The diverse neural crest: from embryology to human pathology.

We review here some of the historical highlights in exploratory studies of the vertebrate embryonic structure known as the neural crest. The study of the molecular properties of the cells that it produces, their migratory capacities and plasticity, and the still-growing list of tissues that depend on their presence for form and function, continue to enrich our understanding of congenital malformations, paediatric cancers and evolutionary biology. Developmental biology has been key to our understanding of the neural crest, starting with the early days of experimental embryology and through to today, when increasingly powerful technologies contribute to further insight into this fascinating vertebrate cell population.

The dependence receptor TrkC triggers mitochondria-dependent apoptosis upon Cobra-1 recruitment.

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1.

The "beginnings" of the neural crest.

FOREWORD: The neural crest has been the main object of my investigations during my career in science, up to now. It is a fascinating topic for an embryologist because of its two unique characteristics: its large degree of multipotency and the fact that its development involves a phase during which its component cells migrate all over the embryo and settle in elected sites where they differentiate into a large variety of cell types. Thus, neural crest development raises several specific questions that are at the same time, of general interest: what are the mechanisms controlling the migratory behavior of the cells that detach from the neural plate borders? What are the migration routes taken by the neural crest cells and the environmental factors that make these cells stop in elected sites where they differentiate into a definite series of cell types? When I started to be interested in the neural crest, in the late 1960s, this embryonic structure was the subject of investigations of only a small number of developmental biologists. Fifty years later, it has become the center of interest of many laboratories over the world. The 150th anniversary of its discovery is a relevant opportunity to consider the progress that has been accomplished in our knowledge on the development of this ubiquitous structure, the roles it plays in the physiology of the organism through its numerous and widespread derivatives and its relationships with its environment, as well as the evolutionary advantages it has conferred to the vertebrate phylum. I wish to thank Pr Marianne Bronner, Chief Editor of Developmental Biology and Special Issue Guest Editor, for dedicating a special issue of this journal to this particular structure of the vertebrate embryo. In the following pages, Elisabeth Dupin and I will report some of the highlights of our own acquaintance with the neural crest of the avian embryo, after retracing the main trends of the discoveries of the historical pioneers.

The issue of the multipotency of the neural crest cells.

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.

The dependence receptor TrkC regulates the number of sensory neurons during DRG development.

The development of the sensory nervous system is the result of fine-tuned waves of neurogenesis and apoptosis which control the appropriate number of precursors and newly generated neurons and orient them toward a specific lineage. Neurotrophins and their tyrosine-kinase receptors (RTK) orchestrate this process. They have long been in the scope of the neurotrophic theory which established that a neuron is committed to die unless a trophic factor generated by its target provides it with a survival signal. The neural death has thus always been described as a "default" program, survival being the major player to control the number of cells. New insights have been brought by the gain of function studies which recently demonstrated that TrkC (NTRK3) is a "dependence receptor" able to actively trigger apoptosis in absence of its ligand NT-3. In order to address the role of TrkC pro-apoptotic activity in the control of sensory neurons number, we generated a TrkC gene-trap mutant mice. We found out that this new murine model recapitulates the sensory phenotype of TrkC constitutive mutants, with reduced DRG size and reduced number of DRG neurons. We engineered these mice strain with a lacZ reporter in order to follow the fate of neurons committed to a TrkC lineage and observed that they are specifically protected from NT-3 mediated apoptosis in NT-3/TrkC double knock-out embryos. Finally, using a chicken model we demonstrated that silencing NT-3 emanating from the ventral neural tube induced apoptosis in the DRG anlage. This apoptosis was inhibited by silencing TrkC. This work thus demonstrates that, during in vivo DRG development, TrkC behaves as a two-sided receptor transducing positive signals of neuronal survival in response to NT-3, but actively inducing neuronal cell death when unbound. This functional duality sets adequate number of neurons committed to a TrkC identity in the forming DRG.

Respective contribution of the cephalic neural crest and mesoderm to SIX1-expressing head territories in the avian embryo.

