Current topics in developmental biology, volume 111

CURRENT TOPICS IN
DEVELOPMENTAL BIOLOGY
“A meeting-ground for critical review and discussion of developmental processes”
A.A. Moscona and Alberto Monroy (Volume 1, 1966)

SERIES EDITOR
Paul M. Wassarman
Department of Developmental and Regenerative Biology
Icahn School of Medicine at Mount Sinai
New York, NY, USA

CURRENT ADVISORY BOARD
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FOUNDING EDITORS
A.A. Moscona and Alberto Monroy

FOUNDING ADVISORY BOARD
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S. Spiegelman
Hewson W. Swift
E.N. Willmer
Etienne Wolff


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CONTRIBUTORS
Youngwook Ahn
Stowers Institute for Medical Research, Kansas City, Missouri, USA
Aria C. Attia
Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital
Medical Center, Cincinnati, Ohio, USA
Tiziano Barberi
Pluripotent Stem Cell Differentiation Laboratory, Southwest National Primate Research
Center, Texas Biomedical Research Institute, San Antonio, Texas, USA, and Department of
Anatomy Neuroscience, The University of Melbourne, Parkville, Victoria, Australia
Linda A. Barlow
Department of Cell and Developmental Biology; Graduate Program in Cell Biology, Stem
Cells and Development, and Rocky Mountain Taste and Smell Center, University of
Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA
Onur Birol
Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, USA
Bianca E. Borchin

Pluripotent Stem Cell Differentiation Laboratory, Southwest National Primate Research
Center, Texas Biomedical Research Institute, San Antonio, Texas, USA, and Australian
Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia
Samantha A. Brugmann
Division of Plastic Surgery, Department of Surgery, and Division of Developmental Biology,
Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio,
USA
Ching-Fang Chang
Division of Plastic Surgery, Department of Surgery, and Division of Developmental Biology,
Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio,
USA
Bharesh Chauhan
Division of Pediatric Ophthalmology and Strabismus, Department of Ophthalmology,
University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
Alwyn Dady*
Laboratoire de Biologie du De´veloppement, Universite´ Pierre et Marie Curie-Paris 6, and
CNRS, Laboratoire de Biologie du De´veloppement, Paris, France

*Present address: Children’s Hospital of Pittsburgh, Rangos Research Building, Pittsburgh, Pennsylvania,
USA

xi


xii

Contributors

Jean-Loup Duband
Laboratoire de Biologie du De´veloppement, Universite´ Pierre et Marie Curie-Paris 6, and

CNRS, Laboratoire de Biologie du De´veloppement, Paris, France
Rene´e K. Edlund
Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, USA
Katherine A. Fantauzzo
Department of Developmental and Regenerative Biology, Icahn School of Medicine at
Mount Sinai, New York, USA
Alessandro Fantin
UCL Institute of Ophthalmology, University College London, London, United Kingdom
Vincent Fleury
Laboratoire Matie`re et Syste`mes Complexes, CNRS et Universite´ Denis-Diderot-Paris 7,
Paris, France
Andrew K. Groves
Program in Developmental Biology; Department of Molecular and Human Genetics, and
Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA
Ophir D. Klein
Departments of Orofacial Sciences and Pediatrics; Program in Craniofacial and Mesenchymal
Biology, and Institute for Human Genetics, University of California San Francisco, San
Francisco, California, USA
Takahiro Kunisada
Department of Tissue and Organ Development, Regeneration and Advanced Medical
Science, Gifu University Graduate School of Medicine, Gifu, and Japan Science and
Technology Agency ( JST), Core Research for Evolutional Science and Technology
(CREST), Tokyo, Japan
Anthony-Samuel LaMantia
George Washington University Institute for Neuroscience, and Department of
Pharmacology and Physiology, The George Washington University, School of Medicine and
Health Sciences, Washington, DC, USA
Richard Lang
The Visual Systems Group, Abrahamson Pediatric Eye Institute, Division of Pediatric
Ophthalmology, Department of Ophthalmology, University of Cincinnati and Children’s

Hospital Research Foundation, Cincinnati, Ohio, USA
Ming Lou
Department of Chemistry and Physics, Lamar University, Beaumont, Texas, USA
Sally A. Moody
Department of Anatomy and Regenerative Biology, The George Washington University,
School of Medicine and Health Sciences, and George Washington University Institute for
Neuroscience, Washington, DC, USA


Contributors

xiii

Tsutomu Motohashi
Department of Tissue and Organ Development, Regeneration and Advanced Medical
Science, Gifu University Graduate School of Medicine, Gifu, and Japan Science and
Technology Agency ( JST), Core Research for Evolutional Science and Technology
(CREST), Tokyo, Japan
William A. Mun˜oz
Stowers Institute for Medical Research, Kansas City, Missouri, USA
Jason M. Newbern
School of Life Sciences, Arizona State University, Tempe, Arizona, USA
Noriko Osumi
Department of Developmental Neuroscience, Centers for Neuroscience, Tohoku
University Graduate School of Medicine, Sendai, Japan
Timothy Plageman
College of Optometry, The Ohio State University, Columbus, Ohio, USA
Alice Plein
UCL Institute of Ophthalmology, University College London, London, United Kingdom
Christiana Ruhrberg

UCL Institute of Ophthalmology, University College London, London, United Kingdom
Gerhard Schlosser
School of Natural Sciences & Regenerative Medicine Institute (REMEDI), National
University of Ireland, Galway, Ireland
Elizabeth N. Schock
Division of Plastic Surgery, Department of Surgery, and Division of Developmental Biology,
Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio,
USA
Philippe Soriano
Department of Developmental and Regenerative Biology, Icahn School of Medicine at
Mount Sinai, New York, USA
Rolf W. Stottmann
Division of Developmental Biology, and Division of Human Genetics, Department of
Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
Jun Suzuki
Department of Developmental Neuroscience, Centers for Neuroscience, and Department of
Otorhinolaryngology-Head and Neck Surgery, Tohoku University Graduate School of
Medicine, Sendai, Japan
Paul A. Trainor
Stowers Institute for Medical Research, Missouri, and Department of Anatomy and Cell
Biology, University of Kansas Medical Center, Kansas City, Kansas, USA


