Elisabeth Dupin
email: elisabeth.dupin@inserm.fr

Giordano W Calloni
email: giordano.calloni@ufsc.br

Juliana M Coelho--Aguiar

Nicole M Le Douarin
email: nicoleledouarin@gmail.com

Nicole M Le

The issue of the multipotency of the neural crest cells

Keywords: neural crest, peripheral nervous system, ectomesenchyme, stem cells, multipotency, cellular plasticity, Running title: Neural crest cell multipotency CNS, central nervous system, E, embryonic day, EDN, endothelin, EMT, epithelial--to-mesenchymal transition, ENS, enteric nervous system, Mab, monoclonal antibody, NC, neural crest, NCC, neural crest cells, PNS, peripheral nervous system, Shh, Sonic hedgehog neural crest, peripheral nervous system, ectomesenchyme, stem cells, multipotency, cellular plasticity

In the neural primordium of vertebrate embryos, the neural crest (NC) displays a unique character: the capacity of its component cells to leave the neural primordium, migrate along definite (and, for long, not identified) routes in the developing embryo and invade virtually all tissues and organs, while producing a large array of differentiated cell types.

The most striking diversity of the NC derivatives is found in its cephalic domain that produces, not only melanocytes and peripheral nerves and ganglia, but also various mesenchymal derivatives (connective tissues, bones, cartilages…) which, in other parts of the body, are mesoderm--derived. The aim of this article was to review the large amount of work that has been devoted to solving the problem of the differentiation capacities of individual NC cells (NCC) arising from both the cephalic and trunk levels of the neural axis. A variety of experimental designs applied to NCC either in vivo or in vitro are evaluated, including the possibility to culture them in crestospheres, a technique previously designed for cells of the CNS, and which reinforces the notion, previously put forward, of the existence of NC stem cells. At the trunk level, the developmental potentialities of the NCC are more restricted than in their cephalic counterparts, but, in addition to the neural--melanocytic fate that they exclusively express in vivo, it was clearly shown that they harbor mesenchymal capacities that can be revealed in vitro. Finally, a large amount of evidence has been obtained that, during the migration process, most of the NCC are multipotent with a variable array of potentialities among the cells considered. Investigations carried out in adults have shown that multipotent NC stem cells persist in the various sites of the body occupied by NCC. Enlightening new developments concerning the invasive capacity of NCC, the growing peripheral nerves were revealed as migration routes for NCC travelling to distant ventrolateral regions of the body. Designated "Schwann cell precursors" in the mouse embryo, these NCC can leave the nerves and are able to convert to a novel fate.

The convertibility of the NC--derived cells, particularly evident in the Schwann cell-melanocyte lineage transition, has also been demonstrated for neuroendocrine cells of the adult carotid body and for the differentiation of parasympathetic neurons of ganglia distant from their origin, the NC. All these new developments attest the vitality of the research on the NC, a field that characterizes vertebrate development and for which the interest has constantly increased during the last decades.

Introduction

The neural crest (NC) has long been designated as a "fourth germ layer" from which multipotent cells migrate and invade the developing embryo. Apart from ectoderm, mesoderm and endoderm, the foundation of the vertebrate body involves this transitory ectodermal structure, which yields neural and mesenchymal cell types, otherwise derived from either the ectodermal or mesodermal germ layers, respectively (for references, Le [START_REF] Le Douarin | The neural crest[END_REF][START_REF] Le Douarin | The neural crest, 2nd ed[END_REF][START_REF] Hall | The Neural Crest and Neural Crest Cells in Vertebrate Development and Evolution[END_REF][START_REF] Dupin | The neural crest, a multifaceted structure of the vertebrates[END_REF]. This view has recently been reinforced by the work of [START_REF] Buitrago--Delgado | Neurodevelopment. Shared regulatory programs suggest retention of blastula--stage potential in neural crest cells[END_REF], which shows that regulatory genes Snail1 and Sox5 are expressed both in the pluripotent blastula cells of amphibian embryos and in NC. In both cell types, they are required for embryonic cell competence to respond to instructive cues and therefore to keep functional multipotency. These findings led the authors to consider the NC a "neotenic" structure, in which part of the ectoderm has retained, over gastrulation, subsets of phenotypes characteristic of the early blastula, which is in sharp contrast with previous studies that favored a molecular model in which NC cells (NCC) reacquire multipotency through instructive signals like Wnt, Fgf and Bmp.

In addition to neurons and glial cells of the peripheral nervous system (PNS), melanocytes and some endocrine derivatives, the NC has the striking capacity of producing a large diversity of mesenchymal cell types in the head and neck, designated as mesectoderm or ectomesenchyme (for references, Le [START_REF] Le Douarin | The neural crest, 2nd ed[END_REF][START_REF] Le Douarin | The neural crest[END_REF]Dupin, 2012, 2016). Such multipotency of the NC challenged the widely accepted von Baer's germ layer theory, according to which mesenchymal cells endowed with skeletal and connective tissue capacities were exclusively produced, all over the body, by the mesodermal germ layer. The production of facial and skull bones from the ectodermally--derived NCC raised the question of the capacity of single NCC to generate both skeletal and neural cell types, or whether this capacity stands for the structure as a whole rather than for its constitutive cells, as proposed by some authors [START_REF] Weston | Pentimento: Neural Crest and the origin of mesectoderm[END_REF]. This problem has prompted us, and several others, to systematically explore the extent of single NCC differentiation abilities, at various stages of their development. The approach of these studies was inspired by the in vitro clonal assays previously devised to address the developmental capacities of murine bone marrow progenitors [START_REF] Metcalf | Concise review: hematopoietic stem cells and tissue stem cells: current concepts and unanswered questions[END_REF] for references), which were instrumental for the understanding of blood cell lineage segregation. This article will consider the work that has been done over the last decades in our and others' laboratories to enlighten this issue, which included in vitro single NCC cultures and in vivo analysis of the fate of NCC individually labeled during the course of their migration from the neural folds. In these lineage experiments, the technologies used to isolate or target single cells and to identify their clonal progeny are critical; these technological issues are reviewed in the first section of this article. The problem of the interpretation of the behavior of cells in vitro versus in vivo will also be considered. The multipotency of the early NCC has been fully evidenced through this large body of work, which extended from the seminal and early contribution of [START_REF] Cohen | A clonal approach to the problem of neural crest determination[END_REF] up to now. In the last decade, it was shown that, in the various locations of the body where the migratory NCC have settled and differentiated during embryonic life, NC--derived multipotent progenitors (or stem cells) persist in adult tissues.

1-Technical considerations about in vitro analysis of NCC differentiation potentials

The NC is a fast evolving structure and important considerations concern when and how it is removed from the embryo, which procedure is used to ensure the clonality of NCC cultures, and what are the in vitro culture conditions appropriate for the growth and differentiation of individual NCC. We briefly describe here the different techniques applied for the preparation of cranial and trunk NCC in in vitro cultures.

The "classical" bi-dimensional culture method for trunk and cranial NCC

Initially devised for the quail NC by Cohen and Konisgberg (1975) and, later on by Sieber--Blum and [START_REF] Sieber--Blum | Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells[END_REF], the "classical" NC culture procedure takes advantage of the property of the NCC to undergo delamination from the neural epithelium at the onset of their emigration, a process that takes place in vitro from isolated neural tubes or neural folds. This general method is now widely used to isolate NCC from avian and rodent embryos and was recently adapted to human embryos (for a technical review, [START_REF] Etchevers | Primary culture of chick, mouse or human neural crest cells[END_REF].

At the trunk level, this technique is applied to avian (quail or chick) embryos at 2 days of incubation (E2); NCC can be recovered after a period of 15 to 24 hr of in vitro migration [START_REF] Lahav | Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Coelho--Aguiar | Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest[END_REF]. Similar methods for trunk NCC isolation were applied to E10.5 rat and E9 mouse embryos [START_REF] Stemple | Isolation of a stem cell for neurons and glia from the mammalian neural crest[END_REF]Ito et al., 1993), and, more recently, to human embryos [START_REF] Thomas | Human neural crest cells display molecular and phenotypic hallmarks of stem cells[END_REF]. At the cranial level, the NCC can be recovered by several methods, which differ essentially according to the stage considered. One is an adaptation from the classical technique described above for trunk NCC, using quail mesencephalon and rhombencephalon [START_REF] Sieber--Blum | Distribution of pluripotent neural crest cells in the embryo and the role of brain--derived neurotrophic factor in the commitment to the primary sensory neuron lineage[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF][START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF]. Another is to remove mesencephalic NCC in the course of their early migration under the superficial ectoderm (Ziller et al., 1983[START_REF] Baroffio | Clone--forming ability and differentiation potential of migratory neural crest cells[END_REF]. Finally, some authors surgically remove the cranial neural folds; in that case, the migratory NCC are produced in vitro (e.g., [START_REF] Abzhanov | Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro[END_REF][START_REF] Mcgonnell | Trunk neural crest has skeletogenic potential[END_REF][START_REF] Ito | Role of retinoic acid in mouse neural crest cell development in vitro[END_REF].

Control of clonality and culture conditions for the growth of single NCC in vitro

The in vitro clonal assays using dissociated NCC suspensions have been initially carried out either by the limit dilution method (e.g., Sieber--Blum and [START_REF] Sieber--Blum | Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells[END_REF][START_REF] Sieber--Blum | Commitment of neural crest cells to the sensory neuron lineage[END_REF], 1991;[START_REF] Sieber--Blum | Distribution of pluripotent neural crest cells in the embryo and the role of brain--derived neurotrophic factor in the commitment to the primary sensory neuron lineage[END_REF]. In our laboratory, we chose a direct cloning procedure, which consisted in selecting single cells with a micropipette, followed by their plating individually, under microscopic control, which ensured true clonal seeding of virtually all the cultures [START_REF] Baroffio | Clone--forming ability and differentiation potential of migratory neural crest cells[END_REF][START_REF] Baroffio | Common precursors for neural and mesectodermal derivatives in the cephalic neural crest[END_REF][START_REF] Dupin | Schwann-cell differentiation in clonal cultures of the neural crest, as evidenced by the anti--Schwann cell myelin protein monoclonal antibody[END_REF][START_REF] Lahav | Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF][START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF][START_REF] Coelho--Aguiar | Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest[END_REF].

