On the road again: Establishment and maintenance of stemness in the neural crest from embryo to adulthood

Abstract Unique to vertebrates, the neural crest (NC) is an embryonic stem cell population that contributes to a greatly expanding list of derivatives ranging from neurons and glia of the peripheral nervous system, facial cartilage and bone, pigment cells of the skin to secretory cells of the endocrine system. Here, we focus on what is specifically known about establishment and maintenance of NC stemness and ultimate fate commitment mechanisms, which could help explain its exceptionally high stem cell potential that exceeds the “rules set during gastrulation.” In fact, recent discoveries have shed light on the existence of NC cells that coexpress commonly accepted pluripotency factors like Nanog, Oct4/PouV, and Klf4. The coexpression of pluripotency factors together with the exceptional array of diverse NC derivatives encouraged us to propose a new term “pleistopotent” (Greek for abundant, a substantial amount) to be used to reflect the uniqueness of the NC as compared to other post‐gastrulation stem cell populations in the vertebrate body, and to differentiate them from multipotent lineage restricted stem cells. We also discuss studies related to the maintenance of NC stemness within the challenging context of being a transient and thus a constantly changing population of stem cells without a permanent niche. The discovery of the stem cell potential of Schwann cell precursors as well as multiple adult NC‐derived stem cell reservoirs during the past decade has greatly increased our understanding of how NC cells contribute to tissues formed after its initial migration stage in young embryos.

array of diverse NC derivatives encouraged us to propose a new term "pleistopotent" (Greek for abundant, a substantial amount) to be used to reflect the uniqueness of the NC as compared to other post-gastrulation stem cell populations in the vertebrate body, and to differentiate them from multipotent lineage restricted stem cells. We also discuss studies related to the maintenance of NC stemness within the challenging context of being a transient and thus a constantly changing population of stem cells without a permanent niche. The discovery of the stem cell potential of Schwann cell precursors as well as multiple adult NC-derived stem cell reservoirs during the past decade has greatly increased our understanding of how NC cells contribute to tissues formed after its initial migration stage in young embryos. The neural crest (NC) is a population of stem cells that arises from the ectodermal germ layer and gives rise to a broad range of derivatives.
Although multipotency is usually described as the ability to give rise to all cell types within a particular lineage, the NC gives rise to cells of several lineages, spanning from cells of the nervous system to cartilage and bone to endocrine cells, representing ectodermal as well as mesodermal-like and endodermal-like cell types. According to the rules set during gastrulation, certain types of derivatives are specific to each germ layer.
The diversity of NC derivatives contradicts this concept and has raised discussions on how the NC possesses such exceptionally high stem cell potential-higher than any other cell type post-gastrulation and is sometimes referred to as the fourth germ layer. 1 It has been suggested, based on similarities on gene expression profiles of certain genes and gene families in the blastula and the NC, that NC stemness is preserved from pregastrula stages. 2 Recent studies have revealed coexpression of generally accepted pluripotency factor genes such as Nanog, Klf4, and Oct4/PouV within a subpopulation of the premigratory NC, a temporary niche, which supports the idea of a substantially higher molecular stem cell signature of the NC than previously anticipated, 3 although the function of these genes in NC cells remains largely unknown. The formation of this temporary NC stem cell niche is poorly understood and further proof is required to determine whether NC formation is enabled via escaping from the earliest fate determining cues provided during gastrulation, or if the high stem cell capacity is gained de novo in the ectodermal germ layer.
Here, we discuss what is currently known about the developmental potential of the NC, the regulation of its stem cell characteristics and how data from decades ago fits with new discoveries published within the recent years. We also discuss how the existing stem cell nomenclature is inadequate to describe the NC stem cell population.
Due to the lack of precise terminology, the NC is commonly referred to as anything between pluripotent stem cells to multipotent progenitors, which from a stem cell perspective represent two very different things (not to mention the problematic usage of the word "progenitor" in this context that as such refers to a cell with restricted proliferation capacity that can differentiate to a few cell types within a single tissue or organ, and would typically represent an oligopotent cell). Finally, we address the maintenance of NC stemness during embryogenesis and adulthood in the form of different NC-derived stem cell populations and briefly discuss how the implications of these findings relate to the regenerative potential of these cells.

