Endothelin signalling in the development of neural crest-derived melanocytes.

In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest-derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early Dct+ cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+ melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-alpha-methylleucyl-D -1-methoxycarbonyltryptophanyl-D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+ melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation.     

Isolation and developmental expression of tyrosinase family genes in Xenopus laevis

The tyrosinase family of genes in vertebrates consists of three related members encoding melanogenic enzymes, tyrosinase (Tyr), tyrosinase-related protein-1 (TRP-1, Tyrp1) and tyrosinase-related protein-2 (Dct, TRP-2, Tyrp2). These proteins catalyze melanin production in pigment cells and play important roles in determining vertebrate coloration. This is the first report examining melanogenic gene expression in pigment cells during embryonic development of amphibians. Xenopus provides a useful experimental system for analyzing molecular mechanisms of pigment cells. However, in this animal little information is available not only about the developmental expression but also about the isolation of pigmentation genes. In this study, we isolated homologues of Tyr, Tyrp1 and Dct in Xenopus laevis (XlTyr, XlTyrp1, and XlDct). We studied their expression during development using in situ hybridization and found that all of them are expressed in neural crest-derived melanophores, most of which migrate through the medial pathway, and in the developing diencephalon-derived retinal pigment epithelium (RPE). Further, XlDct was expressed earlier than XlTyr and XlTyrp1, which suggests that XlDct is the most suitable marker gene for melanin-producing cells among them. XlDct expression was detected in migratory melanoblasts and in the unpigmented RPE. In addition, the expression of XlDct was detected in the pineal organ. The sum of these studies suggests that expression of the tyrosinase family of genes is conserved in pigment cells of amphibians and that using XlDct as a marker gene for pigment cells will allow further study of the developmental mechanisms of pigment cell differentiation using Xenopus.     

Development of Melanocyte Progenitors in Murine Steel Mutant Neural Crest Explants Cultured With Stem Cell Factor,Endothelin-3, or TPA

Stem cell factor (SCF) has been suggested to be indispensable for the development of neural crest cells into melanocytes because Steel mutant mice (i.e., Sl/Sf¹) have no pig-mented hairs. On the other hand, it has been demonstrated that the addition of endothelin 3 (ET-3) or TPA to neural crest cell cultures can induce melanocyte differentiation without addition of extrinsic SCF. In this study, we excluded the influence of intrinsic SCF by using SI/SI mouse embryos to study more precisely the effects of natural cytokines, such as extrinsic soluble SCF or ET-3, or chemical reagents, such as TPA or cholera toxin. We found that SCF is supplied within the wild-type neural crest explants and that ET-3 cannot induce melanocyte differentiation or proliferation without SCF. These results indicate that SCF plays a critical role in survival or G1/S entry of melanocyte progenitors and that SCF initially stimulates their proliferation and then ET-3 accelerates their proliferation and differentiation. TPA has the ability to elicit neural crest cell differentiation into melanocytes without exogenously added SCF but it is not as effective as SCF because many more melanocytes developed in the wild-type neural crest explants cultured with TPA.