BMP-4 regulates melanocyte differentiation in mouse neural crest cells just before they enter the Kit-dependent cycle of melanogenesis

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Genes encoding Kit and Kit ligand (Kit1) play essential roles in the differentiating melanoblasts. We previously established three immortal but distinct cell populations of mouse neural crest (NC) cells. NCCmelb4M5 cells do not express Kit and grow independently of Kit1 ; they have the potential to differentiate into NCCmelb4 cells, which are Kit-positive melanocyte precursors. NCCmelan5 cells show the characteristics of differentiated melanocytes. We investigated the effects of BMP-4 on the differentiation of immature melanocyte precursors. Three cell lines showed BMP-receptor expression. BMP-4 up-regulated Kit protein and mRNA expression in most immature NCCmelb4M5 cells. Noggin, a BMP-4 antagonist, dramatically decreased the Kit expression induced by BMP-4. Western blot analysis revealed that extrinsic BMP-4 leads to the phosphorylation of Smads and activates signaling in NCCmelb4M5 cells. We conclude that in NCCmelb4M5 cells, BMP-4 promotes melanocyte precursor differentiation via Kit expression. We further investigated the influence of BMP-4 in vitro using primary NC cells cultured from wild-type mice. Addition of BMP-4 to the medium increased the number of Kit-positive cells compared to untreated controls. An excess of BMP-4 added to wild-type NC explants abolished the growth of NC cells. Thus, we have identified BMP-4 as a differentiation factor for prepubertal Kit-negative melanoblasts just before they enter the Kit-dependent cycle of melanogenesis.