Neural Crest Genes

Perspective
Insights into neural crest development and evolution

from genomic analysis
Marcos Simões-Costa and Marianne E. Bronner1
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA
The neural crest is an excellent model system for the study of cell type diversification during embryonic development due
to its multipotency, motility, and ability to form a broad array of derivatives ranging from neurons and glia, to cartilage,
bone, and melanocytes. As a uniquely vertebrate cell population, it also offers important clues regarding vertebrate
origins. In the past 30 yr, introduction of recombinant DNA technology has facilitated the dissection of the genetic
program controlling neural crest development and has provided important insights into gene regulatory mechanisms
underlying cell migration and differentiation. More recently, new genomic approaches have provided a platform and
tools that are changing the depth and breadth of our understanding of neural crest development at a ‘‘systems’’ level. Such
advances provide an insightful view of the regulatory landscape of neural crest cells and offer a new perspective on
developmental as well as stem cell and cancer biology.
The neural crest is an embryonic cell population with stem cell-like toward the midline and then in the dorsal aspect of the neural
properties, including multipotency and the ability to self-renew. tube. Shortly thereafter, they lose their intercellular connections,
Unique to vertebrates, neural crest cells contribute to a wide variety undergo an epithelial to mesenchymal transition (EMT), and ac-
of derivatives, including sensory and autonomic ganglia of the quire mesenchymal, migratory characteristics that endow these
peripheral nervous system, adrenomedullary cells, cartilage and cells with the ability to leave the neural tube (Gammill and Bronner-
bone of the face, and pigmentation of the skin. Although similar Fraser 2003; Sauka-Spengler and Bronner-Fraser 2008b).
cell types, such as pigment cells and sensory neurons, already exist Once they have emigrated from the neural tube, neural crest
in nonvertebrate chordates and other multicellular organisms, cells migrate in organized streams to populate different niches
these derivatives arise de novo under the umbrella of the neural throughout the embryo. Depending upon their starting position
crest in the vertebrate lineage. along the body axis and the subsequent path taken, they give rise
Since its discovery by His (1868), the neural crest has occupied to different cell types and contribute to the formation of a variety
a prominent place in developmental biology due to its extensive of tissues and organs (Fig. 2). The cranial neural crest forms a large
migratory properties and remarkable developmental potential. portion of the facial skeleton as well as cranial ganglia, smooth
Interest in this cell population has been further fueled by its muscle, and pigment cells. The vagal neural crest has an important
medical and evolutionary importance. For example, numerous role in cardiac development since it contributes to the valves and
congenital birth defects and neoplastic diseases are linked to ab- septa of the heart and also forms the enteric nervous system that
normal development of the neural crest development and its innervates the entire length of the gut. The trunk neural crest gives
derivatives (Hall 1999). Due to its inherent stem cell properties, rise to dorsal root and sympathetic ganglia of the peripheral ner-
there is great interest in using these cells in regenerative medicine vous system, as well as secretory cells and melanocytes. Finally, the
to treat disorders like familial dysautonomia, cleft palate, and neural crest formed at the sacral region cooperates with the vagal
some heart conditions ( Jones and Trainor 2004; Lee et al. 2009). crest to form a small portion of the enteric nervous system (Le
Furthermore, as the neural crest gives rise to a number of verte- Douarin 1986; Le Douarin and Kalcheim 1999). Although neural
brate-specific traits, it is thought to have played an important role crest cells appear to become progressively restricted as they reach
in chordate evolution (Gans and Northcutt 1983; Northcutt particular targets, many early migrating cells are multipotent, with
2005). the ability to form several derivatives (Bronner-Fraser and Fraser
The initial phases of neural crest formation include some of 1988, 1989). Moreover, many neural crest-derived tissues appear to
the most extensive morphogenetic movements observed during retain neural crest stem cells that are multipotent (Crane and
vertebrate embryonic development (Fig. 1). Initially the prospec- Trainor 2006).
tive neural crest cells reside in a territory known as the neural plate A number of classical experiments were fundamental for
border, which is located at the edges of the neural plate, the em- understanding the main characteristics of the neural crest. For
bryonic region destined to form the central nervous system. example, early studies with amphibian embryos suggested that
Through a process called neurulation, the neural plate invaginates there was regionalization of neural crest populations along the
by elevation of the edges, or neural folds. The end result is the body axis and broad developmental potential (Horstadius 1950).
conversion of the flat neural plate into a cylindrical structure called Importantly, the elegant quail-chick chimera experiments pio-
the neural tube, which will later form the brain and spinal cord. neered by Nicole Le Douarin in the 1960s and 1970s were central
During the process of neural tube closure, premigratory neural in defining the precise contributions of different populations of
crest cells reside first within the neural folds as they converge the neural crest to distinct derivatives in higher vertebrates (Le
Douarin and Kalcheim 1999). Bronner-Fraser and Fraser (1988)
1 tackled the question of the developmental potential of individual
Corresponding author
E-mail [email protected] neural crest by labeling single premigratory cells with a vital dye.
Article is online at http://www.genome.org/cgi/doi/10.1101/gr.157586.113. They showed that the progeny of a single neural crest cell included
23:1069–1080 Ó 2013, Published by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/13; www.genome.org Genome Research 1069
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Simões-Costa and Bronner
a wealth of molecular data that has helped to formulate our views

on how complex processes such as induction, specification, dif-
ferentiation, and migration are regulated. This extensive body of
work has established the neural crest as a prime model for the
exploration of questions central to developmental biology—such
as how complex and diverse tissues arise from a seemingly ho-
mogeneous population of cells.
Currently, genomic approaches are being used to address es-
sential aspects of neural crest development. The extensive work
done on the signaling and transcriptional control of neural crest
development culminated with the assembly of a gene regulatory
network that attempts to explain, in molecular terms, the process
of neural crest formation. Sequencing of chordate genomes has
allowed for identification of novel genes involved in neural crest
formation and provided numerous tools to scrutinize the regula-
tory mechanisms underlying this process. Furthermore, compar-
ative genomic analyses are providing important insights about the
evolution of the neural crest and the origins of the vertebrate clade.