| Brief overview of development of the NC and its derivatives
The NC, a transient population unique to vertebrates, arises in a temporal, anterior to posterior progressing fashion, from the dorsal aspects of almost the entire axial length of the neural tube during the formation of the nervous system. After their initial induction at the neural plate border, the cells are fully specified into bona fide NC stem cells as the neural folds rise and meet each other in the dorsal neural tube. Soon after, these NC stem cells undergo epithelial to mesenchymal transition (EMT) and emigrate from different axial levels of the neural tube to migrate throughout the developing embryo giving rise to a wide array of derivatives, as shown in Figure 1 and Table 1.
Despite their transient nature, NC cells are able to self-renew in order to multiply and give rise to the correct size of the stem cell pool at the premigratory stage 46,47 before emigration and differentiation into ectodermal, mesodermal, and endodermal derivatives, making them the most astonishing stem cell population post-gastrulation. New derivatives and contributions of NC cells, such as phagocytotic activity of the migratory NC cells, 48 are identified even now, over 150 years after the NC was first discovered.
There are some differences in the developmental potential of the NC cells originating along the various axial levels that are broadly subdivided to cranial, vagal, trunk, and lumbosacral NC cells based on the segments of origin. 49 Despite this variance along the axial levels, NC cells at all levels give rise to neurons and glia of the peripheral nervous system and melanocytes (reviewed in Reference 50). Unique to the axial level (as presented in Figure 1 and Table 1), the cranial NC (from forebrain to hindbrain) gives rise to several mesenchymal derivatives of the face and neck such as smooth muscle, odontoblasts, chondrocytes, osteocytes, connective tissue, pericytes, meninges, facial dermis, and adipocytes. 4,[6][7][8] The main contribution of the vagal NC (at the level of somites 1-7), in comparison, is to the neurons and glia of the enteric nervous system 32 and several derivatives in the cardiac system. [34][35][36][37][38] In addition to giving rise to neurons and glia, the trunk NC (somites 8-24 in chick and to 8-27 in mouse) gives rise to adrenal chromaffin cells. 33 Finally, the sacral NC (posterior to the trunk NC) gives rise to a small portion of the enteric nervous system along with the vagal NC. [43][44][45] Taken together, the NC gives rise to more than 30 different cell types, although many derivatives are specific to a certain axial level ( Figure 1; Table 1). The trunk NC has commonly been considered to lack the capacity to form bone and cartilage 49 at least in the more intensely studied model systems for NC development such as the chicken, frog, mouse, and zebrafish. However, contribution of trunk NC cells to some skeletal derivatives has been reported in zebrafish 51 (although contradicting with another report 22 ), turtle 52,53 and skate. 54 A recent molecular level study in the chick elegantly showed that lack of skeletogenic potential in trunk NC cells is due to differences in the gene regulatory network at the premigratory stage, which is reversible by genetic manipulation by activating a set of cranial genes in the trunk. 55 In line with this, a recent spatiotemporal transcriptomics study suggests that, in the

Significance statement
The neural crest is a remarkable embryonic stem cell population that contributes to a wide variety of derivatives ranging from neurons, glia, cartilage, bone, pigment cells, and endocrine cells. As the list of neural crest derivatives has greatly expanded since its identification over 150 years ago, the clinical significance of the neural crest has become more evident, whereas some central questions regarding stemness formation and maintenance remain poorly understood. This concise review discusses studies related to how this postgastrulation cell population maintains its exceptional stem cell potential from embryonic to adult stages.
F I G U R E 1 Neural crest cell populations contribute to a diverse range of cells in adult tissues and organs. Schematic of an adult human showing the tissues and organs derived from the neural crest based on studies in different animal species (chick, mouse, frog, and zebrafish). These neural crest derivatives can be broadly categorized to five main cell groups: craniofacial skeleton (mesenchymal cells), other mesenchymal cells, cells of the peripheral nervous system, endocrine cells, and melanocytes. Please see Table 1 for a detailed overview of these derivatives. Asterisk denotes contradictory evidence between species. Figure created with BioRender T A B L E 1 The diverse range of derivatives to which the neural crest gives rise, based on studies of different animal species: chick, mouse, frog, and zebrafish.  The majority of trunk premigratory and, interestingly, also migratory NC cells were found to be able to generate sensory and autonomic neurons, glia and melanocytes. 60 More recently, advanced profiling of the molecular signature of NC cells using single cell sequencing and spatial transcriptomics has shown that fate biasing of NC is already detectable when these cells delaminate from the neural tube. 56  Even though a majority of the premigratory NC cells were found to be multipotent in the study by Baggiolini and colleagues, they reported differences in the developmental potential of clones, from some giving rise to all possible fates to some only producing two possible fates, suggesting heterogeneity of the stem cell potential of individual cells within the NC population. 60 This is consistent with in vitro clonal analyses, which have shown that NC cells are multipotent and able to self-renew 46,67,68 but, as seen in the in vivo studies of multipotency, show differences in developmental potential (the number of derivatives to which it gives rise). [67][68][69] However, it is important to keep in mind that what a NC cell becomes is not necessarily synonymous with its potential, although a recent study does show that overexpression of the stem cell gene Lin28 significantly increases the existence of multipotent clones in vitro. 70 Further molecular profiling is needed to confirm the differences between NC cells that give rise to different numbers of the possible fates.

