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From head to tail: Regionalization of the neural crest


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The neural crest is regionalized along the anteroposterior axis, as demonstrated by foundational lineage-tracing experiments that showed the restricted developmental potential of neural crest cells originating in the head. Here, we explore how recent studies of experimental embryology, genetic circuits and stem cell differentiation have shaped our understanding of the mechanisms that establish axial-specific populations of neural crest cells. Additionally, we evaluate how comparative, anatomical and genomic approaches have informed our current understanding of the evolution of the neural crest and its contribution to the vertebrate body.
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From head to tail: regionalization of the neural crest
Manuel Rocha
, Anastasia Beiriger
, Elaine E. Kushkowski
, Tetsuto Miyashita
, Noor Singh
Vishruth Venkataraman
and Victoria E. Prince
The neural crest is regionalized along the anteroposterior axis, as
demonstrated by foundational lineage-tracing experiments that
showed the restricted developmental potential of neural crest cells
originating in the head. Here, we explore how recent studies of
experimental embryology, genetic circuits and stem cell differentiation
have shaped our understanding of the mechanisms that establish
axial-specific populations of neural crest cells. Additionally, we
evaluate how comparative, anatomical and genomic approaches
have informed our current understanding of the evolution of the
neural crest and its contribution to the vertebrate body.
KEY WORDS: Neural crest, Ectomesenchyme, Stem cells,
Gene regulatory networks, Patterning
The neural crest (NC) is a transient, multipotent cell population that
exhibits remarkable migratory capacity and gives rise to a vast array
of cell types, including neurons, glia, pigment cells, chondrocytes
and odontoblasts (Le Douarin and Kalcheim, 1999). The NC
has long fascinated developmental biologists, who have used
multi-disciplinary approaches ranging from classic embryological
techniques to single cell transcriptomics to investigate its
development. These studies have furthered our understanding of
the mechanisms that underlie NC cell development (reviewed by
Cheung et al., 2019; Le Douarin and Dupin, 2018; Mayor and
Theveneau, 2013; Prasad et al., 2019; Simões-Costa and Bronner,
2015). However, the broad question of how the NC is regionalized
into distinct cell populations along the anteroposterior (AP) axis
remains a crucial topic for discussion.
The development of the quail-chick chimera system in the early
1970s by Nicole Le Douarin allowed for comprehensive analyses of
NC migration and contributions. This system took advantage of the
fact that the embryos of these closely related avian species are of a
similar size during the early stages of development, yet their cells
exhibit unique nuclear morphologies. Because the nuclei of quail
cells show a large mass of condensed heterochromatin upon
Feulgen-Rossenbeck staining, researchers could use them as
natural, indelible lineage tracers (Le Douarin, 1973; see also Tang
and Bronner, 2020). By generating quail-chick chimeras, Le
Douarin and colleagues (summarized by Le Douarin and
Kalcheim, 1999) and Noden (1975, 1978, 1983) elucidated NC
cell migration pathways and derivatives along the AP axis (Fig. 1).
Based on these and other studies, we now understand that the NC is
regionalized along the AP axis into discrete subpopulations with
distinct differentiation potential: cranial, vagal, trunk and sacral.
Understanding the evolutionary and developmental origin of NC
regionalization is essential, given that defects in NC formation that
affect specific regional populations may lead to devastating
diseases, such as Treacher Collins syndrome (Trainor, 2010) or
Hirschsprungs disease (Bergeron et al., 2013; Butler Tjaden and
Trainor, 2013).
In this article, we highlight how lineage-tracing experiments,
primarily in avians, have revealed axial-specific differences in NC
potential, and discuss how these differences may be explained by
modifications to the gene regulatory network that underlies NC
development. Next, we focus on recent studies of human pluripotent
stem cell (hPSC) differentiation, which suggest that neuromesodermal
progenitors (NMPs) cellsthat form much of thetrunk and tail may
be an important source of trunk NC cells. Finally, we present models
for the evolution of NC regionalization and suggest experimental
approaches to enhance our understanding of NC evolution.
Axial differences in neural crest differentiation potential
The pioneering lineage-tracing experiments using the quail-chick
chimera system showed that NC cells from all levels of the body axis
give rise to pigment cells, Schwann cells and neurons. NC cells that
originate in the head migrate in broad streams and differentiate into
neurons of the sensory and parasympathetic ganglia, as well as a
wide array of ectomesenchymal cell types (Le Douarin and
Kalcheim, 1999). By contrast, trunk NC cells delaminate from the
neural tube to migrate along two distinct pathways: ventrally
between the neural tube and the adjacent somite, or along a
dorsolateral route between the somite and the overlying ectoderm.
NC cells that migrate along the ventral route differentiate into
sensory neurons of the dorsal root ganglia and sympathetic neurons
of the sympathetic chain ganglia, whereas those that migrate along
the dorsolateral pathway form pigment cells (Le Douarin and
Kalcheim, 1999). Subsequently, complexity was added to this
model by the finding that a large number of pigment cells also
originate from Schwann cell precursors, which originate from NC
cells that travel along the ventral pathway (Adameyko et al., 2009).
In chick embryos, NC cells from the level of somites 1-7 also give
rise to the parasympathetic neurons that innervate the gut, which are
collectively termed the enteric nervous system (Le Douarin and
Teillet, 1973). In addition, the sacral NC cells that originate
posterior to somite 28 form enteric neurons, although these are
limited to the posterior-most region of the gut.
One of the striking differences between NC cell populations
along the AP axis is that at least in amniotes only cranial NC cells
give rise to ectomesenchymal derivatives, including cartilage,
connective tissues, dermis, dermal bone and teeth ( for a discussion
on the ectomesenchymal potential of NC cells in other species,
please see the section below on The evolution of neural crest
regionalization). This ectomesenchymal potential of NC cells was
first proposed in the late 19th century by Julia Platt based on her
Committee on Development, Regeneration and Stem Cell Biology, The University
of Chicago, Chicago, IL 60637, USA.
Department of Organismal Biology and
Anatomy, The University of Chicago, Chicago, IL 60637, USA.
Canadian Museum
of Nature, Ottawa, ON K1P 6P4, Canada.
*Author for correspondence (
V.E.P., 0000-0001-5810-7300
© 2020. Published by The Company of Biologists Ltd
Development (2020) 147, dev193888. doi:10.1242/dev.193888
experiments in salamander (Platt, 1893). Her results were highly
controversial at the time, as they countered the prevailing dogma of
germ layer theory, which posited these tissues to be derived from the
mesoderm. However, her findings were eventually confirmed by
several researchers, including Hörstadius and Sellman, who used
experimental embryology to elucidate the migration and
contributions of NC cells (Hörstadius, 1950). Later, radiographic
labeling with tritiated thymidine allowed researchers to characterize
the extensive contributions of the NC to the vertebrate body and
begin to demonstrate differences in potential between NC cells from
various axial levels (reviewed by Le Douarin and Kalcheim, 1999).
These studies also demonstrated that NC cells form embryonic
facial processes and cartilage, and contribute to cranial ganglia
(Johnston, 1966; Noden, 1975). However, as the radiolabel
becomes diluted by cell proliferation, this method was of limited
use in the analysis of late-developing tissues.
The development of the quail-chick chimera system allowed
Le Douarin and colleagues, as well as Noden, to demonstrate that
cranial NC cells contribute to the facial and visceral skeleton, and its
adjacent connective tissue. Briefly, NC cells from the
prosencephalon and the mesenecephalon form the nasal and
periorbital skeleton and contribute to the cranial vault (see
Box 1). Mesencephalic NC cells additionally give rise to the
skeleton of the upper and lower jaws, the palate, and the tongue.
They also contribute to the pre-otic region, alongside
rhombencephalic NC cells. Finally, cartilage of the hyoid and
posterior pharyngeal arches is derived from rhombencephalic NC
cells (Le Douarin and Kalcheim, 1999; Noden, 1978). Cranial NC
cells also produce loose connective tissue of the lower jaw, tongue
and ventrolateral part of the neck, as well as dermis and striated
muscles of the branchial arches (Le Douarin and Kalcheim, 1999;
Noden, 1978).
More-recent lineage-tracing experiments revealed that the cranial
NC migration pathways (Lumsden et al., 1991) and craniofacial
derivatives (Köntges and Lumsden, 1996) maintain the spatial
organization of the rhombencephalon and mesencephalon from
which they derive. Moreover, the contributions of cranial NC cells
are influenced in part by the action of intrinsic factors, including the
Hox genes. Hox genes play a crucial role in patterning the skeletal
derivatives of NC cells arising from the posterior rhombencephalon
[rhombomeres (r)4-r8] (see Fig. 1). By contrast, NC cells that
arise from the prosencephalon, mesencephalon and anterior
rhombencephalon (r1 and r2) which form the bones of the
cranial and facial skull do not express Hox genes (Couly et al.,
1998, 2002; Creuzet et al., 2002). Accordingly, transplanting Hox-
expressing neural folds from r4-5 into the anterior Hox-negative
domains (Couly et al., 1998), or ectopically expressing Hox genes in
the diencephalic neural folds (Creuzet et al., 2002), causes defects in
the lower jaw and facial skeleton. Thus, Hox expression is
incompatible with proper development of the jaw or facial
derivatives of NC cells.