Culture medium and substrate conditions have been optimized for the development of single NCC progeny in two--dimensional cultures. Our experiments were regularly performed by plating single quail NCC on a feeder--layer of growth--arrested mouse 3T3 fibroblasts, according to the procedure devised by [START_REF] Barrandon | Cell size as a determinant of the clone--forming ability of human keratinocytes[END_REF] to generate colonies from single human keratinocytes. The presence of this feeder--layer, together with a "clonal culture medium" enriched with serum, chick embryo extract and a cocktail of hormones and growth factors [START_REF] Baroffio | Clone--forming ability and differentiation potential of migratory neural crest cells[END_REF][START_REF] Baroffio | Common precursors for neural and mesectodermal derivatives in the cephalic neural crest[END_REF][START_REF] Dupin | Schwann-cell differentiation in clonal cultures of the neural crest, as evidenced by the anti--Schwann cell myelin protein monoclonal antibody[END_REF], strongly enhanced single NCC survival and proliferation, while allowing their differentiation along a large array of neural and non--neural NC lineages. However, for serial subcloning of NCC cultures, the 3T3 feeder--layer was replaced by a collagen1-coated substrate [START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF]. Collagens, fibronectin and other extracellular matrix (ECM) components were also routinely employed as a substratum for in vitro cultures of avian and mammalian adherent NCC (reviewed by [START_REF] Dupin | Neural crest progenitors and stem cells: from early development to adulthood[END_REF][START_REF] Dupin | Isolation and differentiation properties of neural crest stem cells[END_REF].

The new "crestosphere" procedure: growing early NCC in tri-dimensional cultures

Recently, Keruoso et al. (2015) devised a method for the in vitro long--term growth of early neuroepithelial NCC in free--floating spheres, the so--called "crestospheres". Sphere-forming assays have been previously instrumental to identify stem cells in many organs, since the initial report of adult brain--derived neurospheres [START_REF] Reynolds | Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[END_REF] for a review, [START_REF] Pastrana | Eyes wide open: a critical review of sphere--formation as an assay for stem cells[END_REF]. Keruoso et al. (2015) implemented such assays to the early NCC, obtained directly from the chick embryo by dissociation of the cranial dorsal neural tube cells and after in vitro derivation from human embryonic stem (ES) cells. On non--adhesive substrate, spherical clusters formed from single NCC and could be serially replated, allowing long--term propagation of premigratory NCC that retained expression of early NC genes and self--renewal ability. When transferred to adhesion-and differentiation--promoting conditions, cells of the "crestospheres" underwent EMT while migrating onto the substratum and they differentiated into neural, melanocytic, myofibroblastic and skeletogenic phenotypes. This clonal sphere assay thus provided evidence of cranial NCC multipotency at single cell resolution (Keruoso et al., 2015). The "crestospheres" therefore represent a valuable tool for further investigations on the regulation of NC stem cell properties, as recently exemplified by the role of c-myc in controlling the size of the multipotent NCC pool (Keruoso et al., 2016).

Identification of NC-derived phenotypes in vitro

In the pioneer work by Cohen and Konisgberg (1975), the analysis of single NCC progeny relied only upon the microscopic detection of melanin, the natural marker of pigment cells, which led to the first evidence for bipotent NCC, generating both pigmented and unpigmented cells. Later on, the characterization of catecholaminergic cells within subsets of NCC colonies (Sieber--Blum and [START_REF] Sieber--Blum | Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells[END_REF], paved the way towards a broader analysis of NC--derived phenotypes with cell type--specific markers.

From the late 1980ies, the production of monoclonal antibodies (Mab) directed against antigens expressed by NCC and NC derivatives, has provided an increasing number of tools to identify cell types in the descent of isolated NCC.

One of the main technical issues in order to decipher the developmental repertoire of individual NCC was the choice and combination of markers, technically compatible for the analysis of a large diversity of phenotypes in each NC colony. With the aim of investigating the main neural and mesenchymal NC lineages, we examined as many as six distinct phenotypes in quail NC--derived clones [START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF]; See next section).

The cells of the glial and pigment cell lineages were labeled using Mabs against the Schwann cell Myelin Protein (SMP) [START_REF] Dulac | A surface protein expressed by avian myelinating and nonmyelinating Schwann cells but not by satellite or enteric glial cells[END_REF] and the MelEM [START_REF] Nataf | Novel markers of melanocyte differentiation in the avian embryo[END_REF] antigens respectively, whereas immunodetection of neurofilament proteins, β3-tubulin, and tyrosine hydroxylase, a marker for catecholaminergic cells identified neuronal cells. Immunoreactivity to α--smooth muscle actin (αSMA) defined smooth muscle/myofibroblastic cells. Chondrocytes and adipocytes were evidenced based on several morphological criteria [START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF][START_REF] Billon | The generation of adipocytes by the neural crest[END_REF]Coelho--Aguiar et al., 2013) and bone cell identification relied on the expression of Runx2, a master gene of osteogenesis [START_REF] Ducy | Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation[END_REF][START_REF] Otto | Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development[END_REF], detected by in situ hybridization [START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF][START_REF] Coelho--Aguiar | Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest[END_REF]. We also used the HNK1/NC1 Mab to detect NCC subsets that did not express the aforementioned lineage markers. HNK1 is considered as a reliable marker of a large subset of migratory NCC in several, including avian, rat (not mouse) and zebrafish species. In the avian embryo, HNK1 labels the majority of undifferentiated migratory NCC and, later on, is retained in the peripheral nervous system (PNS) but lost in melanocytes and mesenchymal NC derivatives [START_REF] Vincent | A cell surface determinant expressed early on migrating avian neural crest cells[END_REF][START_REF] Tucker | Identical reactivity of monoclonal antibodies HNK--1 and NC--1: conservation in vertebrates on cells derived from the neural primordium and on some leukocytes[END_REF].

2-Individual cranial NCC give rise to both mesenchymal/ skeletogenic and neural phenotypes in vitro

Transplantation experiments in avian embryos based on the quail--chick chimera system, and, more recently, genetic fate mapping in the mouse, have shown that ectomesenchymal derivatives of the cranial NC in amniotes include chondrocytes and bone cells, smooth muscle cells and pericytes, tendon, dermal and other connective tissue cells, adipocytes, stromal and endothelial cells of the cornea, and, in mammals, odontoblasts (for references, Le [START_REF] Le Douarin | The neural crest, 2nd ed[END_REF][START_REF] Minoux | Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development[END_REF][START_REF] Le Douarin | The neural crest[END_REF]Dupin, 2012, 2016). The problem was thus raised as to whether, at the time they start migrating, the NCC of the cephalic area were capable to yield a large array of different cell types (neural cells, melanocytes and mesenchymal cells), or if they could comprise mesenchymal restricted progenitors, distinct from those yielding the neural-melanocytic derivatives.

In vitro clonal analysis of quail cephalic NCC to tackle the cellular origin of neural, melanocytic, myofibroblastic and chondrocytic cells

In vitro clonal cultures experiments carried out by ours and Maya Sieber--Blum's laboratories first showed very diverse proliferation and differentiation potentials in cephalic quail NCC during the early migratory phase [START_REF] Baroffio | Clone--forming ability and differentiation potential of migratory neural crest cells[END_REF][START_REF] Baroffio | Common precursors for neural and mesectodermal derivatives in the cephalic neural crest[END_REF][START_REF] Dupin | Schwann-cell differentiation in clonal cultures of the neural crest, as evidenced by the anti--Schwann cell myelin protein monoclonal antibody[END_REF][START_REF] Ito | In vitro clonal analysis of quail cardiac neural crest development[END_REF][START_REF] Sieber--Blum | Distribution of pluripotent neural crest cells in the embryo and the role of brain--derived neurotrophic factor in the commitment to the primary sensory neuron lineage[END_REF][START_REF] Sieber--Blum | In vitro clonal analysis of cardiac outflow tract mesenchyme[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF]. Initial investigations on mesencephalic migratory NCC isolated at 10--somite--stage (10ss), led us to identify multipotent progenitors for Schwann cells, adrenergic neurons, myofibroblasts and/or melanocytes. Noticeably, 2.5% of 842 clone--forming NCC produced both chondrocytes and glial cells, together with neurons, melanocytes and/or myofibroblasts [START_REF] Baroffio | Clone--forming ability and differentiation potential of migratory neural crest cells[END_REF][START_REF] Baroffio | Common precursors for neural and mesectodermal derivatives in the cephalic neural crest[END_REF][START_REF] Dupin | Schwann-cell differentiation in clonal cultures of the neural crest, as evidenced by the anti--Schwann cell myelin protein monoclonal antibody[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF]. Moreover, by in vitro clonal assays, [START_REF] Ito | In vitro clonal analysis of quail cardiac neural crest development[END_REF] found that 17% (13/74) of rhombencephalic NCC could generate chondrocytes together with sensory neurons and/or pigment cells.

At that time, culture conditions and technical limitations to the analysis of multiple phenotypes in the colonies did not permit a more comprehensive characterization of mesenchymal NC progenitors, particularly those forming bones.

More efficient production of skeletogenic cell types and terminal differentiation of cartilage and bone cells were obtained in vitro from cephalic quail NCC isolated after 15 to 17hr of migration from the explanted mesencephalon primordium [START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF][START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF][START_REF] Dupin | The cephalic neural crest of amniote vertebrates is composed of a large majority of precursors endowed with neural, melanocytic, chondrogenic and osteogenic potentialities[END_REF]. Using these settings, [START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF] found that, after 10 days of clonal culture, 14.5% of the cephalic NCC yielded chondrocytes together with neural and/or melanocytic cells. In particular, 6.6% of the clones were derived from a highly multipotent type of NCC, which differentiated into glia (G), neurons (N), melanocytes (M), myofibroblasts (F) and chondrocytes (C) (i.e., GNMFC progenitor).