BACKGROUND: Vertebrate head development depends on a series of interactions between many cell populations of distinct embryological origins. Cranial mesenchymal tissues have a dual embryonic source: - the neural crest (NC), which generates most of craniofacial skeleton, dermis, pericytes, fat cells, and tenocytes; and - the mesoderm, which yields muscles, blood vessel endothelia and some posterior cranial bones. The molecular players that orchestrate co-development of cephalic NC and mesodermal cells to properly construct the head of vertebrates remain poorly understood. In this regard, Six1 gene, a vertebrate homolog of Drosophila Sine Oculis, is known to be required for development of ear, nose, tongue and cranial skeleton. However, the embryonic origin and fate of Six1-expressing cells have remained unclear. In this work, we addressed these issues in the avian embryo model by using quail-chick chimeras, cephalic NC cultures and immunostaining for SIX1. RESULTS: Our data show that, at early NC migration stages, SIX1 is expressed by mesodermal cells but excluded from the NC cells (NCC). Then, SIX1 becomes widely expressed in NCC that colonize the pre-otic mesenchyme. In contrast, in the branchial arches (BAs), SIX1 is present only in mesodermal cells that give rise to jaw muscles. At later developmental stages, the distribution of SIX1-expressing cells in mesoderm-derived tissues is consistent with a possible role of this factor in the myogenic program of all types of head muscles, including pharyngeal, extraocular and tongue muscles. In NC derivatives, SIX1 is notably expressed in perichondrium and chondrocytes of the nasal septum and in the sclera, although other facial cartilages such as Meckel's were negative at the stages considered. Moreover, in cephalic NC cultures, chondrocytes and myofibroblasts, not the neural and melanocytic cells express SIX1. CONCLUSION: The present results point to a dynamic tissue-specific expression of SIX1 in a variety of cephalic NC- and mesoderm-derived cell types and tissues, opening the way for further analysis of Six1 function in the coordinated development of these two cellular populations during vertebrate head formation.

The neural crest, a multifaceted structure of the vertebrates.

In this review, several features of the cells originating from the lateral borders of the primitive neural anlagen, the neural crest (NC) are considered. Among them, their multipotentiality, which together with their migratory properties, leads them to colonize the developing body and to participate in the development of many tissues and organs. The in vitro analysis of the developmental capacities of single NC cells (NCC) showed that they present several analogies with the hematopoietic cells whose differentiation involves the activity of stem cells endowed with different arrays of developmental potentialities. The permanence of such NC stem cells in the adult organism raises the problem of their role at that stage of life. The NC has appeared during evolution in the vertebrate phylum and is absent in their Protocordates ancestors. The major role of the NCC in the development of the vertebrate head points to a critical role for this structure in the remarkable diversification and radiation of this group of animals.

Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest.

The neural crest (NC), an ectoderm-derived structure of the vertebrate embryo, gives rise to the melanocytes, most of the peripheral nervous system and the craniofacial mesenchymal tissues (i.e., connective, bone, cartilage and fat cells). In the trunk of Amniotes, no mesenchymal tissues are derived from the NC. In certain in vitro conditions however, avian and murine trunk NC cells (TNCCs) displayed a limited mesenchymal differentiation capacity. Whether this capacity originates from committed precursors or from multipotent TNCCs was unknown. Here, we further investigated the potential of TNCCs to develop into mesenchymal cell types in vitro. We found that, in fact, quail TNCCs exhibit a high ability to differentiate into myofibroblasts, chondrocytes, lipid-laden adipocytes and mineralizing osteoblasts. In single cell cultures, both mesenchymal and neural cell types coexisted in TNCC clonal progeny: 78% of single cells yielded osteoblasts together with glial cells and neurons; moreover, TNCCs generated heterogenous clones with adipocytes, myofibroblasts, melanocytes and/or glial cells. Therefore, alike cephalic NCCs, early migratory TNCCs comprised multipotent progenitors able to generate both mesenchymal and melanocytic/neural derivatives, suggesting a continuum in NC developmental potentials along the neural axis. The skeletogenic capacity of the TNC, which was present in the exoskeletal armor of the extinct basal forms of Vertebrates and which persisted in the distal fin rays of extant teleost fish, thus did not totally disappear during vertebrate evolution. Mesenchymal potentials of the TNC, although not fulfilled during development, are still present in a dormant state in Amniotes and can be disclosed in in vitro culture. Whether these potentials are not expressed in vivo due to the presence of inhibitory cues or to the lack of permissive factors in the trunk environment remains to be understood.