In addition to the intrinsic functions of Hox genes, extrinsic signals
from the surrounding tissues are instructive in the development of
cranial NC skeletal derivatives. This was elegantly demonstrated by
experimental manipulations of the chick foregut endoderm. When
researchers ablated strips of foregut endoderm, specific cranial NC-
derived skeletal structures failed to develop, while grafts of ectopic
foregut endoderm altered the identity of the skeletal structures (Couly
et al., 2002). Importantly, only anterior, Hox-negative NC cells can
respond to these endoderm-derived cues, whereas posterior,
Hox-expressing NC cells do not form bone and cartilage in
response to anterior foregut endoderm grafts.
Strikingly, these studies indicated that the posterior limit of
skeletogenic NC cells corresponds to the level of the 5th somite
(Le Liè
vre and Le Douarin, 1975), near the transition between the
rhombencephalon and the spinal cord (Fig. 1). To determine
whether this represents an intrinsic feature of the cranial NC or
Hox negative
PG1-5 Hox genes
PG5-9 Hox genes
PG10-13 Hox genes
Pigment cells, neurons and Schwann cells
Connective tissues (including dermis and muscle)
Skeletogenic tissues (including cartilage and bone)
Dorsal root ganglia and sympathetic chain ganglia
Enteric neurons
Fig. 1. Axial regionalization of the neural crest. Hox gene expression
domains and neural crest derivatives are aligned to the body axis of a
schematized amniote embryo. PG, paralog group. Fates of neural crest cells
from all axial levels (yellow), cranial region only (blue, striped), trunk region only
(green) and vagal/sacral regions (purple) are shown. The sacral neural crest
occurs posterior to somite 28 in older embryos and is therefore shown
alongside the unsegmented region of the pre-somitic mesoderm. The
prosencephalon (P), metencephalon (M) and rhombencephalon (R) are
labeled within the central nervous system. The otic vesicle (o), somites (s1-s5)
and cranial neural crest streams migrating to the pharyngeal arches (PA) 1-3
are also indicated.
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results from different signaling environments, Le Douarin and
colleagues performed a series of transplantation experiments
(summarized in Fig. 2). When quail mesencephalic and anterior
rhombencephalic primordia were grafted into the chick neural axis
at the level of somites 18-24, donor quail cells differentiated into
dermis, cartilage and connective tissues (Le Douarin and Teillet,
1974), suggesting that cranial NC are still capable of generating
ectomesenchymal derivatives in an ectopic environment.
Conversely, bilateral grafting of the trunk NC primordium into the
anterior rhombencephalon resulted in the absence of facial and
branchial skeletal elements (Le Douarin et al., 1977; Nakamura and
Ayer-le Lievre, 1982). Similarly, when trunk dorsal neural tube was
grafted to the midbrain, donor NC cells failed to form normal
corneal derivatives, contributed fewer neurons to the trigeminal
ganglion and did not form cartilage, even when grafted directly into
the first branchial arch (Lwigale et al., 2004). These results
demonstrate that chondrogenic potential is an intrinsic and
distinguishing feature of cranial, but not trunk, NC.
Nevertheless, the signaling environment is also important in
directing ectomesenchymal differentiation. Cranial and trunk NC
cells differ in their survival and differentiation in response to various
extracellular signals in vitro (Abzhanov et al., 2003).Yet when quail
trunk NC fragments are unilaterally grafted to the anterior
rhombencephalon of a chick host, donor NC cells migrate
alongside host NC cells. In these chimerea, quail ectomesenchyme
derivatives are detected in connective tissues, dermis and muscle, but
not cartilage or bone (Fig. 2) (Nakamura and Ayer-le Lievre, 1982).
These results suggest that the host cranial NC might provide extrinsic
signals that allow trunk NC cells to give rise to a subset of
ectomesenchymal derivatives. Moreover, when avian trunk NC
cells are cultured in media commonly used for growing bone and
cartilage cells, they generate ectomesenchymal derivatives in
vitro (Coelho-Aguiar et al., 2013; McGonnell and Graham,
2002) and contribute to cranial skeletal components when
transplanted into the head (McGonnell and Graham, 2002).
Thus, although chondrogenic potential is an intrinsic feature of
the cranial NC, the signaling environment contributes to
promoting this fate.
Although much emphasis has been placed on the development of
the cranial NC, it should be noted that trunk NC cells also give rise
to unique cell types and exhibit distinct cellular behaviors. At the
level of somites 18-24 in the chick, some NC cells form chromaffin
cells: the neuroendocrine cells of the adrenal medulla (Le Douarin
and Kalcheim, 1999). It was a commonly held view that
sympathetic neurons and chromaffin cells are derived from a
common lineage of catecholaminergic NC-derived progenitors,
termed sympathoadrenal progenitors, that migrate to the dorsal aorta
(reviewed by Huber et al., 2009). However, recent lineage-tracing
and genetic ablation experiments have revealed that chromaffin cells
are, in fact, largely generated by Schwann cell precursors: a
NC-derived population of peripheral glial progenitors that migrate
along motor nerve fibers (Furlan et al., 2017). Nevertheless, as graft-
derived NC cells are detected in the adrenal medulla following
transplantation of cranial neural primordium to the adrenomedullary
region (Le Douarin and Teillet, 1974), the ability to generate
chromaffin cells is not limited to trunk NC.
Axial-specific gene regulatory networks
A gene regulatory network (GRN) is a powerful tool used to
describe the genetic basis of cell fate specification. Indeed, the
distinct properties of NC cells at various axial levels may be
explained by axial differences in their GRNs (Simões-Costa and
Bronner, 2015). Several transcription factors, including Id2
(Martinsen and Bronner-Fraser, 1998) and Ets-1 (Tahtakran and
Selleck, 2003; Théveneau et al., 2007), are expressed in cranial, but
not trunk, NC cells in chick embryos. However, it should be noted
that chick Id2 is also expressed in cardiac NC cells (Martinsen et al.,
2004) and Ets1 is expressed in zebrafish and hPSC-derived trunk
NC cells (Frith et al., 2018; Gomez et al., 2019a; Martik et al.,
2019). Thus, a greater understanding of the regulatory functions
of these factors, as well as the species-specific variation in the
mechanisms that establish axial identity, is still needed.
Nevertheless, Ets-1 is both necessary and sufficient to confer
cranial-specific delamination properties on NC cells in chick
embryos (Théveneau et al., 2007). In addition, the regulatory
regions of two key NC specifier genes in chick Foxd3 (Simões-
Costa et al., 2012) and Sox10 (Betancur et al., 2010) have
axial-specific enhancers that drive their expression in either the
cranial or the trunk NC (Betancur et al., 2010; Simões-Costa
et al., 2012). Notably, both cranial enhancers are directly
activated by Ets-1.
In recent years, next-generation sequencing approaches,
primarily in amniote model systems, have enabled researchers to
evaluate the hypothesis that axial-specific GRNs pattern the NC.
Specifically, transcriptional profiling has further elucidated the gene
regulatory differences between cranial and trunk NC cell
populations. For example, Simões-Costa and Bronner (2016)
uncovered a cranial-specific transcriptional circuit in chick
embryos. This GRN includes Brn3c,Lhx5 and Dmbx1, which are
expressed in the anterior region of gastrula-stage embryos and
persist throughout NC specification. Subsequently, Tfap2b,Sox8
and Ets-1 are detected in NC progenitors in the cranial neural folds
and in migrating NC cells. Introducing the latter three components
of this network into the trunk is sufficient to reprogram trunk NC
cells to a cranial identity and leads to the acquisition of
Box 1. The contribution of NC cells to the cranial vault
The role of NC cells in the development of the cranial vault including the
frontal and parietal bones has been controversial. Both Noden (1978)
and Le Lievre (1978) reported that the avian frontal bone is of combined
mesodermal and NC origin, while Le Douarin (1982) concluded that both
bones are derived entirely from the mesoderm (Le Douarin, 1982; Le
Lievre, 1978; Noden, 1978). Subsequent fate mapping from an earlier
stage of development the late neurula suggested that the frontal and
parietal bones are derived exclusively from NC (Couly et al., 1993).
However, the most recent chick data indicate a mixed origin of the frontal
bone with the supraorbital region derived from the NC and the calvarial
region from the mesoderm and a mesodermal origin of the parietal
bone (Evans and Noden, 2006). Advances in lineage-tracing
approaches in mice have allowed continued investigation of this issue.
Wnt1-Cre/R26R transgenic-based NC lineage tracing, together with DiI
labeling of the cephalic mesoderm, revealed that the mouse frontal bone
is derived from NC cells, whereas the parietal bone originates from the
mesoderm (Jiang et al., 2002). Recent experiments in amphibians have
revealed yet more complexity (Maddin et al., 2016; Piekarski et al.,
2014). In axolotls, the frontal, but not the parietal, bone originates from
NC cells of the mandibular stream (Maddin et al., 2016; Piekarski et al.,
2014), a pattern that largely reflects that of amniotes. However, Xenopus
embryos exhibit a unique pattern characterized by extensive contribution
of NC cells from the mandibular, hyoid and branchial streams to the
osteocranium, including the frontoparietal bone (Piekarski et al., 2014).