Here, we will present a synopsis of the current state of the field of
neural crest biology from a gene regulatory perspective and will
also discuss how recent technological advances can help shape
future research.
Overview of the current state of the neural crest gene

regulatory network (GRN)
The regulatory machinery controlling neural crest formation and
diversification is comprised of an intricate array of transcription
factors and signaling molecules that act in concert to provide this
cell population with its defining features (Sauka-Spengler and
Bronner-Fraser 2008b). In order to interrogate such a complex
regulatory program, we and others have assembled a multistep
Figure 1. Morphogenetic movements during early neural crest de- neural crest GRN that integrates transcriptional inputs and di-
velopment. (A) Schematic diagram of transverse sections through chick verse environmental signals (Meulemans and Bronner-Fraser 2004;
embryo during neurulation. Prospective neural crest cells reside in the
neural plate border (green), a territory between the neural plate and the Betancur et al. 2010a). A simplified version of the cranial neural
non-neural ectoderm. (B) As neurulation proceeds, the neural plate in- crest GRN is represented in Figure 3. It is composed of a series of
vaginates, resulting in the elevation of the neural folds, which contain regulatory steps arranged hierarchically, which include induction
neural crest precursors. (C ) After neural tube closure, neural crest cells lose of the prospective neural crest, specification of the neural plate
intercellular connections and undergo an epithelial to mesenchymal
transition. Once they have delaminated from the neural tube, they mi- border, specification of the bona fide neural crest cells, and the
grate extensively to populate different niches throughout the embryo. diversification of the neural crest cells through the action of neural
crest effector genes (Sauka-Spengler and Bronner-Fraser 2008b).
Remarkably, these regulatory stages not only define cell identity
cell types as diverse as sensory neurons, presumptive pigment cells, and behavior at a given time point, but also drive seamless tran-
ganglionic supportive cells, and neural tube cells (Bronner-Fraser sitions to the next regulatory state.
and Fraser 1988). Clonal analysis of the neural crest in tissue cul- The first level of the neural crest GRN is comprised of in-
ture further demonstrated multipotency of single neural crest duction events that lead to the formation of the neural plate bor-
cells to form multiple derivatives (Sieber-Blum and Cohen 1980; der. This process is dependent upon the interplay of different sig-
Trentin et al. 2004; Dupin et al. 2010) as well as their ability to self- naling pathways such as Wnt, Fgf, BMP, and Notch/Delta. Secreted
renew (Stemple and Anderson 1992). However, the presence of signaling molecules are produced by adjacent tissues and result in
some clones that formed single derivatives has been interpreted the activation of a particular set of genes in the edges of the neural
as suggesting a possible fate restriction in some subpopulations plate. These neural plate border specifier genes (e.g., Msx, Pax3/7, Zic1,
(Stemple and Anderson 1992). These and innumerous other ex- Dlx3/5) initiate expression prior to the traditional neural crest
periments have characterized cellular properties of the neural marker genes and often are down-regulated once neural crest
crest that can now be tackled in molecular terms due to advances identity is established. Their region of overlap at the neural plate
in molecular biology and genomics. border defines a broad territory of cells competent to respond to
In the last three decades, advances in recombinant DNA neural crest specifying signals and later form migrating neural crest
technology and molecular genetics techniques have made it pos- cells (Meulemans and Bronner-Fraser 2004).
sible to start examining the genetic program controlling neural The next step in the neural crest GRN is initiated when neural
crest development. Identification of a number of tissue-specific plate border specifiers in combination with signaling molecules
transcription factors allowed for the description of the first regu- activate transcription of neural crest specifier genes. These include
latory interactions necessary for establishment of neural crest transcription factors like Snai1/2, Tfap2a, Foxd3, Twist, Id, Myc,
identity. Studies in several model organisms have provided and Sox9/10. Expression of these factors reflects the specification
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Genomic approaches in neural crest biology
Adams et al. 2008). Transcriptome analysis

of pure neural crest populations has un-
covered a variety of new specific transcrip-
tion factors, nuclear receptors and signal-
ing molecules that are expressed during
different stages of development (M Simões-
Costa and ME Bronner, unpubl.). Further-
more, novel techniques for genome-wide
chromatin profiling allow genome-wide
identification of active and poised cis-
regulatory modules (Rada-Iglesias et al.
2012) and facilitate functional analysis for
rapid characterization of epistatic interac-
tions. Such approaches will increase the
complexity of the neural crest GRN by
providing a more accurate and complete
Figure 2. Contributions of different neural crest cell populations to adult tissues and organs. representation of the different regulatory
Depending on their axial level of origin and migratory pathway followed, neural crest cells adopt dif- modules and their interactions.
ferent fates and contribute to distinct tissues and organs. Cranial neural crest forms a large portion of The current version of the neural
the facial skeleton as well as cranial ganglia, most of the dental tissues, and the cornea. Vagal neural crest GRN is grounded in interactions
crest contributes to the valves and septa of the heart, the smooth muscle of the great vessels, and the
enteric nervous system. Trunk neural crest gives rise to dorsal root and sympathetic ganglia of the between transcription factors and signal-
peripheral nervous system and the chromaffin cells of the adrenal gland. Most caudally, the neural crest ing molecules, and does not include extra
formed at the sacral region contributes to a small portion of the enteric nervous system. Melanocytes of levels of regulation such as those im-
the skin and integuments are derived from neural crest at all axial levels. parted by epigenetic, post-transcriptional
and post-translational control. Neverthe-
less, there is increasing evidence pointing
state of the neural crest population and endows them with its to an important role for chromatin modification in the de-
defining features, like the ability to undergo EMT and become velopment of the neural crest. For example, the histone demeth-
migratory. Activation of the neural crest specifiers can be quite ylase JMJD2A directly regulates a number of neural crest specifier
complex and depends upon various inputs from all levels of the genes, thus controlling the time of onset of their expression in the
neural crest GRN. For example, Snai2 activation requires co- neural folds (Strobl-Mazzulla et al. 2010). Furthermore, the neural
operation between elements of the Wnt pathway, Zic1, and Pax3/7 crest specifier, SNAI2, forms a complex with histone deacetylases
(Sato et al. 2005). In addition, SoxE transcription factors are nec- (HDACS) to silence genes such as CAD6B, thus allowing neural
essary for the maintenance of snai2 expression (Honoré et al. crest delamination (Strobl-Mazzulla and Bronner 2012). Epigenetic
2003). Thus, factors from different hierarchical levels of the neural silencing is also crucial for the initial demarcation of the neural
crest gene regulatory network operate in concert to establish and crest territory. Hu and colleagues have shown that DNA methyl-
maintain the neural crest transcriptional state (Sauka-Spengler and transferase3A promotes neural crest specification by repressing
Bronner-Fraser 2008b). neural genes SOX2 and SOX3, thus acting as a fate switch in the
The neural crest specifier genes, in turn, regulate expression of cells of the neuroectoderm (Hu et al. 2012). Recent results also
effector genes involved in cell cycle control, epithelial to mesen- emphasize the role of post-translational modifications in neural
chymal transition, and migration. Neural crest effector genes jump- crest regulation. Lee and colleagues have shown that SUMOylation
start a number of gene batteries that instruct the behavior of the of SoxE factors causes recruitment of the repressor Grg4 (also
newly formed neural crest cells, allowing them to delaminate from known as Tle4) instead of its usual coactivator partners. This study
the neural tube, proliferate and maintain population size, migrate highlights how post-translational regulation can alter gene func-
along different pathways, and finally differentiate into a wide va- tion and drive the same factor to play opposing roles in a context-
riety of derivatives (Meulemans and Bronner-Fraser 2004; Sauka- dependent manner (Lee et al. 2012).