PREFACE
Neural crest cells and placodes give rise to an extraordinary array of cell types
and tissues. Neural crest cells form bone; cartilage; odontoblasts of teeth;
connective tissue; cranial and trunk sensory neurons; peripheral autonomic
neurons; and glia, smooth muscle, pigment, and endocrine cells. Ectodermal
placodes contribute to the major sensory organs including the olfactory epithelium, lens of the eye, inner ear, and teeth and generate most of the cranial
sensory neurons, together with hair and mammary glands. Neural crest cells

and placodes are essential for embryonic development and adult homeostasis
and are increasingly clinically significant. Collectively, they generate many
of the defining characteristics of the craniates and have played major roles in
vertebrate evolution.
Neural crest cells and placodes were discovered independently in the
nineteenth century and in different species. Neural crest cells were first
described by His (1868) in chick embryos, while placodes were described
a little latter by van Wijhe (1883) in sharks. The study of neural crest cells
and placodes exhibits a rich history, serving as important paradigms for vertebrate evolution, cell and tissue induction, epithelial to mesenchymal transformation, migration, and differentiation, while also providing a profound
understanding of the underlying pathogenesis of congenital disorders. The
persistence of neural crest cells and placodes into adulthood serves as important models of stem cell biology and tissue homeostasis and provides insights
into cancer and metastasis.
Recent studies in tunicates and amphioxus point to neural crest cells and
placodes having independent evolutionary origins. However, neural crest
cells and placodes develop similarly in many respects and are mutually
interdependent. This is particularly true with respect to evolution and development of the vertebrate head and more specifically the peripheral nervous
system. For example, cranial neural crest cell-derived glia support placodederived neurons during the formation and function of the cranial sensory
ganglia. Furthermore, cranial neural crest cells establish corridors for the
proper migration of epibranchial placode-derived neurons. These properties
are a reflection of their extensive coevolution.
This issue of Current Topics and Developmental Biology highlights the current state of our knowledge concerning the evolution and development of
neural crest cells and placodes throughout the entire body. Where and when
xv


xvi

Preface

did these specialized cells occur and how are they governed by signaling

pathways and increasingly complex gene regulatory networks? What contributions do these cells make to specific tissues and organs and how are they
integrated? The answers to these questions together with the derivation and
application of stem cell-derived neural crest and placode cells in regenerative
medicine have major implications for understanding and potentially treating
congenital disorders.
PAUL A. TRAINOR
If I have seen further it is by standing on the shoulders of Giants.
Isaac Newton


CHAPTER ONE

Neural Crest Cell Evolution: How
and When Did a Neural Crest Cell
Become a Neural Crest Cell
William A. Muñoz*, Paul A. Trainor*,†,1
*Stowers Institute for Medical Research, Kansas City, Missouri, USA
†
Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, USA
1
Corresponding author: e-mail address:

Contents
1. Introduction
2. Defining Neural Crest Cells
3. Chordate Evolution and Vertebrate Origins
4. Neural Crest Cell Origin
5. Neural Crest Cell Evolution in Vertebrates
6. Cranial Neural Crest Cell Gene Regulatory Network
7. Evolution of Neural Crest Cell Gene Regulatory Networks

8. Conclusions and Perspectives
Acknowledgments
References

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11
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15
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20
20

Abstract
As vertebrates evolved from protochordates, they shifted to a more predatory lifestyle,
and radiated and adapted to most niches of the planet. This process was largely facilitated by the generation of novel vertebrate head structures, which were derived from
neural crest cells (NCC). The neural crest is a unique vertebrate cell population that is frequently termed the “fourth germ layer” because it forms in conjunction with the other
germ layers and contributes to a diverse array of cell types and tissues including the craniofacial skeleton, the peripheral nervous system, and pigment cells among many other
tissues and cell types. NCC are defined by their origin at the neural plate border, via an
epithelial-to-mesenchymal transition (EMT), together with multipotency and polarized
patterns of migration. These defining characteristics, which evolved independently in
the germ layers of invertebrates, were subsequently co-opted through their gene regulatory networks to form NCC in vertebrates. Moreover, recent data suggest that the ability to undergo an EMT was one of the latter features co-opted by NCC. In this review, we
discuss the potential origins of NCC and how they evolved to contribute to nearly all
tissues and organs throughout the body, based on paleontological evidence together
with an evaluation of the evolution of molecules involved in NCC development and their
migratory cell paths.
Current Topics in Developmental Biology, Volume 111
ISSN 0070-2153

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#

2015 Elsevier Inc.
All rights reserved.

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William A. Muñoz and Paul A. Trainor

1. INTRODUCTION
Neural crest cells (NCC) are considered to be a vertebrate innovation
that significantly contributed to the ability of chordates to diversify and radiate to most niches on the planet. Originally identified by Wilhelm His in
1868 (Hall, 2000), NCC have been shown to contribute to almost all tissues
throughout the body. NCC give rise to neurons, glia, Schwann cells, cartilage, bone, smooth muscles, adipocytes, and melanocytes, among many
others (Table 1) (Bronner & LeDouarin, 2012; Dupin, Creuzet, & Le
Douarin, 2006; Le Douarin & Dupin, 2012). Interestingly, many of these
cell types originally arose from the other germ layers, particularly the mesoderm, in vertebrates and nonvertebrate chordates (Bronner & LeDouarin,
2012; Dupin et al., 2006; Etchevers, Vincent, Le Douarin, & Couly,
2001). The function of the NCC and their diversity of cell and tissue
derivatives lent to the idea that NCC constituted a “fourth germ layer”
(Hall, 2000).
One of the most significant accomplishments of the NCC was in contributing to evolution of a “new head” with a hinged jaw, special sense
Table 1 Contributions of NCC to tissues throughout the animal