This pattern may have evolved after anurans diverged from other living
amphibians (Piekarski et al., 2014). Lineage-tracing approaches will no
doubt continue to be an important tool for investigating the role of the NC
in the evolution of the cranium (reviewed by Teng et al., 2019).
REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888
chondrogenic potential (Simões-Costa and Bronner, 2016). A
similar approach identified a transcriptional subcircuit comprising
Tgif1,Ets1 and Sox8 that imparts cardiac NC identity and is
necessary for proper heart development. Ectopic expression of this
subcircuit is sufficient to reprogram trunk NC cells to a cardiac fate
and enables them to rescue defects in heart formation caused by
cardiac NC ablation (Gandhi et al., 2020).
By coupling transcriptional and epigenomic profiling in cranial
NC cells at population and single-cell levels, Williams et al. (2019)
reverse engineered the global NC GRN with remarkable resolution.
Midbrain and
anterior hindbrain
Quail donor
(4-9 somites)
Quail donor
(11-29 somites)
Chick host
(24-26 somites)
Chick host
(4-10 somites)
Quail donor
(15-25 somites)
Chick host
(6-10 somites)
Chick host
(22 somites)
Cell culture donor
to level of
somites 18-24
Produces dermis,
connective tissue,
chromaffin cells
and cartilage
Last 1-6 somites and
unsegmented PSM
to mid- and
Fails to produce
facial and branchial
skeletal elements
Produces connective
tissue, dermis and
muscle, but not
cartilage or bone Trunk NC cells
in bone-promoting media
Transplanted to
first pharyngeal arch
Contributes to
cranial skeletogenic
Last 1-6 somites and
unsegmented PSM
to mid- and
Fig. 2. Transplantation approaches reveal that intrinsic factors and extrinsic signals underlie differences between cranial and trunk NC cells.
Transplantation experiments reveal differences in the contributions of cranial and trunk neural crest. Derivatives of transplanted tissue are in red; quail
embryos are shown in blue, chick embryosin orange. (A) Bilateral and heterotopic transplant of cranial (midbrain and anterior hindbrain) neural primordium from a
quail donor (4-9 somite stage; ss) to the trunk of a chick host (24-26 ss) leads to the formation of skeletogenic derivatives, as well as chromaffin cells, at ectopic
posterior positions. (B) The reciprocal transplant of trunk neural tube from a quail donor (11-29 ss) to the cranial (mid- and hindbrain) region of a chick host
(4-10 ss) shows that trunk NC does not form skeletogenic derivatives. (C) A unilateral version of the transplant experiment shown in B demonstrates that
host tissue can influence the migration and potential of transplanted cells. Donor cells form connective tissues alongside the host NC but cannot form skeletogenic
derivatives. (D) When trunk NC cells cultured in bone-promoting media are transplanted into the mandibular and maxillary primordium of a chick host,
the transplanted cells are able to form skeletogenic derivatives, demonstrating the importance of the NC signaling environment for cell fate decisions.
REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888
Their analysis of chromatin dynamics revealed three distinct classes
of regulatory elements: one that is accessible in premigratory and/or
migratory NC, one that is accessible in both NC and neuroepithelial
cells, and one that is accessible in naive epiblast and premigratory
NC cells but inaccessible at later stages (Williams et al., 2019). This
study also uncovered an early cis-regulatory split between
mesenchymal and neural progenitors, which was confirmed by
single cell transcriptomics (Williams et al., 2019). Relevant to these
findings, Weston and colleagues have posited that the cranial cell
types typically attributed to the NC are in fact derived from two
spatially, temporally and molecularly distinct pools of progenitors.
The first is the metablast, which encompasses non-neural
epithelium that lies lateral to the developing neural folds and
gives rise to the cranial ectomesenchymal lineage. The second is the
more medial, neuroepithelial-derived authenticNC that migrates
at a slightly later stage to produce neurogenic and melanogenic cell
types (Breau et al., 2008; Lee et al., 2013a; Weston and Thiery,
2015; Weston et al., 2004). This model has been disputed by Dupin
et al. (2018), who contend that single NC cells can give rise to both
ectomesenchymal and neural-melanocytic derivatives in vitro.
These competing models do have important implications
regarding the patterning of the NC. The model put forth by Dupin
and colleagues reflects a traditional view in which the potential of
NC cells is axially regionalized. By contrast, the model of Weston
and colleagues would argue for a more uniform authenticNC
being present at all axial levels alongside a metablastthat is
restricted to the cranial region. The results obtained by Williams and
colleagues (2019) highlight how systems-biology approaches may
serve as an important tool for informing this discussion.
Using a similar approach, Ling and Sauka-Spengler (2019) dissected
the GRN that governs the development of the vagal NC. Their study
showed that this heterogeneous cell population can be separated into a
/FoxD3+ sub-population capable of forming neural,
mesenchymal and neuronal derivatives, and a Sox10
population that is restricted to neuronal and mesenchymal fates. By
incorporating chromatin accessibilityandgeneticinteractions,thisstudy
identified the Tfap2, Sox, Hbox andbHLHfamiliesoftranscription
factors as core regulators of the vagal crest GRN and validated their
function by genetic knockout (Ling and Sauka-Spengler, 2019).
Single-cell analyses of mouse embryos have revealed that NC
cells at distinct axial positions exhibit largely similar transcriptional
profiles over time, yet they also have important axial-specific
biases (Soldatov et al., 2019). For example, cranial NC cells are
biased towards a mesenchymal fate, whereas trunk NC cells
are biased towards sensory and autonomic neuronal fates. These
biases emerge during delamination, with mesenchymal fates
resulting from sustained high levels of expression of Twist1 in the
cranial region (Soldatovet al., 2019). Interestingly, cranial and trunk
NC cells become transcriptionally distinct at different times in
mouse and chick: while the mouse cranial program is established
during delamination, the chick cranial GRN initiates during the
early stages of NC specification (Simões-Costa and Bronner, 2016).
It will be important to establish whether this apparent offset in
timing is a technical artefact e.g. due to inconsistent labeling
techniques or inconsistencies in staging or reflects species-
specific biological differences.
Cranial NC cells in zebrafish also express Twist1, which
promotes ectomesenchymal fate at the expense of other genetic
programs (Das and Crump, 2012). In mice, Twist1 mutants show
impaired skeletogenic differentiation and fail to form bones of the
snout, upper face and skull vault (Bildsoe et al., 2009; Soo et al.,
2002). In both species, Twist1 deficiency leads to persistent
expression of Sox10 and a loss of ectomesenchymal differentiation
markers (Bildsoe et al., 2009; Das and Crump, 2012; Soo et al.,
2002). Soldatov and colleagues also showed that loss of Twist1 in
mouse cranial NC results in a reduction of mesenchymal derivatives
and an increase in glial and neuronal fates. Conversely, ectopic
expression of Twist1 in the mouse trunk NC, starting from pre-EMT
stages, results in the expression of a mesenchymal marker (Prrx1)at
the expense of neuronal sensory, autonomic and glial fates
(Soldatov et al., 2019). Together, these results indicate that Twist1
is sufficient to drive the acquisition of some ectomensenchymal
Recent advances in the dissection of genetic circuits and
interrogation of transcriptional profiles have been invaluable in
uncovering the molecular basis of NC axial identity. These
approaches have revealed that intrinsic differences in gene
expression mediate at least some axial-specific properties of NC
cells, including ectomesenchymal potential, and have begun to
establish the regulatory logic that underlies the cranial genetic circuit.
Lessons from stem cells
The ability to differentiate human pluripotent stem cells (hPSCs)
into NC cells in vitro has provided novel insights into the
mechanisms by which the NC is patterned along the AP axis.
Importantly, it has also proven an important tool for studying human
NC biology and NC-associated developmental disorders. Early
methods for deriving NC cells from hPSCs relied on stromal co-
culture (Jiang et al., 2009; Lee et al., 2007; Pomp et al., 2005) or
induction of neural rosettes (Chambers et al., 2009; Lee et al.,
2010). However, these protocols yielded limited numbers of NC
cells and often required FACS isolation using the cell surface
markers HNK-1 and p75. More recently, several protocols have
described feeder-free conditions for generating NC cells with high
efficiency using small molecules and growth factors (Hackland
et al., 2017; Lee et al., 2010; Leung et al., 2016; Menendez et al.,
2011, 2013; Mica et al., 2013).
Remarkably, these protocols yield hPSC-derived NC cells that
possess cranial identity by default, indicated by their ability to give
rise to chondrocytes and their lack of Hox expression (Fig. 3)
(Fukuta et al., 2014; Hackland et al., 2017; Lee et al., 2007, 2010;
Leung et al., 2016; Menendez et al., 2011; Mica et al., 2013).
