Genetic data in the mouse have shown that endothelin 3 (ET3) and its receptor B (ETRB) are essential for the development of two neural crest (NC) derivatives, the melanocytes and the enteric nervous system. We report here the effects of ET3 in vitro on the differentiation of quail trunk NC cells (NCC) in mass and clonal cultures. Treatment with ET3 is highly mitogenic to the undifferentiated NCC population, which leads to expansion of the population of cells in the melanocytic, and to a lesser extent, the glial lineages. The effect of ET3 on these two NC derivatives was confirmed by the quantitative analysis of clones derived from individual NCC subjected to ET3: we found a large increase in the survival and proliferation of unipotent and bipotent precursors for glial cells and melanocytes, with no significant effect on multipotent cells generating neurons. ET3 first stimulates expression of both ETRB and ETRB2 by cultured NCC. Then, under prolonged exposure to ET3, ETRB expression decreases and switches toward an ETRB2-positive melanogenic cell population. We therefore propose that the present in vitro experiments (long-lasting exposure to a high concentration of ET3) mimic the environment encountered by NCC in vivo when they migrate to the skin under the ectoderm that expresses ET3.The neural crest (NC) appears dorsally to the neural primordium as it forms according to a craniocaudal gradient. The presumptive NC cells (NCC) undergo an epithelio-mesenchymal transition and, after a phase of migration, give rise to multiple cell types including melanocytes, neurons, and glial cells of peripheral nerves and ganglia, a large majority of the cephalic mesenchymal structures, and certain endocrine cells (1, 2). Because of its pluripotentiality and the fact that its constitutive cells become localized in various regions of the developing embryo, the NC is an ideal developmental system in which to study the mode of action of factors involved in differentiation choices.The importance of environmental influences on the development of NCC has been demonstrated by in vivo transplantation experiments in the chicken embryo and in vitro culture studies (1-6). At the onset of migration, the NCC population is composed of a mixture of pluripotent and more or less restricted progenitors that have been identified by various cell cloning experiments (7-14). These observations suggest that both selective and instructive mechanisms are involved in NCC diversification. Thus far, differentiation of definite lineages of NC-derived cells in clonal cultures has proved to be favored by factors such as brain-derived neurotrophic factor (15), glial growth factor (16), retinoic acid (17), and members of the transforming growth factor ␤ family (18). Other growth factors (and their receptors) encoded by loci that affect NC derivatives in mice have been shown to have an important role in NC ontogeny but their mode of action is not yet fully understood. Such is the case for the receptor-ligand system constituted by endothelin receptor B (E...

Constitutive activation of the Wnt/␤-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/␤-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing ␤-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.Melanocytes originate in the neural crest as undifferentiated nonpigmented melanoblasts that migrate to the epidermis and hair follicles, where they differentiate and are responsible for skin and hair color. As melanocytes are not essential for survival and as mutations affecting the survival or differentiation of the melanocyte lineage are reflected in an obvious pigmentation phenotype, the melanocyte system represents an excellent model for understanding how signal transduction pathways coordinate the program of gene regulation underlying the genesis of a specific cell type. Importantly, constitutive activation of signaling pathways normally operating during melanocyte development is linked to the transformation of a melanocyte to a malignant melanoma (5, 16), a highly aggressive cancer, the incidence of which is increasing at an alarming rate (33).The Wnt signaling pathway (for reviews of Wnt signaling, see references 3 and 12) is critically required for development of the melanocyte lineage; in both zebrafish and mice, overexpression of components of the Wnt signaling pathway result in an increase in the number of melanocytes at the expense of neurons and glia (9, 11), and disruption of the Wnt-1 and Wnt-3a genes leads to complete loss of melanoblasts (24). Wnt proteins interact with frizzled receptors and lead to the inhibition of serine-threonine kinase glycogen synthase kinase 3␤. Phosphorylation of ␤-catenin by glycogen synthase kinase 3␤ is associated with the destabilization of ␤-catenin. Thus, increased Wnt signaling leads to stabilization of ␤-catenin and its translocation from the cytoplasm to the nucleus, where it can activate transcription via association with the Lef1 and Tcf transcription factors (1,22,29). A key role for Wnt signaling in melanocyte development is the activation of the promoter for the gene encoding the microphthalmia-associated transcription factor Mitf (10, 43). Mitf (19,23) is essential fo...

