Neural crest progenitors of the melanocyte lineage: coat colour patterns revisited

Neural crest-derived melanoblasts are the progenitors of melanocytes, the pigment cells of the skin, hair and choroid. Previous studies of adult chimaeric mice carrying different coat colour markers have suggested that the total melanocyte population is derived from a small number of melanoblast progenitors, each of which generates a discrete unilateral transverse band of colour. This work also suggested minimal mixing of cells between clones. We have used two complementary approaches to assess the behaviour of migrating clones of melanoblasts directly in the developing embryo. First, we made aggregation chimaeras between transgenic Dct-lacZ and non-transgenic embryos, in which lacZ is a marker for melanoblasts. Second, we generated transgenic mice carrying a modified lacZ reporter construct containing a 289 base pair duplication (laacZ) under the control of the Dct promoter. The laacZ transgene is normally inactive, but reverts to wild-type lacZ at low frequency, labelling a cell and all of its progeny at random. Mosaic embryos containing labelled melanoblast clones were generated. In contrast to previous data, chimaeric and mosaic embryonic melanoblast patterns suggest that: (1) there is a large number of melanoblast progenitors; (2) there is a pool of melanoblasts in the cervical region; (3) different cell dispersion mechanisms may operate in the head and trunk regions; and (4) there is extensive axial mixing between clones.     

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.     

Genetic control over the determination and proliferation of melanocyte stem cells in mammals]

Data obtained in mutant mouse strains provide evidence for multilocus control of determination and proliferation of melanocyte stem cells. Mice are known to have five loci (mi, Sp, s, Ls, Dom) controlling melanoblast determination. Locus mi is expressed in pluripotent cells of the neural crest from which melanocyte and neuron clones are formed; it is also expressed in a strain of ectomesenchyme cells. Loci Sp, s, ls and Dom are expressed somewhat later, probably during one of the last quantal cell cycles leading to the determination of unipotent melanocyte stem cells. Mutant genes of these loci impair the development of pigment cells as well as of ganglial neurons. Three loci (W, vs, Sl) control the proliferation of melanocyte stem cells. Mutations of locus W present in a single copy inhibit the proliferative activity of melanoblasts, whereas when present at the double dose they completely block their proliferation. Locus Sl is not expressed in melanocytes but acts in another cell system, which is very important for the proliferation of melanocyte stem cells. Mutant genes Ga, si and vit decrease the lifespan of stem cells for epidermal melanocytes.     

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.