Spengler and Bronner-Fraser 2008b). At the same time, the effector At the present time, the neural crest GRN (Meulemans and
genes activate the expression of receptors and signaling molecules Bronner-Fraser 2004; Betancur et al. 2010a) primarily focuses on
that equip the cells with the capacity to respond to environmental the regulatory interactions of the cranial neural crest. Since im-
cues. This molecular toolkit also allows cell–cell interactions that portant differences exist between distinct subpopulations of the
influence not only other neural crest cells, but also numerous neural crest along the body axis (Le Douarin et al. 2004), it would
embryonic tissues with which they interact during migration. For be naı̈ve to assume that such interactions are common to all
example, neural crest cells instruct somite cells to differentiate into neural crest populations. Indeed, there are likely to be important
muscle precursors (Rios et al. 2011). modifications that help explain regional differences in neural
The neural crest GRN integrates >20 yr of work from many crest cell migratory properties and cell fates (Simões-Costa et al.
research groups performed in different model organisms, and 2012). Thus a more inclusive version of the GRN should include
provides a conceptual framework for the study of the neural crest the molecular circuits that are particular to each of the neural
genetic program. It is, however, not yet complete and will greatly crest subpopulations. Studies scrutinizing the regulatory states of
benefit from the high-throughput genomic approaches that are these distinct populations will reshape the GRN and provide
currently part of the scientific repertoire. For instance, macroarray important clues about the plasticity of the neural crest. Ulti-
screens have uncovered numerous new molecules that are up- mately and optimally, the GRN will have predictive value, which
regulated in the neural crest (Gammill and Bronner-Fraser 2002; will help anticipate the molecular outcome of particular pertur-
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This can only be accomplished through

systematic identification and character-
ization of the cis-regulatory apparatus
that controls expression of specific com-
ponents of the neural crest GRN (Fig. 4).
Tissue-specific enhancers have been
invaluable for investigation of the genetic
program underlying neural crest forma-
tion and can be exploited in several dif-
ferent contexts. This is exemplified by
numerous studies that have taken ad-
vantage of the mouse Wnt1 enhancer to
address the contribution, potential, and
genetic regulation of the neural crest.
This enhancer region, which is a 5.5-kb
element at the 39 region of the Wnt1 lo-
cus, activates transcription in the dorsal
neural tube and migrating neural crest
cells of murine embryos (Echelard et al.
1994). It has been employed extensively
in mapping the contribution of different
neural crest populations ( Jiang et al.
2000; Nakamura et al. 2006; Barraud et al.
2010) through the use of the Rosa26 sys-
tem (Soriano 1999). It has also been
employed for targeted genetic manipula-
tion through conditional knockout ani-
mals (Ito et al. 2003; Akiyama et al. 2004;
Brewer et al. 2004; Dudas et al. 2004;
Nakamura et al. 2006) that have genes
inactivated specifically in the neural
crest. This has allowed for the functional
characterization of a number of genes in
a neural crest cell-autonomous manner.
The initial approach for the identi-
fication of enhancers of neural crest genes
was done by screening noncoding DNA
isolated from the locus of the gene of in-
terest (Fig. 4; Echelard et al. 1994). Frag-
ments from the noncoding DNA were
cloned upstream of a minimal promoter
Figure 3. Cranial neural crest gene regulatory network (GRN). The neural crest gene GRN is com-
plus reporter sequence and tested in vivo
posed of different regulatory modules arranged hierarchically. Each regulatory state defines cell identity
and behavior at a given time and also drives the transition to the next level of the network. This is or in vitro. The sheer size of the genetic
a simplified representation of the cranial neural crest GRN (Betancur et al. 2010a). loci and number of subregions that had to
be tested made this approach laborious
and time consuming, particularly given
bations. This will be useful for etiological studies, of particular the time and expense of using mice. New genomic tools have since
importance due to the large number of neural crest-related birth emerged and revolutionized the way cis-regulatory modules are
defects. A complete neural crest GRN will also inform upon ge- identified. Importantly, the sequencing of complete genomes has
netic reprogramming of induced pluripotent stem (iPS) cells or greatly benefited the search for conserved cis-regulatory elements
embryonic stem (ES) cells into neural crest cells, which will have (Cooper and Sidow 2003; Uchikawa et al. 2004). Comparison of
significant implications for regenerative medicine and cancer. the noncoding regions between multiple species has proved an
efficient approach for uncovering evolutionarily conserved re-
gions (ECRs) that may regulate transcription of neighboring genes
Cis-regulatory analysis of neural crest enhancers (Uchikawa et al. 2003; Betancur et al. 2010b; Simões-Costa et al.