Peripheral nervous system
Cranial sensory ganglia

Sympathetic ganglia
Parasympathetic ganglia
Sensory dorsal root ganglia
Schwann cells
Ensheathing olfactory cells
Satellite cells of PNS ganglia
Central nervous system
Meninges
Enteric nervous system
Ganglia
Glial cells
Enteric neurons
Endocrine system
Carotid body cells
C cells (thyroid gland)
Adrenal-medullary cells

Fat tissue
Adipocytes
Skin and inner ear
Melanocytes
Dermal cells
Blood vessels and heart
Smooth muscle cells
Pericytes
Heart conotruncus
Striated muscles
Connective cells
Tendons
Extraocular muscles

Craniofacial skeleton
Odontoblasts
Osteocytes
Chondrocytes

The tissues with NCC contributions and the terminally differentiated NCC-derived cell types that populate the respective tissues are summarized here.


Neural Crest Cell Evolution

5

organs, and neural circuitry. These novel, predominately NCC-derived
tissues facilitated vertebrates becoming predatory, shifting away from the
filtration feeding lifestyle of their Amphioxus-like ancestors (Gans &
Northcutt, 1983; Northcutt & Gans, 1983). Additionally, NCC have
become integral in the organization of the vertebrate brain, possibly
facilitating its enhanced growth in vertebrates (Creuzet, Martinez, & Le
Douarin, 2006; Le Douarin, Couly, & Creuzet, 2012). Deficiencies in
NCC development are known to result in various birth defects including
craniofacial and heart anomalies, disorders affecting the bowel and other
organs, and loss of pigmentation in the skin and hair. In contrast overproliferation of NCC can result in several aggressive tumor types (Butler
Tjaden & Trainor, 2013; Noack Watt & Trainor, 2014). Therefore, the
innovation of NCC is one of the most significant factors contributing to vertebrate evolution and diversity. Understanding the mechanisms controlling
the specification, migration, and terminal specification of NCC will provide
insights into the evolutionary history of vertebrates and may lead to the
development of therapies for treating disorders of NCC development,
which are known collectively as neurocristopathies.

2. DEFINING NEURAL CREST CELLS

NCC have been the focus of extensive research since their initial
discovery, particularly with respect to the mechanisms underlying their
formation, the signals that determine how and where they migrate, and
to what cell types and tissues they contribute. NCC are induced to form
at the neural plate border, which is the junction between the neural
ectoderm and surface ectoderm (Simoes-Costa & Bronner, 2013). During
neurulation, the neural ectoderm elevates to form neural folds, which then
join to form the neural tube. During this process dorsal neuroepithelial cells
lose their intercellular connections, acquire apicobasal polarity, and undergo
and epithelial-to-mesenchymal transition (EMT). These processes facilitate
the delamination and migration of NCC in streams or in chains (Fig. 1A and
B), which then proceed to their terminal sites of differentiation (Fig. 1C)
(Baker & Bronner-Fraser, 1997; Groves & LaBonne, 2014; Mayor &
Theveneau, 2013).
During their emigration from the neural plate or neural tube, NCC
maintain a stem cell-like, multipotent state with the capacity for self-renewal
(Bronner-Fraser & Fraser, 1988, 1989; Coelho-Aguiar, Le Douarin, &
Dupin, 2013; Crane & Trainor, 2006; Dupin & Sommer, 2012; Le


Figure 1 NCC migration and differentiation in mice. (A–C) Wnt1-Cre YFP mouse section
at E10.5. Green labels NCC, blue labels DAPI stained nuclei. (A) NCC in the neural tube
and start of emigration from the dorsal neural tube. (B) NCC migrating in streams from
the neural tube. (C) NCC populating sites of terminal differentiation including pharyngeal arches (PA). (D) Terminally differentiated NCC-derived neurons in the peripheral
nervous system in an E11.5 mouse stained for Tuj1 (green) and DAPI (blue).
(E) Terminally differentiated NCC-derived neurons in the enteric nervous system of
an E13.5 mouse stained for Tuj1 (red) and DAPI (blue). (F) NCC-derived bone, stained
with alizarin red, and cartilage, stained with alcian blue, of the craniofacial skeleton
of an E18.5 mouse.

Neural Crest Cell Evolution

7

Douarin, Calloni, & Dupin, 2008; Le Douarin, Creuzet, Couly, & Dupin,
2004; McKinney et al., 2013; Prasad, Sauka-Spengler, & LaBonne, 2012;
Trentin, Glavieux-Pardanaud, Le Douarin, & Dupin, 2004). Interestingly,
this stemness is partially retained in adult NCC in stem cell niches which can
be isolated, purified, cultured, and used in neurodegenerative clinical applications (El-Nachef & Grikscheit, 2014; Greiner et al., 2014; Konig et al.,
2014; Sanchez-Lara & Zhao, 2014; Trolle, Konig, Abrahamsson,
Vasylovska, & Kozlova, 2014).
As NCC migrate throughout the developing embryo to their final destinations, they respond to various intrinsic and extrinsic signals promoting
their proliferation, survival, and terminal differentiation into numerous cell
types depending on their axial position (Barlow, Dixon, Dixon, & Trainor,
2012; Bhatt, Diaz, & Trainor, 2013; Walker & Trainor, 2006). NCC can be
categorized as cranial, cardiac, vagal, trunk, and sacral based on their axial
position of origin together with the cells and tissues they contribute to during terminal differentiation. Cranial NCC give rise to most of the bone and
cartilage of the facial skeleton (Fig. 1F) and neurons and glia of the cranial
ganglia (Fig. 1D), as well as smooth muscle and pigment cells. Cardiac NCC
contribute to the valves, septa, and outflow tract of the heart. The vagal
NCC form the enteric nervous system, which innervates the gastrointestinal
tract (Fig. 1E). Trunk NCC differentiate to form melanocytes, secretory
cells, and neurons and glia of the peripheral nervous system through their
formation of dorsal root and sympathetic ganglia. Sacral NCC, which are
a component of trunk NCC, also contribute to the enteric nervous system,
however to a significantly less extent than the vagal NCC (Trainor, 2014;
Simoes-Costa & Bronner, 2013). Despite this compartmentalization, each
axial population of NCC maintains a latent capacity to differentiate into lineages derived from other NCC populations when isolated at the start of their
migration and cultured in vitro, and when heterotypically transplanted in vivo