Furthermore, NCC were responsive to Sonic Hedgehog (Shh), a morphogen previously shown to be crucial for cranial NCC survival and formation of the craniofacial skeleton [START_REF] Chiang | Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function[END_REF]Ahlgren and Bronner--Fraser, 1999;[START_REF] Cordero | Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes[END_REF][START_REF] Jeong | Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia[END_REF][START_REF] Brito | An early role for sonic hedgehog from foregut endoderm in jaw development: ensuring neural crest cell survival[END_REF][START_REF] Brito | Induction of mirror--image supernumerary jaws in chicken mandibular mesenchyme by Sonic Hedgehog--producing cells[END_REF]. In the presence of Shh, chondrocytes--containing clones showed a three--fold increase in frequency and were all derived from multipotent (i.e., at least, bipotent) NCC founders (43% and 14.5% of treated and untreated clones, respectively). Thus, the proportion of GNMFC progenitors reached 18.5% of clone-forming NCC after exposure to Shh. Therefore, neural--melanocytic and chondrocytic NC phenotypes arise from common progenitors that are highly frequent in the cranial NCC isolated at early migratory stages [START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF][START_REF] Le Douarin | The stem cells of the neural crest[END_REF].

In these experiments, the colonies contained from about a few hundred to more than ten thousands of cells in both Shh--treated and untreated conditions. By scoring the total number of cells in the phenotypically distinct categories of clones, we found that the extent of NCC potentialities did not correlate with the total cell number in their progeny (Calloni et al., 2007 and unpublished data). For example, the total number of cells in the clones arising from highest multipotent GNMFC progenitors varied (Figure 1). Likewise, large--sized colonies (of about 5,000 to 20,000 cells) originated from tetra-- and quinta-potent progenitors as well as from more restricted NCC yielding neural and melanocytic cells only (e.g., GNM and GM). These findings indicate that acquisition of multiple fates in the progeny of single NCC is not dependent upon extensive proliferative activity.

In vitro clonal analysis allows to uncovering the developmental potentials of a cell a posteriori, through the phenotypic composition of its progeny, but one drawback of this approach is that the identity of distinct founders remains unknown. In attempt to further define different subsets of cephalic NCC, we took advantage of the HNK1 Mab, which recognizes a surface carbohydrate epitope expressed by the majority, but not all, of early migratory NCC [START_REF] Vincent | A cell surface determinant expressed early on migrating avian neural crest cells[END_REF][START_REF] Tucker | Identical reactivity of monoclonal antibodies HNK--1 and NC--1: conservation in vertebrates on cells derived from the neural primordium and on some leukocytes[END_REF]. At later stages, this marker is not expressed in melanocytic and ectomesenchymal lineages while present in most PNS cells. Hence, the developmental potentials of the early NCC that express HNK1 remained elusive, particularly regarding their capacity to yield mesenchymal derivatives.

In order to know whether HNK1--expressing NCC possess either a restricted or a full array of the differentiation capacities of the NC, we isolated mesencephalic quail NCC as described above, and we immunolabeled them with HNK1. The HNK1+ cells were then selected under the control of fluorescence microscopy for clonal plating in the presence of Shh (Calloni et al., 2007 and unpublished data). After 10 days, 45% of the plated HNK1+ cells had generated a clone. Of these clones, 27.5% contained glial, neuronal, melanocytic, myofibroblastic and chondrocytic cell types, therefore retrospectively identifying highly multipotent GNMFC progenitors (Figure 2). The remaining colonies lacked chondrocytes but contained glia, neurons, melanocytes and myofibroblasts in various combinations. Although preliminary, these data show that cephalic NCC bearing the HNK1 marker comprised a subset of multipotent progenitors capable to produce chondrocytes together with neural--melanocytic cells.

In vitro analysis of the osteogenic potential of the cephalic NCC

Using Runx2 transcription factor as an early bone--specific marker [START_REF] Ducy | Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation[END_REF][START_REF] Komori | Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts[END_REF][START_REF] Karsenty | Genetic control of bone formation[END_REF], we have further investigated the osteogenic potential of cephalic quail NCC in vitro [START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF]. We found that, after 5 days of in vitro culture, numerous NCC start to express Runx2 mRNA. Later on, large clusters of mineralizing bone cells differentiated in these cultures [START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF]. When grown as single cells, cephalic NCC gave rise to variable combinations of the six distinct recorded phenotypes, i.e., glial cells (G), neurons (N), melanocytes (M), myofibroblasts (F), chondrocytes (C) and osteoblasts (O). To our surprise, more than 90% of the clones contained both osteogenic and neural and or melanocytic cell types, and we reported the first evidence of common progenitors for the six cell types under scrutiny (i.e., GNMFCO progenitors) [START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF][START_REF] Dupin | The cephalic neural crest of amniote vertebrates is composed of a large majority of precursors endowed with neural, melanocytic, chondrogenic and osteogenic potentialities[END_REF] (Figure 3). Treatment with Shh resulted in a higher proportion of the clones generated by hexa--and pentapotent progenitors (51.5% versus 23% in controls). While the vast majority of colonies contained osteoblasts but no chondrocytes in untreated NCC cultures, the frequency of osteo--chondrogenic progenitors increased by three--fold in the presence of Shh.

Therefore, the skeletogenic cells derived from the cephalic NC, which rely upon Sox9 gene activity for development in the mouse [START_REF] Mori--Akiyama | Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest[END_REF], originated in vitro from Shh--responsive, highly multipotent progenitors.

In sum, the analyses of single NCC fate in vitro have revealed that large subsets of the early cephalic NCC are progenitors for both mesenchymal (including skeletogenic) and neural--melanocytic NC lineages. By providing an unequivocal demonstration of the origin of head chondrogenic and osteogenic cells from multipotent NCC, these findings further underline the broad multipotency of individual NCC, which thus are endowed with the striking ability to differentiate into both ectodermal and mesodermal lineages.

A recent work reporting the generation and propagation of clonal spheres ("crestospheres") from chicken and human ES cell--derived NCC, further strengthens these conclusions by showing that premigratory cranial NCC are stem cells that exhibit self--renewal and can produce both mesenchymal and neural cell types (Keruoso et al., 2015). Together, these data obtained at single cell resolution contradict the contention that "cranial skeletal mesenchyme and other mesectodermal derivatives originate evolutionarily and developmentally from a distinct epithelial domain of the neural fold, the Metablast, rather than from the Neural crest" [START_REF] Weston | Pentimento: Neural Crest and the origin of mesectoderm[END_REF].

Nevertheless, a common cellular origin of mesectoderm and neural--melanocytic derivatives at the single NCC level does not preclude some heterogeneity in the neural folds. Along this line, distinct subdomains were recently evidenced in the chick midbrain NC regarding multiplex gene expression [START_REF] Lignell | Identification of a neural crest stem cell niche by Spatial Genomic Analysis[END_REF], providing novel insights into the gene regulatory networks underlying early NCC heterogeneity.

3) Trunk NCC progenitors are multipotent and harbor dormant mesenchymal potentials

As opposed to cephalic NCC, the in vivo differentiation repertoire of trunk NCC in amniote vertebrates was so far considered to be limited to PNS neuronal and glial cells, melanocytes and adrenomedullary cells, until mouse transgenesis experiments by [START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF] led to the discovery that the NC, via Schwann cell precursors along the sciatic nerve, gave rise to a subset of endoneurial fibroblasts [START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF].

Using in vitro clonal cell culture approaches, we have shown that avian trunk NCC comprise various subsets of multipotent progenitors, including those endowed with the capacity to clonally produce glia (G), neurons (N), melanocytes (M) and myofibroblasts (F) (i.e., GNMF progenitors), as well as more restricted NCC, such as glial--melanocytic (GM) and glial--myofibroblastic (GF) progenitors [START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF]. These bipotent GM and GF progenitors exhibited self--renewal capacity after serial in vitro subcloning in response to endothelin--3 (EDN3) and FGF2, respectively [START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Bittencourt | Fibroblast growth factor 2 promotes the self--renewal of bipotent glial smooth muscle neural crest progenitors[END_REF]. The in vitro culture methods of avian NCC adapted to mammalian cells likewise led to identify multipotent progenitors in the mouse and rat trunk NC [START_REF] Ito | In vitro clonal analysis of mouse neural crest development[END_REF][START_REF] Stemple | Isolation of a stem cell for neurons and glia from the mammalian neural crest[END_REF][START_REF] Shah | Glial growth factor restricts mammalian neural crest stem cells to a glial fate[END_REF][START_REF] Shah | Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members[END_REF][START_REF] Paratore | Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling[END_REF]. For the first time, [START_REF] Stemple | Isolation of a stem cell for neurons and glia from the mammalian neural crest[END_REF] isolated precursors from the rat NC through their expression of p75 neurotrophin receptor, which gave rise to autonomic neurons, glial cells and myofibroblasts and exhibited self--renewal in vitro, thus deserving to be considered as NC stem cells. Futhermore, Anderson and co--workers identified NC stem cells of similar developmental capacities in the fetal rat sciatic nerve [START_REF] Morrison | Prospective identification, isolation by flow cytometry, and in vivo self--renewal of multipotent mammalian neural crest stem cells[END_REF]. Several signaling pathways are at work to regulate the differentiation choice of these mammalian NC stem cells: neuregulin--1 and Delta-mediated Notch receptor activation triggered glial fate [START_REF] Shah | Glial growth factor restricts mammalian neural crest stem cells to a glial fate[END_REF][START_REF] Morrison | Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells[END_REF], while BMP signaling promoted autonomic neuronal outcome at the expense of glial fate, and TGFβ drove NCC to adopt a myofibroblastic phenotype [START_REF] Shah | Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members[END_REF].