Neural crest progenitors and stem cells: from early development to adulthood.

In the vertebrate embryo, the neural crest forms transiently in the dorsal neural primordium to yield migratory cells that will invade nearly all tissues and later, will differentiate into bones and cartilages, neurons and glia, endocrine cells, vascular smooth muscle cells and melanocytes. Due to the amazingly diversified array of cell types it produces, the neural crest is an attractive model system in the stem cell field. We present here in vivo and in vitro studies of single cell fate, which led to the discovery and the characterization of stem cells in the neural crest of avian and mammalian embryos. Some of the key issues in neural crest cell diversification are discussed, such as the time of segregation of mesenchymal vs. neural/melanocytic lineages, and the origin and close relationships between the glial and melanocytic lineages. An overview is also provided of the diverse types of neural crest-like stem cells and progenitors, recently identified in a growing number of adult tissues in animals and humans. Current and future work, in which in vivo lineage studies and the use of injury models will complement the in vitro culture analysis, should help in unraveling the properties and function of neural crest-derived progenitors in development and disease.

Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos.

Epithelial-mesenchymal interactions are crucial for the development of the endoderm of the pharyngeal pouches into the epithelia of thymus and parathyroid glands. Here we investigated the dynamics of epithelial-mesenchymal interactions that take place at the earliest stages of thymic and parathyroid organogenesis using the quail-chick model together with a co-culture system capable of reproducing these early events in vitro. The presumptive territories of thymus and parathyroid epithelia were identified in three-dimensionally preserved pharyngeal endoderm of embryonic day 4.5 chick embryos on the basis of the expression of Foxn1 and Gcm2, respectively: the thymic rudiment is located in the dorsal domain of the third and fourth pouches, while the parathyroid rudiment occupies a more medial/anterior pouch domain. Using in vitro quail-chick tissue associations combined with in ovo transplantations, we show that the somatopleural but not the limb bud mesenchyme, can mimic the role of neural crest-derived pharyngeal mesenchyme to sustain development of these glands up to terminal differentiation. Furthermore, mesenchymal-derived Bmp4 appears to be essential to promote early stages of endoderm development during a short window of time, irrespective of the mesenchymal source. In vivo studies using the quail-chick system and implantation of growth factor soaked-beads further showed that expression of Bmp4 by the mesenchyme is necessary during a 24 h-period of time. After this period however, Bmp4 is no longer required and another signalling factor produced by the mesenchyme, Fgf10, influences later differentiation of the pouch endoderm. These results show that morphological development and cell differentiation of thymus and parathyroid epithelia require a succession of signals emanating from the associated mesenchyme, among which Bmp4 plays a pivotal role for triggering thymic epithelium specification.

Isolation and differentiation properties of neural crest stem cells.

A wide array of neural and non-neural cell types arises from the neural crest during vertebrate embryogenesis. The neural crest forms transiently in the dorsal neural primordium to yield migratory cells that will invade nearly all tissues and later, differentiate into bones and cartilages, vascular smooth muscle cells, connective tissues, neurons and glial cells of the peripheral nervous system, endocrine cells, and melanocytes. Due to the amazingly diversified array of cell types they generate, the neural crest cells represent an attractive model in the stem cell field. We review here in vivo and in vitro studies of individual cells, which led to the discovery and characterization of neural crest progenitors endowed with multipotency and stem cell properties. We also present an overview of the diverse types, marker expression, and locations of the neural crest-derived stem cells identified in the vertebrate body, with emphasis on those evidenced recently in mammalian adult tissues.

High frequency of cephalic neural crest cells shows coexistence of neurogenic, melanogenic, and osteogenic differentiation capacities.