(Calloni, Glavieux-Pardanaud, Le Douarin, & Dupin, 2007; Coelho-Aguiar
et al., 2013; Le Douarin & Teillet, 1974; Le Lievre, Schweizer, Ziller, & Le
Douarin, 1980; McGonnell & Graham, 2002).
H€
orstadius posited the multipotency and regionalization of neural crest
populations along the body axis in amphibians (H€
orstadius, 1950, 1973).
This was subsequently refined by Nicole Le Douarin through quail–chick
chimeras, which demonstrated that specific regions of NCC would differentiate and contribute to adult tissues, and furthermore that NCC contributed to many more tissues than previously thought (Le Douarin &
Kalcheim, 1999). As cell-labeling technologies improved, it was definitively


8

William A. Muñoz and Paul A. Trainor

demonstrated that an individual NCC is multipotent and capable of differentiating into very diverse cell types (Bronner-Fraser & Fraser, 1988; Chai
et al., 2000; Jiang, Rowitch, Soriano, McMahon, & Sucov, 2000; Matsuoka
et al., 2005).
Thus the defining features of NCC are their origin at the neural plate
border, multipotency, formation via EMT, and acquisition of polarity
and migratory ability, together with their regulation by a conserved gene
regulatory network (GRN). These features must be considered collectively
and individually in any analysis of the evolutionary origins of NCC.

3. CHORDATE EVOLUTION AND VERTEBRATE ORIGINS
For over a century, the origin of vertebrates and thus NCC has been
debated based on shared structural homology between primitive vertebrates
and protochordates (urochordates and cephalochordates) (Fig. 2), which had
separated by the early Cambrian period (Dupret, Sanchez, Goujet,

Tafforeau, & Ahlberg, 2014; Gai, Donoghue, Zhu, Janvier, &
Stampanoni, 2011; Hall & Gillis, 2013; Mallatt & Chen, 2003). All animals
in these subphyla have a similar body plan at a particular stage in their life,
which includes a dorsal hollow neural tube and an axial structure, the

Figure 2 Cladogram showing the position of commonly studied model organisms of
NCC development and evolution. Key evolutionary events contributing to NCC formation and function are indicated.


Neural Crest Cell Evolution

9

notochord, which collectively defines them as chordates (Hall & Gillis,
2013; Holland & Chen, 2001). Vertebrates are further characterized by
the addition of a head and skeletal tissues both of which are absent in
protochordates.
Comparative embryology suggests that the cephalochordates are the sister
clade to vertebrates, and consistent with this idea, Amphioxus has been used
extensively as a model to explore the origins of vertebrates (Bertrand &
Escriva, 2011; Delsuc, Brinkmann, Chourrout, & Philippe, 2006;
Holland, 2013; Holland et al., 2008). Consequently, the epidermal nerve
plexus of protochordate-like ancestors resembling amphioxus was hypothesized to have evolved to produce the anterior parts of the head (Gans &
Northcutt, 1983). Mechanistically, this was thought to be achieved through
additions to the anterior regions of protochordates rather than a transformation resulting in evolution of a “new head.” This addition of a “new head” is
evident in Amphioxus by a notochord that extends to the most anterior
region of the body. In contrast, in vertebrates the forebrain and sense organs
lie rostral to the notochord. Additionally, a significant majority of the tissues
that constitute the “new head” are derived from NCC (Gans & Northcutt,
1983; Le Douarin & Dupin, 2012). This strongly suggests that NCC were

necessary for “new head” formation and therefore vertebrate evolution.
Interestingly, decades of extensive work in Amphioxus has not revealed
the presence of bona fide primitive NCC-like cells by either morphology or
molecular markers (Hall & Gillis, 2013; Yu, 2010). This missing evolutionary link may be partially explained by the recent genomic discovery that
urochordates are in fact more closely related to vertebrates than cephalochordates (Fig. 2) (Delsuc et al., 2006). This change in subphyla organization provides new avenues for exploring NCC innovation and vertebrate
evolution, as urochordates comprise a very diverse extant group with little
known about their embryology. Recently, it was provocatively proposed
that rudimentary NCC-like cells may exist in at least one genus of urochordates (Abitua, Wagner, Navarrete, & Levine, 2012).

4. NEURAL CREST CELL ORIGIN
The origin and diversification of NCC and ectodermal placodes were
proposed by Gans and Northcutt to be associated with the shift to active predation in vertebrate evolution (Gans & Northcutt, 1983). With the evolution of NCC, vertebrates became quadroblastic with ectoderm, mesoderm,
endoderm and NCC contributing to an enormous increase in cell diversity.


