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

编码Kit和Kit配体（Kit1）的基因在黑素母细胞的分化中起重要作用。我们先前建立了三种不朽但不同的小鼠神经嵴（NC）细胞群。NCCmelb4M5细胞不表达Kit，独立于Kit1生长；它们有可能分化成NCCmelb4细胞，这是kit阳性的黑素细胞前体。NCCmelan5细胞具有分化黑色素细胞的特征。我们研究了BMP-4对未成熟黑素细胞前体分化的影响。3株细胞系表达bmp -受体。BMP-4上调大多数未成熟NCCmelb4M5细胞中Kit蛋白和mRNA的表达。Noggin是一种BMP-4拮抗剂，可显著降低BMP-4诱导的Kit表达。Western blot分析显示，外源性BMP-4可导致Smads磷酸化并激活NCCmelb4M5细胞中的信号传导。我们得出结论，在NCCmelb4M5细胞中，BMP-4通过Kit表达促进黑素细胞前体分化。我们利用野生型小鼠原代NC细胞进一步研究了BMP-4的体外影响。与未处理的对照组相比，在培养基中添加BMP-4增加了kit阳性细胞的数量。在野生型NC外植体中添加过量的BMP-4可抑制NC细胞的生长。因此，我们已经确定了BMP-4是青春期前kit阴性黑素细胞进入kit依赖性黑素形成周期之前的一个分化因子。

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.