Treatment of hPSCs with retinoic acid (RA) during differentiation
yields a subpopulation of NC cells with characteristics of cardiac
and/or vagal NC, including expression of paralog group (PG) 1-5
Hox genes (Figs 1 and 3) (Frith et al., 2018; Fukuta et al., 2014;
Mica et al., 2013). In particular, these conditions yield cultures with
the potential to form enteric neurons, a cell type that defines the
vagal NC (Barber et al., 2019; Fattahi et al., 2016; Workman et al.,
2017). Huang et al. (2016) reported that, when combined with both
TGFβinhibition and Wnt signaling activation, treatment with RA
generates NC cells that express PG6-9 Hox genes in addition to
PG2-5 Hox genes (Figs 1 and 3). These NC cells activate the
Sox10E1 enhancer, which is expressed in both vagal and trunk NC,
and they are capable of differentiating into TH
cells (Huang et al., 2016). However, the expression of PG6-9 in
these cells is relatively low and they are unlikely to efficiently
generate trunk NC cells. Finally, when NC cells are derived from
stem cells in the presence of RA, they give rise to enteric neurons
when grown together with human intestinal organoids, or colonize
the foregut when transplanted into chick embryos (Workman et al.,
2017). These findings are consistent with the known role of
endogenous RA, which is necessary for proper development and gut
colonization of the enteric NC (Niederreither et al., 2003; Uribe
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et al., 2018). Together, these studies indicate that treatment with RA
during differentiation yields vagal NC cells.
In recent years, increasing evidence has suggested that the
production of bona fide trunk NC cells from hPSCs requires cells to
pass through an intermediate state that resembles neuromesodermal
progenitors (NMPs). NMPs are bipotent stem cells found in the
primitive streak and tailbud that produce much of the trunk and tail
(see Fig. 4 and Box 2). Initial studies suggested that hPSC-derived
NMP-like cells, marked by robust co-expression of Sox2 and
Brachyury/T, can be differentiated into trunk NC cells capable of
differentiating into chromaffin cells in vitro, as well as in vivo upon
transplantation into chick embryos (Abu-Bonsrah et al., 2018;
Denham et al., 2015). Subsequently, Frith and colleagues
demonstrated that hPSC-derived NMPs (Gouti et al., 2014) can
differentiate into trunk NC cells and their derivatives (Frith and
Tsakiridis, 2019; Frith et al., 2018). Interestingly, several known
markers of neural plate border and early NC identity are also
detected in these NMPs (Frith et al., 2018). Under differentiation
conditions, the hPSC-derived NMPs give rise to NC cells that
express PG5-9 Hox genes typical of the thoracic neurectoderm
and can also give rise to sympathoadrenal cells (Frith et al., 2018).
Other protocols for generating trunk NC cells defined by HoxC9
expression, limited mesenchymal potential, and the ability to
produce sympathoadrenal cells again report the presence of an
NMP-like intermediate state (Gomez et al., 2019b; Hackland et al.,
2019). Notably, these trunk NC cell cultures exhibit a wider
developmental potential than do avian trunk NC cells, as they are
capable of forming smooth muscle and osteoblasts (Gomez et al.,
2019b; Hackland et al., 2019). Finally, NMP-derived pre-neural
progenitors give rise to trunk NC cells with progressively more
posterior identity over increasing passages (Cooper et al., 2020
preprint), perhaps reflecting the co-linear expression of Hox genes
observed in vivo.
Additional experiments have revealed that Wnt and Fgf
signaling, which are necessary for maintaining the NMP niche
in vivo (reviewed by Wilson et al., 2009) are also critical for
specifying the axial identity of hPSC-derived NC cells in vitro. Wnt
signaling levels are crucial for determining cranial versus trunk fate
of hPSC-derived NC cells (Gomez et al., 2019b; Hackland et al.,
2019). hPSCs exhibit a bimodal response to Wnt signaling, whereby
low Wnt signaling leads to anterior Hox-negative NC cells, and high
Wnt signaling results in posterior Hox-expressing NC cells (Gomez
et al., 2019b). Furthermore, the magnitude of Wnt stimulus dictates
the degree of NC posterior identity based on Hox gene expression,
suggesting a rheostat response. Within the trunk compartment, Fgf
signaling determines axial identities: treatment of hPSC cultures
with Fgf2 during the first 2 days of NC induction leads to expression
of the sacral HoxA10-13 genes (Figs 1 and 3), whereas the Fgf
inhibitor PD17 abrogates all Hox expression (Hackland et al.,
The finding that NMPs produce trunk NC cells in vitro is
consistent with the results of lineage-tracing studies in vivo. Based
on their analyses of chick and mouse embryos, colleagues
(Schoenwolf and Nichols, 1984; Schoenwolf et al., 1985) first
proposed that cells in the tail bud might give rise to NC cells in the
tail. This hypothesis was later substantiated by grafting quail tissue
into the tailbud of 25-somite stage chick hosts, which revealed that
the cells in the chordoneural hinge region of the tailbud contribute
not only to the spinal cord and somitic mesoderm as expected of
NMPs but also to the NC and its derivatives (Catala et al., 1995).
More recently, fate mapping of the mouse primitive streak and
tailbud, either by grafting GFP-labeled cells or by permanent
genetic cell labeling, has also shown that NMPs give rise to trunk
and tail NC cells (Javali et al., 2017; Rodrigo Albors et al., 2018;
Tzouanacou et al., 2009; Wymeersch et al., 2016), as well as NC-
derived sensory neurons of the dorsal root ganglia in the sacral
Trunk NC cell
PG6-9 Hox genes
Sacral NC cell
PG10-13 Hox genes
Vagal/cardiac NC cell
PG1-5 Hox genes
+ RA
High Wnt4
High Wnt
Low FGF5
High Wnt
High FGF6
Anterior NC progenitor
Cranial NC cell
Hox negative
Pigment cells, neurons and Schwann cells
Connective tissues
Skeletogenic tissues (including cartilage and bone)
Dorsal root ganglia and
sympathetic chain ganglia
Enteric neurons
High Wnt
Active FGF3
Intermediate BMP,
Wnt1 and FGF2
Fig. 3. Differentiation of hSPCs into distinct axial
subpopulations of NC. An undifferentiated human
pluripotent stem cell (hPSC) passes through different
intermediate states en route to a cranial or trunk neural crest
cell fate. Each cell type and its characteristic gene
expression is in red. The signals needed to promote each
cell type are indicated next to the arrows. Derivatives formed
by each cell type are color coded.
Hackland et al., 2017;
Lee et al., 2007;
Frith et al., 2018;
Fattahi et al., 2016;
Gomez et al., 2019b;
Hackland et al., 2019.
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region (Shaker et al., 2020 preprint). However, the extent to which
NMPs contribute to the NC remains unclear, as these analyses are
based on only a small number of clones. Cre-based lineage tracing
driven by Tbx6 and Nkx2 regulatory sequences, which are expressed
in NMPs and early mesodermal and neural progenitors, labeled NC
cells in the anterior trunk (Javali et al., 2017; Rodrigo Albors et al.,
2018), although Javali et al. (2017) reported a higher contribution to
NC cells at the sacral level. Clonal analysis using the R26nlaacZ
system in which an inactive variant of LacZ is driven by the
ubiquitous Rosa26 promoter and rare spontaneous deletions restore
β-galactosidase activity in single cells yielded clones that labeled
the NC along the entire extent of the trunk and tail (Tzouanacou
et al., 2009). Collectively, these results provide compelling
evidence that NMPs contribute to at least a subset of trunk NC
cells in vivo.
Despite the evidence for a role for NMPs in generating trunk NC
cells, it remains unclear whether NMP intermediates are necessary
for trunk NC identity in vivo. In fact, Gomez et al. (2019b) also
showed that cells exposed to high Wnt can still adopt a trunk NC fate
even when expression of NMP markers is compromised following
inhibition of Fgf signaling. Additionally, it is unclear how the NMP
state might imbue trunk-specific features of NC cell development,
such as the restriction of ectomesenchymal potential. Thus, it will be
important to better elucidate the regulatory link between the NMP
state and trunk NC identity. To address this issue, it will be valuable
to use systems biology approaches to determine how components of
the NMP genetic circuit establish trunk NC cell fate. Moreover,
while the contribution of NMPs to axial elongation and NC cell
populations has been well documented in amniotes, whether
equivalent cells play a role in the development of non-amniotes,
such as zebrafish (Attardi et al., 2019; Kanki and Ho, 1997; Martin
and Kimelman, 2012) and Xenopus (Gont et al., 1993), remains
controversial. Nevertheless, some have proposed that the molecular
mechanisms governing posterior extension of the embryo
including the gene regulatory state that defines NMPs are
conserved across vertebrates (Kimelman, 2016; Steventon and
Martinez Arias, 2017; Wilson et al., 2009). As current experimental
evidence for this assertion remains inconclusive, it will be important
to investigate the cell lineages that give rise to trunk NC cells in
zebrafish and Xenopus, and determine whether they pass through an
NMP-like state.
The evolution of neural crest regionalization
Our understanding of NC regionalization along the AP axis is
largely based on studies from a small set of model organisms. An
evolutionary framework is therefore required to compare
regionalization mechanisms between these organisms and
reconstruct the ancestral state of NC development. In this context,
Gans and Northcutt (1983) hypothesized that the vertebrate head is
an evolutionary novelty, the emergence of which was facilitated by
the acquisition of NC cells and neurogenic placodes in a vertebrate
ancestor. New lines of research continue to test predictions of this
hypothesis. Fortunately, the ectomesenchymal derivatives of NC
cells are preserved in the fossil record as a diverse array of skeletal
structures (Smith and Hall, 1990). This provides a dataset that
complements findings from living embryos. In addition, different
approaches such as the comparative analysis of ectomesenchymal
tissues and the examination of genetic circuits have shed light on
the evolutionary pattern of NC fates and potential regulatory
processes underlying these patterns. Below, we highlight results
derived from both these lines of questioning and discuss additional
tests that could be used to validate emerging models and synthesize
new hypotheses.
