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Evidence for NCC multipotency in vivo

The remarkable ability of NCC to give rise to a wide variety of cell types stands out from any other structure in the vertebrate embryo. Major questions in the understanding of NCC diversification are whether single NCC are endowed with multiple differentiation potentials in early stages and also to what extent this potentiality is maintained during and after NCC migration (Le Douarin and Kalcheim, 1999). Conversely, another scenario would argue for a heterogeneous NCC population composed of a mosaic of restricted progenitors, already determined towards specific cell types (Krispin et al., 2010). Nonetheless, a significant number of in vivo and in vitro studies tend to favor the NCC multipotency model (Figure 7).
In heterotopic transplantation experiments in avian embryos, the replacement of the chick trunk NC, at the adrenomedullary level, by a quail vagal NC, promoted the differentiation of vagal NCC into the appropriate NC-derived cell types of the trunk, including the chromaffin cells of the host adrenal glands (Le Douarin and Teillet, 1974). On the other hand, when thoracic quail NCC were transplanted to the vagal level of the NC, thoracic NCC colonized the gut and differentiated there into enteric ganglia (Le Douarin and Teillet, 1974; Fontaine-Perus et al., 1982; Le Douarin et al., 1975). These experiments represented a strong evidence that some degree of plasticity exists in premigratory NCC, the fate of which could be reprogrammed by changing their embryonic environment. However, in some cases, the donor NCC were unable to produce all the cell types normally derived from the host NC. One remarkable example is the NC differential ability to yield mesenchymal derivatives observed along the rostrocaudal axis: the trunk NC transplanted into the cephalic NC domain failed to yield skeletal derivatives (Chibon, 1967; Nakamura and Ayer-le Lièvre, 1982).
Fate plasticity in the avian NC was also identified by heterochronic, or back-transplantation, experiments where postmigratory NCC, or fragments of tissues containing postmigratory NCC, were grafted into the NC migration pathway of younger host embryos. In these conditions, the non-neuronal NCC present in grafted PNS ganglia from E4-15 quail embryos were still able to generate PNS neurons and glia in a younger chick host embryo (Ayer-Le Lievre and Le Douarin, 1982; Dupin, 1984). Furthermore, the back-transplantation of E4 quail gut pieces, containing NCC, into the NCC migratory route at the trunk level, led to the differentiation of all thoracic NC cell types, except melanocytes, from the donor NC tissue (Rothman et al., 1990). These studies revealed that, at migratory and postmigratory stages, the NCC population has broader differentiation potentials than those expressed in normal development; however, these studies also point out some gradual restrictions of NCC potentialities as, in many cases, the replacement of all NC derivatives by the donor tissue was not completely achieved (Le Douarin and Kalcheim, 1999). For a better understanding of the timing and mechanisms of segregation of different NC lineages, it is important to analyze the progeny of an individual NCC. Single-cell analyses, in vivo and in vitro, are ideal to solve issues regarding multipotentiality and heterogeneity of the NC population.
The first experiments of NC lineage tracing in vivo were performed by intracellular injection of a fluorescent dextran into individual premigratory NCC in chicken embryos (Bronner-Fraser and Fraser, 1988). Two days after NCC injection, the progeny of individual labeled cells could be detected in different trunk NC-derived structures, such as the dorsal root and sympathetic ganglia, the adrenal medulla, and in Schwann cells lining spinal nerves, and migrating melanocytic cells (Bronner-Fraser and Fraser, 1988, 1989).
Similar experiments were also performed in other species, such as Xenopus (Collazo et al., 1993) and mouse (Serbedzija et al., 1994), which confirmed that at least a fraction of the premigratory NCC are multipotent. Nevertheless, in zebrafish embryos, the descendants of individually labeled NCC were detected in only one NC-derived structure, suggesting NC early fate restrictions in this particular model (Raible and Eisen, 1994; Schilling and Kimmel, 1994). Recently, the issue of NCC fate restriction was revisited in amniotes. By single-cell labeling in semi-open trunk neural tube preparations, Krispin and colleagues have shown that the initial ventrodorsal position of a premigratory NCC in the dorsal neural tube could predict its fate (Krispin et al., 2010). Moreover, they found that NCC progeny was restricted to a single rather than multiple NC derivatives, suggesting that the fate of NCC would be determined before cell delamination. These results contradict previous findings in cranial NCC: when grafted at the head level, late-migrating mesencephalic NCC generated the same cranial NC derivatives as early-migrating ones, and vice versa (Baker et al., 1997). In other words, cranial NCC in different migratory stages are not fate restricted. Recently, McKinney and colleagues challenged the results from Krispin and co-workers, by using time-lapse imaging of photo-converted cells labeled in the intact chick trunk neural tube (McKinney et al., 2013). They observed that dynamic rearrangements occurred in the premigratory NC, and thus, their position within the dorsal neuroepithelium was not strictly related to their exit time from the neural tube. Furthermore, except for the first migrating NCC that form the sympathetic ganglia, most of the premigratory NCC, issued from different dorsoventral levels in the neural tube, yielded multiple NC derivatives (McKinney et al., 2013). In sum, albeit Krispin .