10

William A. Muñoz and Paul A. Trainor

The significance of NCC in vertebrate evolution has made the identification
of NCC-like structures during development and their derivatives in adults of
nonvertebrate chordates of fundamental scientific importance.
Although, significant work has focused on Amphioxus in the pursuit of
NCC precursors, no such cells have been identified to date. This lack of
NCC-like candidates in cephalochordates could be a result of a true lack
of this cell type in the subphyla or due to a lack of species diversity with
so few extant species within the subphyla. It is highly unlikely that
NCC-like cells will be found given the lack of typical NCC derivatives
in amphioxus normally found in vertebrates, such as peripheral pigment cells
and dentine-, bone-, or cartilage-forming cells. Interestingly, dorsal root

nerves in these animals are ensheathed by Schwann cell-like glial cells
(Bone, 1960; Peters, 1963). However, the origin for these glial cells has been
shown to be ectodermal. Furthermore, numerous invertebrate animals also
possess peripheral glial cells despite a complete absence of NCC-like cells
(Coles & Abbott, 1996; Hall & Gillis, 2013). This suggests that the glial cell
fate of ectodermal cells evolved prior to the divergence of NCC from ectoderm. Furthermore, the GRN for glial cell development may have not
arisen separately in NCC, but rather been conserved in ectoderm-derived
NCC and lost in the remainder of the ectodermal cells.
Recently, potential rudimentary NCC precursors in the form of pigment
cells were identified in the mangrove tunicate, Ciona intestinalis, which
belongs to the larger urochordate group. These cells originate near, though
not necessarily from, the neural plate in blastomere pair a7.6. These cells
migrate as individual cells either through the mesoderm or between the
mesoderm and epidermis, eventually populating the siphon and body wall
( Jeffery, Strickler, & Yamamoto, 2004). These cells and their derivatives
express HNK-1, a carbohydrate epitope found in migrating NCC in many
species together with Zic3, a transcription factor expressed in the vertebrate
neural plate border but not in migrating NCC. Furthermore, HNK-1 positive cells in ascidians also express enzymes necessary for melanogenesis. This
led to speculation that the evolutionary origins of a NCC precursor resided
in cells fated to become pigment cells ( Jeffery, 2006, 2007). However, there
are significant discrepancies within these studies including but not limited to
the fact that DiI lineage tracing labeled large populations of cells near the
neural plate not just the a7.6 blastomere pair. Furthermore, the molecular
markers used label multiple cell types including neural plate, not just
NCC ( Jeffery et al., 2004). Blastomere a7.6 derivatives include a number
of mesendodermal cell types (Hirano & Nishida, 1997), and in other animals


Neural Crest Cell Evolution

11

pigment cells may arise from mesodermal origins (Gibson & Burke, 1985).
These studies highlight the difficulties associated with identifying potential
NCC-like cells outside the vertebrate subphyla, given NCC mimic some
behaviors of the mesoderm-derived cells in vertebrates. For example,
NCC migrate significant distances with large protrusions and differentiate
into various cell types in numerous tissues, including cell types that prior
to the innovation of NCC were derived from the mesoderm such as chondroblasts and osteoblasts. Interestingly, this also suggests that the NCC may
have co-opted developmental programs from other cell types during
evolution.
More recently, it was found that blastomere a9.49 found near the neural
plate border of C. intestinalis expresses various neural plate border markers
and perhaps some NCC specification signals (Abitua et al., 2012; Imai,
Levine, Satoh, & Satou, 2006; Jeffery et al., 2008; Squarzoni, Parveen,
Zanetti, Ristoratore, & Spagnuolo, 2011). Interestingly, cells derived from
blastomere a9.49 give rise to the gravity sensing otolith and the melanocytes
in light-sensing tissues (Nishida & Satoh, 1989). Furthermore, ectopic
expression of Twist in the a9.49 blastomere endows the lineage with migratory ability. This suggests that a GRN of the type that might be expected in
rudimentary NCC was indeed already present in this lineage, and furthermore, that it evolved before the divergence of urochordates and vertebrates.
More importantly, the identification and characterization of this NCC-like
cell lineage that gives rise to pigment cells in a urochordate suggest that the
vertebrate NCC specification GRN was partially achieved by co-opting
existing differentiation networks together with an EMT GRN to endow
rudimentary NCC-like cells with migratory ability.
The identification of blastomere a9.49 of C. intestinalis as a very strong
potential candidate of primitive NCC-like cells is already a major step in the
direction of identifying when and where NCC originated. It remains to be
seen whether, given the extensive diversity of extant urochordates, that
more NCC-like cells will be identified in this subphylum. If there were,

it would suggest that NCC began to evolve in the Precambrian period.
In addition, primitive NCC-like cells in urochordates may reveal what cell
types NCC evolved from, together with their early functions.

5. NEURAL CREST CELL EVOLUTION IN VERTEBRATES
While protochordates have been important in identifying the potential origins of NCC and their influence on evolution and development, it is


12

William A. Muñoz and Paul A. Trainor

equally important to characterize how NCC have shaped the vertebrate subphyla. Numerous vertebrate model systems have been used including but
not limited to mice, chick, amphibians, zebrafish, and the jawless basal vertebrates, lamprey and hagfish. Despite highly conserved GRNs, vertebrate
NCC are able to create many diverse structures from the shell of a turtle to
the heart outflow tract of a mammal (Cebra-Thomas et al., 2007; Gilbert,
Bender, Betters, Yin, & Cebra-Thomas, 2007; Trainor, 2014).
Analyses of NCC development were first carried out in amphibians and
fish, and later in avians and other amniotes (H€
orstadius, 1950; Le Douarin,
1969), and much of the early focus centered on the contributions of NCC to
the craniofacial skeleton. NCC contribute to the cranium, facial skeleton,
connective and adipose tissues, dermis of the face, ventral part of the neck,
and several sense organs (Couly, Coltey, & Le Douarin, 1993; Creuzet et al.,
2006; Le Douarin & Kalcheim, 1999; Le Lievre et al., 1980). However, it
has become apparent that during vertebrate evolution, NCC have evolved
to participate in numerous other functions providing specific advantages
suited to an animal’s environment. The most influential evolutionary effects
of the NCC on vertebrates were through contributions that improved
metabolism, circulation, and respiration with significant changes from one