| Exceptionally high stem cell potentialpleistopotent?
NC cells have a higher stem cell potential than what is typically understood as multipotent, which is defined by having the ability to differentiate into all cell types within one particular lineage (e.g., hematopoietic stem cells that give rise to a myriad of blood cells-stem cells that do not coexpress pluripotency factors). However, so far there is no evidence of a NC cell having the capacity to form a whole new embryo with all its cell types, either, which would be considered as true pluripotency. Thus, as noticed within the NC stem cell field for decades, the existing stem cell nomenclature lacks an intermediate version of these two states, which would adequately describe the potency of NC cells being higher than "just" multipotent but lower than pluripotent, which is different from any other stem cell population described to date, except for neuromesodermal progenitors that could fall into a similar category. [71][72][73][74][75][76] Although the diverse outcomes of what the NC cells ultimately become have been known for decades, only recent work has provided molecular level proof that may explain the exceptionally high stem cell potential. A recent theory based on observations in the frog embryo suggests a shared regulatory program between the blastula and the NC. 2 The study hypothesizes that, unlike all other cells in the vertebrate body that go through gastrulation, the high stemness of the NC may be preserved from pre-gastrula stages by a yet unknown protection mechanism in the particular ectodermal domain. 2 The hypothesis is based on data which shows several NC regulatory transcription factors such as Id3, Sox5, TF-AP2, Ets1, primary determination of a NC domain in the epiblast prior to gastrulation has also been recently suggested. 79 Lineage tracing followup studies combined with single cell transcriptomics will be needed to elucidate the differences and similarities as well as the steps in between these two developmental stages. In line with the hypothesis, however, a recent study identified a transcriptionally and spatially distinct subdomain of premigratory cranial NC cells in the chicken embryo that indeed coexpress the generally accepted pluripotency factors Nanog, Oct4/PouV, and Klf4, which forms the core of a temporary NC stem cell niche discussed in more detail in the next section. 3 Since then, Oct4/PouV expression has also been shown in the NC in frog together with Ventx2, and in zebrafish. 71,72 Although, currently, there is not enough evidence to fully specify how similar the potency of NC cells is to pluripotent blastula cells, the existing gene expression and lineage tracing data certainly verifies NC stem cell potential to be far beyond what we understand by multipotency and necessitates a reconsideration of traditionally used terms to describe the potency of these unique NC cells (Table 2). For this, we contacted a linguistics expert in Latin and Greek who suggested NC potency to be referred to as pleistopotent (pleisto is Greek for "abundant, a substantial amount"), which we suggest to refer to stem cells that produce progeny that display features of more than one germ layer. Neu-  in the dorsal neural tube as the NC starts to prepare for emigration. [82][83][84][85][86] Substantial amount of work has also been invested in revealing regulatory links between these transcription factors. 87,88 In comparison, signaling mechanisms and molecules required for establishing and controlling self-renewal and/or developmental conditions with FGF, IGF, and retinoic acid were found to maintain chick and human NC cells in a self-renewing state that reflects their premigratory character 46 and addition of BMP4 was found to enhance expression of some NC marker genes to maintain trunk NC cells as self-T A B L E 2 Stem cell nomenclature for different stem cell potential. Current, existing stem cell nomenclature is missing a category that would adequately represent the neural crest cells, which have higher stem cell potential than multipotent cells but are not pluripotent. For this reason, we suggest the addition of an intermediate stage, pleistopotent (in bold), which describes stem cells that give rise to cell types that display features of "more than one germ layer," and thus would adequately describe the potential of neural crest cells and also neuromesodermal progenitors.  116 In line with this, a recent multiplex spatial genomics study revealed unbiased hierarchical clustering of Sox9, FoxD3, and Snail2 genes in premigatory NC cells in chicken embryos suggesting a high probability of coexpression and thus possibility of the three acting as co-binding factors in the gene regulatory network. 3 In comparison, Sox10 and Sox8 clustered further away from this core cluster F I G U R E 2 Neural crest stemness consists of multiple cellular functions driven by several genes in the dorsal neural tube. Schematic of a cross section through the neural tube showing the premigratory cranial neural crest containing a stem cell niche (dark orange region) where the pluripotency factor genes Nanog, Oct4, and Klf4 are coexpressed with neural crest markers. Current knowledge from different animal species suggests that several of the known neural crest specifier genes have different roles in maintaining neural crest stemness-whereas some regulate the size of the stem cell pool to ensure that the correct amount of neural crest cells will be produced in accordance with proper specification and preparation for emigration, some promote a particular cell fate such as neural, glial, chondrocytic, osteogenic, and/or melanocytic. As different transcription factors bias the cell for different lineages, the cell is maintained in a pleistopotent stage as long as all these lineage-promoting genes are actively coexpressed suggesting that although there may be some redundancy in the SoxE genes in NC cells, there may also be some independent functions to each of the genes, and these roles may differ depending on the devel-