The first version of the neural crest gene regulatory network was 2012). The activity of the ECRs subsequently can be investigated
based on data assembled from a combination of gene expression by stable or transient transgenesis. In this context, the chicken
patterns and functional studies obtained from several model or- embryo has proved to be an excellent amniote model for cis-reg-
ganisms (Meulemans and Bronner-Fraser 2004). Although this ulatory analysis in higher vertebrates, due to highly efficient
approach provided a conceptual framework for investigating transient transgenesis accomplished through electroporation
neural crest identity, further resolution of the GRN requires the techniques (Sauka-Spengler and Barembaum 2008; Takemoto et al.
identification of direct epistatic interactions among its players. 2011). Similarly, tol2-mediated transgenesis in zebrafish is an ef-

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Figure 4. Strategies for cis-regulatory analysis of the neural crest. (A) Early approaches for identifying enhancers of neural crest genes included screening
long stretches of noncoding DNA. Fragments from the locus of the gene of interest were cloned upstream of a minimal promoter and a reporter gene and
tested in vivo by cell transfection or transgenesis. (B) Sequencing of vertebrate genomes facilitated the search for cis-regulatory modules. Computational
comparison between different species reveals evolutionary conserved regions (ECRs) that are putative regulators of nearby genes. The ECRs are sub-
sequently tested for activity by stable or transient transgenesis in different model organisms. (C ) Novel approaches allow for genome-wide analysis of the
cis-regulatory modules. Profiling of histone modification through ChIP-seq allows mapping of chromatin mark patterns and identification of active and
poised enhancers. This method was used to annotate enhancers that are active in human neural crest cells induced from human embryonic stem cells
(Rada-Iglesias et al. 2012). Once enhancers are validated in vivo, they become valuable tools that can be exploited in different contexts. (MP) Minimal
promoter; (REP) reporter gene; (TF) transcription factor.
ficient way to test enhancers, including those that work across the cis-regulatory apparatus (Degenhardt et al. 2010). Similar to the
distant species (Fisher et al. 2006; Rada-Iglesias et al. 2012). case in mice, a study in zebrafish by Garnett and colleagues shows
A number of neural crest-specific enhancers have been iden- that the zebrafish pax3a gene expression is also controlled by two
tified in the past few years in different model organisms. Com- distinct enhancers. Although manipulations in BMP, Wnt, and
parative genomics was used to identify neural crest-specific en- FGF signaling disrupt the activity of the individual enhancers,
hancers for Zic3, Sox9, Foxd3, Sox10, Snai2, and Twist, among when combined in the same construct, they are less susceptible to
others (Smith et al. 1997; Bagheri-Fam et al. 2006; Betancur et al. environmental perturbations. Thus, synergistic activity of differ-
2010b; Garnett et al. 2012; Simões-Costa et al. 2012). For example, ent regulatory modules can bring a level of robustness to tran-
cis-regulatory analysis of murine Pax3 revealed intriguing aspects scriptional control, particularly in the case of genes downstream
of how neural crest genes are regulated. This analysis uncovered from signaling pathways (Garnett et al. 2012).
two distinct enhancers that are capable of driving very similar Tissue-specific enhancers for neural crest derivatives have also
neural crest-specific expression. Mutation of either region by itself been identified. For example, evolutionarily conserved regions of
does not disrupt gene expression, and thus these enhancers con- the gene Mef2c (Agarwal et al. 2011), and tyrosinase-related family
stitute an interesting case of redundancy at the cis-regulatory member Tyrp1 (Murisier et al. 2006), can drive specific reporter
level (Milewski et al. 2004; Degenhardt et al. 2010). The lack expression in melanocytes. Interestingly, both enhancers seem to
of similarity between the two regions also suggests that they re- be regulated by the transcription factor Sox10, a key regulator of
quire different inputs, highlighting not only the complexity of the melanocytic lineage. Similarly, a Hoxb3 enhancer that is active
the regulatory code but also the necessity for thorough analysis of in the vagal neural crest drives reporter expression in the enteric

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nervous system (Chan et al. 2005). Such regulatory modules are rently emerging in the field of gene regulation that may reshape
useful tools for targeted functional studies, particularly in chicken our views of the neural crest gene regulatory network and bring an
and zebrafish embryos, given the new approaches for efficient and extra layer of complexity to the proposed regulation of this cell
rapid integration of transgenes in these embryos (Fisher et al. 2006; population. Increasing evidence indicates that a complete un-
Yokota et al. 2011). derstanding of transcriptional events will require taking into
Cis-regulatory analysis can also provide insights into how consideration nuclear architecture and dynamics (Cremer and
differences between distinct neural crest populations arise. The cis- Cremer 2001; Mateos-Langerak et al. 2007). In the context of the
regulatory analyses of both SOX10 (Betancur et al. 2010b) and neural crest, a recent study has shown that mutations of histone
FOXD3 (Simões-Costa et al. 2012) point to distinct genetic programs H3.3 cause down-regulation of neural crest specifiers and loss
controlling gene expression at different axial levels of the chick of crest-derived ectomesenchyme (Cox et al. 2012). This is par-
embryo. Although both SOX10 and FOXD3 are pan-neural crest ticularly interesting since the H3.3 variant histones tend to be as-
markers, different enhancers are responsible for driving transcrip- sociated with permissive chromatin structures and have been
tion in the cranial versus trunk neural crest populations. Further- described as regulators of transcription and specification of germ
more, cranial and trunk enhancers are activated by different inputs cells. The results highlight how chromatin rearrangements can
(Simões-Costa et al. 2012), which suggests an intrinsic heteroge- impact the transcriptional state of the neural crest and their im-
neity in the neural crest population that precedes specification. For portance for its developmental potential (Cox et al. 2012). In-
example, two enhancers, NC1 and NC2, were uncovered, which terestingly, transcription factors also have been shown to interact
collectively drive reporter expression that recapitulates the en- with repressive molecules involved in epigenetic silencing (Wang
dogenous pattern of FOXD3 in the neural crest but separately et al. 2011; Strobl-Mazzulla and Bronner 2012). Thus, increasing
control expression in nonoverlapping neural crest populations. evidence points to a new paradigm in which transcription factor
NC1 drives initial expression in the cranial neural crest but is not complexes are constantly rearranging chromatin structure, result-
active in the trunk region. In contrast, NC2 mediates initial ex- ing in dynamic shifts of the regulatory state.