evolutionary branch to the next (Green & Bronner, 2014; Landacre,
1921; Lievre, 1974; Mongera et al., 2013).
NCC contribute to skeletal structures through their differentiation into
chondrocytes that form primary and secondary cartilage, osteoblasts forming
dermal and endochondral bone, and odontoblasts producing teeth and scale
dentine. Of these cell types only dentine secreting cells are exclusively a
NCC derivative. All the other cell types can also arise from the mesoderm
(Hall, 2009; Hall & Gillis, 2013). This makes dentine a definitive marker of
NCC in fossils and in tissues today. Congruent with studies that NCCderived bone formation is important for vertebrate evolution, it has been
suggested that the first mineralized skeletal tissues of the vertebrate subphylum were of NCC origin (Fig. 2). Early vertebrates, such as pteraspidomorphs agnathans, possessed ossified armor consisting of dentine
produced by odontoblasts ( Janvier, 1996; Le Douarin & Dupin, 2012;
Smith, 1991). Interestingly, these animals are devoid of well-characterized
vertebrae suggesting the presence of a cartilaginous endoskeleton most likely
derived from the somites (Ota, Fujimoto, Oisi, & Kuratani, 2011). As vertebrate evolution proceeded, gnathostomes began to develop an endoskeleton made of somite-derived cellular bone, while still retaining their
dentine-based exoskeleton. Later, however, as the endoskeleton became


Neural Crest Cell Evolution

13

fully developed, the putatively NCC-derived exoskeleton was lost and only
the NCC-derived skull, facial bones, and cartilages remained ( Janvier,
2011). Interestingly, while trunk NCC generally no longer contribute to
skeletal formation, they still maintain a latent ability in vitro to produce mesenchymal derivatives including skeletal tissues from both birds and mammals
(Calloni et al., 2007; Coelho-Aguiar et al., 2013; Ido & Ito, 2006;
McGonnell & Graham, 2002; Nakamura & Ayer-le Lievre, 1982).
In addition to these skeletal studies, extensive work has focused on the
most basal extant vertebrate, the jawless lamprey. The lamprey is used to
understand the role of NCC in the progressive evolution of vertebrates

after their separation from protochordates. Interestingly, the NCC GRN,
which is discussed later in this review, is mostly conserved in lamprey when
compared to gnathostomes suggesting that it has an ancient origin
(Medeiros, 2013).
Histological studies of lamprey revealed the presence of NCC in
embryos and their contributions to numerous tissues in adults (Hardisty,
1979; Medeiros, 2013). Furthermore, transplantation experiments showed
that cranial NCC grafted to the flank of lamprey or newts generated neurons, pigment cells, and cartilage nodules, while ablation experiments demonstrated that removal of NCC resulted in reductions of typical NCC
derivatives (Langille & Hall, 1986, 1988; Newth, 1951, 1956). Vital dye
labeling experiments subsequently confirmed the primary observations
and conclusions drawn from NCC transplantations and ablations
(Horigome et al., 1999; McCauley & Bronner-Fraser, 2003). However,
despite the significant conservation between lamprey and gnathostomes,
there are some differences in the NCC GRN, which will be discussed later.
Nonetheless, these studies in lamprey show that the NCC have an ancient
origin predating the divergence of lamprey and gnathostomes despite a complete lack of jaw structures in lamprey.

6. CRANIAL NEURAL CREST CELL GENE REGULATORY
NETWORK
NCC development comprises several distinct steps and events including specification and induction at the neural plate border, delamination
through EMT, migration, and finally terminal differentiation. To help
understand these complex processes, molecular manipulation has been used
to identify, characterize, and describe interactions between a considerable
number of genes involved in these processes of NCC development in species


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William A. Muñoz and Paul A. Trainor

spanning the vertebrate subphyla from jawless vertebrates to mice (Ota,
Kuraku, & Kuratani, 2007; Sauka-Spengler, Meulemans, Jones, &
Bronner-Fraser, 2007). These data have subsequently been assembled into
a putative conserved GRN to help explain the complex events of NCC
development (Betancur, Bronner-Fraser, & Sauka-Spengler, 2010a,
2010b; Meulemans & Bronner-Fraser, 2004; Sauka-Spengler & BronnerFraser, 2008). It is important to note that the proposed cranial GRN cannot
account for all NCC processes along the body axis as each region of NCC
exhibits a distinct repertoire of cell fates with some variations between species as well. Examples of these variations are exhibited by Snail and Twist,
which are expressed in postmigratory cells in lamprey while they are
expressed in premigratory and migratory NCC of gnathostomes (Rahimi,
Allmond, Wagner, McCauley, & Langeland, 2009; Sauka-Spengler et al.,
2007). Additional variations in the NCC GRN nodes exist between less
basal species. Mouse for example does not require Snail1 or Snail2 for
NCC formation (Murray & Gridley, 2006) nor Pax3 and Pax7, because
individual or combined loss-of-function does not affect NCC development
(our unpublished results). Furthermore, in Xenopus laevis, Snail and Twist
regulate mesoderm formation in addition to NCC formation (Shi,
Severson, Yang, Wedlich, & Klymkowsky, 2011).
The NCC GRN is subdivided into a series of temporal steps, with each
step governed by a distinct regulatory module. This network is initiated at
the neural plate border by several ligands secreted from the neuroectoderm,
nonneural ectoderm, and underlying mesoderm, including Wnts, BMPs,
FGFs, and retinoic acid. These extracellular signals act during gastrulation,
activating a transcriptional program that specifies the neural plate border,
making it competent to produce NCC (Groves & LaBonne, 2014;
Munoz, Sater, & McCrea, 2014).
BMPs form a gradient in the ectoderm, with low levels specifying the neural plate, high levels the surface ectoderm, and intermediate levels the neural
plate border. The intermediate levels of BMP are thought to work together
with ectoderm-derived Wnt signaling and underlying mesoderm-derived Fgf
signaling, which leads to the activation of transcription factors such as Pax3/7,

Zic1, and Msx1 in the neural plate border and the competency to form NCC
(Basch, Bronner-Fraser, & Garcia-Castro, 2006; Betancur et al., 2010a;
Huang & Saint-Jeannet, 2004; Munoz et al., 2014).
The neural plate border transcription factors are expressed not only in the
neural plate border but also in the immediate surrounding areas as well
where ectodermal placodes will form. The neural plate border becomes