Paleontology and comparative anatomy suggest ectomesenchymal
potential is ancestral
Histological examination of the vertebrate dermal skeleton in fossils
and extant vertebrates led Smith and Hall (1990, 1993) to postulate
that the ancestral NC possessed ectomesenchymal potential at all
axial levels. Both fossil and extant vertebrates show a remarkable
B Mouse E8.5A Mouse E7.5 C Mouse E10.5
Node streak border
Caudal lateral epiblast
Chordoneural hinge
Fig. 4. Bipotent NMPs in the tailbud give rise to posterior
tissues. (A) Gastrulation stage (E7.5) mouse embryo
showing the primitive streak and the location of
neuromesodermal progenitors (NMPs) within the node streak
border (NSB) and caudal lateral epiblast (CLE). Gastrulation
movements through and away from the primitive streak are
shown using dashed arrows, while the anterior (A) to posterior
(P) axis of the developing body is labeled with a blue arrow.
(B) Early somite-stage mouse embryo (E8.5). Arrows show
contributions of bipotent NMPs located in the NSB and CLE to
both neuroectodermal tissues, such as the spinal cord (1),
and mesodermal tissues, such as the somitic mesoderm (2)
and notochord (3). (C) At later stages (E9.5-14.5), NMPs
located in the chordoneural hinge (CNH) of the developing
tailbud continue to contribute to both mesodermal and
neuroectodermal tissues. A, anterior; FB, forebrain; HB,
hindbrain; MB, midbrain; NC, notochord; P, posterior; PSM,
pre-somitic mesoderm; SC, spinal cord.
REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888
diversity of mineralized scales and dermal bones along the AP axis.
The mineralized dermal tissues in vertebrates are derived from
different combinations of odontogenic (dentine-forming) and
mesodermally derived osteogenic (bone-forming) units (Sire
et al., 2009; Smith and Hall, 1990). These two cell condensations
are primarily distinguished based on their position within the
dermis, the state of polarization of the extracellular matrix and their
modes of development (Fig. 5).
The role of cranial NC cells in the formation of dentine in oral
teeth has been well established in amphibians (Graveson et al.,
1997; Smith and Hall, 1990) and mice (Chai et al., 2000; Lumsden,
1988). Experimental embryology approaches in amphibians
revealed that the dentine-producing dental mesenchyme is derived
from cranial NC cells, while the overlying enamel develops from
oral ectodermal epithelium. In addition, tissue recombination
studies in mice showed that teeth can form when cranial NC cells
are co-cultured with ectodermal epithelium from the mandibular
arch, but not with limb epithelium (Lumsden, 1988). More recently,
genetic-based lineage tracing using the Wnt1-Cre system
demonstrated that cranial NC cells contribute to the condensed
dental mesenchyme, dental papilla, odontoblasts, dentine matrix,
pulp, cementum and periodontal ligaments (Chai et al., 2000).
Because dentine found in oral teeth is derived from NC cells, Smith
and Hall (1990) concluded that the odontogenic tissues of dermal
scales are similarly derived from post-cranial NC cells. This
conclusion is further supported by reports that post-cranial NC cells
can form dentine under appropriate signaling conditions (Graveson
et al., 1997; Lumsden, 1988).
Differential losses and elaborations of osteogenic and
odontogenic components have led to the variety of dermal tissues
found in extinct and extant vertebrates (Fig. 5). Chondrichthyan
species cartilaginous fishes including sharks and skates have a
complete dermal armor of small dentinous scales and have lost the
osteogenic layer (Gillis et al., 2017; Sire et al., 2009). Thus, they
offer tractable models for elucidating the relationship between oral
teeth and scales. Oral teeth and trunk scales in sharks display similar
expression patterns of Dlx transcription factors during development
(Debiais-Thibaud et al., 2011), shedding light on the gene-
regulatory basis of the long-recognized histological and
morphological similarities between skin denticles and oral teeth.
Consistent with this, lineage-tracing experiments suggest that trunk
NC cells give rise to odontoblasts of trunk dermal denticles in the
little skate (Gillis et al., 2017). However, the extended
developmental period of skate embryos in this case requiring
analysis 4-5 months after dye injections (Gillis et al., 2017)
presents a formidable challenge to precise and comprehensive
labeling, and to securing robust controls that could rule out a
mesodermal contribution.
In contrast to the findings in chondrichthyans, lineage-tracing
studies in teleosts indicate a mesodermal origin for trunk scales.
Contrary to early reports that zebrafish trunk NC cells contribute to
fin ectomesenchyme (Kague et al., 2012; Smith et al., 1994) and
scales (Sire and Akimenko, 2004; Smith and Hall, 1990), recent
analyses have clarified that these tissues are derived exclusively
from the mesoderm in both zebrafish (Lee et al., 2013b,c; Mongera
and Nüsslein-Volhard, 2013) and medaka (Shimada et al., 2013).
However, the superficial odontogenic layer has been highly reduced
or eliminated in teleosts, implying that the analysis of teleost scale
development may be of limited use for evaluating the broader
evolutionary pattern of ectomesenchymal potential of trunk NC
cells across vertebrates.
In summary, the results from comparative anatomy approaches
suggest that the ancestral NC possessed ectomesenchymal capacity
throughout the body axis, but that this potential was restricted to the
head as a result of evolutionary loss (Fig. 6A). In fact, the trend
towards reduction and restriction of ectomesenchymal potential of
NC cells to cranial and oral domains is observed across multiple
lineages, including cyclostomes (discussed below) and ray-finned
fishes, and within lobe-finned fishes (Fig. 5). Nevertheless, it is
important to note that although the regulatory framework
underlying odontogenesis by NC cells has been studied in
mammals and amphibians (Chai et al., 2000; Graveson et al.,
1997; Lumsden, 1988; Smith and Hall, 1990), it has not been well
characterized in cartilaginous and non-teleost bony fishes, which
have a clearly identifiable dentine layer in their scales (Sire and
Huysseune, 2003). If dentine in scales is not derived from NC cells,
then a fundamental assumption of the hypothesis that ancestral NC
possessed ectomesenchymal potential along the entire body axis is
Comparative genetics suggests that ectomesenchymal potential was
restricted by modifying subcircuits
Recent studies of the NC GRN in a broad variety of extant
vertebrates support an increased degree of axial regionalization and
elaboration of the NC in jawed (versus jawless) vertebrates. Such
molecular axial regionalization may provide an explanatory
mechanism for the restriction of ectomesenchymal potential to
cranial NC in some lineages. Key insights have come from a jawless
(agnathan) species, the lamprey a member of the early diverging
vertebrate cyclostome lineage. Lampreys express core genes of the
NC GRN (Hockman et al., 2019; Nikitina et al., 2008; Sauka-
Spengler et al., 2007). In fact, it has been suggested that some
components of the NC GRN may predate vertebrate origins (York
and McCauley, 2020). Yet multiple components of the avian cranial
Box 2. Neuromesodermal progenitors contribute to the
post-cranial body
In mouse and chick, tissues posterior to the head are in large part
generated by multipotent stem cells, termed neuromesodermal
progenitors (NMPs). This idea was first supported by lineage tracing of
Hensens node in the chick embryo, which showed that single cells can
contribute to more than one tissue type (Selleck and Stern, 1991).
Similarly, labeling of small groups of cells in the caudal lateral epiblast
yielded clones that contribute to the neural tube and somitic mesoderm
(Brown and Storey, 2000). Clonal analyses of mouse somites (Nicolas
et al., 1996) and the spinal cord (Mathis and Nicolas, 2000), using the
LaacZ system (see main text) uncovered long clones spanning many
segments, suggesting that these progenitors must persist over extended
periods. Later, Cambray and Wilson showed that cells located in several
discrete regions of the mouse primitive streak and the adjacentepibl ast
the caudal lateral epiblast and the node-streak border give rise to both
neural and mesodermal derivatives (Fig. 4), and can be serially
transplanted into younger hosts. The descendants of cells from these
regions are later found within the chordoneural hinge of the tailbud
(Fig. 4) and exhibit similar properties (Cambray and Wilson, 2002, 2007).
In chick embryos, tailbud progenitors are capable of resetting their Hox
gene expression to match the surrounding tissue upon heterochronic
transplantation into younger hosts, indicating that NMPs change their
Hox gene expression profile over time and that this process is reversible
(McGrew et al., 2008). NMPs are characterized by co-expression of the
mesodermal marker Brachyury/T and the neural marker Sox2 (Cambray
and Wilson, 2007; Garriock et al., 2015; Wymeersch et al., 2016), and
recent studies have begun to elucidate the genetic circuits that govern
NMP formation, differentiation and maintenance (Amin et al., 2016; Gouti
et al., 2017).