| Sox8 (cell survival and migration and possibly a negative regulator of the osteogenic lineage)
Sox8, the third member of the SoxE family, has also been shown to play a role in NC specification, although some differences in the spatiotemporal expression pattern occur between different species (reviewed in Reference 117). In frog, loss of Sox8 results in defects in cranial and trunk NC derivatives, 123 and in mouse Sox8 is required for the maintenance of the vagal NC population and is thought to have a role in cell survival. 124 Sox8 has also been identified to be a negative regulator of osteoblast differentiation, whereas there is no effect on chondrogenic differentiation. 125 Although this study did not particularly focus on NC-derived craniofacial bone development, Sox8 could potentially have a similar role in NC cells, negatively regulating the osteogenic lineage.

| Sox2 (promotes cell survival and glial fate)
Sox2, a member of the SoxB family, is highly expressed at the neural plate and well known for its role for maintaining the neural stem cell population required for proper formation of the central nervous system. 126,127 However, lower Sox2 levels are also expressed in the NC cells in the neighboring domain, 3,80,128 which may function to maintain glial lineage as it is known that Sox2 expression is upregulated in NCderived cells that contribute to the peripheral nervous system and is restricted to Schwann cells and satellite glia later in development. 129 3.1.5 | Sox5 (regulator of the size of NC domain in the early ectoderm; promoter of chondrogenesis and gliogenesis) Sox5, a transcription factor of the SoxD family, has two isoforms in mammals, a short SSox5, only expressed in testis, and a long isoform LSox5, which in the mouse in conjunction with LSox6 and Sox9 drives activation of Col2a to promote chondrogenesis. 130,131 In the chicken embryo, LSox5 regulates the size of the NC domain in the dorsal neural tube. 132 The study also found that in addition to promoting chondrogenesis, Sox5 is expressed in the cranial glia, Schwann cells and satellite glia, and could promote formation of the glial lineage as well. 132 Similarly, Sox5 is linked to early ectodermal patterning in the early frog as well, where it was shown to be a DNA-binding cofactor essential for recruitment of Smad1/4 to BMP regulatory elements. 133  Future studies blocking Snail genes in NC cells will help clarify its specific role in maintaining stem cell potential.

| Lin28 (pluripotency/stem cell maintenance)
Lin28 is an established pluripotency factor that has been identified to have a role in maintaining stemness and drive reprogramming of somatic cells. 99,102,159 Lin28 is expressed in chicken NC cells throughout their development, from specification stages at the neural plate border to late migration stages, with highest relative expression levels marked during the premigratory stage, which led A recent study in amphibians also showed that Nanog paralogue Ventx2 is broadly expressed in the neural plate border. 170 Knockdown of Ventx2 selectively affected expression of the NC specifiers Snail2, Sox9, Sox10, and Twist1 whereas Myc, Tfap2a, and Foxd3 levels were unaffected. 170 This altered expression profile resulted in reduced migration to the pharyngeal arches and reduced craniofacial skeleton whereas the neuronal lineage was unaffected in vivo and in vitro (in comparison, the melanocytic lineage was affected in vitro but not in vivo). 170 Furthermore, ectopic NC cells that were not biased toward any certain fate were formed in the ectoderm lateral to the NC domain in a process that required Ventx2, thus suggesting a role for Ventx2 in enabling the NC with a broader potential than just being restricted to neural derivatives, which would be the default for an ectodermal cell population. 170