Neural Crest Cell Evolution

15

further refined as the extracellular signals continue to enhance the neural
plate border specifiers eventually activating “NCC specifiers.” Included
in these specifiers are conserved transcription factors Snail/Slug, Id, FoxD3,
cMyc, and Sox9/10. These genes will begin to repress epidermal and neural
tube markers, such as Sox2 and E-cadherin, while upregulating factors necessary for migration, such as cadherin-7 and various matrix
metalloproteinases. This facilitates EMT and the delamination of NCC from
the neuroepithelium. These genes also play reiterated roles in the terminal
differentiation of several NCC lineages (Bronner & LeDouarin, 2012;
Hall & Gillis, 2013; Milet & Monsoro-Burq, 2012).
This presumptive NCC GRN is well conserved across multiple species
and continues to evolve. Novel genomic high-throughput methodologies
continue to reveal additional transcription factors, as well as epigenetic, posttranscriptional, and posttranslational regulators, broadening the network
(Attanasio et al., 2013; Brunskill et al., 2014; Simoes-Costa & Bronner,
2013; Simoes-Costa, Tan-Cabugao, Antoshechkin, Sauka-Spengler, &
Bronner, 2014).

7. EVOLUTION OF NEURAL CREST CELL GENE
REGULATORY NETWORKS

NCC formation, migration, and differentiation are controlled by a
complex GRN consisting of both intrinsic and extrinsic input. How these
transcriptional regulators and signals were first incorporated into a GRN during evolution is beginning to elucidate the origin of NCC. In early vertebrate
evolution, there were two whole-genome duplication events (WGD), not
found in closely related invertebrates (Fig. 2) (Green & Bronner, 2013;
Holland, 1999; Ohno, 1999; Simoes-Costa & Bronner, 2013; Taylor &
Raes, 2004). These two WGD appear to coincide with the evolutionary
origin of NCC that are found in all vertebrates. This suggests that WGD
may have contributed to or facilitated the diversification of NCC from the
potentially rudimentary neural crest-like cells found in nonvertebrate chordates (Abitua et al., 2012). WGD could have accomplished this through several mechanisms including alterations in gene expression patterns, creation of
novel genes through domain shuffling, the appearance of novel sequence
motifs in transcription factors, or the co-option of genes and their functions
leading to the generation of new cell types (Fig. 3).
Co-option occurs when a feature or molecule that performs a function is
repurposed for another use. Despite Amphioxus lacking NCC, they possess


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William A. Muñoz and Paul A. Trainor

Figure 3 The potential mechanisms contributing to the molecular evolution of NCC
GRN components. (A) Evolution of cis-regulatory elements of NCC specifiers. Shapes
indicate tissue-specific, cis-regulatory elements on the DNA with colors corresponding
to expression in the tissues of the embryo section on right. As vertebrates evolved from
Amphioxus, loss-of-function mutations in regulatory elements (loss of circle) resulted in
loss of expression in corresponding tissue. Concurrently, a novel cis-regulatory element
(triangle) evolved to promote expression of the NCC specifier in the evolving NCC. NT,
neural tube; NO, notochord; SO, somites. (B) Evolution of novel gene product via domain
shuffling of chromosomes. Several gene products unspliced do not bind to regulatory

elements of NCC specifier genes. Following domain shuffling, a novel gene, consisting
of regions of each original gene, is created that can bind and activate transcription of
the NCC specifier promoting NCC development. (C) Evolution of novel protein motifs in
existing molecules. Proteins evolve either a novel motif allowing DNA binding or interactions with additional transcription factors promoting transcription of a NCC specifier.

homologs of most genes known to be necessary for neural crest specification,
migration, and terminal differentiation (i.e., Snail1/2, AP-2, FoxD3, Twist, Id,
etc.). Interestingly however, none of these molecules are expressed at the neural plate border in Amphioxus except Snail (Holland, 2013; Holland et al.,
2008; Meulemans & Bronner-Fraser, 2005, 2007; Meulemans,
McCauley, & Bronner-Fraser, 2003). This indicates that NCC evolved in part
by co-opting genes into a primitive gene network that was already present in
the neural plate border of the ancestral chordate.


Neural Crest Cell Evolution

17

It has been assumed that for a gene to be expressed in a new domain, it must
have acquired new cis-regulatory control enhancers. One study supporting this
hypothesis involves the vertebrate transcriptional repressor FoxD3. FoxD genes
are members of the Fox family of transcription factors with only one FoxD gene
identified in amphioxus. In amphioxus, FoxD is expressed in the forebrain,
somites, and notochord, while in vertebrates, five duplicates have independent
regions of expression. FoxD3 is the only duplicate that is expressed in NCC, but
FoxD3 is also expressed in the somites and spinal cord. This demonstrates that
FoxD3 acquired a new domain of activity in NCC, where it maintains NCC
potential at the neural plate border and prevents melanocyte differentiation
(Ignatius, Moose, El-Hodiri, & Henion, 2008; Teng, Mundell, Frist,
Wang, & Labosky, 2008; Thomas & Erickson, 2008). To investigate if a regulatory region for FoxD3 had evolved since a common ancestor, a previously

identified regulatory region of Amphioxus FoxD (Yu, Holland, & Holland,
2004) was introduced into chick where it directed expression to the somites,
notochord, and neural tube, but not to neural crest (Yu, Meulemans,
McKeown, & Bronner-Fraser, 2008). These studies demonstrate that evolution of a regulatory region was necessary for FoxD3 to be expressed in the neural
plate border region and potentially become part of the NCC GRN (Fig. 3A).
However, this work does not address whether the spatiotemporal expression
was sufficient for FoxD homologs to contribute to NCC specification.
Given the significant evolutionary distance between vertebrates and
Amphioxus, it is expected that there would be differences in the protein
structure as well as in the regulatory regions. This made it a distinct possibility that FoxD3 had also developed a novel protein function allowing it to
regulate neural crest specification beyond Amphioxus FoxD activity. To test
this idea numerous chimeric versions of Amphioxus FoxD with zebrafish
FoxD3, which alone induces ectopic NCC in the neural tube, were tested
for their ability to induce ectopic NCC. The chimeras and full-length proteins, including the FoxD3 paralogs, were assayed for their ability to induce
ectopic NCC when electroporated into chick neural tubes. The FoxD3
paralogs and Amphioxus FoxD could not induce ectopic NCC; however,
a chimeric construct replacing the first 39 amino acids of Amphioxus FoxD
with the first 39 amino acids of Xenopus FoxD3 was able to induce ectopic
NCC. This study identified a necessary amino-terminal region of FoxD3
that has evolved specifically allowing FoxD3 to induce NCC when
expressed in the neural tube (Fig. 3C). These two studies demonstrate that
neural crest specifiers may not require just one evolutionary advantage over
their cephalochordate homologs.