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NC GRN (discussed above) are curiously absent in lamprey pre-
migratory and migratory cranial NC, although they are present at
later stages in the pharyngeal arches (Martik et al., 2019).
Hierarchical clustering of transcriptional profiles revealed that
early lamprey cranial NC shares more similarities with chick trunk
NC than with chick cranial NC (Martik et al., 2019). Finally,
lineage-tracing experiments have revealed that lampreys lack vagal
NC, and the enteric nervous system is instead derived from late-
migrating trunk NC cells (Green et al., 2017). Together, these
findings suggest that axial regionalization of the NC may have been
a gradual ongoing process during vertebrate evolution.
To better understand how the cranial NC GRN emerged during
evolution, Martik and colleagues examined the expression of
components of the avian cranial GRN in embryos of skate
(a cartilaginous fish) and zebrafish (a bony fish). In both species,
NC cells express two cranial GRN genes, Tfap2b and Ets-1;
however, these genes are expressed in trunk NC as well as cranial
NC in these taxa. Additionally, zebrafish express: (1) lhx5 and
dmbx1 in the early cranial NC, but not in the pharyngeal arches; and
(2) sox8b at all axial levels, rather than exclusively in the cranial NC
(Martik et al., 2019). Moreover, axial-specific enhancers of Sox10
are conserved in avians and mammals, but are absent in amphibians
and teleosts (Betancur et al., 2010). In comparison, the cranial
enhancer of Foxd3 is highly conserved in chick, human, mouse and
Xenopus, but not zebrafish (Simões-Costa et al., 2012). These
results suggest that NC patterning may have evolved via the
progressive addition of a cranial-specificcircuit onto an ancestral,
generalized, trunk-like GRN that is retained in lampreys (Fig. 6B).
Based on these and other findings, Martik and colleagues (2019)
suggested that the primitive pan-axial distribution of
ectomesenchymal tissues seen in fossil vertebrates and to the
best of our current understanding retained in living cartilaginous
fishes (discussed above) has become restricted to cranial axial
levels during evolution by changes to the spatiotemporal expression
of network components. This is best illustrated by the amniote-
specific restriction of Ets-1 to the cranial NC. However, this model
Jawless fish
Jawed vertebrates
Cartilaginous fishes
Bony fishes
Ray-finned fishes
Ectomesenchymal derivatives
Mineralized dermal tissues
Odontogenic Osteogenic Both
Arches Teeth Scales
and osteogenic
Fig. 5. Distribution of mineralized dermal tissues and neural crest derivatives across the vertebrate evolutionary tree. (A) Mineralized dermal
tissues can be odontogenic (green) or osteogenic (gray), or both (striped). The extent of mineralized dermal tissues along the body axis of each species is
indicated by the distribution of color. Contributions of the neural crest to cartilage in the pharyngeal arches and to odontogenic tissues of the teeth and scales are
represented by symbols located above each species silhouette. Representative species are depicted for each branch of the tree. Extant groups (left to right):
hagfish, lampreys, cartilaginous fishes, teleosts, non-teleost ray-finned fishes (e.g. Polypterus), lungfishes, anamniotes (e.g. Xenopus) and amniotes.
Extinct groups (left to right): ostracoderms, placoderms, fossil ray-finned fishes (e.g. Cheirolepis), fossil lungfishes (e.g. Dipterus) and fossil tetrapods
(e.g. Osteolepis). Asterisks indicate that ostracoderms and placoderms are both paraphyletic. (B) Representative cross-section of dermal skeletal elements
with both odontogenic and osteogenic layers (left), odontogenic layer only (middle) and osteogenic layer only (right).
REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888
does have some caveats. First, it is based solely on expression data
and requires functional validation. Second, despite the absence of a
cranialGRN in early cranial NC cells of lamprey, these cells
nevertheless form pharyngeal cartilage (McCauley and Bronner-
Fraser, 2003). Thus, even with an apparently simple GRN, lampreys
exhibit distinctions between cranial and trunk NC. Third, the
lamprey NC GRN may be secondarily simplified. Although extant
lampreys lack scales altogether, extinct taxa from the cyclostome
stem possessed dentinous scales (Fig. 5) (Keating and Donoghue,
2016; Miyashita et al., 2019). This fossil evidence implies that the
ability to form dentine preceded the jawless-jawed vertebrate split,
and that the absence of ectomesenchyme in the lamprey trunk is a
secondary loss (Fig. 6A).
Conclusions and perspectives
Since the early realization that the NC is regionalized along the body
axis, the issue of how distinct axial NC cell populations are
established has fascinated researchers. Recent systems biology
approaches have uncovered the gene regulatory basis for the unique
ectomesenchymal potential exhibited by cranial NC cells, while
advances in stem cell differentiation and lineage-tracing methods
have revealed the importance of NMPs as a source of trunk NC
cells. Moving forward, we propose that multidisciplinary
approaches that integrate distinct subfields and incorporate
evolutionary data could further our understanding of NC
Although analyses of GRNs are invaluable, subsequent
functional characterization is necessary to explain how GRNs
confer disparate differentiation potential along the AP axis. As
illustrated by Simões-Costa and Bronner (2016) and Soldatov et al.
(2019), experimental manipulations that couple early transcriptional
differences with readouts of NC fate are especially informative.
Therefore, future analyses must be complemented by experimental
approaches to evaluate the functions of axial-specific genetic
circuits in a variety of species. Although zebrafish appear to have a
simpler cranial GRN than do chicks, this does not preclude the
Ancestral vertebrate NC
has global EM potential
EM potential
lost in trunk
of modern
jawless fish
Ancestral NC of jawed vertebrates
has global EM potential
EM potential lost
in trunk NC
Ancestral NC has
trunk-like GRN
Progressive elaboration of
GRNs restricts EM potential
to cranial NC
Cartilaginous fishes
Jawless fishes
Bony fishes
Cartilaginous fishes
Jawless fishes
Bony fishes
A Ectomesenchyme as an ancestral vertebrate trait
B Ectomesenchyme restricted to the head via elaboration of NC GRN
Fig. 6. Evolutionary models for axial
regionalization of the neural crest.
(A) Paleontological data on the location of
mineralized dermal tissues along the body
axis suggest that ectomesenchymal (EM)
potential originated either: (1) at the base of
the vertebrate tree (orange); or (2) in the last
common ancestor of jawed vertebrates (blue).
Circles indicate acquisition of a trait, while
lines indicate a loss. In both cases, EM
potential would have been lost in an ancestor
of modern teleosts and modern tetrapods.
(B) Genetic data from a variety of species,
including lamprey, shark, zebrafish and chick,
suggest that multiple heterochronic shifts in
transcription factor expression may have led
to the restriction of EM potential to the cranial
neural crest. In this scenario, the ancestral
vertebrate neural crest would have resembled
that found in the modern amniote trunk (pink).
REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888
existence of a distinct, teleost-specific cranial NC GRN. Indeed, the
zebrafish GRN is simplifiedonly with respect to chick, and both
model species represent equally ancient lineages that diverged from
their last common ancestor.
Understanding the evolution of ectomesenchymal potential also
remains acentral topic for future investigation. Important insights into
this may come from exploring whether trunk NC cells exhibit
ectomesenchymal potential in non-teleost fishes with dentinous
scales. Specifically, the developmental origin of scales in non-teleost
ray-finned fishes such as sturgeons, gars and bichirs warrants
examination. The bichir Polypterus is especially interesting, both
because its scales have an extensive layer of dentine (Sire and
Huysseune, 2003), and because its embryos have recently become
accessible (Stundl et al., 2019), potentially paving the way for lineage
tracing and evaluating whether trunk NC cells give rise to dermal
scales. Such experimental data would complement our understanding
of tissue development from the fossil record (Giles et al., 2013; Sire
et al., 2009) and help bridge the evolutionary gap of more than 425
million years since the last common ancestor of zebrafish and
amniotes. Similarly, the teleost group itself warrants more-detailed
comparative studies. For example, investigation of the development
of dentinous dermal armor in some catfish (Sire and Huysseune,
1996), which may represent retention or redeployment of an
ectomesenchymal program, would be of significant value.
Axial patterning of the NC provides a rich system for integrating
the genetic, morphological and evolutionary underpinnings of
vertebrate development and diversity. The results presented here
demonstrate how integrating findings from disparate subfields may
illuminate complex questions in developmental biology. We have
highlighted the importance of integrating findings from novel
sequencing methods with classic experimental embryology and
demonstrated how in vitro approaches enrich discoveries from
in vivo models. Expanding this interdisciplinary approach to include
paleontological evidence will be crucial for uncovering the
mechanisms by which the NC is regionalized along the AP axis
and understanding its contribution to the vertebrate body plan.
We thank Megan Martik and Marianne Bronner for their thoughtful comments on the
manuscript, and Lily Tang and Marianne Bronner for sharing an unpublished draft of
their accompanying article. We are grateful to Thomas Frith for his comments on the
stem cell section of the manuscript, and to Michael Coates for sharing his deep
knowledge of evolutionary biology with us. We also thank the reviewersof the article,
both for their careful reading and for their helpful recommendations. Finally,this work
benefitted from the resources of the ZFIN database (
Competing interests
The authors declare no competing or financial interests.
