plate (D)
neural plate (not shown). As more cells migrate
inward, the primitive streak regresses. (D) The epiblast neural ectoderm
cells of the epiblast that remain at the surface
form epidermal (yellow) and neural (blue) notochord
ectoderm, with the neural tissue forming epidermal ectoderm
anterior to the primitive streak. (E) A sagittal Hensen’s node
section through the embryo reveals the
orientation of the neural tissue and primitive primitive
streak as well as the prechordal and notochord streak Hensen’s node
regions. The notochord lies beneath the neural
plate tissue. The prechordal plate is formed primitive streak
where the surface ectoderm and underlying
endoderm form a tight junction. The
(E) prechordal neural plate
prechordal plate limits the anterior migration
plate tissue
of notochord cells. Gastrulation in humans and
other mammals that form a blastodisc is similar ectoderm primitive streak
to that of chick. (A, adapted from Patten BM
[1958] Foundations of Embryology. McGraw- endoderm notochord
Hill; C, E adapted from Balinsky BI [1975] An
Introduction to Embryology, 4th ed. Saunders.)

DevNeuro_Chapter02.indd 32 13/10/17 2:42 pm

 EARLY DISCOVERIES IN THE STUDY OF NEURAL INDUCTION 33

(A) epiblast mesoderm (B) (C) neural epidermal

ectoderm ectoderm
extraembryonic anterior
region visceral
endoderm posterior
embryonic visceral
region endoderm
primitive
streak
primitive
streak
migrating
cells epiblast mesoderm notochord

mesoderm ANTERIOR endoderm POSTERIOR

node

visceral definitive endoderm

endoderm

are not part of the embryo proper, but contribute to the maintenance of the Figure 2.5 Gastrulation in mouse embryos
embryo. Similar to amphibian embryo, a specified group of cells migrates is similar to that in the chick. If the blastocyst
of a mouse embryo is flattened out, the
inward along the midline to form the notochord. As the notochord elongates movements of cells appear similar to those
and the germ layers are established, the primitive streak regresses (Figure of the blastodisc. (A) The blastocyst with part
2.4C, E). At the anterior region of the epiblast, the prechordal plate forms of the posterior region cut away to reveal the
(Figure 2.4A, C, E). This is an area where the surface ectoderm adheres to the orientation of the forming cell layers. If the
dn N2.100/2.05 blastocyst were cut open along the dashed
underlying endoderm. This tight association prevents cells of the notochord
lines and flattened out, the epiblast would
from migrating further anteriorly (Figure 2.4E). The epiblast cells remaining at be on the surface and the migration of cells
the surface form neural ectoderm and epidermal ectoderm. The neural tissue (arrows) would be appear similar to the those
forms as a sheet of cells that arises and extends from the anterior portion of of the chick blastodisc. (B) A sagittal section
through the cup-shaped blastocyst of the
the primitive streak. Like the dorsal blastopore lip, Hensen’s node, a group
mouse embryo reveals the epiblast in the
of cells at the anterior (cephalic) end of the primitive streak, is necessary inner region. The cells of the epiblast migrate
for development of neural ectoderm (Figure 2.4D, E), as is the node, the through the primitive streak that begins at the
equivalent structure in mammals. junction of the extraembryonic and embryonic
regions. The migrating epiblast cells begin to
Gastrulation in mice is a bit more difficult to envision because the
form germ layers between the epiblast and
epiblast is found inside the blastocyst, a cuplike structure (Figure 2.5). If visceral endoderm. The visceral endoderm
one imagines the blastocyst cut open (dashed line, Figure 2.5A) and flat- does not form part of the definitive endoderm
tened out, the similarities between gastrulation in a blastodisc and blasto- of the embryo, but is an extraembryonic
tissue that is replaced by cells that form the
cyst are more apparent. The mouse blastocyst is surrounded by visceral
endoderm layer, similar to the hypoblast in the
endoderm (VE). Similar to cells of the blastodisc, the epiblast cells of the chick. The node is located at the anterior end
blastocyst migrate through the primitive streak to form the mesoderm and of the primitive streak. (C) A section through
endoderm layers between the epiblast and VE (Figure 2.5A, B). The VE does the blastocyst at a later stage of development
not become part of the definitive endoderm and is similar to the hypoblast in reveals the orientation of the neural ectoderm,
epidermal ecdoderm, and notochord. The
that it becomes displaced by the forming endoderm germ layer. As described anterior visceral endoderm underlies the
in Chapter 3, the anterior visceral endoderm (AVE) comes to lie below the anterior portion of the neural tissue. (Adapted
anterior portion of the neural ectoderm and plays a role in defining regions from Nagy A, Gertsenstein M, Vinterstan K &
of neural tissue (Figure 2.5C). Epiblast cells that remain at the inner surface Behringer R [2003] Manipulating the Mouse
Embryo, 3rd ed. Cold Spring Harbor Press.)
of the blastocyst will form either the neural or epidermal ectoderm (Figure
2.5C). As noted above, it is during a specified period in the late gastrula-stage
embryo that ectoderm can give rise to either neural or epidermal tissues.
Precisely how different ectoderm cells are designated to become either neu-
ral or epidermal has been an active area of research for over a century.

EARLY DISCOVERIES IN THE STUDY OF

NEURAL INDUCTION

By the early twentieth century, embryologists had established that dorsal

ectoderm forms neural plate tissue, whereas ventral ectoderm forms
epidermal tissues. What was not clear was how the ectoderm in these
two regions became specialized as one tissue or the other. As scientists

DevNeuro_Chapter02.indd 33 13/10/17 2:42 pm

 34 Chapter 2 Neural Induction

began to focus greater effort into identifying the origins of neural tissue,
a groundbreaking discovery was made that indicated specific cells in one
embryonic region can signal, or induce, dorsal ectoderm to become neural.

Amphibian models were used in early neuroembryology

research and remain popular today

Many of the first studies of neural induction were conducted using amphibian
embryos, particularly those from salamanders and frogs. Frogs remain a
popular animal model today, in part because of the comparatively large size
of their eggs and early-stage embryos. For example, the frog egg is about
2–3 mm in diameter compared to the 0.1–0.2 mm diameter of a human egg.
This larger size makes cellular regions easier to identify and experimentally
manipulate. Although such manipulations are more easily accomplished with
the larger eggs and embryos of amphibians, early experiments still required
considerable creativity (for example, designing tiny glass needles to use as
surgical knives and using a baby’s hair to separate blastomeres), tremendous
patience, and very steady hands. The technical accomplishments of these
first experiments, now almost a century old, are nearly as impressive as the
scientific hypotheses that resulted from the studies.
A popular experimental method refined in the early twentieth century,
and still used today, relies on grafting tissues from one embryo to another.
This approach has proved critical to advancing our understanding of nervous
system formation. Among the first studies conducted to evaluate neural
specification of embryonic tissues were those in which scientists harvested
tissue from one region of an early gastrula-stage amphibian embryo (the
donor) and grafted it to another region of a second embryo at the same
developmental stage (the host). The host embryo continued to develop
and the effects of the donor and host tissues on subsequent developmental
events were then evaluated. It was one such grafting experiment that first
revealed the source of signals needed to induce neural tissue.
Figure 2.6 The classic grafting experiment
performed by Hans Spemann and Hilde A region of the dorsal blastopore lip organizes the
Mangold. The dorsal region of a blastopore amphibian body axis and induces the formation of
lip from a pigmented donor embryo was neural tissue
grafted onto a nonpigmented host embryo’s
ventral surface, an area of ectoderm that does In 1924 Hans Spemann and Hilde Mangold published what has become
not give rise to neural tissue. As development
proceeded, a neural plate and body axis from one of the most cited studies in developmental neurobiology. In this study,
the host embryo developed along the dorsal the researchers completed grafting studies using two different species of
surface as expected. In addition, a second newts. The pigmentation of the embryos from these two newts differed,
neural plate and body axis formed from the allowing scientists to visualize the fate of donor and host tissues following
ventral tissue of the host. This result indicated
that the donor dorsal blastopore lip did not surgical manipulations. Spemann and Mangold grafted the dorsal
expand to form a new body axis, but instead blastopore lip (DBL) of a pigmented, early gastrula-stage embryo to the
produced signals that induced host ventral ventral side of a nonpigmented embryo (the host) at the same stage of
ectoderm to form an entirely new body axis development (Figure 2.6). The ventral side of the embryo normally gives
complete with neural tissue.