pression in trunk neural crest and only drives reporter expression The development of novel techniques such as ChIA-PET, 5C,
later in migrating cranial crest cells that no longer have NC1 ac- and Hi-C have allowed the identification of transcription factor
tivity. Whereas PAX7 and MSX1/2 are common inputs to both modules that are comprised of trans-regulators complexed with
NC1 and NC2, ETS1 is a direct input into NC1 and ZIC1 into NC2. different cis-regulatory regions and transcription start sites (Sanyal
Consistent with axial level specific inputs, ETS1 is a cranial-specific et al. 2011; Lan et al. 2012). These innovative approaches allow for
transcription factor, whereas ZIC1 is expressed in a graded fashion, the characterization of chromosome looping, interaction between
from posterior to anterior along the body axis (Simões-Costa et al. different enhancers and promoter regions, and provide a three-
2012). This differential regulation is consistent with the differences dimensional assessment of the state of transcriptional regulation.
observed in the migratory patterns, potential, and behavior of the Recent studies using chromosome conformation capture carbon
trunk versus cranial neural crest cells (Le Douarin and Kalcheim copy (5C) in different human cell lines have pointed to a large
1999). Further characterization of unique regulatory modules in number of long-range interactions between enhancers and pro-
distinct neural crest subpopulations hold the promise of providing moters and indicate that only a minority of such interactions occur
important insights into neural crest heterogeneity and develop- with the nearest gene (Sanyal et al. 2012). Furthermore, ChIA-PET
mental potential. analysis highlights a large number of promoter–promoter inter-
Currently, an array of new technologies allows high- actions between often-distant genes, suggesting the formation of
throughput identification of cis-regulatory modules. In the future, multigene complexes that are coregulated by the same transcrip-
these will allow a systems biology-level analysis of gene regulation tional machinery (Li et al. 2012). These findings infer a different
during neural crest formation. Importantly, novel approaches such model of gene regulation in which promoters and enhancers co-
as ChIP-seq allow for genome-wide analysis of the cis-regulatory alesce in transcription ‘‘factories’’ assembled at particular compart-
apparatus at specific times and axial levels, promising to yield new ments in the nuclei (Sandhu et al. 2012). Such emerging concepts in
insight into the regulatory events underlying neural crest de- eukaryotic gene regulation will surely impact how the neural crest
velopment. This technique can be used to map the genome-wide genetic program is interpreted and scrutinized in the years to come.
occurrence of histone modifications as well as active binding sites
for transcription factors and coactivators (Valouev et al. 2008;
Creyghton et al. 2010). In a remarkable effort to describe the epi-
Evolutionary conservation of the neural crest GRN
genomic landscape of neural crest cells, Rada-Iglesias et al. (2012) The neural crest gives rise to a number of vertebrate-specific features
mapped the chromatin mark patterns and transcription factor such as the craniofacial skeleton and peripheral nervous system. As
occupancy in induced neural crest cells produced from human a vertebrate synapomorphy, neural crest cells are thought to have
embryonic stem cells. This study identified TFAP2A and nuclear been crucial for the early evolution of vertebrate body plan and
receptors NR2F1 and NR2F2 as key players in neural crest de- predatory lifestyle. This idea was put forward in the ‘‘New Head’’
velopment, as the regulators occupy active enhancers character- hypothesis by Gans and Northcutt, which postulated that the
ized by high levels of EP300 and H3K27ac occupancy (Rada-Iglesias emergence of neural crest derivatives allowed for remodeling of the
et al. 2012). It also revealed thousands of active enhancers func- chordate head and resulted in a shift from filter feeding to active
tioning during neural crest development. Moreover, the result- predation (Gans and Northcutt 1983). Although the main premise
ing epigenomic annotation of these cells constitutes an invalu- of the New Head hypothesis is still supported by evidence from the
able resource for further investigation of the neural crest genetic fossil record and developmental studies, tracing the origin of the
program. neural crest to an invertebrate precursor cell type has proven to be
Such studies provide a wealth of information and highlight a challenge, as invertebrate chordates lack migratory cells that are
the extraordinary complexity of the regulatory events controlling formed at the neural plate border (Northcutt 2005; Hall and Gillis
neural crest formation. Not surprisingly, new paradigms are cur- 2013; Medeiros 2013). Assessment of the regulatory identity of the

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neural crest and its putative evolutionary precursors holds the to determine unequivocally if these cells are derived from the neural
promise of shedding light on the evolutionary origins of the verte- plate border, neural tube, or other tissue (Hall and Gillis 2013). In an
brate neural crest. intriguing recent study, Abitua et al. (2012) showed that a lineage
Genomic studies have been key in informing scenarios that a9.49 in Ciona intestinalis expresses ID, Snail, ETS, and FoxD, as well
attempt to explain the origins of neural crest and its role in ver- as melanogenic genes like MITF, TYR, and TYRP. This lineage nor-
tebrate evolution. Sequencing of chordate genomes allowed for the mally contributes to the formation of the otolith and the ocellus
systematic characterization of expression and function of the and does not migrate extensively. However, misexpression of Twist
neural crest GRN homologs in the chordate lineage (Yu et al. 2008). causes these cells to adopt a mesenchymal fate and exhibit long-
Studies from basal vertebrates such as the lamprey have offered range migration (Abitua et al. 2012). Because these lineages share
interesting insights pertaining to the origin and evolution of the some common gene signatures with vertebrate neural crest cells,
neural crest (Sauka-Spengler et al. 2007). Finally, whole genome they may hold important clues to neural crest origins.