Related work in the Prince lab was funded by a National Science Foundation award
(1528911) and by the Chicago Biomedical Consortium with support from the Searle
Funds at Chicago Community Trust (C-070 to V.E.P. and Ankur Saxena). M.R. and
A.B. were supported by the Eunice Kennedy Shriver National Institute of Child
Health and Human Development (NICHD) of the National Institutes of Health
(T32HD055164). This material is additionally based upon research supported by a
grant from the NICHD (F31HD097957 to M.R.) and by the National Science
Foundation Graduate Research Fellowship Program (DGE-1144082 to A.B.). Any
opinions, findings, and conclusions or recommendations expressed in this material
are those of the authors and do not necessarily reflect the views of the National
Institutes of Health or the National Science Foundation. Deposited in PMC for
release after 12 months.
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... Later ostracoderms and placoderms (jawless and jawed members of the gnathostome stem group) show a definite odontoosteogenic arrangement of dermal armor with reduction or elaboration varying in individual lineages (9,38,39) (Fig. 4). Based on lineage tracing of dermal denticles of cartilaginous fishes and highly derived elasmoid scales of teleost fishes, it has been suggested that the odontogenic component of the ancient dermal armor is trunk NC derived, whereas the osteogenic component is mesoderm derived (13). ...
Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.
... The neural crest is a transient and multipotent cell population. 35,36 The neural crest cells in caudal regions derive from the tail bud. [37][38][39] In human embryos, a secondary neural tube arises from the mesenchyme of caudal eminence (tail bud). ...
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Background: Limited dorsal myeloschisis (LDM) and intramedullary infantile hemangioma rarely coexist in the spinal cord. Observations: The authors describe the case of a 3-month-old girl who, despite lacking neurological symptoms or signs, had a cigarette burn-like mark at the lumbosacral area and skin dimpling in the gluteal area. Magnetic resonance imaging showed a low-set conus due to a thickened filum and an abnormal subcutaneous stalk connected to the conus medullaris. In combination with the skin lesions, these findings strongly implied nonsaccular-type LDM. An intramedullary mass in the conus medullaris was also shown on magnetic resonance imaging and was homogenously enhanced with isointensity on T1- and T2-weighted images. We prophylactically untethered the spinal cord and partially removed the intramedullary mass, which had no clear borders, for a safe surgical dissection. Histologically, the intramedullary mass was an infantile hemangioma, and the subcutaneous stalk was a lesion associated with LDM. The patient remained neurologically intact after surgery, and then 2 years later, there was spontaneous regression of the residual tumor. Lessons: Although rare, nonsaccular type LDM may appear concurrently with intramedullary infantile hemangioma at the conus medullaris. The authors present a possible mechanism behind this concurrent presentation in the same area.
... Cell type diversification of the sclerotome is remarkably similar to that of the neural crest lineage. Previous studies have shown that migrating neural crest cells are multipotent (Baggiolini et al., 2015), and the ultimate cell fate is determined by their axial positions as well as the migration timing and direction (Rocha et al., 2020). ...
Fibroblasts play an important role in maintaining tissue integrity by secreting components of the extracellular matrix and initiating response to injury. Although the function of fibroblasts has been extensively studied in adults, the embryonic origin and diversification of different fibroblast subtypes during development remain largely unexplored. Using zebrafish as a model, we show that the sclerotome, a sub-compartment of the somite, is the embryonic source of multiple fibroblast subtypes including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, fin mesenchymal cells, and interstitial fibroblasts. High-resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Long-term Cre-mediated lineage tracing reveals that the sclerotome also contributes to cells closely associated with the axial skeleton. Ablation of sclerotome progenitors results in extensive skeletal defects. Using photoconversion-based cell lineage analysis, we find that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single-cell clonal analysis combined with in vivo imaging suggests that the sclerotome mostly contains unipotent and bipotent progenitors prior to cell migration, and the fate of their daughter cells is biased by their migration paths and relative positions. Together, our work demonstrates that the sclerotome is the embryonic source of trunk fibroblasts as well as the axial skeleton, and local signals likely contribute to the diversification of distinct fibroblast subtypes.
... The pigment cells of the head are also neural crest derivatives, although they arise from a distinct cell population: the cranial neural crest. Pigment cells of the trunk and tail are derivatives of the trunk neural crest, and these two populations differ in several ways, but perhaps most obviously by lack of an association or interaction with somites (Ferguson and Graham 2004;Kuratani et al. 2018;Rocha et al. 2020). The stripe-forming model of Murray et al. (Murray et al. 1990) begins only at the nape of the neck and extends along the length of the body to the tip of the tail, so the mechanisms underlying pigmentation patterning of the alligator head are currently unknown, but are clearly affected by incubation temperature. ...
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Considerations of the impact climate change has on reptiles are typically focused on habitat change or loss, range shifts, and skewed sex ratios in species with temperature-dependent sex determination. Here, we show that incubation temperature alters stripe number and head colouration of hatchling American alligators (Alligator mississippiensis). Animals incubated at higher temperatures (33.5˚C) had, on average, one more stripe than those at lower temperatures (29.5˚C), and also had significantly lighter heads. These patterns were not affected by estradiol-induced sex reversal, suggesting independence from hatchling sex. Therefore, increases in nest temperatures as a result of climate change have the potential to alter pigmentation patterning, which may have implications for offspring fitness.
... The neural crest was reported to contribute to the facial skeleton in vertebrates and could be directed toward mesenchymal lineages [20,21]. Here, we tested whether the DS-NCSCs had mesenchymal differentiation potential similar to that of the control group. ...
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Down syndrome (DS) is the most common chromosomal abnormality in live-born infants and is caused by trisomy of chromosome 21. Most individuals with DS display craniofacial dysmorphology, including reduced sizes of the skull, maxilla, and mandible. However, the underlying pathogenesis remains largely unknown. Since the craniofacial skeleton is mainly formed by the neural crest, whether neural crest developmental defects are involved in the craniofacial anomalies of individuals with DS needs to be investigated. Here, we successfully derived DS-specific human induced pluripotent stem cells (hiPSCs) using a Sendai virus vector. When DS-hiPSCs were induced to differentiate into the neural crest, we found that trisomy 21 (T21) did not influence cell proliferation or apoptosis. However, the migratory ability of differentiated cells was significantly compromised, thus resulting in a substantially lower number of postmigratory cranial neural crest stem cells (NCSCs) in the DS group than in the control group. We further discovered that the migration defects could be partially attributed to the triplication of the coxsackievirus and adenovirus receptor gene (CXADR; an adhesion protein) in the DS group cells, since knockdown of CXADR substantially recovered the cell migratory ability and generation of postmigratory NCSCs in the DS group. Thus, the migratory deficits of neural crest cells may be an underlying cause of craniofacial dysmorphology in individuals with DS, which may suggest potential targets for therapeutic intervention to ameliorate craniofacial or other neural crest-related anomalies in DS.
The pharyngula stage of vertebrate development is characterized by stereotypical arrangement of ectoderm, mesoderm, and neural tissues from the anterior spinal cord to the posterior, yet unformed tail. While early embryologists over-emphasized the similarity between vertebrate embryos at the pharyngula stage, there is clearly a common architecture upon which subsequent developmental programs generate diverse cranial structures and epithelial appendages such as fins, limbs, gills, and tails. The pharyngula stage is preceded by two morphogenetic events: gastrulation and neurulation, which establish common shared structures despite the occurrence of cellular processes that are distinct to each of the species. Even along the body axis of a singular organism, structures with seemingly uniform phenotypic characteristics at the pharyngula stage have been established by different processes. We focus our review on the processes underlying integration of posterior axial tissue formation with the primary axial tissues that creates the structures laid out in the pharyngula. Single cell sequencing and novel gene targeting technologies have provided us with new insights into the differences between the processes that form the anterior and posterior axis, but it is still unclear how these processes are integrated to create a seamless body. We suggest that the primary and posterior axial tissues in vertebrates form through distinct mechanisms and that the transition between these mechanisms occur at different locations along the anterior-posterior axis. Filling gaps that remain in our understanding of this transition could resolve ongoing problems in organoid culture and regeneration.
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The vertebrate eye develops as an intricate collaboration among multiple tissue types derived from the neural tube (neuroectodermal), mesenchyme (mesodermal and neu-ral crest origin), and the preplacodal regions originating in the surface ectoderm. Developmental studies suggest tightly orchestrated mutual signaling and cell surface interactions among these codeveloping tissues that underlie specification, patterning, differentiation, and maturation of specialized tissues of the eye. Periocular mesen-chyme (majorly neural crest-derived) emanating Wnt signals pattern posterior regions of the developing optic vesicle as retinal pigment epithelium (RPE) over the neural
The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors in vitro and in vivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.