In the mouse, Sommer and colleagues found that a combination of Wnt and BMP factors regulates the maintenance of multipotent NC stem cells [START_REF] Kleber | Neural crest stem cell maintenance by combinatorial Wnt and BMP signaling[END_REF]. In the absence of BMP, Wnt/β--catenin signaling exerts a temporal control on NCC fate: at premigratory stages, by promoting sensory neuron generation; and when activated later on, in migratory NCC, by triggering melanocyte production (Lee et al., 2004;[START_REF] Hari | Lineage--specific requirements of ß--catenin in neural crest development[END_REF][START_REF] Hari | Temporal control of neural crest lineage generation by Wnt/beta--catenin signaling[END_REF].

As already mentioned, the presence of myofibroblasts/smooth muscle cells was recurrently recorded in avian and mammalian trunk NCC in vitro progeny. In the recent years, it became clear that, besides myofibroblasts, trunk NCC in amniote vertebrates do have the capacity to give rise to diverse mesenchymal phenotypes, including skeletal cells. In appropriate culture conditions, avian trunk NCC differentiated into adipocytes [START_REF] Billon | The generation of adipocytes by the neural crest[END_REF][START_REF] Coelho--Aguiar | Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest[END_REF] and chondrocytes [START_REF] Mcgonnell | Trunk neural crest has skeletogenic potential[END_REF][START_REF] Abzhanov | Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro[END_REF][START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF][START_REF] Ramos--Hryb | Matrigel supports neural, melanocytic and chondrogenic differentiation of trunk neural crest cells[END_REF]. As observed for the cephalic NCC, trunk NCC exposed in vitro to Shh exhibited markedly increased chondrogenesis, and we could record a rare type of multipotent trunk NCC yielding glial cells, myofibroblasts and chondrocytes [START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF]Le Douarin et al., 2008).

Recently, we further investigated the ability of trunk NCC to differentiate into bone and fat cells [START_REF] Coelho--Aguiar | Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest[END_REF]. When using a cocktail of hormones and growth factors that enhanced terminal differentiation of adipogenic and osteogenic cells, quail trunk NCC isolated after 15--18 hr of migration from explanted neural tubes, exhibited a high ability to differentiate in vitro into chondrocytes, lipid--laden adipocytes and mineralizing osteoblasts (Figure 4 These data suggest that most of the trunk NCC, which adopt neural and melanocytic phenotypes in vivo, possess dormant potentials to differentiate into connective, osteogenic and adipogenic cells. Together with several reports that mammalian trunk NCC also displayed a limited ability to differentiate in vitro into skeletogenic cells [START_REF] Ido | Expression of chondrogenic potential of mouse trunk neural crest cells by FGF2 treatment[END_REF][START_REF] John | Transforming growth factor beta-mediated Sox10 suppression controls mesenchymal progenitor generation in neural crest stem cells[END_REF], these findings therefore show that, albeit reduced compared to cranial NCC, mesenchymal potentials are indeed present in the trunk NC.

The reason for which these potentialities are not expressed in vivo remains unknown.

4) Evolutionary considerations on trunk versus cephalic NCC potentials

Taken together, the in vitro clonal assays have shown that, in both cephalic and trunk NCC, the mesenchymal phenotypes and the neural--melanocytic ones arise from common progenitors. In an evolutionary perspective, one attractive possibility is that such multipotent neural--melanocytic--mesenchymal progenitors, as recorded in the avian NC, might represent a primitive type of NCC. In early vertebrates, like extinct jawless fishes (Ostracoderms) of the Ordovician, a superficial armor of calcified dermal bone containing dentine covered the head and trunk. Since dentine is synthesized by NCC in all extant vertebrates (i.e., by the NC--derived odontoblasts in mammals), it has been assumed that both cranial and trunk skeleton of the early vertebrates originated from the NC [START_REF] Janvier | Early vertebrates[END_REF][START_REF] Smith | Development and evolutionary origins of vertebrate skeletogenic and odontogenic tissues[END_REF][START_REF] Smith | Putative skeletal neural crest cells in early late ordovician vertebrates from Colorado[END_REF]. During evolution, this superficial skeleton regressed in the trunk while substituted for by an internal endochondral vertebral skeleton of mesoderm origin. The dermal skeleton in the distal part of the rays in dorsal and caudal fins of teleosts might be a remnant of the primitive superficial skeleton of extinct vertebrates, although its origin, from either the trunk NC or the somitic mesoderm in zebrafish is still debated [START_REF] Smith | Trunk neural crest origin of caudal fin mesenchyme in the zebrafish Brachydanio rerio[END_REF][START_REF] Kague | Skeletogenic fate of zebrafish cranial and trunk neural crest[END_REF]Lee et al., 2013a,b). In higher vertebrates, the NC skeletogenic capacity has been retained in the craniofacial region, whereas it vanished, but did not completely disappear in the trunk since it can be evidenced in appropriate in vitro culture conditions.

Much unknowns remain about the mechanisms and molecular determinants which underlie the differential expression of mesenchymal potencies between the cephalic and trunk NC. However, the expression domains of the genes belonging to the Hox clusters, which have the capacity to pattern the body of all bilateria, are remarkable in this respect: Hox genes are not expressed in the area where the vertebrate head skeleton develops [START_REF] Couly | Determination of the identity of the derivatives of the cephalic neural crest: incompatibility between Hox gene expression and lower jaw development[END_REF] and, more strikingly, their ectopic expression in the cephalic region completely inhibits the development of the head skeleton [START_REF] Grammatopoulos | Homeotic transformation of branchial arch identity after Hoxa2 overexpression[END_REF][START_REF] Couly | Interactions between Hox--negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head[END_REF][START_REF] Creuzet | Negative effect of Hox gene expression on the development of the neural crest--derived facial skeleton[END_REF]. In the mouse, similar severe defects of craniofacial structures resulted from conditional deletion in the NC of ezh2, which encodes a subunit of the Polycomb Repressive Complex 2 (PRC2) that catalyzes trimethylation of histone H3 at Lys27 (Schwarz et al., 2014). The craniofacial phenotype of ezh2 mutant embryos was correlated to de--repression of Hox genes in the cephalic but not the trunk NC, suggesting that epigenetic regulation of Hox genes controls head NC mesenchymal fate [START_REF] Schwarz | Ezh2 is required for neural crest--derived cartilage and bone formation[END_REF].

The role of intrinsic determinants in NC mesenchymal fate was further highlighted in a recent work, showing that a defined set of transcription factors (TFAP2b, Sox8 and Ets1) acts positively to establish the "cephalic character" of the NC (Simoes--Costa and Bronner, 2016). Expression of this set of genes was found to be enriched in the transcriptome of cranial versus trunk avian NCC (Simoes--Costa et al., 2014), while these two NCC populations were defined by, and isolated through, activation of distinct Sox10 enhancers at the onset of migration [START_REF] Betancur | Genomic code for Sox10 activation reveals a key regulatory enhancer for cranial neural crest[END_REF]. If co--electroporated in ovo at early stages of NC formation, TFAP2b, Sox8 and Ets1 conferred a cephalic NC--type identity to the trunk NCC, at least regarding their transcriptomic profile and their capacity to produce chondrocytes after in vitro culture. Nevertheless, head mesenchymal tissue formation, which represents assessment of a complete trunk to cranial--like NCC conversion, was reported after grafting the reprogrammed trunk NCC in a cranial, favorable, location but was not achieved in situ in the trunk (Simoes--Costa and Bronner, 2016). Therefore, specific transcription factors act as positive regulators of the cranial NC fate and, together with yet undetermined environmental cues, can promote acquisition of cephalic NC features by the trunk NCC.

5) Multipotency versus restrictions of the early NCC in vivo

First attempts to tackle the pivotal question of NCC commitment and multipotency in vivo was reached through the development of methods to mark single premigratory NCC with a fluorescent intracellular dye allowing to trace their fate after migration in early embryos. Following the microinjection of individual cells in the dorsal neural tube of the chicken embryo with a lipophilic fluorescent dye, Bronner-- [START_REF] Bronner--Fraser | Cell lineage analysis reveals multipotency of some avian neural crest cells[END_REF] provided the first sound evidence of NCC multipotency in vivo. After 2 days of migration, about half of the labeled trunk NCC produced descendent cells that populated more than one NC derivative (e.g. skin, DRG, sympathetic ganglia, peripheral nerves and adrenal medulla) and adopted distinct fates, including glial cells, sympathetic and sensory neurons, melanocytes and adrenomedullary cells (Bronner--Fraser [START_REF] Bronner--Fraser | Cell lineage analysis reveals multipotency of some avian neural crest cells[END_REF][START_REF] Bronner--Fraser | Developmental potential of avian trunk neural crest cells in situ[END_REF]. By similar experiments, they showed multipotency of subsets of individual trunk NCC in Xenopus [START_REF] Collazo | Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration[END_REF] and mouse [START_REF] Serbedzija | Developmental potential of trunk neural crest cells in the mouse[END_REF]. These pioneer studies have revealed the heterogeneity of early NCC with respect to their developmental fate and uncovered the presence of multipotent as well as more restricted NCC in vivo before the onset of migration.

However, in zebrafish embryos, the labeling of individual premigratory cranial and trunk NCC, in most cases, gave rise to a lineage--restricted progeny homing to single locations (DRG, sympathetic ganglia, ventral root nerves and skin), suggesting earlier NCC fate restrictions in teleosts than in higher vertebrates [START_REF] Raible | Restriction of neural crest cell fate in the trunk of the embryonic zebrafish[END_REF][START_REF] Schilling | Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo[END_REF]. The hypothesis of an early determination of the chick NCC was put forwards recently by Krispin et al. (2010a,b), after microinjection of a fluorescent dye or discrete GFP plasmid electroporation into small numbers of trunk NCC, before their emigration from the dorsal midline. These experiments lend support to a model in which NCC fate could be predicted prior to delamination, depending on their relative dorso--ventral position in the neural tube and their temporal order of exit from the neural tube; accordingly, the progeny of NCC labeled at an early emigration stage was restricted to sympathetic ganglia, whereas at the same level of the neural axis, later--emigrating NCC populated exclusively more dorsal derivatives and sequentially produced DRG cells and melanocytes (Krispin et al., 2010a, b).

