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...

Functional signaling of endothelin 3 (ET3) and its receptor B (ETRB)has been shown to be required for the development of neural crest (NC)-derived pigment cells in mouse, but the precise role of ET3 is not completely understood. Using the avian embryo as a model, we previously reported that ET3 promotes the survival and proliferation of unipotent melanocyte and bipotent glia-melanocyte precursors in trunk NC cultures. Here we investigated whether, at later stages, embryonic pigment cells respond to ET3. Such a possibility is supported by the previous finding that, in vivo, avian melanocytes express endothelin receptor B2 (ETRB2) during migration and after their differentiation in the skin. We found that in vitro ET3 exerts a dose-dependent stimulation of proliferation and melanogenesis in NC cells that had homed to the epidermis of embryonic quail dorsal skin. Moreover, in clonal cultures of skin-derived pigment cells, ET3 induces rapid cell divisions of clonogenic melanocytes that generate a mixed progeny of melanocytes and cells devoid of pigment granules and expressing glial markers in more than 40% of the colonies. It can therefore be concluded that ET3 is strongly mitogenic to embryonic pigment cells and able to alter their differentiation program, leading them to recapitulate the glial-melanocyte bipotentiality of their NC ancestors.epidermis ͉ quail embryo ͉ clonal cultures ͉ transdifferentiation ͉ Schwann cell myelin protein T he pigment cells of the body are one of the numerous derivatives of the neural crest (NC), a pluripotent structure of the vertebrate embryo that forms from the lateral ridges of the dorsal neural primordium. In addition to the melanocytes, the NC is the source of the neurons and glia of the peripheral nervous system, of endocrine cells and, at the cephalic level, of mesenchymal cells (the mesectoderm) that differentiate into connective tissues and smooth muscles and form most of the head skeleton (see refs. 1 and 2 for reviews). These multiple cell types arise from multipotent precursors, whose existence was demonstrated in avian and rodent NC at the premigratory and migratory stages (3-9). The population of migratory NC cells also includes lineage-restricted precursors, which increase in number as development proceeds (5, 10-15). Finally, the phenotype of the various NC derivatives is controlled by exposure of the cells to environmental signals during their migration and at their homing sites. A number of these signals have been characterized in mice by analyzing spontaneous mutations or by gene targeting (see refs. 16-18 for reviews).Thus the spontaneous or experimental inactivation of the murine genes encoding endothelin 3 (ET3) and its receptor (endothelin receptor B) (ETRB) causes defects of body pigmentation and posterior gut innervation (19,20). To understand the role for ET3͞ETRB signaling in the development of NC-derived cells, we have studied these ligand͞receptor system in avian NC ontogeny. Avian ETRB and ET3 genes were shown to have complementary expression patterns in...