| LINEAGE RESTRICTION OF NC CELLS
As NC development proceeds, stem cell mechanisms that support the highly multipotent/pleistopotent features at the premigratory stage need to be silenced or modified to allow for differentiation to take place. After the cells exit the dorsal neural tube, exposure to environmental cues, molecular as well as mechanical, likely play important roles in lineage restriction. 82,[171][172][173] As discussed in section 2.2, current data suggest that fate determination does not occur before delamination, 3,56,59,60 even though differences in axial level specific transcriptional profiles are detectable at the premigratory stage. 55 The acquisition of a fully differentiated fate thus likely involves cell-intrinsic state and extrinsic signaling during NC migration. To note, the development of melanocytes derived from chicken trunk NC cells, the last population to emigrate from the dorsal neural tube, may represent an exception to this rule as some studies suggest they may become fate restricted before or shortly after delamination. 145,174,175 After trunk NC cells delaminate from the dorsal neural tube, the ventrally migrating trunk NC cells follow a path between the somites and neural tube, and give rise to neurons, glia, and some endocrine cells, whereas the dorsolaterally migrating cells follow a path between the dermamyotome and ectoderm and become melanocytes in the skin. 174 By culturing single migratory NC cells isolated immediately after emergence from the neural tube and at intervals as development proceeds, it was observed that some isolated cells gave rise to mixtures of precursors (of neurons, glia, and melanocytes), whereas others gave rise to precursors of only one fate. 62 The results thus suggest the existence of fate restricted neurogenic and melanogenic sublineages and a broad time frame and heterogeneity in the ways the NC cells ultimately commit to their future fates. 62 Recent data based on genome-wide transcriptomic analysis derived pseudo-time trajectories in mouse embryos shows that newly migrating NC cells start to display a fate-biased transcriptional profile soon after delamination. 56 This is followed by lineage specification following a certain pattern in which future neurons and glial cells of the sensory lineage separate first from bipotent autonomic-mesenchymal progenitors, which then further divide into progenitors of the neurons and glia of the autonomic nervous system as well as mesenchymal cells. 56 However, a recent quantitative live imaging based study of trunk NC cells of the dorsoventral migration pathway in chick embryos suggests that the movement of individual cells is stochastic and unpredictable. 176 Although the overall direction of the movement is eventually toward the dorsal aorta, there was no correlation between the original position and total displacement of the individual cells, with some cells even moving backward. 176 The stochastic cell motion also led to sister cells ending up on different trajectories and final destinations with distinct fates, for example, sensory vs sympathoadrenal. 176 Finally, the confetti system was also applied to study cranial NC development during migratory stage. 177    .
FoxD3 is also expressed in a subset of glial cells, most likely a (sub) population of SCPs, as shown in a recent single cell transcriptomic study comparing different stages of mouse NC development. 56 [196][197][198] In vitro studies also corroborated their stem cell nature by showing that isolated boundary cap cells from mouse embryos self-renew and give rise to neuronal and nonneuronal derivatives. 199,200 As mentioned, comparison of the gene expression profile of NC cells, the different subpopulations of SCPs and boundary cap cells in more detail may help identify a common stem cell potency network in these cells.

| NC-derived stem cell reservoirs in neonatal and adult tissues
In addition to the broad developmental potential of the NC-derived nerve-associated cells during embryonic stages described above, in animal models NC-derived stem cells have been identified in developed, postnatal tissues such as dorsal root ganglia, 201  carotid body, 10 skin and hair follicles, [208][209][210] and palatal tissue 211,212 ( Figure 3). The human adult dental pulp also contains an odontoblast derived (and thus NC-derived) subpopulation of progenitors with a limited capacity to form dentin and pulp-like structures, 213 and has osteogenic potential 214 and possibly some neuroregenerative capacity as well. 215 Furthermore, NC-derived forebrain pericytes have also shown potential to differentiate to neural and nonneural cells following ischemia. 216 The discovery of NC-derived stem cells in skin has been particularly exciting considering this could be an easily accessible source of the cells for autologous transplantation for treatment of disease conditions. Since this discovery, several studies have isolated NC-derived progenitor or stem cells from human skin. [220][221][222][223] A recent study also showed a high level of stem cell potency of NC-derived nerve associated adult mesenchymal stem cells, which upon tissue repair have the capacity to form bone and dermis. 224

| Regenerative potential of NC cells
With the multitude of derivatives to which the NC gives rise, this stem cell population could have profound significance in regenerative medicine. Making a certain cell type via the correct differentiation pathway that mimics normal development as faithfully as possible is considered beneficial for obtaining the best possible outcome for therapeutic purposes. As such, for transplantation of NC derivatives into patients, it may be essential to first differentiate induced pluripotent stem cells (iPSCs) to NC cells before directing them to a particular NC derivative.
However, before applying NC-derived stem cells for regenerative purposes, we need a better understanding of human NC development.

| CONCLUSIVE REMARKS AND PERSPECTIVE
The data presented here bring together our current knowledge about the range of the diverse derivatives to which the NC gives rise, either directly from the initial embryonic NC cells or through an intermediate

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
S.N.P., L.K.: conceived overall review content and wrote the paper.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.