The differentiation potentials of the NCC in vitro

Cohen and Konigsberg were the pioneers to perform clonal assays in avian trunk NCC (Cohen and Konigsberg, 1975), inspired by previous in vitro single cell analysis developed to unveil the diversity of murine bone marrow progenitors (Bradley et al., 1967; for references, Metcalf et al., 2007). Firstly, they devised a technique to obtain isolated trunk NCC, which migrated in vitro from the neural primordium explanted at developmental time preceding NCC exit. With this methodology, they cultured NCC at low-density conditions (limit dilution method) and observed that single NCC expanded in vitro gave rise to clonal populations containing non-pigmented and pigmented cells, indicating a mixed fate adopted by a single NC progenitor (Cohen and Konigsberg, 1975; Sieber-Blum and Cohen, 1980). Afterward, part of the non-pigmented cells present in the NC colonies was defined as sensory and adrenergic neuroblasts, showing that the clonogenic NCC were indeed multipotent in vitro (Sieber-Blum, 1989).
In vitro clonal assays were further improved with the identification of various phenotypic markers and the optimization of culture conditions. For instance, culturing avian NCC on 3T3 feeder-layers, according to a method adapted from single-cell cultures of human keratinocytes (Barrandon and Green, 1985), greatly improved NCC cloning efficiency, i.e. the percentage of single seeded cells that actually yields a clone. Moreover, the direct and microscopically controlled plating of single cells taken from NCC suspensions helped to ensure clonality of the NC cultures (Baroffio et al., 1988). As a consequence, clonal assays carried out by Dupin and colleagues using quail NCC isolated from the entire neural axis and at different developmental stages, confirmed the heterogeneity of NC progenitors in terms of proliferation, survival and differentiation capacities and further demonstrated the existence of highly multipotent NCC (Baroffio et al., 1988, 1991; Trentin et al., 2004; Calloni et al., 2009; Coelho-Aguiar et al., 2013; Lahav et al., 1998; Dupin et al., 1990). Among all the advances obtained by avian NC clonal assays, one of the most interesting discoveries was that some cephalic NC progenitors are endowed with both neural and mesenchymal potentialities (Baroffio et al., 1988, 1991; Ito and Sieber-Blum, 1991). For instance, Baroffio and collaborators have shown that neural (neuronal and glial) cells, melanocytes and cartilage nodules could originate from the same NC progenitor, therefore suggesting that neural and mesenchymal progenitors may not be segregated at early stages of NC development (Baroffio et al., 1988, 1991). Nevertheless, in these initial experiments, the neural-mesenchymal clones represented only a small fraction of the resulting clones. However, optimization of the cell culture conditions and isolation of mesencephalic NCC at earlier time points of in vitro migration (15 hours instead of 24 hours), later on led Calloni and colleagues to obtain a larger proportion of cephalic NC-derived clones containing both cartilage and neural/melanocytic cells (Calloni et al., 2007). Moreover, osteoblasts were also observed in these clonal cultures, in approximately 94% of the colonies (Calloni et al., 2009). Interestingly, the authors also showed that a highly multipotent progenitor was able to give rise to six different cell types, that is, glial, neuronal, melanocytic, myofibroblastic, chondrocytic and osteoblastic cells. In summary, these in vitro results revealed the high ability of single cephalic NCC to produce diverse cell types, as previously shown in vivo at the whole cell population level. Also, they evidenced that the cranial NC is composed of highly multipotent NC stem cells in early stages of development.