It is presently unclear whether discrepancies between these data and the earlier in vivo experiments showing chick NCC multipotency (Bronner--Fraser [START_REF] Bronner--Fraser | Cell lineage analysis reveals multipotency of some avian neural crest cells[END_REF][START_REF] Bronner--Fraser | Developmental potential of avian trunk neural crest cells in situ[END_REF][START_REF] Fraser | Migrating neural crest cells in the trunk of the avian embryo are multipotent[END_REF] are due to temporal and/or technical differences in single NCC labeling procedures. Recently, [START_REF] Mckinney | Evidence for dynamic rearrangements but lack of fate or position restrictions in premigratory avian trunk neural crest[END_REF] further explored the dynamics of NCC migratory behavior using targeted photoconversion in single cells of the dorsal neural tube and time--lapse imaging in the chicken embryo in vivo and in slice cultures. In contrast to Krispin et al. (2010a,b) who found that NCC emigration was limited to the dorsal midline, they observed that NCC exited in a stochastic manner from the 25% dorsal most region of the neural tube. Moreover, except for the first NCC to emigrate, which preferentially populated the sympathetic ganglia, the remaining, later-emigrating NCC exhibited an unfixed fate, showing similar ability to contribute multiple NC derivatives, in the ventral root nerve, the DRG and the melanocytic subectodermal pathway [START_REF] Mckinney | Evidence for dynamic rearrangements but lack of fate or position restrictions in premigratory avian trunk neural crest[END_REF].

The precise time and pattern of NCC diversification remain ill defined and vary according to the diverse NC sublineages. Yet altogether, the analyses of NCC fate in vivo have pointed to the presence of distinct progenitors in the early NC population, comprising several types of multipotent cells together with NCC generating a more limited repertoire of cell types. This is in agreement with the results obtained by in vitro clonal NC cultures. Some subpopulations of avian NCC can undergo lineage specification very early, at the onset of migration or a few hours after exiting the neuroepithelium, as observed in chick neural tube explant cultures [START_REF] Henion | Timing and pattern of cell fate restrictions in the neural crest lineage[END_REF]. Early fate restriction of NCC was documented particularly for the sensory neuronal phenotype. At the neural fold stage or soon after the onset of migration, the avian cranial and trunk NC comprises a set of apparently committed precursors that can readily differentiate in vitro into sensory neurons (Ziller et al., 1983(Ziller et al., , 1987;;[START_REF] Bader | Development of electrical membrane properties in cultured avian neural crest[END_REF]. In transgenic mice, expression of definite marker genes (eg., neurogenin2, GDF7 and neuropilin2) in premigratory NCC subpopulations could be linked to a predictable sensory fate (Zirlinger et al., 2002;[START_REF] Lo | Late--emigrating neural crest cells in the roof plate are restricted to a sensory fate by GDF7[END_REF][START_REF] Lumb | Neuropilins define distinct populations of neural crest cells[END_REF]. However, it should be emphasized that, amongst the large diversity of sensory neuron subtypes, most of them arise from multipotent NCC (for references, [START_REF] Marmigere | Specification and connectivity of neuronal subtypes in the sensory lineage[END_REF], as shown by in vitro and in vivo single cell studies [START_REF] Sieber--Blum | Commitment of neural crest cells to the sensory neuron lineage[END_REF]Bronner--Fraser andFraser, 1988, 1989;[START_REF] Frank | Lineage of neurons and glia in chick dorsal root ganglia: analysis in vivo with a recombinant retrovirus[END_REF][START_REF] Kleber | Neural crest stem cell maintenance by combinatorial Wnt and BMP signaling[END_REF]. Collectively, these data illustrate the complex pattern whereby sequential waves of cell specification occur in the NCC, hence the challenging task to dissect how diversity of developmental lineages is created in the heterogenous population of the early NCC.

Providing a way to circumvent the inherent technical drawbacks of intracellular tracer injections or electroporation into single cells in vivo, novel genetic fate mapping approaches have been devised to trigger heritable multicolor labeling of many clones in the intact embryo [START_REF] Livet | Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system[END_REF][START_REF] Snippert | Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells[END_REF][START_REF] Loulier | Multiplex cell and lineage tracking with combinatorial labels[END_REF][START_REF] Weissman | Brainbow: new resources and emerging biological applications for multicolor genetic labeling and analysis[END_REF]. In this respect, [START_REF] Baggiolini | Premigratory and migratory neural crest cells are multipotent in vivo[END_REF] have used the R26R-Confetti reporter combined with inducible Wnt1-Cre and Sox10-Cre mouse lines to drive multicolor reporter expression in single premigratory and early migratory trunk NCC, respectively. Quantitative phenotypic analysis of the labeled clones showed that, both before and during migration, the vast majority of trunk murine NCC are multipotent [START_REF] Baggiolini | Premigratory and migratory neural crest cells are multipotent in vivo[END_REF]. Meanwhile, several types of clones were also recorded, which attested to the presence of distinct multipotent progenitors in the mouse trunk NC, in accordance with the results from in vitro clonal cultures of avian NCC [START_REF] Sieber--Blum | Role of the neurotrophic factors BDNF and NGF in the commitment of pluripotent neural crest cells[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF][START_REF] Dupin | Neural crest progenitors and stem cells: from early development to adulthood[END_REF]. Adameyko and colleagues (2009) have discovered an unexpected important role of Schwann cell precursors in the development of pigment cells. In the course of Schwann cell differentiation, "Schwann cell precursors", corresponding to an intermediate early step between NCC and "immature Schwann cells", are defined in mammalian peripheral nerves as Sox10+ NCC located along developing nerves, which strictly depend for survival on nerve--derived factor neuregulin1, and which express early Schwann cell lineage markers (Dhh, PLP, P0 Protein) [START_REF] Jessen | Schwann Cells: Development and Role in Nerve Repair[END_REF]. In chicken and mouse embryos, significant subsets of Schwann cell precursors were found to detach from the axons of peripheral nerves growing to the skin, lose glial traits while acquiring expression of the melanocytic lineage master gene MITF, then migrate to the skin and eventually differentiate into pigment cells [START_REF] Adameyko | Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin[END_REF][START_REF] Adameyko | Sox2 and Mitf cross--regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest[END_REF][START_REF] Ernfors | Cellular origin and developmental mechanisms during the formation of skin melanocytes[END_REF]. This glial to melanocytic conversion was restricted to Schwann cell precursors and could occur at later stages of Schwann cell development only in experimental contexts of disrupted association between axons and Schwann cells like after nerve transection. The production of melanocytes from nerve--adjacent cells required downregulation of the expression of Foxd3, a transcription factor involved in maintenance of NCC multipotency and inhibition of melanogenesis [START_REF] Mundell | Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates[END_REF][START_REF] Thomas | FOXD3 regulates the lineage switch between neural crest--derived glial cells and pigment cells by repressing MITF through a non--canonical mechanism[END_REF]Nitzan et al., 2013a,b).

6) Lineage promiscuity of Schwann cells and melanocytes

Besides the melanocytes that first differentiate from NCC that migrate dorso--laterally from the neural primordium to the skin, this second later source of pigment cells was estimated to contribute at least 50% of whole pigmentation in adult mouse hair follicles [START_REF] Adameyko | Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin[END_REF]. Of note, a similar strategy of the deployment of nerve-associated NC progenitors seems to operate for the generation of colored stripes in the adult zebrafish [START_REF] Singh | Pigment Cell Progenitors in Zebrafish Remain Multipotent through Metamorphosis[END_REF].

The propensity by cells of the Schwann cell lineage to generate melanocytes was previously evidenced in vitro in Schwann cells isolated from embryonic quail sciatic nerves. In the presence of EDN3, a cytokine expressed in the embryonic skin, Schwann cells proliferated and reverted to early NCC--like multipotent progenitors that clonally produced melanocytes and myofibroblasts in addition to glial cells [START_REF] Dupin | Reversal of developmental restrictions in neural crest lineages: transition from Schwann cells to glial--melanocytic precursors in vitro[END_REF]Real et al., 2005) (Figure 5). Schwann cells isolated from adult rat palatal ridges also behaved as multipotent NC--like cells in neurosphere cultures [START_REF] Widera | Schwann cells can be reprogrammed to multipotency by culture[END_REF]. Strikingly, EDN3, which is highly mitogenic for avian melanocytic cells [START_REF] Lahav | Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture[END_REF][START_REF] Lahav | Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro[END_REF], triggered the reverse in vitro conversion, from pigmented melanocytes to Schwann cells [START_REF] Dupin | Endothelin 3 induces the reversion of melanocytes to glia through a neural crest--derived glial--melanocytic progenitor[END_REF]Real et al., 2006). When analyzed in single pigment cell cultures, this conversion took place by the proliferation and dedifferentiation of pigment cells into oligopotent, self--renewing NC--like stem cells, such as bipotent glial-melanocytic ones, which were previously identified in the early avian NC [START_REF] Lahav | Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro[END_REF][START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF]Real et al., 2006) (Figure 5). The capacity of producing multipotent NCC in vitro, was also recently highlighted in rodent melanoblasts cocultured with stromal cells [START_REF] Motohashi | Unexpected multipotency of melanoblasts isolated from murine skin[END_REF][START_REF] Motohashi | Neural crest cells retain their capability for multipotential differentiation even after lineage--restricted stages[END_REF][START_REF] Kunisada | The stemness of neural crest cells and their derivatives[END_REF] Therefore, peripheral glial cells and melanocytes have a common direct ancestor in the NCC hierarchy and are highly prone to dedifferentiating and recapitulating an early multipotent NC state.