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William A. Muñoz and Paul A. Trainor

As NCC specifiers were modified and co-opted into the NCC GRN,

various activities distinct from the surrounding ectoderm and neurectoderm
were acquired, including the ability to undergo EMT, migrate away from
the neural tube and differentiate into novel cell types. Among these, it
has been suggested that molecules governing EMT were the last to be
co-opted into the GRN. In Amphioxus, many of the NCC specifiers are
expressed in mesenchymal structures, with only the conserved NCC specifier Snail also being expressed at the neural plate border (Imai, Hino, Yagi,
Satoh, & Satou, 2004). Interestingly in Ciona, the NCC-like cell (blastomere
a9.49) derivatives that form pigment cells express many of the NCC specification genes, including Id, FoxD, Snail, and Ets but do not migrate as mesenchymal cells. However, a9.49 derivatives can be transformed into
mesenchymal-like cells through ectopic expression of Twist, which is
required for the EMT of mesodermal cells in Ciona (Abitua et al., 2012).
This demonstrates that much of the molecular machinery to transform these
NCC-like cells into mesenchymal cell types is present in Ciona, but it still
lacks key regulators to promote EMT.

8. CONCLUSIONS AND PERSPECTIVES
In this chapter, we have briefly summarized our knowledge regarding
the properties of NCC, a vertebrate innovation that facilitated the extensive
evolutionary diversity and expansion of vertebrates. Recent work has begun
to identify and characterize the coincident origins of NCC and vertebrates.
The data obtained in various species and phyla provide an overview of NCC
and how they are defined by their differing regulatory states, position at particular developmental time points, and ability to form a wide variety of cell
types. The regulatory state referenced here as the cranial NCC GRN still
requires significant future work to further define additional interactions that
have not been identified yet and continue to characterize the interactions
that have already been established. However, there are still significant gaps
in our knowledge of the NCC GRN with little known about the earliest
NCC specifiers that commit NCC to their fates. Furthermore, as NCC
migrate and undergo differentiation, our understanding of what signals they
receive and how they are processed remains incomplete. Within the already
defined NCC GRN there remain undefined interactions with recent work

describing crosstalk between several known components and potential posttranslational regulation that has yet to be considered in these models. It will
be important to understand these mechanisms, so comparisons with other


Neural Crest Cell Evolution

19

networks can be established to illuminate how various cellular pathways
were adapted and integrated into the GRN.
Of key importance to furthering our understanding of the NCC regulatory states will be continued efforts to understand the GRN of NCC at
each axial level during their formation and migration. This will require comparable studies to those accomplished with the cranial NCC already, across
multiple species and at various times of development possibly using the cranial NCC GRN as a guidepost. Overlap with cranial NCC is expected, but
so too is significant variance and specificity within each axial region, illustrating a much broader involvement of the genome. It will be important
in these studies to monitor and identify changes outside the defined
GRN with respect to epigenetic, posttranscriptional, and posttranslational
modifications. High-throughput sequencing combined with various molecular perturbations has already begun to provide insight into these questions
highlighting large changes in transcriptional profiles as NCC progress
through each regulatory state. Epigenetic profiling is a maturing field and
can now be easily performed in parallel to RNA-seq experiments to understand the effects of various perturbations on chromatin structure and function. Finally, proteomics can provide another method to monitor whole
proteome changes in NCC at distinct axial levels and stages of development.
These high-throughput methods will continue to evolve and become major
resources for expanding the GRN of NCC.
Aside from the GRN, there is still much to be understood concerning
how NCC evolved from primitive NCC-like cells. With a shift in research
from cephalochordates to urochordates, it is likely that NCC-like cells will
be identified in a wide variety of these diverse extant subphyla. The identification of additional NCC-like cells and variation between the cells, their
derivatives, and active pathways will promote our understanding of the steps
that were necessary for NCC formation prior to the divergence of vertebrates from protochordates. These steps will hopefully reveal how NCC
specifiers came to be expressed in the neural plate border and how they came

to regulate gene products necessary for NCC specification and differentiation. Furthermore, they may also help to reveal the factors necessary for the
induction of mammalian NCC. Although we understand many of the signals required for NCC formation in avian and aquatic species, the counterparts underpinning mammalian NCC formation remain to be functionally
determined. What is clear is that the origins of NCC are closely linked to
evolution of the vertebrate lineage, and disruptions of NCC development
result in neurocristopathies, demonstrating their significance in vertebrates.


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William A. Muñoz and Paul A. Trainor

Despite the phenomenal work that has been accomplished to date to
understand the origins, mechanisms, and evolution of NCC, the field is still
relatively young with numerous questions and gaps in our knowledge.
Technological advances will provide exciting opportunities to address these
outstanding questions and significantly impact the field of developmental
biology.

ACKNOWLEDGMENTS
The authors thank Annita Achilleos, Naomi Tjaden, and George Bugarinovic for images used
in Fig. 1A–C, E, and F, respectively. Research in the Trainor laboratory is supported by the
Stowers Institute for Medical Research and the National Institute of Dental and Craniofacial
Research (DE 016082).

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