phylogenetic analysis also has had considerable impact on how The expression of neural crest orthologs also has been in-
vertebrate evolution and neural crest evolution are surveyed. vestigated in the amphioxus (Branchiostoma floridae) (Meulemans
Molecular phylogenetic studies have placed tunicates as sister and Bronner-Fraser 2002; Meulemans et al. 2003; Yu et al. 2008),
groups of vertebrates (Philippe et al. 2005; Delsuc et al. 2006) and a cephalochordate. Although the amphioxus body plan shares
also pointed to a potential role for genome-wide duplications in important morphological traits with vertebrates, molecular phy-
facilitating the origins of the neural crest (Green and Bronner logeny has now placed cephalochordates at the base of the chor-
2013). date tree (Philippe et al. 2005). A systematic analysis of neural crest-
The use of the lamprey Petromyzon marinus as a model or- related genes indicates conservation of specification mechanisms
ganism has provided important information about the ancestral at the neural plate border between cephalochordates and verte-
state of the neural crest. Lampreys are basal vertebrates that lack brates. The transcription factor Snail is expressed specifically in the
several neural crest derivatives, including a neural crest-derived lateral portion of the neural tube in the neurula, although no other
jaw and sympathetic ganglia (Nicol 1952; Häming et al. 2011). neural crest-specifier genes are coexpressed at this location (Yu
Nevertheless they possess a SoxE expressing population of mi- et al. 2008). Thus, it is tempting to speculate that this transient
grating neural crest cells (McCauley and Bronner-Fraser 2006) that expression of Snail may reflect the beginnings of the assembly of
contribute to branchial arch cartilage, as well as the formation of a neural crest-specifier module.
pigment cells, cranial, and dorsal root ganglia. In an extensive Co-option of regulatory elements is likely to have played an
analysis of the lamprey neural crest genetic program, Sauka- important role in the origins of the neural crest in the vertebrate
Spengler et al. (2007) described the expression patterns of ortho- lineage. As an example, Manzanares et al. (2000) performed
logs of numerous genes occupying different hierarchical positions transgenesis experiments in which they expressed cis-regulatory
within the neural crest GRN. The results from this study defin- regions encoding amphioxus Hox gene expression in vertebrates.
itively show conservation of most of the neural crest genetic pro- Surprisingly, despite the fact that amphioxus lacks neural crest,
gram in this basal vertebrate with genes maintained in the same these enhancers drove spatially localized expression in vertebrate
spatial and temporal relationships as those observed in jawed neural crest and placode derivatives like cranial ganglia and bran-
vertebrate models (Sauka-Spengler et al. 2007). This was corrobo- chial arches (Manzanares et al. 2000). This study demonstrates that
rated by functional studies demonstrating that the epistatic re- cis-regulatory elements capable of driving gene expression in
lationships between neural plate border genes and neural crest neural crest cells are already present in the cephalochordate ge-
specifiers are largely conserved (Nikitina et al. 2008). Although nome. Such elements might have been co-opted to control ex-
these results point to a strong conservation of the neural crest GRN pression of neural crest-specifier genes, thereby facilitating emer-
across vertebrates, some intriguing differences have emerged from gence of this cell type.
this work. For example, the neural crest specifier Twist, which is Although invertebrate cell lineages described above share
thought to be crucial in the acquisition of the mesenchymal state some regulatory and behavioral traits with the neural crest, it is
in higher vertebrates, is only expressed in lamprey neural crest cells difficult to ascertain their position with respect to the evolutionary
at a later time, when they populate the branchial arches rather precursors of neural crest cells. If one defines the neural crest by
than within the ‘‘neural crest specifier’’ module. Similarly, Ets1, virtue of its specification kernel, that is, the network module that
which acts as a critical neural crest specifier gene in jawed verte- comprises the neural crest specifier genes, then a bona fide neural
brates, functioning directly upstream of both Sox10 and Foxd3, is crest must have factors that control multipotency, EMT, and di-
only employed in the effector module in the lamprey GRN (Sauka- verse lineage specification. As highlighted by Davidson (2009),
Spengler et al. 2007; Sauka-Spengler and Bronner-Fraser 2008c). there are numerous instances in which a given regulatory gene
Such differences suggest that shifts in the circuitry of the specifier functions in different levels of the GRN, often within the com-
module within the neural crest GRN might result in species-spe- mitment and differentiation of the same cell type. This is observed
cific traits. repeatedly in neural crest development with genes such as Foxd3,
Since comparative genomic analyses support the position of SoxE, and Msx1/2, being employed early for specification purposes,
tunicates as the sister group of vertebrates, Ciona intestinalis or other and again later to activate differentiation of various cell types (e.g.,
ascidians now occupy a key phylogenetic position for examining melanocytes, cartilage, and neuronal cell types). Further work will
the origins of vertebrate features (Schubert et al. 2006). Studies of the be necessary to define whether the regulatory similarities observed
development of the ascidian, Ecteinascidia turbinate, led to the between neural crest cells and intriguing lineages in invertebrate
identification of pigmented cells from the A7.6 lineage that arises chordates are a consequence of conservation of ancient differen-
near the neural tube and undergoes migration ( Jeffery et al. 2004). tiation programs or if they indeed are part of the novel regulatory
This cell lineage, which is known as the trunk lateral cells, expresses module that characterizes the neural crest.
the HNK-1 epitope as well as the Twist, FoxD, AP2, and Myc ortho- Taken together, these studies indicate that the proximal levels
logs ( Jeffery 2006; Jeffery et al. 2008). Further studies are necessary of the neural crest gene regulatory network, such as induction of

www.genome.org
the neural plate border, are primitive features shared amongst quence of increased apoptosis and reduced proliferation of mi-
chordates, whereas the core neural crest specification modules of grating cells (Dixon et al. 2006).