7) Pre-glial NCC as a mean for the spreading of NC-like stem cells at postmigratory and adult stages

The first evidence that plasticity is retained at NCC postmigratory stages was provided by back--transplantations of PNS ganglia into the early NC migration path in quail--chick chimeras (for references, Le Douarin and Kalcheim, 1999). One of the most striking findings was that embryonic quail sensory ganglia contain precursors able to give rise to sympathetic neurons and glial cells. Such precursors belong to the non--neuronal lineage of PNS ganglia, as shown unequivocally by tracing the fate of transplanted chimeric nodose sensory ganglia, in which either only the placode--derived neurons or the NC-derived glial cells harbored the quail marker (Ayer--Le Lièvre and Le Douarin, 1982).

In the last two decades, genetic fate mapping studies have revealed that virtually any tissues and organs of the fetal and adult mice harbor NC--derived cells, which exhibit multipotency and stem cell properties when isolated in vitro (for references, [START_REF] Dupin | Neural crest progenitors and stem cells: from early development to adulthood[END_REF][START_REF] Dupin | Isolation and differentiation properties of neural crest stem cells[END_REF]. Adult sources of these NC stem cells encompass NC derivatives (e.g., peripheral nerves, DRG, enteric ganglia, corneal stroma and dental pulp) as well as tissues that are not of NC origin but contain NC--derived cells, such as bone marrow and hair follicles [START_REF] Sieber--Blum | Pluripotent neural crest stem cells in the adult hair follicle[END_REF][START_REF] Fernandes | A dermal niche for multipotent adult skin--derived precursor cells[END_REF]Wong et al., 2006;[START_REF] Nagoshi | Ontogeny and multipotency of neural crest--derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad[END_REF], Glejzer et al., 2011; for recent reviews, see [START_REF] Motohashi | Extended multipotency of neural crest cells and neural crest--derived cells[END_REF][START_REF] Liu | Neural crest stem cells and their potential therapeutic applications[END_REF]. In most of these tissues however, the precise localization, specific markers and renewal capacity of resident NC-like stem cells remained undefined, with the notable exception of the carotid body, a small organ located near carotid artery bifurcation, which contains catecholaminergic neuron--like cells responsive to variations of arterial oxygen pressure. [START_REF] Pardal | Glia--like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body[END_REF] provided evidence that NC--derived stem cells in the adult rodent carotid body belong to the glial cell compartment and were multipotent in vitro; in response to chronic hypoxemia, these cells produced myofibroblasts and new neuroendocrine glomus cells in vivo.

A particular type of peripheral glial cells, the boundary cap (BC) cells, turned out to assume an unsuspected role in the production of several NC--derived differentiated cell types. BC cells, derived from late--emigrating NCC, form clusters at the limit between peripheral nerve roots and the neural tube [START_REF] Niederlander | Late emigrating neural crest cells migrate specifically to the exit points of cranial branchiomotor nerves[END_REF][START_REF] Golding | Border controls at the mammalian spinal cord: late-surviving neural crest boundary cap cells at dorsal root entry sites may regulate sensory afferent ingrowth and entry zone morphogenesis[END_REF] and they exhibit NC stem cell properties in vitro [START_REF] Hjerling--Leffler | The boundary cap: a source of neural crest stem cells that generate multiple sensory neuron subtypes[END_REF]. Taking advantage of BC--specific Krox20 expression during early development, [START_REF] Maro | Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS[END_REF] genetically traced the fate of BC cells and showed that they gave rise to sensory nociceptive neurons and satellite cells in the DRG, in addition to Schwann cells in the dorsal roots. Moreover in the adult mouse, BC--derived cells migrated as far as the dermis and produced Schwann cells, terminal glia and endoneurial fibroblasts in cutaneous nerves [START_REF] Gresset | Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin[END_REF].

By using selective ablation of cranial nerves and genetic lineage tracing in mice, two independent teams recently unveiled another neurogenic function of Schwann cell precursors, in the building of the parasympathetic ganglia [START_REF] Espinosa--Medina | Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors[END_REF]Dyachuk et al., 2014). Located at distal sites along growing visceral motor cranial nerve fibers, Schwann cell precursors assumed a dual glial--parasympathetic neuronal identity, before undergoing transition to differentiated neurons in the parasympathetic ganglia.

Thus, pre--Schwann cells dwelling from cranial nerve VII contributed neurons in submandibular and pterygopalatine ganglia while formation of the otic ganglia required the presence of presynaptic cranial nerve IX and its associated glia.

Neurogenesis from nerve--associated cells also occurred in the murine enteric nervous system (ENS), wherein a late--differentiating subset of neurons originated from Schwann cell precursors that invaded the gut at perinatal stages alongside the terminals of extrinsic sympathetic nerve fibers [START_REF] Uesaka | Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System[END_REF]Heuckeroth and Schäfer, 2016).

Moreover, in the oesophagus and stomach, enteric neurons developed from pre--Schwann cells that traveled along the vagus nerve [START_REF] Espinosa--Medina | Dual origin of enteric neurons in vagal Schwann cell precursors and the sympathetic neural crest[END_REF]. One of the remaining questions is how long Schwann cell precursors maintain their neurogenic capacity after a functional ENS has been established. At postnatal stages, when murine neurogenesis continues, multipotent NC--derived stem cells persist in the ENS [START_REF] Kruger | Neural crest stem cells persist in the adult gut but undergo changes in self--renewal, neuronal subtype potential, and factor responsiveness[END_REF][START_REF] Bondurand | Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures[END_REF][START_REF] Heanue | Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies[END_REF]. In the adult gut however, neurogenesis seems very limited and is still a matter of debate [START_REF] Liu | 5--HT4 receptor--mediated neuroprotection and neurogenesis in the enteric nervous system of adult mice[END_REF][START_REF] Joseph | Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut[END_REF][START_REF] Laranjeira | Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury[END_REF]. Nevertheless, the ENS of adult mammals harbors glial progenitors with latent neurogenic potential, which can be activated after in vitro culture and in vivo after injury (reviewed by [START_REF] Coelho--Aguiar | The enteric glia: identity and functions[END_REF].

The plasticity of PNS glial precursors remarkably extends to the production of endocrine chromaffin cells in the murine adrenal medulla [START_REF] Furlan | Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla[END_REF] and even to the generation of mesenchymal cell types. As already mentioned, nerve--associated mouse NCC generated endoneurial fibroblasts in vivo in the sciatic and cutaneous nerves [START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF][START_REF] Gresset | Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin[END_REF]. Furthermore, using the model of the continuously growing mouse incisor, [START_REF] Kaukua | Glial origin of mesenchymal stem cells in a tooth model system[END_REF] recently uncovered that Schwann cell precursors in nerve terminals at tooth apex were at the origin of dental pulp cells and odontoblasts. Moreover, these nerve--derived precursors displayed mesenchymal stem cell characteristics and were capable to regenerate dentine after trauma of the adult tooth.

Taken together, these data underline the remarkable plasticity of the Schwann cell lineage in various peripheral sites. They also suggest that, as in the CNS, in which radial glia and astrocytes function as neural stem cells in developmental and adult neurogenesis [START_REF] Kriegstein | The glial nature of embryonic and adult neural stem cells[END_REF] for a review), NC--derived stem cells in the PNS assume a pre--glial identity. These pre--glial NC stem cells are transported via the peripheral innervation network, thus ensuring a nearly ubiquitous distribution in the body of NC--like progenitors endowed with multiple differentiation options.

Concluding remarks

The NC, a specific embryonic structure of vertebrates, has attracted the interest of many researchers in developmental and stem cell biology, due to the remarkable migration properties of its component cells and their large contribution to multiple cell types in various organs and tissues. Here, we have focused on the question of NCC multipotency and fate diversification.

Collectively, the available data show that, before and during migration from the neural folds, the NCC exhibit a certain level of heterogeneity regarding their differentiation capacities. This heterogeneity and the evidence of NCC multipotency in the premigratory NCC, both at the level of the population and of single cells, was an undisputable result drawn from several and complementary approaches.

Early multipotency may be lost rapidly in particular NCC subpopulations, such as in those giving rise to some sensory neuronal subtypes. However, multipotent cells are present in subsets of all migratory NCC, isolated in avian and mammalian species. NC-derived cells isolated at postmigratory stages still comprise oligopotent progenitors, such as bipotent progenitors for Schwann cells and pigment cells, which are functional in vivo during development and in regenerating hair follicles.

More generally, an unexpected and important function of peripheral nerves and nerve terminals is to provide a niche for multipotent NC--like stem cells. Activation of these nerve--associated NCC represents a parcimonious strategy at play to take over and amplify the role of NC stem cells. Nerve--associated NCC insure the production of diversified cell types, at late developmental stages and in remote sites relative to their initial source. In the adult, they can replenish NC derivatives in tissues with high cellular turnover (mouse incisor, carotid body) or during injury repair (e.g., in the teeth).

One future goal will consist in molecularly defining the heterogeneity and plasticity of NCC and NC--derived cells. Recent advances in single--cell technologies (for a review, [START_REF] Hoppe | Single--cell technologies sharpen up mammalian stem cell research[END_REF], such as single cell RNA--sequencing, will likely be beneficial to unveil distinct state identities and developmental lineage trajectories of the NCC, as shown recently in embryonic and neural stem cells [START_REF] Klein | Droplet barcoding for single--cell transcriptomics applied to embryonic stem cells[END_REF][START_REF] Dulken | Single--Cell Transcriptomic Analysis Defines Heterogeneity and Transcriptional Dynamics in the Adult Neural Stem Cell Lineage[END_REF], and during the diversification of mesodermal tissues [START_REF] Loh | Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types[END_REF] and the segregation of neuro--mesodermal lineages [START_REF] Gouti | A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development[END_REF]. Ziller, C., Dupin, E., Brazeau, P., Paulin, D., Le Douarin, N.M., 1983. Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically defined medium. Cell 32, 627--638.