dorsal blastopore lip

DORSAL DORSAL

blastopore VENTRAL VENTRAL neural plates

PIGMENTED NONPIGMENTED
DONOR HOST

DevNeuro_Chapter02.indd 34 13/10/17 2:42 pm

 EARLY DISCOVERIES IN THE STUDY OF NEURAL INDUCTION 35

rise to epidermal but not neural tissue. At this early stage of gastrulation,
however, the regions of epidermal and neural ectoderm have not yet been
specified, so the investigators were able to evaluate any inductive effects
of the DBL. Spemann and Mangold discovered that the host embryos
continued to develop and also formed a second body axis, complete with
neural tissue. The key observation from this experiment was that the
new body axis formed primarily from the nonpigmented, host embryo.
This indicated that the ventral tissue of the host—ectoderm that normally
becomes epidermal—was induced to form neural tissue when provided
with a signal originating in the DBL. Thus, the second body axis did not
result by a simple expansion of the pigmented donor dorsal lip tissue, as
was presumed to be the case based on earlier grafting studies conducted
by other investigators.
The band of DBL that induced the formation of new neural tissue
came to be called Spemann’s organizer because it organized the entire
body axis. Spemann and his students, as well as investigators in many
other labs, continued to explore the mechanisms that governed neural
induction. Spemann ultimately received the Nobel Prize in Physiology or
Medicine in 1935 for his work in this area. Tragically, Hilde Mangold died
following a household explosion in 1924, when she was only 26 years old,
and was unable to witness the continuing impact of her work. Her husband,
Otto Mangold, also a student of Spemann’s, went on to make additional
contributions regarding the mechanisms underlying the processes of cell
determination and induction of embryonic tissues.
The intriguing discovery of a neural inducer originating in the
organizer—that is, the DBL of amphibians as well as its equivalent in other
species, such as Hensen’s node of the chick—led to numerous studies
investigating the nature of this signal. Additional grafting experiments
conducted in the 1930s noted that the signal arising from the organizer was
not species-specific. For example, when grafts of organizer tissue from duck
were transplanted to chick, neural tissues formed. Similarly, when grafts
of chick organizer tissue were transplanted to either duck or rabbit, neural
tissues were induced. Thus, the signal appeared to be universal in its ability
to induce the formation of neural tissue from early-stage ectoderm. Other
grafting experiments in the 1930s revealed that the medial or lateral portions
of the amphibian DBL preferentially gave rise to head or tail structures,
respectively. This suggested the possibility of multiple signals available to
direct the formation of specific neural regions along the anterior–posterior
body axis of the embryo (see Chapter 3).

The search for the organizer’s neural inducer took

decades of research

For almost 100 years, scientists have investigated the signals governing
neural induction, using an ever-expanding array of experimental
approaches. It is hard to capture in just a few paragraphs the extensive
efforts made and the number of scientific debates and alternative
hypotheses put forth during the search for putative neural inducers.
One serious obstacle to identifying neural inducers is the very small
size of the organizer region. The small tissue size prevented scientists
from isolating the signaling molecule directly; to overcome this problem,
researchers screened other tissues for larger and more plentiful sources
of inductive activity, tested other substances for inductive properties, and
developed new ways to determine how the signal reached the ectoderm.
In the course of various studies, scientists determined that the neural
inducer was diffusible and that, surprisingly, any number of biological and
nonbiological substances induced competent undifferentiated ectoderm to
form neural tissue. For example, tissues that were first boiled to denature

DevNeuro_Chapter02.indd 35 13/10/17 2:42 pm

Another random document with
no related content on Scribd:
should infer, they conceived white people lived, like humming birds,
upon suction.
On leaving the rajah’s place, my guides took me again to the
bazaar, where it appears to be a custom to take strangers: this I
attribute to their Mahometan prejudices, of not being desirous of
receiving Christians under their roofs. Here mats were placed, so
that I might be seated, and gazed at, like a curious animal, by a
large crowd of natives of all classes and orders, who, from the
eagerness they evinced, and the crowds which assembled around us
upon these occasions, seemed to regard Europeans as curiosities.
However, instead of waiting to be gazed at, I amused myself by
wandering over the bazaar, which was plentifully supplied with
sugar-canes, plantains, rice, cucumbers, dried fish, sere, (the leaf of
the piper betel,) the Areka nut, or Pinong, cut up ready for
mastication, and a quantity of live stock, as small bullocks, ducks,
fowls, &c. &c.
From the bazaar I walked down by the banks of the river, upon
the raised paths which intersected the numerous marshes, which
now, during the dry season, abounded in luxuriant grass and other
herbaceous plants, affording fine feeding for the numerous bullocks
(of the small hunch-backed Bengal breed) and buffaloes, which
roamed about. During the rainy season the whole of this flat is
planted with rice, which, together with the scattered picturesque
habitations, and groups of palms and other trees, form, by their
combination, a very pleasing landscape. Upon the banks of the river
was the Acrostic humaureum, or “Ongpi” of the natives, as well as
the “Ba, jurugu,” or Acanthus ilicifolius, covered with a profusion of
blue flowers; and brilliant butterflies and other insects flew about the
rich vegetation, which was so profusely strewed about. Surrounding
a hut near the river was the “Sekar,” a species of Pandanus, the
younger leaves of which several women were engaged in collecting:
they are bleached by soaking in water, and afterwards exposing
them to the heat of the sun. Being thus prepared, they manufacture
them into various kinds of coarse mats.
 The Thespesia populnea, profusely covered with its large yellow
flowers, and called “Onseran” by the natives, was very common
about their habitations, forming usually a portion of the fence
around their gardens. A leafless species of the Euphorbiaceæ family,
which they named “Bugar,” was also growing plentifully in the
hedges: they did not use it medicinally, but said, if the juice was
taken internally, it would produce violent pain and excessive
vomiting. Having arrived at a fisherman’s station, we crossed over a
creek in one of the large fishing boats, in which the seine was very
large, and manufactured from the fibres of the trunk of a palm,
(which I shall hereafter have occasion to mention,) this fibrous
material is known by the common name of “black coir;” it is strong,
elastic, and very durable.