The neural crest (NC) is a vertebrate innovation that distinguishes vertebrates from other chordates and was critical for the development and evolution of a "New Head and Brain." In early vertebrates, the NC was the source of dermal armor of fossil jawless fish. In extant vertebrates, including mammals, the NC forms the peripheral nervous system, melanocytes, and the cartilage and bone of the face. Here, we show that in avian embryos, a large majority of cephalic NC cells (CNCCs) have the ability to differentiate into cell types as diverse as neurons, melanocytes, osteocytes, and chondrocytes. Moreover, we find that the morphogen Sonic hedgehog (Shh) acts on CNCCs to increase endochondral osteogenesis while having no effect on osteoblasts prone to membranous ossification. We have developed culture conditions that demonstrate that "neural-mesenchymal" differentiation abilities are present in more than 90% of CNCCs. A highly multipotent progenitor (able to yield neurons, glia, melanocytes, myofibroblasts, chondrocytes, and osteocytes) comprises 7-13% of the clonogenic cells in the absence and presence of Shh, respectively. This progenitor is a good candidate for a cephalic NC stem cell.

Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials.

In the vertebrate embryo, the cephalic neural crest cells (CNCCs) produce cells belonging to two main lineages: the neural [including neurons, glial cells of the peripheral nervous system (PNS), and melanocytes] and the mesenchymal (chondrocytes, osteoblasts, smooth muscle cells, and connective tissue cells), whereas the trunk NCCs (TNCCs) in amniotes yield only neural derivatives. Although multipotent cells have previously been evidenced by in vitro clonal analysis, the issue as to whether all of the mesenchymal and neural phenotypes can be derived from a unique NC stem cell has remained elusive. In the present work, we devised culture conditions that led us to identify a highly multipotent NCC endowed with both neural and mesenchymal potentials, which lies upstream of all the other NC progenitors known so far. We found that addition of recombinant Sonic Hedgehog (Shh) increased the number of CNCC progenitors yielding both mesenchymal and neural lineages and promoted the development of such precursors from the TNCC. Shh decreased the neural-restricted precursors without affecting the overall CNCC survival and proliferation. By showing a differential positive effect of Shh on the expression of mesenchymal phenotypes (i.e., chondrocytes and smooth muscle cells) by multipotent CNCCs, these results shed insights on the in vivo requirement of Shh for craniofacial morphogenesis. Together with evolutionary considerations, these data also suggest that the mesenchymal-neural precursor represents the ancestral form of the NC stem cell, which in extinct forms of vertebrates (the ostracoderms) was able to yield both the PNS and superficial skeleton.

Multipotentiality of the neural crest.

Multiple neural and non-neural cell types arise from the neural crest (NC) in vertebrate embryos. Recent work has provided evidence for multipotent stem cells and intermediate precursors in the early NC cell population as well as in various NC derivatives in embryos and even in adult. Advances have been made towards understanding how cytokines, regulatory genes and cell-cell interactions cooperate to control commitment and differentiation to pigment cells, glia and neurone subtypes. In addition, NC cell fates appeared to be unstable, as differentiated NC cells can reverse to multipotent precursors and transdifferentiate in vitro.

Neural crest progenitors and stem cells.

In the vertebrate embryo, multiple cell types originate from a common structure, the neural crest (NC), which forms at the dorsal tips of the neural epithelium. The NC gives rise to migratory cells that colonise a wide range of embryonic tissues and later differentiate into neurones and glial cells of the peripheral nervous system (PNS), pigment cells (melanocytes) in the skin and endocrine cells in the adrenal and thyroid glands. In the head and the neck, the NC also yields mesenchymal cells that form craniofacial cartilages, bones, dermis, adipose tissue, and vascular smooth muscle cells. The NC is therefore a model system to study cell diversification during embryogenesis and phenotype maintenance in the adult. By analysing the developmental potentials of quail NC cells in clonal cultures, we have shown that the migratory NC is a collection of heterogeneous progenitors, including various types of intermediate precursors and highly multipotent cells, some of which being endowed of self-renewal capacity. We also have identified common progenitors for mesenchymal derivatives and neural/melanocytic cells in the cephalic NC. These results are consistent with a hierarchical model of lineage segregation wherein environmental cytokines control the fate of progenitors and stem cells. One of these cytokines, the endothelin3 peptide, promotes the survival, proliferation, and self-renewal capacity of common progenitors for glial cells and melanocytes. At post-migratory stages, when they have already differentiated, NC-derived cells exhibit phenotypic plasticity. Epidermal pigment cells and Schwann cells from peripheral nerves in single-cell culture are able to reverse into multipotent NC-like progenitors endowed with self-renewal. Therefore, stem cell properties are expressed by a variety of NC progenitors and can be re-acquired by differentiated cells of NC origin, suggesting potential function for repair.

Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

The contribution of the neural crest to the vertebrate body.

As a transitory structure providing adult tissues of the vertebrates with very diverse cell types, the neural crest (NC) has attracted for long the interest of developmental biologists and is still the subject of ongoing research in a variety of animal models. Here we review a number of data from in vivo cell tracing and in vitro single cell culture experiments, which gained new insights on the mechanisms of cell migration, proliferation and differentiation during NC ontogeny. We put emphasis on the role of Hox genes, morphogens and interactions with neighbouring tissues in specifying and patterning the skeletogenic NC cells in the head. We also include advances made towards characterizing multipotent stem cells in the early NC as well as in various NC derivatives in embryos and even in adult.

The instability of the neural crest phenotypes: Schwann cells can differentiate into myofibroblasts.

In the vertebrate embryo, the neural crest cells (NCCs) that migrate out from the neural primordium yield multiple phenotypes, including melanocytes, peripheral neurones and glia and, in the head, cartilage, bone, connective cells and myofibroblasts / vascular smooth muscle cells (SMCs). The differentiation of pluripotent NCCs is mainly directed by local growth factors. Even at postmigratory stages, NC-derived cells exhibit some fate plasticity. Thus, we reported earlier that pigment cells and Schwann cells are able in vitro to interconvert in the presence of endothelin 3 (ET3). Here, we further investigated the capacity of Schwann cells to reprogram their phenotype. We show that purified quail Schwann cells in dissociated cultures produce alpha smooth muscle actin ((alpha)SMA)-expressing myofibroblasts through the generation of a pluripotent progeny. This transdifferentiation took place independently of ET3, but was promoted by transforming growth factor beta1 (TGF(beta)1). Moreover, when implanted into chick embryos, the Schwann cells were found to contribute with host cephalic NCCs to perivascular SMCs. These data provided the first evidence for the acquisition of an NC-derived mesenchymal fate by Schwann cells and further demonstrate that the differentiation state of NC-derived cells is unstable and capable of reprogramming. The high plasticity of Schwann cells evidenced here also suggests that, as in the CNS, glial cells of the PNS may function as NC stem cells in particular circumstances such as repair.

The Pluripotency of Neural Crest Cells and Their Role in Brain Development.

The neural crest (NC) is, in the Chordate phylum, an innovation of vertebrates, which exhibits several original characteristics: its component cells are pluripotent and give rise to both ectodermal and mesodermal cell types. Moreover, during the early stages of neurogenesis, the NC cells exert a paracrine stimulating effect on the development of the preotic brain.

Development of melanocyte precursors from the vertebrate neural crest.

Pigment cells that differentiate in the vertebral skin arise from the neural crest (NC), a transitory structure formed at the dorsal borders of the neural plate and which gives rise to migratory cells of multiple fates. How NC cells become committed to the melanocytic lineage and what factors control the survival, proliferation and differentiation of melanocyte precursors remain largely unknown. These issues are of great importance for understanding the mechanisms of several pigment cell pathologies including melanomas. Recent in vivo and in vitro analyses of the fate of single NC cells have indicated that multipotent cells yield melanocyte precursors that become spatially and temporally segregated from other, non melanogenic, NC-derived cell types. The proper development of subsets of NC precursors is governed by environmental local cytokines acting in a paracrine manner. The conjunction of recent studies in mammals and birds reviewed here focuses on the action of endothelin 3 in controlling both the emergence and the maintenance of the NC-derived melanocyte phenotype.