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Schwann cells are glial cells of the peripheral nervous system. They exist in several subtypes and perform a variety of functions in nerves. Their derivation and culture in vitro are interesting for applications ranging from disease modeling to tissue engineering. Since primary human Schwann cells are challenging to obtain in large quantities, in vitro differentiation from other cell types presents an alternative. Here, we first review the current knowledge on the developmental signaling mechanisms that determine neural crest and Schwann cell differentiation in vivo. Next, an overview of studies on the in vitro differentiation of Schwann cells from multipotent stem cell sources is provided. The molecules frequently used in those protocols and their involvement in the relevant signaling pathways are put into context and discussed. Focusing on hiPSC- and hESC-based studies, different protocols are described and compared, regarding cell sources, differentiation methods, characterization of cells, and protocol efficiency. A brief insight into developments regarding the culture and differentiation of Schwann cells in 3D is given. In summary, this contribution provides an overview of the current resources and methods for the differentiation of Schwann cells, it supports the comparison and refinement of protocols and aids the choice of suitable methods for specific applications.
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The vertebrate body forms by continuous generation of new tissue from progenitors at the posterior end of the embryo. In mice, these axial progenitors initially reside in the epiblast, from where they form the trunk; and later relocate to the chordo-neural hinge of the tail bud to form the tail. Among them, a small group of bipotent neuromesodermal progenitors (NMPs) are thought to generate the spinal cord and paraxial mesoderm to the end of axis elongation. The study of these progenitors, however, has proven challenging in vivo due to their small numbers and dynamic nature, and the lack of a unique molecular marker to identify them. Here, we report the generation of the Nkx1.2CreER T2 transgenic mouse line in which the endogenous Nkx1.2 promoter drives tamoxifen-inducible CreER T2 recombinase. We show that Nkx1.2CreER T2 targets axial progenitors, including NMPs and early neural and mesodermal progenitors. Using a YFP reporter, we demonstrate that Nkx1.2 -expressing epiblast cells contribute to all three germ layers, mostly neuroectoderm and mesoderm excluding notochord; and continue contributing neural and paraxial mesoderm tissues from the tail bud. This study identifies the Nkx1.2 -expressing cell population as the source of most trunk and tail tissues in the mouse; and provides a key tool to genetically label and manipulate this progenitor population in vivo .
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The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by Wnt/FGF signals in the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient and unable to produce the full range of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity by co-expressing the transcription factors SOX2 and CDX2, and they lose the mesodermal potential by downregulating TBXT. Over 10 passages these cells divide to self-renew and to make trunk neural crest, while gradually adopting a more posterior identity by activating colinear HOX gene expression. Rostrocaudal identity can be prolonged in a thoracic identity for up to 15 passages by modulating TGFβ, and PNPs can be ventralised by Hedgehog signalling.
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The neural crest is a vertebrate-specific migratory stem cell population that generates a remarkably diverse set of cell types and structures. Because many of the morphological, physiological and behavioural novelties of vertebrates are derived from neural crest cells, it is thought that the origin of this cell population was an important milestone in early vertebrate history. An outstanding question in the field of vertebrate evolutionary-developmental biology (evo-devo) is how this cell type evolved in ancestral vertebrates. In this review, we briefly summarize neural crest developmental genetics in vertebrates, focusing in particular on the gene regulatory interactions instructing their early formation within and migration from the dorsal neural tube. We then discuss how studies searching for homologues of neural crest cells in invertebrate chordates led to the discovery of neural crest-like cells in tunicates and the potential implications this has for tracing the pre-vertebrate origins of the neural crest population. Finally, we synthesize this information to propose a model to explain the origin of neural crest cells. We suggest that at least some of the regulatory components of early stages of neural crest development long pre-date vertebrate origins, perhaps dating back to the last common bilaterian ancestor. These components, originally directing neuroectodermal patterning and cell migration, served as a gene regulatory ‘scaffold' upon which neural crest-like cells with limited migration and potency evolved in the last common ancestor of tunicates and vertebrates. Finally, the acquisition of regulatory programmes controlling multipotency and long-range, directed migration led to the transition from neural crest-like cells in invertebrate chordates to multipotent migratory neural crest in the first vertebrates.
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The vertebrate skull varies widely in shape, accommodating diverse strategies of feeding and predation. The braincase is composed of several flat bones that meet at flexible joints called sutures. Nearly all vertebrates have a prominent ‘coronal’ suture that separates the front and back of the skull. This suture can develop entirely within mesoderm-derived tissue, neural crest-derived tissue, or at the boundary of the two. Recent paleontological findings and genetic insights in non-mammalian model organisms serve to revise fundamental knowledge on the development and evolution of this suture. Growing evidence supports a decoupling of the germ layer origins of the mesenchyme that forms the calvarial bones from inductive signaling that establishes discrete bone centers. Changes in these relationships facilitate skull evolution and may create susceptibility to disease. These concepts provide a general framework for approaching issues of homology in cases where germ layer origins have shifted during evolution.
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The enteric nervous system (ENS) predominantly originates from vagal neural crest (VNC) cells that emerge from the caudal hindbrain, invade the foregut and populate the gastrointestinal tract. However, the gene regulatory network (GRN) orchestrating the early specification of VNC remains unknown. Using an EdnrB enhancer, we generated a comprehensive temporal map of the chromatin and transcriptional landscape of VNC in the avian model, revealing three VNC cell clusters (neural, neurogenic and mesenchymal), each predetermined epigenetically prior to neural tube delamination. We identify and functionally validate regulatory cores (Sox10/Tfap2B/SoxB/Hbox) mediating each programme and elucidate their combinatorial activities with other spatiotemporally specific transcription factors (bHLH/NR). Our global deconstruction of the VNC-GRN in vivo sheds light on critical early regulatory mechanisms that may influence the divergent neural phenotypes in enteric neuropathies.
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The neural crest, an embryonic stem-cell population, is a vertebrate innovation that has been proposed to be a key component of the ‘new head’, which imbued vertebrates with predatory behaviour1,2. Here, to investigate how the evolution of neural crest cells affected the vertebrate body plan, we examined the molecular circuits that control neural crest development along the anteroposterior axis of a jawless vertebrate, the sea lamprey. Gene expression analysis showed that the cranial subpopulation of the neural crest of the lamprey lacks most components of a transcriptional circuit that is specific to the cranial neural crest in amniotes and confers the ability to form craniofacial cartilage onto non-cranial neural crest subpopulations³. Consistent with this, hierarchical clustering analysis revealed that the transcriptional profile of the lamprey cranial neural crest is more similar to the trunk neural crest of amniotes. Notably, analysis of the cranial neural crest in little skate and zebrafish embryos demonstrated that the transcriptional circuit that is specific to the cranial neural crest emerged via the gradual addition of network components to the neural crest of gnathostomes, which subsequently became restricted to the cephalic region. Our results indicate that the ancestral neural crest at the base of the vertebrate lineage possessed a trunk-like identity. We propose that the emergence of the cranial neural crest, by progressive assembly of an axial-specific regulatory circuit, allowed the elaboration of the new head during vertebrate evolution.
This 1999 edition of The Neural Crest contains comprehensive information about the neural crest, a structure unique to the vertebrate embryo, which has only a transient existence in early embryonic life. The ontogeny of the neural crest embodies the most important issues in developmental biology, as the neural crest is considered to have played a crucial role in evolution of the vertebrate phylum. Data that analyse neural crest ontogeny in murine and zebrafish embryos have been included in this revision. This revised edition also takes advantage of recent advances in our understanding of markers of neural crest cell subpopulations, and a full chapter is now devoted to cell lineage analysis. The major research breakthrough since the first edition has been the introduction of molecular biology to neural crest research, enabling an elucidation of many molecular mechanisms of neural crest development. This book is essential reading for students and researchers in developmental biology, cell biology, and neuroscience.
Since its discovery 150 years ago, the neural crest has intrigued investigators owing to its remarkable developmental potential and extensive migratory ability. Cell lineage analysis has been an essential tool for exploring neural crest cell fate and migration routes. By marking progenitor cells, one can observe their subsequent locations and the cell types into which they differentiate. Here, we review major discoveries in neural crest lineage tracing from a historical perspective. We discuss how advancing technologies have refined lineage-tracing studies, and how clonal analysis can be applied to questions regarding multipotency. We also highlight how effective progenitor cell tracing, when combined with recently developed molecular and imaging tools, such as single-cell transcriptomics, single-molecule fluorescence in situ hybridization and high-resolution imaging, can extend the scope of neural crest lineage studies beyond development to regeneration and cancer initiation.
The cardiac neural crest arises in the hindbrain, then migrates to the heart and contributes to critical structures, including the outflow tract septum. Chick cardiac crest ablation results in failure of this septation, phenocopying the human heart defect persistent truncus arteriosus (PTA), which trunk neural crest fails to rescue. Here, we probe the molecular mechanisms underlying the cardiac crest’s unique potential. Transcriptional profiling identified cardiac-crest-specific transcription factors, with single-cell RNA sequencing revealing surprising heterogeneity, including an ectomesenchymal subpopulation within the early migrating population. Loss-of-function analyses uncovered a transcriptional subcircuit, comprised of Tgif1, Ets1, and Sox8, critical for cardiac neural crest and heart development. Importantly, ectopic expression of this subcircuit was sufficient to imbue trunk crest with the ability to rescue PTA after cardiac crest ablation. Together, our results reveal a transcriptional program sufficient to confer cardiac potential onto trunk neural crest cells, thus implicating new genes in cardiovascular birth defects.