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Stem cell properties of NCC

Given the plasticity of NCC, associated with their multipotential feature, it is of great importance to assess whether NCC can be considered as true stem cells with self-renewal capacity, that is the capacity of a cell to generate, in addition to a differentiated progeny, daughter cells that remain undifferentiated and preserve its differentiation potentials (i.e. stemness). The self-renewing ability of quail trunk and cephalic NCC was tested by successive subcloning experiments in vitro, which revealed that glial-melanocytic and glial-myofibroblastic bipotent progenitors act as stem cells, and can be propagated in vitro upon the influence of endothelin-3 and FGF2, respectively (Trentin et al., 2004; Bittencourt et al., 2013). In mammals, self-renewing trunk NCC, that yield autonomic neurons, glial cells and myofibroblasts, were first isolated by sorting a rat NC subpopulation expressing p75 receptor (Stemple and Anderson, 1992). The latter receptor, together with the HNK1 marker, also led to the isolation of multipotent human cranial NC-like stem cells from ESC cultures (Lee et al., 2007). Recently, it was reported a new procedure to maintain NCC self-renewal for long periods in culture, inspired by sphere-forming assays classically used to identify stem cells in many tissues, such as brain-derived neurospheres (Reynolds and Weiss, 1992). Individual early NCC, derived from chick embryos or human ESC, were grown in low-attachment conditions, which favor the formation of free-floating spheres, herein named crestospheres (Kerosuo et al., 2015). With this methodology, cells inside the crestospheres could be maintained for several weeks in culture in a premigratory NC state expressing early NC markers such as Sox10, Foxd3, Snail, and AP2a. In pro-differentiation conditions, cells in the crestospheres gave rise to neural cells, melanocytes and mesenchymal cells (myofibroblasts and osteoblasts). Additionally, individual crestosphere clones could generate new crestospheres, showing that a subpopulation of NCC could maintain its stemness. The development of this technique will contribute to further investigations regarding NCC potency and self-renewal. For instance, by using this technique, it was recently described that c-myc, a “pluripotency gene” with an established role in embryonic and adult stem cell maintenance (Chappell and Dalton, 2013, for a review), regulates the size of the premigratory NCC pool in vivo and in vitro (Kerosuo and Bronner, 2016).
In summary, these experiments show that NCC possess many features of true stem cells.

Maintenance of NC stem cells in adult tissues

Accumulating evidence shows that multipotent and self-renewing NCC, the so-called NC-derived stem cells (NCSC), are present even in postmigratory embryonic and adult stages. Hence, many tissues containing NCSC have been described in rodents and human, by either culturing the adult tissue and further identification of NCSC, via NC-specific markers (e.g. p75, Sox10, Sox9, Nestin, among others) or by in vivo lineage tracing of NCC in adult murine tissues, through the use of NC-specific conditional mice. In this way, NCSC were identified in the sciatic nerve (Morrison et al., 1999), intestine (Bixby et al., 2002), dorsal root ganglia (Li et al., 2007), skin (Fernandes et al., 2004; Sieber-Blum et al., 2004; Wong et al., 2006), heart (El-Helou et al., 2008), bone marrow (Nagoshi et al., 2008), cornea (Yoshida et al., 2006; Brandl et al., 2009), teeth (Janebodin et al., 2011; Kaukua et al., 2014) and craniofacial tissues (Kaltschmidt et al., 2012), among others (for further references, Dupin and Coelho-Aguiar, 2013; Dupin and Sommer, 2012). However, in most of these tissues and organs, the localization and marker identity of the NCSC remained rather obscure. Yet, in the carotid body, a small neuroendocrine organ involved in blood oxygen pressure regulation, genetic fate mapping of mouse NCC led to characterize multipotent NCSC in the adult organ as supportive cells expressing the glial marker GFAP, which can reversibly produce new neuron-like glomus cells in vivo for adaptation to hypoxia (Pardal et al., 2007).

