ArticlePDF AvailableLiterature Review

From head to tail: Regionalization of the neural crest

October 2020
Development 147(20):dev193888

DOI:10.1242/dev.193888

Authors:

Canadian Museum of Nature / Musée canadien de la nature

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.

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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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).

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REVIEW

From head to tail: regionalization of the neural crest

Manuel Rocha

, Anastasia Beiriger

, Elaine E. Kushkowski

, Tetsuto Miyashita

2,3

, Noor Singh

Vishruth Venkataraman

and Victoria E. Prince

1,2,

ABSTRACT

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

Introduction

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

Hirschsprung’s 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 (vprince@uchicago.edu)

V.E.P., 0000-0001-5810-7300

Development (2020) 147, dev193888. doi:10.1242/dev.193888

DEVELOPMENT

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

PA1

PA2

PA3

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

KEY

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.

REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888

DEVELOPMENT

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

DEVELOPMENT

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

Transplanted

to level of

somites 18-24

Produces dermis,

connective tissue,

chromaffin cells

and cartilage

Last 1-6 somites and

unsegmented PSM

Transplanted

to mid- and

hindbrain

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

elements

Last 1-6 somites and

unsegmented PSM

Transplanted

to mid- and

hindbrain

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

DEVELOPMENT

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 ‘authentic’NC 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 ‘authentic’NC

being present at all axial levels alongside a ‘metablast’that 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

Sox10

high

/FoxD3+ sub-population capable of forming neural,

mesenchymal and neuronal derivatives, and a Sox10

low

/FoxD3

−

sub-

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

fates.

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

sympathoadrenal

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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DEVELOPMENT

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.,

2019).

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

intermediate

Cranial NC cell

Hox negative

Undifferentiated

hPSC

NMP

intermediate

Pigment cells, neurons and Schwann cells

Connective tissues

Skeletogenic tissues (including cartilage and bone)

Dorsal root ganglia and

sympathetic chain ganglia

Enteric neurons

Key

Sox2+

Brachyury/T+

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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DEVELOPMENT

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

MB FB

PSM

B Mouse E8.5A Mouse E7.5 C Mouse E10.5

Node streak border

(NSB)

Caudal lateral epiblast

(CLE)

Chordoneural hinge

(CNH)

Key

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.

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DEVELOPMENT

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

violated.

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

Hensen’s 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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DEVELOPMENT

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-specific’circuit 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

Lobe-finned

fishes

Tetrapods

Ectomesenchymal derivatives

Mineralized dermal tissues

Odontogenic Osteogenic Both

Arches Teeth Scales

Odontogenic

Enamel

Dentine

Elasmodine

Odontogenic

and osteogenic

Osteogenic

Key

Ostracoderms*

Placoderms*

Lungfishes

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).

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DEVELOPMENT

does have some caveats. First, it is based solely on expression data

and requires functional validation. Second, despite the absence of a

‘cranial’GRN 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

regionalization.

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

Amniotes

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

Amphibians

Amniotes

Cartilaginous fishes

Jawless fishes

Bony fishes

Amphibians

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).

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DEVELOPMENT

existence of a distinct, teleost-specific cranial NC GRN. Indeed, the

zebrafish GRN is ‘simplified’only 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.

Acknowledgements

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 (zfin.org).

Competing interests

The authors declare no competing or financial interests.

Funding

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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REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888

DEVELOPMENT

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REVIEW Development (2020) 147, dev193888. doi:10.1242/dev.193888

DEVELOPMENT

Ancient vertebrate dermal armor evolved from trunk neural crest

Article

Jul 2023
P NATL ACAD SCI USA

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.

Limited dorsal myeloschisis associated with intramedullary infantile hemangioma in the conus medullaris: illustrative case

Article

Full-text available

Apr 2023

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.

Origin and diversification of fibroblasts from the sclerotome in zebrafish

Article

Mar 2023
DEV BIOL

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.

Incubation temperature alters stripe formation and head colouration in American alligator hatchlings and is unaffected by E2-induced sex reversal

Article

Full-text available

Mar 2023
J EXP BIOL

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.

Migration deficits of the neural crest caused by CXADR triplication in a human Down syndrome stem cell model

Article

Full-text available

Dec 2022

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.

Scales, scutes, and embryonic origins of the vertebrate dermal skeleton

Article

Aug 2023

J Andrew Gillis

Constructing the pharyngula: Connecting the primary axial tissues of the head with the posterior axial tissues of the tail

Article

Jun 2023

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.

Leveraging neural crest pluripotency to extend retinal and craniofacial niches for building neurovascular organoids-a theranostic and drug development perspective

Chapter

Full-text available

Mar 2023

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

hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung’s disease

Article

Mar 2023
CELL STEM CELL

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.

Development and In Vitro Differentiation of Schwann Cells

Article

Full-text available

Nov 2022

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.

Spatiotemporal contribution of neuromesodermal progenitor-derived neural cells in the elongation of developing mouse spinal cord

Article

Full-text available

May 2020

Lineage tracing axial progenitors using Nkx1.2CreER T2 mice defines their trunk and tail contributions

Preprint

Full-text available

Feb 2018

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 .

Rostrocaudal Patterning and Neural Crest Differentiation of Human Pre-Neural Spinal Cord Progenitors in vitro

Preprint

Full-text available

Jun 2020

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.

The origin and evolution of vertebrate neural crest cells

Article

Full-text available

Jan 2020

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.

Resolving homology in the face of shifting germ layer origins: Lessons from a major skull vault boundary

Article

Full-text available

Dec 2019
eLife

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.

Early chromatin shaping predetermines multipotent vagal neural crest into neural, neuronal and mesenchymal lineages

Article

Full-text available

Dec 2019
NAT CELL BIOL

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.

Evolution of the new head by gradual acquisition of neural crest regulatory circuits

Article

Full-text available

Oct 2019
NATURE

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.

The Neural Crest

Book

Nov 1999

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.

Neural crest lineage analysis: from past to future trajectory

Article

Oct 2020

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.

Reprogramming Axial Level Identity to Rescue Neural-Crest-Related Congenital Heart Defects

Article

May 2020
DEV CELL

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.

From head to tail: Regionalization of the neural crest

Abstract and Figures

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