The vagal neural crest is the origin of majority of neurons and glia that constitute the enteric nervous system, the intrinsic innervation of the gut. We have recently confirmed that a second region of the neuraxis, the sacral neural crest, also contributes to the enteric neuronal and glial populations of both the myenteric and the submucosal plexuses in the chick, caudal to the level of the umbilicus. Results from this previous study showed that sacral neural crest-derived precursors colonised the gut in significant numbers only 4 days after vagal-derived cells had completed their migration along the entire length of the gut. This observation suggested that in order to migrate into the hindgut and differentiate into enteric neurons and glia, sacral neural crest cells may require an interaction with vagal-derived cells or with factors or signalling molecules released by them or their progeny. This interdependence may also explain the inability of sacral neural crest cells to compensate for the lack of ganglia in the terminal hindgut of Hirschsprung's disease in humans or aganglionic megacolon in animals. To investigate the possible interrelationship between sacral and vagal-derived neural crest cells within the hindgut, we mapped the contribution of various vagal neural crest regions to the gut and then ablated appropriate sections of chick vagal neural crest to interrupt the migration of enteric nervous system precursor cells and thus create an aganglionic hindgut model in vivo. In these same ablated animals, the sacral level neural axis was removed and replaced with the equivalent tissue from quail embryos, thus enabling us to document, using cell-specific antibodies, the migration and differentiation of sacral crest-derived cells. Results showed that the vagal neural crest contributed precursors to the enteric nervous system in a regionalised manner. When quail-chick grafts of the neural tube adjacent to somites 1-2 were performed, neural crest cells were found in enteric ganglia throughout the preumbilical gut. These cells were most numerous in the esophagus, sparse in the preumbilical intestine, and absent in the postumbilical gut. When similar grafts adjacent to somites 3-5 or 3-6 were carried out, crest cells were found within enteric ganglia along the entire gut, from the proximal esophagus to the distal colon. Vagal neural crest grafts adjacent to somites 6-7 showed that crest cells from this region were distributed along a caudal-rostral gradient, being most numerous in the hindgut, less so in the intestine, and absent in the proximal foregut. In order to generate aneural hindgut in vivo, it was necessary to ablate the vagal neural crest adjacent to somites 3-6, prior to the 13-somite stage of development. When such ablations were performed, the hindgut, and in some cases also the cecal region, lacked enteric ganglionated plexuses. Sacral neural crest grafting in these vagal neural crest ablated chicks showed that sacral cells migrated along normal, previously described hindgut pathways and formed isola...

The canonical Wnt signalling pathway induces the β-catenin/LEF transcription factors. It is activated in various cancers, most characteristically carcinomas, in which it promotes metastatic spread by increasing migration and/or invasion. The Wnt/β-catenin signalling pathway is frequently activated in melanoma, but the presence of β-catenin in the nucleus does not seem to be a sign of aggressiveness in these tumours. We found that, unlike its positive role in stimulating migration and invasion of carcinoma cells, β-catenin signalling decreased the migration of melanocytes and melanoma cell lines. In vivo, β-catenin signalling in melanoblasts reduced the migration of these cells, causing a white belly-spot phenotype. The inhibition, by β-catenin of migration was dependent on MITF-M, a key transcription factor of the melanocyte lineage, and CSK, a Src-inhibitor. Despite reducing migration, β-catenin signalling promoted lung metastasis in the Nras-driven melanoma murine model. Thus, β-catenin may play conflicting roles in the metastatic spread of melanoma, repressing migration while promoting metastasis. These results highlight that metastasis formation requires a series of successful cellular processes, any one of which may not be optimally efficient.

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.

SUMMARYWe aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain-and lossof-function b-catenin mutants in the melanocyte lineage. We found that any alteration of b-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through b-catenin.

Studying the development of melanoblasts, precursors of melanocytes, is challenging owing to their scarcity and dispersion in the skin embryo. However, this is an important subject because diverse diseases are associated with defective melanoblast development. Consequently, characterizing patterns of expression in melanoblasts during normal development is an important issue. This requires isolating enough melanoblasts during embryonic development to obtain sufficient RNA to study their transcriptome. ZEG reporter mouse line crossed with Tyr::Cre mouse line was used to label melanoblasts by enhanced green fluorescent protein (EGFP) autofluorescence. We isolated melanoblasts by FACS from the skin of E14.5-E16.5 embryos, and obtained sufficient cells for large-scale transcriptomic analysis after RNA isolation and amplification. We confirmed our array-based data for various genes of interest by standard quantitative real-time RT-PCR. We demonstrated that phosphatase and tensin homolog was expressed in melanoblasts but BRN2 was not, although both are involved in melanomagenesis. We also revealed the potential contribution of genes not previously implicated in any function in melanocytes or even in neural crest derivatives. Finally, the Schwann cell markers, PLP1 and FABP7, were significantly expressed in melanoblasts, melanocytes, and melanoma. This study demonstrates the feasibility of the transcriptomic analysis of purified melanoblasts at different embryonic stages and reveals the involvement of previously unreported genes in melanoblast development.

Altogether, these results show that mouse lemurs undergo deep physiological and genomic seasonal changes, without ever reaching a pathological stage. Further investigation is needed to decipher the underlying mechanisms, which may well be highly relevant for human therapeutic strategies.