A number of natives were fishing upon the banks of the river with
their peculiar hand-nets, called “Gniap:” this net is of a similar
appearance, but of course smaller, to that used in the “Sarambeau
fishing rafts,” at Manilla, of which there is a very correct figure in the
Voyage of La Perouse, 8vo. Engl. ed. vol. ii. p. 322. On examining
the contents of the baskets, which were rudely formed from the
spathe of the Areka palm, they were found to contain only a few
small fish, prawns, and biongs, or crabs. On their success, my native
attendants informed me, the fishermen depend for their daily meal.
During the rice and betel nut harvest, they earn their subsistence by
cutting and threshing the former, and gathering and shelling the
latter; but when the season for those productions has passed, they
depend upon the fish caught with the hand-net, as a subsistence for
themselves and families.
I stood by one of them to see “a haul:” after a short time had
elapsed, the heavy net was raised, and contained only a solitary fish
and a few crabs. The nets were baited with crabs’ claws, tied about
different parts. On a marsh near this spot a flock of two kinds of
crane was feeding; one species small and white, and named “Ecuar,”
the other much larger, of a greyish colour, and named “Ngnar,
ngnar,” by the natives.
 Fruit was at this season scarce, a few guavas, plantains, and
“jack,” was all that could be procured; but during the season,
mangoosteens, a variety of plantains and bananas, oranges, pine-
apples, mangoes, and other tropical fruits, could be procured in
abundance. Having ranged about the Pedir Rajah’s district, near the
sea coast, I returned on board in the evening with the collection I
had made.
Among the natives that occasionally came on board with the cargo
boats, as well as those seen on shore, consisting of different races of
Hindoostan, Malays, &c. there were several with the African features
and hair; none of whom, however, were well-formed or handsome
men, but still seemed to possess great muscular power. They were
of that African race designated the “Black Arabs,” who are shipped
as seamen on board vessels at Bushire and other places in the
Persian Gulf. When I was looking at this variety of the human race,
one of the rajah’s followers said he was the property of the rajah,
and he would sell him to me, if I wanted him. As I did not require a
specimen of that kind, I declined this very obliging offer. The land
and sea breezes were for some days very regular, and at others
extremely irregular, varying also in their degree of strength. The
range of the thermometer, during the short period I remained on this
coast, was from 79° to 88°.[150]
Early one morning, a party was formed, to endeavour to obtain a
view of the country further inland. On landing at the village of Pedir,
we were met by the old trading minister, who accompanied us. The
houses of the natives were constructed of bamboo, raised, like all
the Malay residences, upon strong posts, a short distance from the
ground, and the ascent to the rooms above, was by bamboo ladders.
The habitations are covered with a thatch, formed from the leaves of
different kinds of palms; and the dwellings are cool. This quality, so
desirable in sultry climates, is given to them by gardens surrounding
the habitations, filled with trees, imparting a refreshing verdure; and
from the blossoms delightful odours were exhaled. Among the more
elevated kinds, were the graceful and majestic cocoa-nut, and the
straight Areka palm, (Areka catechu,) surmounted by its tuft of dark-
green foliage, and its long pendent clusters of orange-coloured fruit,
of an oval form.
The Artocarpus incisa, or Jack-tree, the broad-leaved plantain, the
mango, orange, lime, and occasionally, but rarely, the bread-fruit
trees, (A. integrifolia,) ornamented the garden. That most elegant as
well as largest of the gramineous plants, the bamboo, (“Triang” of
the natives,) was abundant, as fences about many of the dwellings,
(as well as the Erythrina corollodendron, or Mangkudu of the
natives; the Jatropha curcas, or “Bánawa” of the natives,[151]) and
in distinct clumps; the Piper betel trailed up some of the trees, and
the Abrus precatorius, (Anasagar of the natives,) with its pods,
containing small, but beautiful crimson seeds, hung in festoons from
the bushes in the jungle, and a Diosma, called Un grupuum by the
natives, was abundant and fragrant; the Manihot (Jatropha manihot)
was also seen; and although I was informed the root was prepared
and eaten, the shrub did not seem to be extensively planted. The
Carambola-trees (Averrhoa carambola) were numerous, and called
Boslemang. A quantity of the fruit was observed laid upon a raised
bamboo platform, spread out to dry in the sun, and the natives
appeared fond of eating them in a raw state, as well as using them
in many of their curries, and other dishes.
About some of the native habitations, that large and elegant palm,
the Borossus gomutus of Loureiro, the Saguerus pinnatus of the
Batavian Transactions, and the Cleophora of Gœrtner, was planted: it
is the “Anau” of the Sumatrans; was called at this place “Eju” and
“Doh” by the Javanese: it is valued on account of excellent toddy
being extracted from it; but more especially for the black fibres
collected from the trunk, about the bases of the petioles of the
fronds; which fibrous substance resembles somewhat in its
appearance, as well as elasticity, horsehair; and it is highly esteemed
for the manufacture of rope used for their seines, vessels, &c.; the
very thick fibres, the natives say, the Moormen resident here use as
pens, and call them “Puré Eju:” it is probably the same tree from
which the fibres, called Cabo-negro by the Spaniards, are procured
at Manilla, and from which they also manufacture rope.
We continued our ramble over a fine plain, terminated in the
distance by palms: bamboos, the broad-leaved plantain, and other
elegant trees were seen, ornamenting some lonely habitation, the
roof just appearing above the dense foliage. This plain at one season
of the year is covered with rice-fields; but was now dried up: the
stubble of the former harvest remained, and the whole was covered
by an abundance of herbage, affording feeding for herds of cattle. A
number of various species of Grylli were hopping about the fields,
and were caught by the native boys for my entomological collection:
they called them, in the language of the country, “Daruar,” and these
insects are eaten by the natives.
 CHAPTER XXI.
Country about Pedir—“White Lions”—The rajah’s habits—A decision—
Ornaments for the ear—Female curiosity—The rajah’s horses—War
between the rajah of Acheen and the rajah of Trumong—A native’s
account of the quarrel—Purchase of betel-nut—The Areka-nut—Trade
in that article—Anecdote—A Chittagong brig—Dried fish—Beautiful
appearance of the Golden Mountain—Assemblage of the mountains—
Tornados—The fire king and his demons—Yamora—Burial-ground—
Large tree—Small crabs—Game called Mein Achu—Leprosy—Party of
natives—The Viverra musanga—Applications for medicine—Rajah of
Putu—His retinue—Object of his visit.

