Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro

Abstract

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.

Figures

Figure 1

Expression of ETRB2 and ETRB by quail NCC cultured in the absence (Left) and presence (Right) of ET3. In situ hybridization with ETRB2 (A, B, E, and F) and ETRB (C, D, G, and H) dig-labeled riboprobes. (A–D) At d3 ET3 promotes increase in ETRB2 (A and B) and ETRB (C and D) transcripts. (E–H) At d6, ET3-treated cultures show enhanced expression of ETRB2 (compare F to E: in F, the positive cells in blue occupy most of the microscopic field) and down-regulation of ETRB (G and H). (Bar = 90 μm.)

Figure 2

Melanogenesis and ETRB2 expression in d9-NCC cultures. (A) View of the culture dishes showing increased pigmentation of ET3-treated (Upper), as compared with control (Lower) cultures. (B) In situ hybridization of control cultures shows that pigment cells (with brown melanin) express ETRB2 (blue staining). (Bar = 40 μm.)

Figure 3

Expression of ETRB and ETRB2 by individual NCC after double in situ hybridization. D3-cultures hybridized with both radioactive ETRB and dig-labeled ETRB2 probes are viewed under epipolarized illumination. Individual cells express either ETRB2 (A) or ETRB (B) alone, or both (C), as detected by the presence of antidig staining and silver grains. (Bar = 16 μm.)

Figure 4

Differentiation of melanoblasts and glial cells in d9-NCC cultures in the absence (Left) and presence (Right) of ET3. (A and B) The population of melanoblast/cyte early marker-positive melanoblasts increases after ET3 treatment (B) as compared with controls (A). (C–F) SMP-immunoreative glial cells also expand in response to ET3 (compare C and D); pigment cells are visible in E (arrow) and F (same fields as C and D, respectively). A–D, UV light; E, phase-contrast; F, bright-field. (Bars = 260 μm in A and B; 83 μm in C–F.)