the network might have been added during early vertebrate evo- Genomic approaches have greatly benefited the dissection of
lution. This is expected since delimitation of the neural plate mechanisms leading to congenital disease. Genome-wide associa-
border is necessary for development of the central nervous system. tion studies have been pivotal to the identification of a number of
The terminal differentiation programs of the neural crest GRN also loci and genetic variants associated to craniofacial anomalies
are deeply conserved in both cephalochordates and tunicates, (Grant et al. 2009; Beaty et al. 2010; Mangold et al. 2010; Dixon
particularly the control of pigment cell differentiation (Yu et al. et al. 2011) and also suggest genes linked to normal variation in
2008; Davidson 2009). This is not surprising as most of the dif- facial structure (Liu et al. 2012). Similarly, recent studies using
ferentiation batteries are thought to be ancient (Davidson 2009). exome sequencing have been used to map the causes of craniofa-
Placement of the origin of the specification kernel of the GRN still cial defects in conditions such as Miller syndrome (Ng et al. 2010)
demands further studies, but it is clear that it has a high degree of and Nager syndrome (Bernier et al. 2012). Current transcriptome
conservation among vertebrates, as supported by studies in the and epigenomic analysis should accelerate the dissection of the
lamprey (Sauka-Spengler et al. 2007; Sauka-Spengler and Bronner- genetic basis of neural crest-related syndromes through regulatory
Fraser 2008a). network comparisons between normal and disease states. Such
studies promise to uncover more complex aspects of disease such
as variable penetrance and multifactorial etiology. In this context,
Neural crest cells and disease centralized databases of genomic information containing data sets
The above data show that scrutinizing the regulatory mechanisms of high-throughput studies such as Facebase and ENCODE will be
controlling neural crest formation can expand our understanding key (The ENCODE Project Consortium 2004; Hochheiser et al.
of essential cellular processes such as delamination, migration, and 2011). Such integrated databases and user-friendly bioinformatic
differentiation, as well as shed light on important evolutionary software will become increasingly important in the neural crest
questions pertaining to vertebrate origins and adaptation. Be- field as it continues to shift toward genomic approaches.
yond its relevance for basic biological questions, the neural crest The medical importance of neural crest cells is not restricted
is also of remarkable medical importance for its role in birth de- to congenital birth defects. This cell population has an important
fects and malignant diseases. Studies on the plasticity of the link to metastatic conditions due to its migratory and invasive
neural crest and its fate switches also offer clues for strategies for properties. Neural crest-derived cancers, such as melanoma and
cell reprograming and stem cell manipulation. In addition, the neuroblastomas, tend to be particularly aggressive and prone to
persistence of neural crest stem cells in adult tissues offers po- metastasis. This has led to the assumption that malignancy in
tential targets for therapies aiming to utilize endogenous pro- neural crest-derived cell types involves the reactivation of regula-
genitors for repair and regeneration. tory circuits important for embryonic development but that are
Given the sheer number of cell types and derivatives formed normally silenced once cell differentiation has occurred. Indeed,
by the neural crest progenitor cell population and the complexity evidence from studies of malignant melanoma cell lines shows
of its developmental program, it is not surprising that a large anomalous transcriptional reactivation of genes such as Snai,
fraction of congenital birth defects can be traced back to the neural Twist, Sox10, and Myb, all of which are important neural crest
crest (Hall 1999). Neural crest developmental anomalies, or neuro- specifier genes in the early GRN (Shakhova et al. 2012; Shirley et al.
cristopathies, are responsible for the vast majority of craniofacial 2012; Weiss et al. 2012). Interestingly, transplanting melanoma
malformations, and more than 700 different syndromes have cells to chicken embryo causes ‘‘regulation’’ of some of the cells
been described (Trainor 2010). In addition to the skeletal ele- that migrate along neural crest pathways, although some do lo-
ments of the face, abnormal neural crest development can also calize in ectopic sites (Kulesa et al. 2006). This suggests that mel-
affect the heart, adrenal medulla, pigment cells, and the periph- anoma cells can respond to cues in the embryonic environment
eral nervous system. Although treatment of neurocristopathies similar to neural crest cells. Thus, the embryonic environment may
has improved considerably over the years, dissection of the mo- be able to control or inhibit the metastatic state (Hendrix et al.
lecular mechanisms underlying many of these disorders could 2007), at least under some circumstances. Although this is an in-
bring considerable improvements to management and preven- triguing possibility, further work that compares the genetic pro-
tion of these conditions. grams controlling neural crest and neural crest-derived tumor cells
In the past few decades, the molecular basis of several neural is needed to inform upon the link between neural crest de-
crest-related syndromes has been investigated through genetic velopment and metastatic behavior in malignant cells.
linkage as well as the use of animal models. One example is The neural crest has also been an important model for the
Treacher Collins disease, an autosomal dominant disorder that is study of EMT, which is central for metastatic processes. A wealth of
marked by severe hypoplasia of skeletal elements of the face and knowledge regarding the genetics and cell biology underlying EMT
irregularities in otic and ophthalmic development (The Treacher has been obtained through the use of the neural crest as a model
Collins Syndrome Collaborative Group 1996; Trainor 2010). Ge- (Barrallo-Gimeno and Nieto 2005; Lee et al. 2006; Yang and
netic mapping and gene expression analysis identified mutations Weinberg 2008; Acloque et al. 2009). Interestingly, recent evidence
in the Tcof1 gene as the cause of the disease. Mapping of TCOF1 suggests that cells undergoing EMT seem to acquire stem-like
expression in mouse embryos shows that it is enriched in neural properties. Induction of EMT in different cell lines has been shown
crest cells and facial mesenchyme, raising the intriguing possibility to up-regulate totipotency markers such as POU5F1 (also known as
that the mutations could be interfering with neural crest de- OCT4) and NANOG in conjunction with transition to a mesen-
velopment (Dixon et al. 2006). In fact, TCOF1 is important for ri- chymal state (Mani et al. 2008). Such findings highlight similari-
bosome biogenesis and disruption of this process results in TP53- ties between the metastatic process and neural crest development.
mediated apoptosis, and analysis of Tcof1 null mice reveals strong They also point to interconnectivity between cell behavior and
reduction in the migratory neural crest population as a conse- developmental potential. In fact, it is likely that advances in the

www.genome.org
assembly of the neural crest regulatory network will highlight diseases that involve failure or impairment of neural crest-derived
common nodes in the genetic circuits controlling EMT, plasticity, tissue (Crane and Trainor 2006; Barraud et al. 2010). For instance,
and migratory potential. Such events are likely to depend on the transplantation of induced ‘‘neural crest-like cells’’ to diabetic mice
shared regulatory circuits that interconnect to define neural crest resulted in improvement of the impaired nerve and vascular
identity. functions that result from diabetic neuropathy (Okawa et al. 2012).