The country about Pedir, as far as I had an opportunity of seeing

it, was very picturesque, abounding in a luxuriant, natural
vegetation, as well as in a state of cultivation. The native habitations
are almost hidden by cocoa-nut, plantain, areka, eju, jack, and other
trees; fragrant odours were exhaled from the multitude of flowers
which strewed the surface of the ground; and a variety of profuse
vegetation was spread over the face of nature. The soil is rich, and
the numerous vegetables (among which the purple and white yams
are abundant) planted in the gardens of the natives, are most
prolific.[152] The habitations, as I have before noticed, are raised
upon posts, which I should suppose, in these marshy situations, are
intended to guard against the miasmata which must rise from the
surface of the ground after the rains, and to the influence of which
the inhabitants would be much exposed, if their dwellings were not
placed on an elevated site. The plain is beautiful, and the back-
ground of the landscape is terminated by mountains, varying in
elevation, and extending in a direction principally from east to west;
[153] sometimes covered by fleecy clouds, and at others, glowing in
the varying and beautiful tints of a setting sun, which cast its
expiring rays, undimmed by a cloud, over the towering masses.
 After walking in the vicinity of the village,—for our guides evinced
no desire of taking us further inland,—we were desired to enter a
house to rest ourselves: by an invitation to enter, is only meant
being seated in the verandah; for we did not, or rather were not
permitted to, intrude ourselves into any other parts of the dwelling.
At this place cocoa-nut water was again offered as a refreshment.
We requested to be taken further in the interior of the country; but,
although a refusal was never given, yet we found we were invariably
taken, by other paths, back to the place from whence we came. We
became at last, from this and other circumstances, convinced that
our Moor friends were fearful of exposing themselves to the krisses
of the “Hill people,” from whom they appear to have conquered
some portion of the country, establishing themselves as traders.
We returned after a short ramble, and were conducted into the
bazaar, and seated with a semicircle of the natives before us, all
staring quietly and decorously at the “white lions.” From this place
we adjourned to the fort, near the rajah’s residence, where we
waited for the appearance of his highness, who had not yet risen
from his couch. The old minister gave us some account of the rajah’s
habits; one of which was, that he lies in bed until three p.m., except
when there is any particular business, such as the arrival of a ship,
to induce him to rise earlier; and he does not retire to rest until
three a.m., after smoking a pipe of opium. The old gentleman must
have been guilty of an exaggeration, when he stated that the rajah
would smoke a ball of opium in four days. His highness is only
eighteen years of age, and has not at present the appearance of an
opium smoker: it must have been the quantity consumed by the
rajah and his numerous followers that was meant, the whole of
which was placed to the rajah’s account. Pipes of opium were
offered to us to regale ourselves, but of course were refused.[154]
After some delay, the rajah came to visit us, having just risen from
his couch, unwashed, and attired in unclean garments. He shook
hands, in the European manner, with the party; and then, having but
little to say, from want of some other employment, he amused
himself with my insect boxes, and the insects placed in them
transfixed by pins: this led to an explanation of my professional
pursuits, and its collateral branches; but as the subject was rather
beyond his comprehension, he became attracted from it to a cloth
cap worn by one of the party, about which there was much
discussion, the result of which was, that the rajah and his followers
came to the important decision, that it would make a very good
pocket or case for containing betel-nut, and the accompanying
articles required to be used with it. Being heartily tired, we were
happy to escape from the royal presence; and the boat being ready,
we returned on board.
All the women had the lobes of the ears enormously distended,
from wearing, when very young, round pieces of wood, polished and
ornamented, or rolls of leaves in them: the richer classes wear large
ornaments of gold and silver: the old women have the lobes hanging
down to a great length, but without ornament; that they formerly
had placed them in the lobes was evident by the distended orifices,
which, having lost their elasticity, prevented their retention as
before. The poorer classes are content with neatly polished and
ornamented round pieces of wood, or a roll of the plantain or some
other kind of leaf, as a substitute for those of gold and silver worn
by the higher and richer classes. The lower class of females were
usually attired in cotton cloth sarongs, and the cabaya, passing over
the head, of a black colour, or other dark patterns. As we passed
their dwellings, they came forth, with the usual feminine curiosity, to
view the strangers: indeed, we appeared to be as much objects of
curiosity among them, as I had before been when landing upon
many of the unfrequented islands of the Polynesian Archipelago; and
the natives, that arrived in the boats with Areka-nut, from the
villages on the coast, seemed to regard us as wonders, and
surrounded the entrance of the poop-cuddy at meal-times, as if to
satisfy themselves how such animals fed.
We had an offer of some of the rajah’s horses to ride about the
village: at first it was thought that some dun cows, with horns cut
off close to the head, and a preternatural erection of the ears, were
the animals offered; but it appears they were real ponies: if we had
ridden them, however, it must have been without any saddle or
bridle, for there were no articles of that description to be procured at
Pedir.
The barque at present at anchor in Pedir roads, under the
Acheenese flag, was captured from the rajah of Trumong, on the
west coast of Sumatra, by the man-of-war grab belonging to the
rajah of Acheen: the cause of it was this:—the Trumong rajah is
tributary to the king, or rajah, of Acheen: he had not paid tribute for
three years; and on its being demanded, the Trumong rajah
returned for answer, that he intended paying it with iron balls; war
was therefore declared against this rebellious rajah, and the barque
was captured by the following stratagem: the commander of his
Acheenese majesty’s grab fell in with the barque at sea, assured her
commander that all differences had been adjusted between the two
rajahs, and requested him to come on board. The captain of the
barque unsuspectingly accepted the invitation, taking presents with
him. On stepping upon the deck of the grab, himself, crew, and
presents were detained, and a boat, with a number of men well
armed, sent on board the barque; and having secured the guns,
hauled down the Trumong rajah’s colours, and hoisted those of his
Acheenese majesty, the vessels will sail, in company, for Acheen in a
few days.
A Madras native, who spoke a little English, amused us with his
version of the affair. “I belong, and barque belong, to the rajah of
Trumong. Acheen rajah and my rajah make war; Trumong rajah
plenty dollars, and go buy ships at Pulo Penang, to fight rajah
Acheen. Acheen rajah very poor, one day buy ship, in a month want
sell, because very poor—Acheen rajah no good, no pay Lascars—
Trumong rajah, my king, pay well, plenty dollars. My barque got
seven guns and twelve Lascar men. The grab send a boat and ask
‘whose barque this?’ My captain say, ‘rajah Trumong’s;’ then grab’s
men take prisoners, and say, ‘barque belong now to Acheen rajah;’
so he pull down colours—our colours before white and black—now
Acheen colours red and white.”
 The quantity of betel-nut agreed for (three thousand peculs) was
sent on board; and a further agreement entered into for three
thousand peculs more, to be delivered in a few days after. Opium, at
nine hundred dollars the chest, was taken in part payment: this was
a high price, but netted to the sellers a profit of only ninety-five Java
rupees, or forty-seven dollars; from the large quantity in the market
at this place, it was with the greatest difficulty it could be disposed
of even at that price: the dollars to be given in addition must have
been the principal inducement, for opium had been purchased from
the Penang brig, “Calder Bux,” at seven hundred and seventy dollars
the chest; but we afterwards found only one dollar the pecul, or
rather laxar, had been paid by that vessel, which will account for
their giving in barter a higher price for the opium.
On the second agreement being made, the rajah and suite came
on board to ratify it, which, after some disputes and discussions with
all parties, was effected by the supercargo of the ship.
The principal article of exportation from this coast is Areka-nut,
and a small quantity of rice; the latter, however, appeared of an
inferior quality, and one-and-a-half dollar a pecul was demanded as
the lowest price; the vessel would be required to furnish bags for the
rice, as there are none manufactured on the coast, and a delay of
the vessel would be also required to procure it. Areka-nut must,
therefore, be regarded as the principal article of trade, as it is to be
purchased cheap, and of a quality as excellent as in any part of the
Eastern islands, or Cochin China.
The Areka palm is the Areka catechu of botanists; it is a palm of
elegant growth, rising with a very erect and small stem to the height
of forty or even sixty feet, the summit terminating in a tuft of dark-
green foliage; the circumference of the trunk is seldom more than
one-and-a-half to two feet, when of early growth of dark-green, and
when old of a dark-grey colour; the circles formed by the clasping
petioles of the fronds being very visible upon it: the tree bears fruit
only once during the year, at which period the tree, with its long
bunches of orange oval-shaped fruit, pendent from the upper part of
the trunk, contrasted by the dark-green foliage, has a beautiful
appearance. The Areka-nut, when planted, takes three years to
arrive at a sufficient size to produce fruit; the wood of this palm is
used at this place for a variety of purposes.
The fruit grows in long pendulous clusters, each about the size of
a small hen’s egg; the external covering is thick, fibrous, covered by
an orange-coloured epidermis; and on the thick fibrous husk being
cleared away, the nut is discovered surrounded by its own immediate
epidermis, which often proves difficult of removal. The nut is conical,
but varies in some, having an elevated apex and small base, and
others a large base and very slightly elevated apex. One nut is the
natural produce of each fruit, although sometimes double or triple
nuts are found, anomalies often met with in the vegetable kingdom.
[155]

