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...
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...
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...
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