Aknowledgements

Zirlinger, M., Lo, L., McMahon, J., McMahon, A.P., Anderson, D.J., 2002. Transient expression of the bHLH factor neurogenin--2 marks a subpopulation of neural crest cells biased for a sensory but not a neuronal fate. Proc Natl Acad Sci U S A 99, 8084--8089. of cephalic NCC (CNCC) using either the bulk population of CNCC [START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF] or only CNCC immunoreactive to HNK1 surface epitope. For this purpose, CNCC suspension was labeled with HNK1 and then, under epifluorescence microscopy, individual fluorescent HNK1+ cells were isolated from the suspension using a micropipette and clonally seeded in culture medium supplemented with Shh. In both cases, the resulting colonies were analyzed after 10 days for differentiated cell types (G, glial cells; N, neurons; M, melanocytes; F, myofibroblasts; C, chondrocytes). Data reveal the presence of three main categories of progenitors, those producing only G, N, and/or M cell types ("neural only" progenitors shown in dark grey), those yielding only F and/or C mesenchymal cell types ("mesenchymal only" progenitors, shown in light grey) and the "neural--mesenchymal" progenitors that generate several combinations of both neural and mesenchymal cell types; the latter progenitors are splitted in two, chondrogenic (C+ in blue) and non--chondrogenic, subgroups (C--in yellow). The frequency (% of total clones) of each progenitor type is indicated. Noticeably, single plated HNK1+ CNCC (n=64) include multipotent GNMFC progenitors that were previously identified in the clones derived from the bulk CNCC population (n=150, [START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF], although the diversity of progenitors was reduced compared to CNCC, probably due to the lower number of plated HNK1+ cells. [START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF]. Progenitors (n=109 control and n=146 +Shh) are classified according to the number of the distinct cell types they clonally produced and their respective frequencies (percentage of total clones) are indicated; red asterisks indicate statistically significant differences in the clone type frequency between control and +Shh cultures (P<0.05). Cellular composition of the colonies identifies three main categories of progenitors as defined in legend of Fig. 2, i.e., "neural only" progenitors for G, N, M (dark grey), "mesenchymal only" progenitors (light grey) yielding only F, C and/or O cell types, and "neural--mesenchymal" progenitors producing both neural and mesenchymal cell types. In both medium conditions, 93% of the clones arise from neural-mesenchymal progenitors; Shh significantly increases the frequency of multipotent NCC endowed with chondro--osteogenic potential (GNMFCO, GMFCO and GFCO progenitors).

Of note, the bipotent GM and GF progenitors exhibited self--renewal capacity (indicated by a curved arrow) after serial in vitro subcloning [START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Bittencourt | Fibroblast growth factor 2 promotes the self--renewal of bipotent glial smooth muscle neural crest progenitors[END_REF]. Adapted from [START_REF] Calloni | High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities[END_REF]. [START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF]Coelho--Aguiar et al., 2013). TNCC progenitors were classified according to the extent of their phenotypic repertoire. Most of the progenitor types yielded "neural only" colonies (containing G, N and/or M only) as well as various types of "neural--mesenchymal" clones, which in addition to "neural" cell types, contained one or two of the mesenchymal cell phenotypes (F, C, O, A). Skeletogenic cells and adipocytes thus arose in vitro from multipotent TNCC. Self--renewal capacity (indicated by a curved arrow) of bipotent GM and GF progenitors of the trunk NC was shown by serial in vitro subcloning [START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Bittencourt | Fibroblast growth factor 2 promotes the self--renewal of bipotent glial smooth muscle neural crest progenitors[END_REF]. Adapted from [START_REF] Coelho--Aguiar | Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest[END_REF]. antibody and grown in single cell culture as described [START_REF] Dupin | Reversal of developmental restrictions in neural crest lineages: transition from Schwann cells to glial--melanocytic precursors in vitro[END_REF]; after 17 days in vitro, they generated heterogenous colonies containing Schwann cells, melanocytes and/or smooth muscle cells--myofibroblasts. Such in vitro transition of single Schwann cells to the melanocytic and fibroblastic lineages was promoted by exposure to EDN3 and TGFβ, respectively [START_REF] Dupin | Reversal of developmental restrictions in neural crest lineages: transition from Schwann cells to glial--melanocytic precursors in vitro[END_REF]Real et al., 2005). (Bottom panel) Pigmented

  A--C). Strikingly, these mesenchymal cell types coexisted with PNS derivatives in trunk NCC clonal progeny (Coelho--Aguiar et al., 2013); for example, the majority of the colonies grown on 3T3 cell feeders comprised Runx2-positive osteoblasts (O) accompanied by autonomic neurons (N) and glial cells (G) (GNO; 57% of total clones) (Figure 4 D).

  This work was supported by Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM) and Université Pierre et Marie Curie (UPMC). G.W.C. and J.M.C.--A. were supported by doctoral fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasil). Sommer, L., 2006. Neural crest--derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J Cell Biol 175, 1005--1015.

Figure 1 :

 1 Figure 1: Small-and medium-size clones from multipotent GNMFC progenitors identified in single mesencephalic NCC cultures. Colonies derived from single quail mesencephalic NCC cultures were analyzed after 10 days for the differentiation of glial cells (G), neurons (N), melanocytes (M), myofibroblasts (F) and chondrocytes (C) and for their total number of cells (see Calloni et al., 2007 for details). Multiphenotypic colonies such as those derived from GNMFC progenitor type exhibited various sizes regarding their number of cells. (A--B, same microscopic field) Micrographic views of a small--size (about 250 cells) GNMFC multiphenotypic colony that includes several dense nodules of cartilage cells (chondroitin sulfate, green in A and B), surrounded by a network of glial cells (HNK1+, green in A); this colony also contains neurons labeled with tyrosine hydroxylase (TH) and Tuj1 (red, thin arrow, in B), melanoblasts (MelEM+, in red, thick arrow in B) and myofibroblasts (expressing α--smooth muscle actin, in red, arrowhead in B). (C--F, same microscopic field) Micrographs of a medium--size (about 1000 cells) GNMFC clone showing the presence of a large cartilage nodule (chondroitin--sulfate green fluorescence in C--E and star in C--F), neurons (labeled with TH and Tuj1, red fluorescence in D and F, thin arrow in D), myofibroblasts (α--smooth muscle actin+, arrowhead in D), glial cells (HNK1+, in E) and melanoblasts (MelEM+, green fluorescence in F). Phenotypes were identified by immunocytochemistry. Cell nuclei were visualized by Hoechst staining (blue in A, C--F). Magnification, X140.

Figure 2 :

 2 Figure 2: Single HNK1+ NCC give rise to both neural and skeletogenic cell types in vitro. Schematic representation of the types of progenitors identified in clonal cultures

Figure 3 :

 3 Figure 3: Cephalic NC progenitors for neural and chondro-osteogenic lineages are favored in clonal cultures treated with Shh. Scheme of the diverse types of progenitors recorded in clonal cultures of mesencephalic NCC grown in the absence (control CNCC) and presence of the morphogen Shh (+Shh) and analyzed for the presence of six main NC phenotypes: glial cells (G), neurons (N), melanocytes (M), myofibroblasts (F), chondrocytes (C) and Runx2--expressing osteoblasts (O) (Data from

Figure 4 :

 4 Figure 4: Skeletogenic and adipogenic in vitro differentiation capacity of trunk NC progenitors. (A--C, same microscopic field) Quail trunk NCC (TNCC) cultured for 21 days under medium condition appropriate for mesenchymal cell terminal differentiation give rise to mineralized bone cells (A, alizarin red staining), mature chondrocytes (B, alcian blue staining) and lipid--storing adipocytes (C, oil red O staining). (D) Single TNCC cultures performed in various conditions were analyzed for the differentiation of glial cells (G), neurons (N), melanocytes (M), myofibroblasts (F), chondrocytes (C), osteoblasts (O) and adipocytes (A) (Data from[START_REF] Trentin | Self--renewal capacity is a widespread property of various types of neural crest precursor cells[END_REF][START_REF] Calloni | Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials[END_REF]

Figure 5 :

 5 Figure 5: Reversion of differentiated Schwann cells and pigmented melanocytes to multipotent NC-like progenitors. Schematic model for in vitro reprogramming of avian NC--derived Schwann cells and melanocytes. (Upper panel) Schwann cells from E10.5 quail sciatic nerves were purified by FACS using the Schwann cell Myelin Protein

melanocytes obtained from dissociated epidermal cells of the quail embryonic skin were individually seeded under microscopic control and cultured for 11 days as described (Real et al., 2006). Up to 90% of the pigment cells grown in the presence of EDN3 formed clones containing Schwann cells and/or fibroblasts in addition to melanocytes.

Serial subcloning of melanocyte progeny showed that the in vitro reprogramming of melanocytes induced by EDN3 involved the maintenance of oligopotent NC--like stem cells endowed with self--renewal (curved arrow). (Adapted from [START_REF] Dupin | Endothelin 3 induces the reversion of melanocytes to glia through a neural crest--derived glial--melanocytic progenitor[END_REF]Real et al., 2006).


Rafael C Deptulski

Bennacer Rachid
email: rachid.bennacer@ens-paris-saclay.fr

Isotropic and anisotropic heat transfer in active wall porous media foam type

Keywords: active wall, porous media foam type, multi-structured porous media, thermal transfer characterization, thermal conductivity

Positive buildings in energy are, nowadays, a recurrent objective of many researches in the construction and energetic efficiency domain. Furthermore, to achieve this objective, some studies about active and reactive walls have been carried out employing porous medium as a main structure. Nevertheless, transfer characterization in a foam type sample is not fully understood. The goal of this study is to improve the characterization of heat transfer in isotropic and anisotropic configurations of a porous medium. Thus, a finite volume method was implemented to study a heat transfer through these media, in the interest of achieving their ratio equivalent to fluid thermal conductivity (i.e. Nusselt number). Finally, the results indicate a notable influence of the ratio of the contact and the total inlet area on the isotropic configuration as well as strong influence given by the different axis on the anisotropic model. Moreover, the analysis shows that in an active wall constituted by two solid phases, these effects will be preponderant for their characterization.