Many of the common drinking and baling utensils in the boats are
made from the spathe of the Areka palm; and I have frequently seen
a vessel for holding water made from it, which was not dissimilar to
those made by the Australian natives from the bark of the Eucalypti
trees; they use the flower spathe also for nailing upon the bottoms
of their boats. May, June, and July, are the months for collecting the
nuts. They had loaded nine ships this season; but forty vessels, of all
sizes, have been freighted in one season, for Pinang, &c., from
whence it is exported to China, Madras, and other parts of
continental India.
The nuts vary in size; their quality, however, does not at all
depend upon this property, but upon their internal appearance when
cut, intimating the quantity of astringent matter contained in them.
If the white, or medullary portion, which intersects the red or
astringent part be small, has assumed a bluish tinge, and the
astringent part is very red, the nut is considered of good quality; but
when the medullary portion is in large quantity, the nut is considered
more mature, and not possessing so much astringency, is not
esteemed so valuable.
 The quantity of nut produced on this coast is stated to be eighty
thousand peculs. When there is no immediate demand for this
article, it is not shelled, but preserved in the husk, as it is considered
not to be so liable to be destroyed by the worm in that state; but
although this is the opinion of the natives on the coast, yet I have
seen nuts destroyed totally by the worm while in the husk, in the
space of two months. The produce of the first month, or month-and-
a-half, amounting usually to forty thousand peculs, the natives
informed us is exported; and the second gathering, amounting to
about the same quantity, is consumed in the country. The nuts were
brought on board the ship in large boats, (originally built and
employed as fishing vessels, except when required for this
employment, they are from three to four tons burthen each, and are
to be purchased for twenty or twenty-five dollars,) in bulk, and
Manilla mat-bags, and are taken on board the ships in bulk. The
quantity of Areka-nut imported by the Chinese, amounts to forty-five
or forty-eight thousand peculs annually, exclusive of that brought
from Cochin China, the amount of which is not known; in 1832, from
a failure of the usual supply of nuts from Cochin China, forty-eight
thousand peculs, imported from other places, sold so high as four
dollars and three-quarters the pecul; the price it usually fetches in
the China market is from two to three and three-quarter dollars the
pecul. The principal consumption of the nut as a masticatory (in
conjunction with the leaf called betel, produced from a vine, the
Piper betel) is in the provinces of Quang, ton, (Canton, of
Europeans,) Quang, si, and Che, keang, and may be seen, exposed
for sale, on little stalls about the suburbs of Canton, with the other
additional articles used in the preparation; it is also used as a
mordant for coarse dyes. The Areka-nuts brought from Cochin China
are considered by the Chinese the best imported. This may, however,
arise from prejudice in favour of the production of a country so
nearly allied to them, to that introduced by foreigners. In the central
provinces of Hoo, kwang, and Kang, si, the nut is, after being
bruised and pounded, mixed with the green food of horses as a
preventive against a diarrhœa, to which that kind of food sometimes
subjects them. It was likewise mentioned to me by a Chinese, that it
is used as a domestic medicine in the north of China, small pieces
being boiled; the decoction is administered in various visceral
affections.
A cargo of this article generates so much heat as to raise the
thermometer in the hold forty degrees above that on the deck; and
from this circumstance, and the quantity of steam generated, the
crew are prevented from sleeping between decks.
The Areka-nut is commonly known by the very prevailing Malay
name of Pinang, or Pinong, but in the Acheenese language it is
called Pénu, and the tree Ba, penu; Ba, signifying tree, is usually
prefixed to the specific name, as Un, signifying plant, is prefixed to
the name of a plant.
The ripe Areka-nut is called also Pénu, massa, and the green
Pénu, mudr; the Gambir, used with the Betel, Gambé; the Betel-leaf,
Ránu; the Chunam, Gapu; the tobacco, Bákun.
The rajah of Pedir claims ten per cent. as a duty levied in kind
upon all the Areka-nut disposed of to ships arriving in his territory;
and, besides this, his subjects are obliged to dispose of the nuts to
the ship, at the price he or his ministers have agreed upon with the
supercargo.[156]
The red colour, produced by chewing the Betel-leaf, in combination
with the Areka-nut, lime, &c., is not produced by them when used
separately. The mastication of the “Betel” is considered very
wholesome by those who are in the habit of using it: it may be so,
but the black appearance it gives to the teeth, although it is said to
be an excellent preserver of them, together with the brick-red lips
and mouth, give any thing but an agreeable appearance.[157] Its use
certainly does not impart additional beauty to the native females,
who habituate themselves to an equal extent to those of the
opposite sex.[158]
There was an old native of Madras, a Moorman, forming one of
the rajah’s attendants on board, whose duty it was to visit ships, and
report their arrival to his highness, and also to attend the ship daily
to see the cargo taken on board; this individual had one of his hands
rendered useless by a blow from a Klawang, or Malay sword. Besides
the scar resulting from this wound, there was an unreduced
dislocation of the carpal extremity of the ulna, and a fracture of the
radius, which, being disunited, an artificial joint had been formed:
the cause of this personal injury, he said, proceeded from an attack
made upon a vessel he was in, by one belonging to the rajah of
Acheen, in which several people were killed. This individual, being on
board one morning, although professing in external appearances the
Mahometan creed, expressed a desire of having a tumbler of the
stimulating beverage denominated “grog,” if it could be administered
without its being seen; proving that his religious scruples were not
so strong in private as his veneration for public opinion, or a fear of
losing caste. A stiff glass of grog was, therefore, prepared for him,
which the old withered disciple of Mahometanism regarded with
glittering eyes. There was also on board another Mussulman, whose
duty it was to take account of the cargo as it came to the ship, and
report the quantity to the rajah; the sinner was about to raise the
glass to his mouth, exclaiming, “What would the other man say if he
was to see me now?” when the old saying was verified, of “there is
many a slip between the cup and the lip;” for the scribe was, at the
same instant, seen descending, and there was only time to conceal
the glass before he was close to him. The old fellow stroked his
whiskers, and began seriously to talk about opium; and as the white
turbaned man saw him clear out from below, the long-sought
enjoyment was obliged to be postponed.
A Chittagong brig, commanded by a black Portuguese, anchored in
the roads, on the 10th of July, from the Maldive islands, bound to
Penang, with a cargo of dried fish and some tortoiseshell, which had
been procured in exchange for rice; his object was to dispose of his
cargo in exchange for dollars and Betel-nut at this place. The dried
fish was the Bonito cut into small pieces. The Maldive natives
prepare it in the following manner:—A long slice is cut from each
side of the fish, and these again are divided into two parts, so that
each fish is divided into four pieces; it is then boiled for a short time
in salt water, after which it is smoked and placed in the sun to dry; it
then becomes extremely hard, and resembles, when broken, a piece
of wood, having a reddish appearance at the fractured parts: after it
has been soaked, it is used for curries and other native dishes.
The “Golden Mountain” is a very conspicuous and beautiful object
from the anchorage; but it ought to be mentioned, that, from this
position, two mountains are seen to the westward, one towering to
a peak, and densely wooded, the other, anterior to it, is a lofty
rounded hill: the first is the one known to Europeans as the “Golden
Mountain;” the second, or rounded mountain, is not named in the
charts, but it may be called the “Pedir Mountain.” The “Golden
Mountain” is called by the natives Yamori, and the other Yamora; the
first the natives designate as the father, the second the mother
(probably of all the little mountains about them).
The natives state, that once every year the mountains come
together, occasioning rain, thunder, lightning, earthquakes, and
violent storms; the Urong Salle, or Fire King, then sits upon the
mountain, surrounded by hideous demons, enjoying the noise and
uproar occasioned by the conflict of the elements; the winds blow in
violent tornados; the thunder is so loud as to occasion the earth to
tremble under the feet of the terrified inhabitants: the rain causes
tremendous mountain torrents, inundating habitations and
plantations, carrying all before them in their impetuous course, and
spreading devastation around. In the midst of this dreadful conflict
of the elements, the mountains meet with a horrible crash. As the
forked lightning plays around them, the Urong Salle, or Fire King,
surrounded by his satellites, laughs and sports in the scene; the
mountains remain united for a minute, when they again separate,
regaining their former position. No person dare ascend the hill at any
time, for there sits the Fire King and his demons, and should any
mortal cast his eyes upon him, that instant he would be struck with
blindness.
 Yamora is stated to be distant, inland from Pedir, two days’
journey travelling on foot, and Yamori is the same distance from the
other mountain; not, however, as the crow flies, but it would take
that period of time to reach it, from the winding and difficulties of
the road. From this account there is every reason to suppose that
earthquakes and volcanic eruptions are occasionally felt upon this
coast. As far as we could ascertain, there was no appearance of a
burning volcano existing in either of the mountains just mentioned;
they were both densely clothed with vegetation, more especially the
“Golden Mountain.”
Near the banks of the river, a short distance up, is an uninclosed
native burial-ground; the graves had a stone or piece of wood
placed both at the head and foot: there were several trees of
Hibiscus tiliaceus, Tamarindus Indica, and a very large one, called
Ba, Glumpong by the natives, (Sterculia fœtida, Linn.) which was