Glia-related postmigratory neural crest stem cells

Recent studies have since put forwards the notion that NC-derived glial cells, particularly Schwann cell precursors, behave as NC-like stem cells in diverse tissues, largely contributing to development and regeneration of different cell types in the many niches they reside (Petersen and Adameyko, 2017, for a review). A striking discovery was that immature Schwann cell precursors in mammalian and avian PNS nerves give rise to a significant part of melanocytes in the body (Adameyko et al., 2009, 2012). Of note, the generation of pigment cells from Schwann cells was previously evidenced in vitro (Dupin et al., 2003; Real et al., 2005; Widera et al., 2011). Besides, the reverse in vitro phenotype conversion, from pigmented melanocytes to Schwann cells, has also been reported in the quail and occurred through a multipotent NC-like intermediate (Dupin et al., 2000; Real et al., 2006). Recently, interesting findings were obtained in zebrafish regarding establishment of the colored stripes of the adult fish: during metamorphosis, the adult pigment cells were generated from post-migratory NCC located along spinal nerve fibers and within the dorsal root ganglia, which also gave rise to PNS neurons and Schwann cells (Dooley et al., 2014; Singh et al., 2016). Other recent findings support a role for nerve-associated glial progenitors in the development of significant subpopulations of PNS neurons in the cranial parasympathetic ganglia (Espinosa-Medina et al., 2014; Dyachuk et al., 2014) and the ENS of mammals (Uesaka et al., 2015). Furthermore, NCSC belonging to the NC glial lineage can adopt phenotypes that exceed neuroglial and melanocytic fates. As already mentioned, Schwann cell precursors in vivo are at the origin of endoneurial fibroblasts along the sciatic nerve (Joseph et al., 2004). In addition, transdifferentiation of Schwann cells into myofibroblasts has been previously described in in vitro culture (Dupin et al., 2003; Real et al., 2005) and in response to infection of the adult nerve by leprosy bacilli (Masaki et al., 2013). Recent findings from long-term genetic tracing and clonal color-coding in mice have shown that, in the model of continuously renewing incisor tooth, PNS nerve-associated glial cells are at the origin of mesenchymal stem cells involved in the renewal and repair of pulp cells, odontoblasts and osteoblasts during development and adult life (Kaukua et al., 2014).

Table of contents :

I.1. Overview of the NC development
I.2. The NC migratory routes and derivatives
I.3. Early steps in NC development: the gene regulatory network of NC induction and specification
I.4. Molecular regulation of the EMT and NCC migration
II.1. Evidence for NCC multipotency in vivo
II.2. The differentiation potentials of the NCC in vitro
II.3. Stem cell properties of NCC
II.4. Maintenance of NC stem cells in adult tissues
II.5. Glia-related postmigratory NC stem cells
III.1. Endochondral and intramembranous ossification
III.2. Bone matrix mineralization
III.3. Molecular aspects of osteogenesis and chondrogenesis
III.4. Adipogenesis
IV.1. NC and mesoderm interactions in the cranial mesenchyme: role of Six1
IV.2. Evolutionary aspects
IV.3. Regulation of the skeletogenesis of NCC
IV.3.1 Early signals involved in NC mesenchymal fate
IV.3.2 Overview of upstream regulators of Runx2
IV.3.3 Epigenetic mechanisms
V.1. Overview of Hox genes effects on development
V.2. Hox genes and the NC


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