Introduction

Porous medium foam type is generally considered as an efficient heat exchanger. Following preceding study [START_REF] Deptulski | Active wall through a porous media foam type: flow and transfer characterization[END_REF], the coupling between the heat and the mass transfer in an orthogonal reference is suitable to form an active wall. The desirable structure here studied is an active and reactive wall, formed by a continuous solid thermal conductive matrix that provides a structural achievement, then, a second solid phase that coats the skeletal phase to get a perfect insulator in a thermal activated mode and, lastly, a void phase that has an important volumetric fraction in an inactivated configuration.

Many research projects pursue to explain the flow and the heat behaviour in porous media [START_REF] Bennacer | Sustainability of Construction Materials. Chapter: Intrinsic properties controlling the sustainability of constructions[END_REF] in order to predict their equivalent properties in different compositions such as metallic, polymeric or mixed samples foam type [START_REF] Zhang | Constitutive modeling of polymeric foam material subjected to dynamic crash loading[END_REF][START_REF] Jiang | Ultralight metal foams[END_REF][START_REF] Fang | Mesoscopic investigation of closed-cell aluminum foams on energy absorption capability under impact[END_REF]. Different applications in this domain are increasingly performed thanks to its high mass and thermal transfer capacity given by an impressive specific surface area and permeability in addition to their significant mechanical resistance [START_REF] Kornev | Foam in porous media: thermodynamic and hydrodynamic peculiarities[END_REF][START_REF] Ghosh | Heat transfer correlation for high-porosity open-cell foam[END_REF]. Hence, the goal is to explore this thermomechanical potential to construct an active wall in porous media. Therefore, this system has many functions and can be combined to work with several thermo-activate applications such as deformable porous polymers membranes [START_REF] Azra | Effect of polymer nanofibers thermoelasticity on deformable fluidsaturated porous membrane[END_REF] in our case or phase change materials [START_REF] Arıcı | Melting of nanoparticle-enhanced paraffin wax in a rectangular enclosure with partially active walls[END_REF] in an energy storage framework.

Analysis and modelling

This part covers the generating process of a 3D porous medium framework divided into three points: generating a random porous structure, controlling its morphology in order to create an anisotropic configuration, as well as numerical modelling.

Here, different structures were presented, firstly in two phases (i.e. solid-fluid configuration) and then in three phases (i.e. solid-solid-fluid configuration). These samples were originally generated in a numerical device, based on correlated Gaussian random fields [START_REF] Adler | The Geometry of Random Fields[END_REF], in a cube with a triple correlation length of 10%, a standard deviation of 1%, an expectation of 0 and 250 eigenmodes retained (Figure 1 (a)). Thus, the designed structure results in a Representative Elementary Volume (REV) of a foam-like porous with 90% voids (Figure 1 (b) and (c)). Hither, the two-phase model is analysed in order to achieve an isotropic and anisotropic transfer characterization, besides that, the three-phase model is presented to illustrate the operation of an active and reactive wall through a porous medium. This proposed structure is still little studied and consisting of a rigid solid matrix which takes the heat energy to the second non-rigid solid matrix and, therefore, result in a system of expansion-shrinkage which gives to this couple an inhomogeneous nature in order to get a perfect insulator. In this case, after an initial request, an inhomogeneous structure will be formed and its response to external requests will no longer have the expected standard nature. Consequently, in order to understand this new heat transfer behaviour, an inhomogeneous structure has been generated based on the first homogeneous structure (Figure 2) by applying a dilatation threshold gradient from -30% to +30% at the two opposite sides in the vertical direction, as shown in Figure 3 and Figure 4. To analyse these different structures and understand their behaviours, a finite volume method is implemented, as the presented scheme in Figure 5, in order to discretize a heat flux through porous media and it has been performed solving the momentum and energy equations.

∇V ⃗ ⃗ = 0 ( 1 )
Momentum equation:

∂V ⃗⃗ ∂t + (V ⃗ ⃗ • ∇)V ⃗ ⃗ = -∇P + 1 𝑅𝑒 ∇(μ * ∇V ⃗ ⃗ ) + 1 𝐷𝑎 V ⃗ ⃗ + 𝑅𝑎 𝑃𝑟 𝑅𝑒 2 (θ + NS)𝑘 ⃗ ( 2 )
Energy equation:

∂θ ∂t + V ⃗ ⃗ • ∇θ = 1 Pr ∇(𝜆 * ∇θ) ( 3 )
The continuity of both temperatures the heat flux on the solid-fluid interface is employed:

θ 𝑠 = θ 𝑓 ∴ 𝜕θ 𝑓 𝜕𝑛 = 𝜆 * 𝜕θ 𝑠 𝜕𝑛 ( 4 )
To reach a stable state solution, a convergence criterion based on the temperature field is adopted. This norm is defined by the following conditions:

∑ |𝜑 𝑖,𝑗 𝑚+1 -𝜑 𝑖,𝑗 𝑚 | 𝑖,𝑗 ∑ |𝜑 𝑖,𝑗 𝑚+1 | 𝑖,𝑗 ≤ 𝜖 = 10 -5 ( 5 )
Where 𝑚 is an interaction counter and 𝜖 is the relative error. Furthermore, some validation tests have been performed in Figure 6 and this method has been presented good accuracy conforming to reference results published in several preceding works [START_REF] Ghosh | Heat transfer correlation for high-porosity open-cell foam[END_REF][START_REF] Azra | Effect of polymer nanofibers thermoelasticity on deformable fluidsaturated porous membrane[END_REF][START_REF] Arıcı | Melting of nanoparticle-enhanced paraffin wax in a rectangular enclosure with partially active walls[END_REF], as well as qualitatively experimental results validation [START_REF] Corvaro | An experimental study of natural convection in a differentially heated cavity through a 2D-PIV system[END_REF] and instability verification [START_REF] Kalla | Double diffusive convection within a horizontal porous layer salted from the bottom and heated horizontally[END_REF]. Moreover, the criterion of validity concerning this method is considered by 0.1 ≤ 𝜆 * ≤ 200 and 𝑅𝑎 = 0.

Results and discussion

In a homogeneous foam type configuration, heat transfer is significantly affected by the continuous nature of the skeletal phase. Moreover, here we analyse the isotropic nature of this medium and the influence of the ratio of the contact and the total inlet area 𝑅 on the Nusselt number (i.e. λ 𝑒𝑞 /λ 𝑓𝑙𝑢𝑖𝑑 in this porous configuration) after heating the sample by the three different axis x, y and z (Figure 7). Hither, this section search to achieve the isotropic nature of heat transfer in the study domain. Figure 7 shows some small variations between the different solicitations axis according to the increase of λ * , despite that, this structure is still considered as isotropic. Thus, according to the same figure, it is evident that the heat transfer in an isotropic model varies according to its ratio of the contact and the total inlet area 𝑅 (i.e. 13%, 12% and 9% to x, y, and z respectively). Such connection between the material and the solid surface is a key point equivalent (in opposing way) to the thermal resistance effect. Besides that, we observe the heat transfer behaviour in an inhomogeneous configuration (Figure 3) following their Nusselt number after heating the sample by the three different axis x, y and z (Figure 8). In this case, the result shows that on the x and y-axis, a continuous solid matrix is present and connecting the opposed side, that provided an important heat conduction. On the other hand, on the z-axis, there is no more direct connection (i.e. absence of percolation effect) between their opposite sides, consequently, the heat transfer is considerably less important. These analyses prove that this structure presents an anisotropic heat transfer vector as well as its morphology.

Conclusions

This review explored the heat transfer behaviour in a porous medium subjected to both morphology configurations: isotropic and anisotropic. Then, using a stochastic generation of 3D structures based on correlated random fields, a foam-like porous medium was constructed with a continuous skeletal solid matrix filled with nonregular voids for the purpose of illustrating the operating role of an active wall.

Thus, a finite volume discretization method has been performed in order to characterize the thermal conductivity in a non-linear system. The Nusselt number, for the proposed 90% voids isotropic model, increases according to the ratio of the contact and the total inlet area of which solicitation axis. In addition, the results of the anisotropic configuration showed that if the solid matrix is not continuous between inlet and outlet sides the equivalent thermal properties are significantly affected.

Lastly, understanding the issue about the isotropic and anisotropic thermal behaviour is a preponderant step towards the construction of an active and reactive wall. This development will be useful to open up new research possibilities and to further investigate the abilities to this structure manages its transfer properties by thermoactivation.

Nomenclature

Figure 1 :

 1 Figure 1: Development stages to generate a porous structure based on Gaussian random draws. Correlated Gaussian random field (a) and 3D structure 90% voids (b) and (c)Hence, Figure2shows the 3D model, at the pore scale, of an active and reactive wall in a considered homogeneous structure by their solid skeletal matrix (Phase 1) and a coat solid phase (Phase 2) that involves this first phase.

Figure 2 :

 2 Figure 2: 3D view of an active and reactive wall in an homogeneous porous medium with skeletal and coat phases

Figure 3 :Figure 4 :

 34 Figure 3: 3D slice (a) and isometric view (b) of an inhomogeneous porous medium by applying a dilatation gradient from -30% to +30% in vertical axis

Figure 5 :Figure 6 :

 56 Figure 5: Porous media heat exchanger configuration

Figure 7 :

 7 Figure 7: Nusselt number for the three-different axis x, y and z and their respective contact surface areas in an isotropic structure

Figure 8 :

 8 Figure 8: Nusselt number for the three-different axis x, y and z in an anisotropic structure

This work does not seek beyond the study of thermal transfer in isotropic and anisotropic porous media, however, a multiphasic model of an active wall will be present to contextualize the structure. Firstly, the methodology for creating the porous medium in its different configurations is presented, followed by the numerical method of heat transfer used. Finally, the results and conclusions are presented in order to answer the degree of influence of the non-linearity of a porous medium for its heat transport.