described by them as being poisonous, producing violent vomiting
and pains in the head, if the fruit be eaten. I subsequently saw it,
planted about the fences in the village. There were two of these
fine, lofty, and spreading trees in the burial-ground, and I procured
specimens both of the flowers and fruit: the former grew in clusters
upon erect spikes, with the corolla of a dark red, mixed with
yellowish green. They have a handsome appearance, but diffuse so
fœtid a smell around, as soon to fill a room with the exceedingly
disagreeable effluvia. The fruit is kidney-shaped; the trees were sixty
or seventy feet in height, and from eight to ten feet in
circumference.
A piece of sandal wood, of good quality, was brought off to the
ship by one of the natives; he stated that large quantities of it could
be procured, as the tree grew abundantly in the mountains. He gave
it the usual Indian appellation of Chandana.[159]
In some brackish pools I collected several small living species of
the Cerethium; and about the banks a great number of a small crab,
remarkable from one of the claws being greatly disproportioned to
the size of the other parts of the animal, and entirely different in
colour. When I first beheld them, I mistook them for small crabs
running away with the claws of larger ones. They are difficult to
catch, from the exceeding rapidity of their motions, and escape, on
the slightest movement or noise being made, into their
subterraneous dwellings in the sand. The body and feet of the
animal are bluish black, with a few white marks across, and the
large claw is of a light or occasional darker red colour. The natives
call them Biong, po. They are seen in great numbers about the
pools, but are not eaten by the natives. I procured several
specimens, which I preserved in spirits. On being placed in strong
rum, they survived for the space of full three minutes; and if more
than one was placed in the same bottle, they would fight and pull
the claws off each other in their death agonies.
Near the village, several boys were playing a game with Areka-
nuts, called Mein-achu, in some degree resembling our game at
marbles. Four nuts were piled up in form of a pyramid, twelve such
forming a row; a nut was then fillipped off with some degree of
force against the heaps, from a distance of about three yards. If the
thrower succeeds in destroying one of the pyramids, he renews his
throw at the others, always at the distance where his nut remained,
until he misses, when the next player takes his turn: the game thus
continuing until all the pyramids are thrown down.
I was much surprised a few days since, while passing a house in
the vicinity of the village, to see apparently a European lad, of about
six years of age; and on examining him closer, found his skin of a
white colour, thinly scattered over with small light-brown patches.
On passing the same house again, I made inquiry on the subject,
and then had an opportunity of seeing two others, who were
females,—one about sixteen or eighteen years of age, the other an
infant just able to run about. They were described to us as children
of native Malay parents, of the usual colour of their race; but we did
not see them, as they had gone a short distance into the country.
The children were named Ceté, Theté, and Cebreté. They had a
plump appearance; flaxen hair, light-blue eyes; and the boy and
young woman were slightly covered with scattered small brown
patches; but the infant had not a blemish on its integument. The
natives could give no reason for this variety; they looked upon it as
curious, but did not seem, as far as I could ascertain, to regard it as
a disease. They have the flat nose of the Malay, but otherwise would
be considered the offspring of European parents, the skin being in
some degree freckled. It ought certainly to be regarded as a variety
of, if not actually the disease called, leprosy.[160]
I met several natives going into the interior; they were all well
armed with krisses, klawangs or Malay swords, spears, and
blunderbusses or musquets; the country in the interior being
described as in a very unsettled state. Some of the spears were
about six feet long, resembling walking-staves, covered above by a
wooden sheath, similar to the other part of the weapon, and
ornamented with rims of silver; the upper part, or sheath, being
taken off, displays the head of the spear.
I purchased a specimen of the Viverra musanga, similar to one I
had before procured at Java, for half a rupee; although very wild
with strangers, it was exceedingly domesticated with its master,
following him like a cat, as he walked along the path: they called
him, on this coast, as at Java, “Mussang.”
This specimen was very little larger than one I had before
procured; but they attain, I was informed, the size of our domestic
cat, living, in the wild state, upon the summits of the trees, eating
fruit, and catching birds as their food. The animal is very fond of
sugar-cane, plantain, rice, and the flesh of fowls, and will also kill
and eat those troublesome insects, the cock-roaches. It, however,
became so very savage on board, that I was obliged at last to
destroy it.
I was frequently applied to by the natives, when sick, to
administer medicines to them. There were several suffering from
different kinds of tumours; one, near the nose, I offered to remove;
but although the person promised to come on board for the
purpose, I afterwards heard he was afraid, and altered his mind.
Among many patients was a little girl, belonging to a Moorman,
suffering from Diarrhœa mucosa: her body had been rubbed entirely
over with a mixture of turmeric, sandal-wood, and oil, as a remedy
for the disease. The yellow appearance—the usual indication of
sickness—was not the result, as may have been expected, of some
disease, but merely a daubing over the body of the above-
mentioned composition,—this being the remedy for all diseases. The
common Hindoo application of cow-dung and turmeric is frequent for
external wounds or bruises, and considered a very efficacious
remedy. Cutaneous diseases were very numerous, and the native
applications proved very inefficacious in removing them.
I had an opportunity of seeing another rajah—the rajah of Putu (a
village and district not far distant, on the sea coast). He was ill-
looking in person, and carried with him the appearance of being
addicted to opium-smoking. He was attired in a sarong of a
handsome pattern, the borders of which were woven with gold
threads. These sarongs are the manufacture of the country, and are
sold at high prices. The rajah was tall and young, and was attended
by a numerous retinue, attired in red cloth jackets ornamented with
gold lace, and handsome sarongs: others could only wear a cotton
baju, or jacket. They were armed with spears, klawangs, krisses,
and old rusty blunderbusses. The object of his visit to this place, was
to pay his respects to the old queen (grandmother of the present
rajah) of Acheen, who was residing at Pedir, and was about to
embark in a few days in the Acheenese grab for Acheen, and was
described as being an excellent old lady.
 NOTE.
(See page 13, vol. i.)
A method has since been mentioned to me, by which the colours
of the flowers of plants are well preserved. The process was this:—
The paper being first heated before the fire, or in an oven, the plant
recently gathered is placed between the hot sheets, and pressed. It
is requisite, however, that the paper, in the same heated state, be
renewed at intervals, on account of the expressed juices from the
stalks and leaves fermenting, which might otherwise injure the
plants.
There is also a method of preserving plants in flower, by which
their natural form, as well as colours, can be preserved. It consists in
placing the plant in a jar, and pouring fine sand upon it, until the
whole plant is covered: it is then to be placed, still kept in the jar,
into an oven; after which, being taken out, and the sand removed,
the plant is found preserved both in its form and colour.
 FOOTNOTES
[1] Madeira signifies, in the Portuguese language, “woody;”
and the island was so named from the very wooded appearance it
had on its discovery.
[2] In summer, Horsburgh states that the north-east winds
prevail, and a south-west current sets through the channel,
between Madeira and the Desertas. The current along the south
side of Madeira and the Desertas mostly sets to the leeward in
strong gales; but at the conclusion of a gale, it sometimes
changes suddenly, and sets contrary to the wind.
[3] They are called “Guinea Ships” by the old navigators, from
their floating like a vessel on the water, and from having very
probably been first seen in great numbers about the coast and
gulf of Guinea.
[4] Mr. John Fuge, of Plymouth, informed me that he captured
a specimen of the Physalia pelagica, in the Catwater, (Plymouth
Sound,) a few years since, in the month of August; it was floating
upon the surface of the water, and living when caught; he placed
it in a glass globe of sea water, and preserved it for three weeks.
The only motion he observed in the animal, was an occasional
contraction and elongation of the beaked end of the bladder
portion of the animal, and the tentaculæ were also drawn up and
thrust forward.
[5] Physalis tuberculosa, P. megalista, P. elongata, and P.
pelagica, are the species given by Lamarck. (Sur les Animaux
sans Vertèbres, tom. ii. p. 478.)
[6] On the 5th of April, 1834, in latitude 29° 17′ north, and
longitude 42° 57′ west, temperature of the atmosphere 68° to
72°, I caught in my towing net a very fine specimen of Physalis
pelagica, adorned with the usual beautiful tints, but not so vivid
as I have usually seen them. The specimen was the largest I had
before witnessed. During the month of April, 1834, I observed
specimens of this mollusca as far north as latitude 38° 32′ north,
and longitude 34° 30′ west. The lowest range of the thermometer
being 58°, and highest 72°. In March, 1831, I had seen them as
far north as the latitude of the Azores or Western Islands. Often
when we had very strong westerly winds, with a heavy sea
running at the time, I saw them; yet not, to use a nautical
expression, “furling sail” and sinking; this was sufficient to prove
the absurdity of the opinion that they collapse and sink during
stormy breezes. I have frequently seen them capsized by a wave,
but almost instantly after regain their natural position.
[7] “Praya” signifies, in the Portuguese language, “a beach or
shore.”
[8] “The largest tree in the world is the Adansonia or Baobab
tree, the trunk of which has been found with a diameter of thirty
feet; but its height is not in proportion. It is emollient and
mucilaginous in all its parts. The leaves dried and reduced to
powder constitute Lalo, a favourite article with the Africans, which