The question of plasticity has been central to neural crest bi- One would imagine that engineered neural crest cells could be used
ology, and interest in the topic of self-renewal and developmental in such fashion to treat neurodegenerative disorders that affect
potential has driven efforts to isolate neural crest stem cells. Work peripheral nervous system, among others.
by several investigators has led to the identification and purifica- Although it is clear that iNCCs are a powerful tool that can
tion of neural crest stem cells—cells with the potential to self- reveal novel molecular mechanisms and hold promise in treat-
renew and also to give rise to the diverse population of derivatives ment of disease, it is important to bear in mind that neural crest
that are generated by the neural crest. The first neural crest pro- development is intrinsically linked to the embryonic environ-
genitor cells were isolated in vitro by clonal analysis of quail cells ment. In vitro conditions do not necessarily reflect the complexity
and shown to be multipotent (Sieber-Blum and Cohen 1980). The and dynamism of a developing embryo and thus are unlikely to
ability of neural crest cells to self-renew was first demonstrated in recapitulate in vivo conditions. This is especially true for the neural
elegant studies by Stemple and Anderson using clonal cultures of crest, which is continuously interacting with different cell types
murine neural crest cells purified by cell sorting based on expres- and embryonic environments. Therefore, in vivo approaches con-
sion of the NGFR (p75) cell surface epitope (Stemple and Anderson tinue to represent the gold standard for neural crest biology, and
1992, 1993). These cells have the ability to self-renew and also to findings obtained with isolated neural crest cells or iNCCs should
form neurons or glia, thus demonstrating true stem cell properties. be validated with model organisms when possible.
More recently, clonal analysis of cranial neural crest in quail has
revealed that this population has the potential to differentiate into
Conclusions
osteoblasts (Calloni et al. 2009), and that the majority of the cra-
nial neural crest is comprised of progenitors with both osteogenic Over one hundred years of investigation of the neural crest has
and neural-melanocytic potential. Remarkably, this study also produced a remarkable body of knowledge about neural crest cell
identified precursors that can give rise to all neural crest-derived behavior, derivatives, and plasticity. The ingenuity and creativity of
phenotypes analyzed, which indicates that part of the migratory classical embryologists helped to frame the very questions that re-
cranial crest remains multipotent (Calloni et al. 2009). Although main integral to the field of neural crest biology today. The in-
isolated neural crest stem cells represent a good model for studies troduction of recombinant DNA technology in the 1990s has finally
of plasticity and commitment, there are possible caveats. Because it allowed these questions to be addressed at the mechanistic level by
is likely that in vitro culture conditions introduce changes that do analysis of the components of the genetic machinery that imbues
not reflect in vivo behavior, embryological observations of these the neural crest with its fascinating properties. In the last twenty
cells in their normal environment will be critical to uncovering the years, several aspects of the genetic circuitry controlling these cells
mechanism underlying maintenance and renewal of the latent have been identified and functionally characterized, providing im-
neural crest stem cell population. portant insights into the logic of neural crest development.
Neural crest-like cell populations have been derived from Yet we are still at the ‘‘tip of the iceberg’’ in terms of under-
human embryonic stem cells by a number of different methods standing this intriguing cell population from an integrated geno-
(Chimge and Bayarsaihan 2010). For instance, neural crest cells mic perspective. Further transcriptome and enhancesome analysis
were isolated from neural rosettes through fluorescence activated of neural crest cells at various stages and states of development will
cell sorting (FACS) using markers NGFR (p75) and B3GAT1 (pre- greatly increase our understanding of the neural crest GRN. Since
viously HNK1,) and subsequently express neural crest specifiers, regulatory states are dynamic and interconnected, this should be
such as SOX10 and SNAI1 (SNAIL). These have been shown to expanded to encompass more axial levels and time points of de-
differentiate into a wide range of neural crest derivatives, including velopment, ranging from the neural plate border to the final dif-
sensory and autonomic neurons, Schwann cells, myofibroblasts, ferentiated state. The formulation of computational models that
adipocytes, cartilage, and bone cells (Lee et al. 2007). Another can predict outcomes within the GRN and account for species-
study identified early migrating neural crest cells in neurospheres specific differences across vertebrates hold the promise of greatly
cultured on fibronectin plates. These cells express the neural crest increasing our understanding of deuterostome evolution (Peter
specifier SOX10 and clonal analysis indicates they are multipo- et al. 2012). Finally, additional levels of control have been shown
tent. Importantly, when these cells are injected in chick embryos, to act in the control of neural crest behavior and identity, including
they migrate normally and contribute to the same derivatives as epigenetic (Hu et al. 2012), post transcriptional ( Jayasena and
endogenous neural crest cells (Curchoe et al. 2010). Bronner 2012), and post-translational regulation (Lee et al. 2012).
Induced neural crest cells (iNCCs) from biopsied human tis- Future research along these lines has the potential to reveal the se-
sue may be useful in different contexts for clinical purposes. First, crets of the neural crest from its inception at the neural plate border
iNCCs can be used as a model for the study of human neuro- to their acquisition of specific differentiated fates.
cristopathies, as cells from patients can be reprogrammed to adopt Another exciting feature of the neural crest is its position at
a neural crest fate (Lee et al. 2009). This approach essentially allows the convergence of developmental, cancer, and stem cell biology.
the observation of an embryonic cell population from repro- As such, neural crest cells serve as a model for the investigation of
grammed adult cells, which can be used for transcriptomic, epi- processes as diverse as the epithelial to mesenchymal transition, to
genomic, or behavioral investigations that can uncover molecular cell motility and migration, and transition from stemness to the
mechanisms of disease and possibly also expand our knowledge of differentiated state. Genomic analyses provide tools and insights
the neural crest gene regulatory network. There is also the promise that are poised to reveal the complexities of these events with
that induced neural crest cells could be used for cell therapy for unprecedented depth and breadth. Given the inherent inter-

www.genome.org
connections that exist between diverse biological processes, the Cooper GM, Sidow A. 2003. Genomic regulatory regions: Insights from
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We would like to thank Tatiana Hochgreb, Sujata Bhattacharyya, developmental state. Proc Natl Acad Sci 107: 21931–21936.
and Stephen Green for critical reading of the manuscript and Curchoe CL, Maurer J, McKeown SJ, Cattarossi G, Cimadamore F, Nilbratt
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