they mix daily with their food, for the purpose of diminishing the
excessive perspiration to which they are subject in those climates;
and even Europeans find it serviceable in cases of diarrhœa,
fevers, and other maladies. The fruit is, perhaps, the most useful
part of the tree. Its pulp is slightly acid and agreeable, and
frequently eaten; while the juice is expressed from it, mixed with
sugar, and constitutes a drink which is valued as a specific in
putrid and pestilential fevers.”—Hooker’s Bot. Mag. 2792.
“The dried pulp is mixed with water, and administered in Egypt
in dysentery. It is chiefly composed of a gum, like gum senegal, a
sugary matter, starch, and an acid, which appears to be malic.”—
Delile Cent. 12. Quoted in Lindley’s Int. to the Nat. Syst. of
Botany.
[9] (In June, 1831.) “Canary orchilla fetches in the London
market from 270l. to 290l. per ton, while that which is brought
from Madeira fetches only 140l., and Barbary not more than from
30l. to 45l. The total quantity imported in 1829, amounted to
1,813 cwt. or 90½ tons.”—“Archil is generally sold in the form of
cakes, but sometimes in that of moist pulp.”—M’Culloch’s Dict. of
Commerce.
[10] At the time of our arrival a Portuguese brig was lying in
the bay, having a cargo of this weed on board, which was
estimated at a low calculation to be worth 30,000l.
[11] “The dyer’s lichen was first exported from the islands of
the Archipelago to Venice, Genoa, France, and England, for the
use of the dyers. Towards the commencement of the last century
it was discovered in the Canary Islands, and was soon placed
among the regalia of the Spanish crown. This excited the
attention of the Portuguese, who collected it without restriction in
the Cape de Verd Islands, Madeira, Porto Santo, and the Azores.
In the year 1730, the Jesuits asked of King John V. the privilege
of collecting the Hervinha secca; but the crown took the
advantage into its own hands, and farmed the right of collecting
it. At a later period the lichen was ceded to the mercantile
company of Gram Pará and Maranhâo; and, lastly, in the year
1790, the government again took this branch of commerce under
its own care, because it had declined considerably under the bad
management of the company. At present the exportation is small;
but more considerable, however, from the Cape de Verd Isles.”
(See I. Da Silva Feijó, in the Memorias Economicas da Acad. de
Lisboa, vol. v. 1815, p. 143.)—Spix and Martius Travels in Brazil,
vol. i. p. 125.
[12] Abel’s Voyage to, and journey into the interior of, China.
4to. p. 6.
[13] Captain Basil Hall. See Fragments of Voyages and Travels.
[14] It would be interesting, but at the same time difficult, to
ascertain where one particular species commences and another
terminates, and the extent of their range. In the summer season
they are found off the Cape of Good Hope, Port Jackson, and
even on the banks of Newfoundland; and I have good authority
for asserting that in the month of August, in even more than one
year, they have been seen in Plymouth Sound.
[15] My journal remarks the atmosphere to have been very
chilly during the day, but much milder in the evening; the range
of the thermometer during the day being from 49° to 56°.
[16] How will this accord with the geographical distribution of
the mollusca by Péron and Leseur? After studying the Holothuria
Medusæ, and other congeners of delicate and changeable forms,
they came to the conclusion that each kind has its place of
residence determined by the temperature necessary to support its
existence. Thus, for example, they found the abode of Pyrosoma
atlanticum to be confined to one particular region of the Atlantic
Ocean.—Voy. aux Terres Aust. tom. 1, p. 492, quoted in Lyell’s
Principles of Geology, vol. ii. pp. 111, 112.
[17] Albicores, bonitos, and dolphins, often follow the ship for
several days in succession; we had occasion to note an albicore
that was marked on the back by some sharp instrument, leaving
a large sear by which it could readily be recognized. It was first
seen in 3° north latitude, and following the ship to latitude 11°
south, a distance of eight hundred and forty miles.
[18] This petrel is said to be found from 24° to 60° south
latitude.
[19] Respecting the name given to this bird, it has been
observed, that the first Portuguese navigator called them, the
boobies, and other sea-birds, alcatros or alcatras. Dampier
applied this name to an actual kind; Grew changed it to albitross,
and Edwards into albatross. The French name these birds mouton
du cap. There are a number of species enumerated; but it will
require frequent and cautious observation previous to the
determination of a new one, as they vary so much in plumage
from sex and age.
[20] The condor is supposed by some to be the “Roc” of the
Arabian Nights.
[21] The other species I have seldom known to measure more
than eight feet across the expanded wings.
[22] This bird is evidently aided by its long wings as well as tail
in directing its flight: they are never seen to soar to any great
height, and are often observed to change their course, by turning
the wings and body in a lateral direction, and oftentimes, when
raising themselves, would bend the last joint of the wings
downwards.
[23] Cuvier enumerates five species; but at the same time
says, “On a observé divers albatrosses plus ou moins bruns ou
noirâtres, mais on n’a pu encore constater jusqu’à quel point ils
forment des variétés ou des espèces distinctes.”—Regne Animal,
tom. i. p. 555.
[24] The building was originally erected as a theatre, at a very
great expense, and after its completion the governor, at that time
General Darling, refused to grant a licence for dramatic
performances, in consequence of which it was fitted up as a
spacious hotel. On the present Governor, General Burke, granting
permission for theatrical entertainments, a portion of the building
has reverted to the original purpose for which the whole had
been erected.
 [25] “It is at least certain that on this microscopic character of
the equal existence of cutaneous glands on both surfaces of the
leaf, depends that want of lustre which is so remarkable in the
forests of New Holland.”—Sketch of the Botany of the Vicinity of
Swan River, by R. Brown, Esq. F.R.S., published in the Journal of
the Royal Geographical Society of London, vol. i. 1830, 1831.
[26] The dried cones of the Banksia are used by the aborigines
for retaining fire, as they will keep ignited for a considerable
length of time.
[27] The analysis of the chemical properties of this gum is
mentioned in Decandolle’s Organographie Végétale, tom i.
[28] I remarked that the wood of a species of Banksia, (I
believe dentata,) which was used for firewood, was of a beautiful
red colour, and when split in a longitudinal direction displayed a
curious interlaced appearance; it had an astringent taste when
chewed, staining the saliva of a dark reddish colour, and I think it
would be worth trying if a dye would be furnished by it.
[29] The Kennedia is called the “woodbine” by some of the
shepherds in the colony, who use a decoction of its leaves as a
lotion for scabby sheep, and they declare it is a cure for that
disease; but their declarations of the curative properties of the
plant is not borne out by the experience of others, who have
found it quite useless as a remedy for that disease.
[30] Among the Psittaceæ tribe is the Psittacus Novæ
Hollandiæ, curious as being one of the parrot tribe, seen and
mentioned by Captain Cook, but is a very rare species in the
present known parts of the colony,—(it is, more correctly, a
species of cockatoo, and which, I believe, Mr. Vigors has; or
intends, to place in a new genus,)—and has not been seen even
in those portions of the colony visited by Cook. The specimen in
the collection, is one among a few of this species that was seen
at Wellington Valley a few years since, during a prevailing
drought, and since that period they have not been seen in that or
any other known part of the colony. I heard at Yas Plains, that it
was not uncommon at some seasons of the year to observe birds,
before unknown to the colonists, appear, and soon after again
disappear, and are, perhaps, never seen again until years after,
and often not at all.
[31] It would also be desirable to have the cases made in such
a manner, as to be opened if required, and a closer inspection of
the specimens obtained, which is often requisite for scientific
examinations. To George Macleay, Esq. the museum is indebted
for many valuable species of birds, which he had collected during
his arduous journey in the exploration of the course of the
Murrumbidge river, in the expedition under Captain Sturt.
[32] Captain Cook observes, “Of this plant, there are two sorts;
the leaves of both resemble those of flags, but the flowers are
smaller, and their clusters more numerous: in one kind, they are
yellow; and in the other, a deep red.” This plant is also indigenous
to Norfolk Island, which, in its vegetation, partakes more of New
Zealand than the Australian continent. Captain Cook observes,
that at Norfolk Island, “we observed many trees and plants,
common at New Zealand, and, in particular, the flax plant, which
is rather more luxuriant here than in any part of the country.”
[33] Captain George Harris, R. N., C. B., and member for
Grimsby, in the present parliament, has recently been
manufacturing rope and cables of the phormium tenax, or New
Zealand flax; and instead of tar, substitutes a solution of gum, or
some such substance, (principally, we suspect, the caoutchouc or
Indian rubber,) by which, it is contended, the rope is rendered
stronger, more pliant, and less liable to part in short bends, turns,
or clinches, and being stronger, smaller ropes than those now in
use will answer for ships’ rigging; the consumption of hemp, of
course, diminishes in proportion—we say hemp, because the
solution will also impart to the hemp the qualities we have
named. If, however, a substitute is to be found for hemp and tar,
we are rendered independent of the Russian trade in these
articles;—a most desirable object, should the state of Europe at
any time involve us in a difference with that nation. The bogs and
rough ground of Ireland, all our African possessions and West
Indian islands, and New South Wales, are particularly adapted to
the culture of the phormium tenax. Captain Harris was here on
Monday, and superintended the making of a 14½ inch cable,
which is to be tried on his Majesty’s ship Rainbow. A trial is also
to be made of the relative strength of the phormium tenax and
hemp in this yard, in a few days, for which a piece of 14½ inch
cable has been expressly manufactured. The price of hemp per
ton is £38; that of the phormium tenax £28. Of the experiments
that have been made at Woolwich, by order of the Admiralty, the
following are the results:—

T. cwt. lbs.
A 4½ inch rope of the old sort broke at a strain 3 8 40
of
4 inch phormium, with the solution 5 10 0
4 inch bolt rope, Italian yarns, present sort 4 15 0
4 inch ditto, with the same yarns, with the 6 8 56
preserving solution
4 inch common rope 5 7 56
4 inch hempen rope, with coal or mineral tar 3 7 56
4 inch phormium, with the solution 5 16 70

The strongest proof is thus given of its strength. Its power,

however, to resist wet, and its durability, are yet to be
ascertained.—Hampshire Telegraph.
[34] The following was mentioned to me as the origin of the
name given to this point. Governor Phillip, at an early period of
the colony, formed a pic-nic party to proceed up the Paramatta
river, and a person was sent on before to prepare kangaroo
steaks. They landed at this point, and having regaled themselves,
the gentlemen, following the maxims of John Hunter, laid down
upon the grass, and aided digestion by falling asleep; the ladies
finding themselves deserted began to propose winning gloves,
and therefore kisses were taken, and on their awaking the forfeit
was demanded, and of course not refused. Before leaving the
place the governor wished some name to be bestowed upon the
point, and one of the ladies being requested to do so, in
consequence of the occurrence just mentioned, named it “Kissing
Point.”
[35] This creek, commonly called the Paramatta river, is a creek
or inlet of the sea from Port Jackson; the true river, which is very
small, falls into this creek at Paramatta.
[36] Boyams are the roots of different genera and species of
the Orchideæ family; some are called, by the colonists, “double or
single boyams,” according to the appearance of the roots, and
they all form an article of food among the aboriginal tribes.
[37] The dimensions of the large specimen were as follows:

Feet. Inch.
From the vertex of the head to the tip of the tail 2 3
Breadth across the shoulders 0 3
Length of the tail 0 11½
 Breadth of the loins 0 3²⁄₈
Length of the fore-leg to the claws 0 6⅝
hind-leg to do. 0 7⅜
Length of the head to the snout 0 4
Length of the ear 0 2½

The tail is naked underneath from its extremity to within five

inches of the base, and is prehensile.
The colour of the male specimen was greyish; a short fine fur
covers the back, being also continuous of the same colour to
within four inches of the tail; after which the fur becomes longer,
more glossy, and of a black colour; the fur on the abdomen was
of a yellowish white colour; near the feet the fur is short, of a
dirty yellow colour, with brownish patches; the colour is similar
under the chin, throat, and angles of the jaw; the upper part of
the ears is nearly bare; the thumbs of the hind feet have no claw,
but the fore-feet are pentadactyle, and armed with sharp claws;
the four toes of the hind-feet are also armed with claws, the first
dividing into two phalanges, each having a claw. The young
specimen differed from this only in having a yellowish tinge mixed
with the grey over the back, legs, and abdomen; angles of the
jaw and throat of a brownish yellow; the under portion about the
eyes and upper part of the head of a yellowish colour.
[38] The Hobart Town Colonist of Oct. 12, 1832, contains the
following paragraph respecting the capability of the opossum fur
being used in manufactures.
“We have been favoured with the sight of a pair of mittens
spun and knit by Mrs. M’Kenzie, of the Lower Clyde, from the fur
of the opossum. In texture and appearance they very much
resemble the best sort of Angola mittens, but to us they appear
of superior quality. The pair that we saw are now in the
possession of Mr. Gordon, of Forcett, to whom they were
presented by Mr. M’Kenzie.”
[39] Besides the vine, other fruit-tree cuttings blossom and
even bear fruit in a very short period of time. I saw a peach
cutting, in a garden near Sydney, about six inches long, which
had been planted only ten days, and was covered with a
profusion of blossoms.
[40] The box-tree of the colonists (Eucalyptus, sp.) is used in
the colony for the spokes and fellies of wheels, and the “apple-
tree” (Angophora lanceolata) for the naves.
[41] The “turpentine tree” attains the elevation of from sixty to
ninety feet, and a diameter of three feet.
[42] Mount York, according to Oxley, is 3,292 feet above the
level of the sea.
[43] “Pi” signifies “to hit or break,” and “cobera,” “head.”
[44] It has been stated frequently to me, that the females
destroying their offspring allege as a reason, that they are too
much trouble to carry about: however, it is well known, that, as
their children become older, they evince much attachment
towards them.
[45] This is not confined to the Australian natives, for it also
occurs in Polynesia. Spix and Martius also observe, in their Travels
in Brazil, (Eng. Trans. 8vo. vol. ii. p. 241,) “We did not meet with
any deformed persons or cripples among the Indians; for which
reason, some people believe that they put them to death
immediately after their birth.”
[46] “Netbul,” (the net-bag of the aborigines,) is a corrupted
native word; “culy” is one of the native appellations.
[47] Those philanthropic individuals who think to change the
habits of these savage tribes, expecting those who have lived
from the earliest period of their existence on the produce of the
chase, to abandon their wandering life, and settle down to
cultivate the soil—an employment to which they are quite
unaccustomed—can never have reflected how difficult, even in
our boasted civilized state, it is to change habits acquired in early
childhood. “Men,” observes Hartley, in his Essays on Man, (page
190,) “are brought to any thing almost sooner than to change
their habit of life, especially when the change is either
inconvenient or made against the force of natural inclination, or
with the loss of accustomed indulgences. It is,” he continues, “the
most difficult of all things to convert men from vicious habits to
virtuous ones, as every one may judge from what he feels in
himself as well as from what he sees in others.” “It is almost,”
says Paley, after making the above quotation in his Evidences of
Christianity, “like making men over again.”
[48] At New Zealand the placenta is named “fenua,” which
word signifies land. It is applied by the natives to the placenta,
from their supposing it to be the residence of the child: on being
discharged, it is immediately buried with great care, as they have
the superstitious idea that the priests, if offended, would procure
it; and, by praying over it, occasion the death of both mother and
child, by “praying them to death,” to use their own expression.
[49] At New Zealand the women are attended, during labour,
by their husbands; but, if it is a difficult labour, they suppose the
spirits to be angry, and therefore send for the Tohunga, or priest.
On the arrival of the Tohunga, he strides over and breathes on
the woman, and then, retiring to a short distance, sits down and
prays to the spirits; if the labour terminates favourably, it is
looked upon as resulting from the influence of the Tohunga in
averting the anger of the spirits; but should the termination be
fatal, the priest is considered to have incurred the displeasure of
the spirits, and lost his influence.
[50] This animal is called “Goribun” by the Yas natives.
[51] Distance of miles in travelling in the interior of the colony
is nominal, and the time occupied in riding the distance is usually
taken into consideration; some stages seem often to be over and
others under-calculated. “Shepherd’s miles,” it is a saying in the
colony, “are short, those of stockmen long.”
[52] The different trees of the Eucalyptus genus are confused,
and require botanical arrangement: many, termed species, are
merely varieties; and the botanical characters of but few species
are accurately known.
[53] The “wire-grass” is said to indicate good soil, being found
growing in alluvial soil, in clumps, upon flats, swamps, &c.
[54] Sedge-grass is used for thatching, as well as beds for the
sheep during shearing time, after they have been washed.
[55] The “swamp oak” bears much resemblance to the larch. I
know not why this and other species of the casuarina trees have
received the colonial appellation of “oaks,” as forest-oak, swamp-
oak, she oak, &c., as they have not the slightest resemblance to
that tree in external character, unless the name may have been
given from some similarity in the wood.
[56] The granite soil at Bolam is said to injure the teeth of the
sheep, the teeth of young sheep being as much worn down by it
as in other soils is often seen in the old sheep.
[57] In February, 1833, the ship “Prince Regent” arrived at Port
Jackson, from England, with emigrants and a general cargo; she