Hou L, Panthier J J, Arnheiter H
Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Development. 2000 Dec;127(24):5379-89. doi: 10.1242/dev.127.24.5379.
Genetic and cell culture analyses have shown that the development of melanocytes from neural crest-derived precursor cells critically depends on the tyrosine kinase receptor KIT and the basic-helix-loop-helix-leucine zipper transcription factor MITF. KIT and MITF show complex interactions in that MITF is needed for the maintenance of Kit expression in melanoblasts and KIT signaling modulates MITF activity and stability in melanocyte cell lines. Using primary neural crest cell cultures from embryos homozygous for a Kit null allele marked by an inserted LacZ gene (Kit(W-LacZ)), we show that the onset of Mitf expression in melanoblasts does not require KIT. In fact, provided that the melanocyte growth factor endothelin-3 is present, a small number of MITF/beta-Gal-positive cells can be maintained for at least 2 weeks in Kit(W-LacZ)/Kit(W-LacZ) cultures. These cells express several pigment cell-specific genes that are thought or have been shown to be activated by MITF, including dautochrome tautomerase, pMel 17/Silver and tyrosinase-related protein-1, but lack expression of the MITF target gene tyrosinase, which encodes the rate-limiting enzyme in melanin synthesis. Consequently, the cells remain unpigmented. Addition of cholera toxin, which elevates cAMP levels and mimics part of the KIT signaling pathway, increases the number of MITF-positive cells in Kit(W-LacZ)/Kit(W-LacZ) cultures, leads to tyrosinase expression, and induces the differentiation of melanoblasts into mature, pigmented melanocytes. Even when added on day 5-6 of culture, cholera toxin still rescues tyrosinase expression and differentiation. The results thus demonstrate that the presence of MITF is not sufficient for tyrosinase expression in melanoblasts and that KIT signaling influences gene expression during melanocyte development in a gene-selective manner.
基因和细胞培养分析表明，神经嵴衍生的前体细胞发育为黑素细胞关键依赖于酪氨酸激酶受体KIT和碱性螺旋-环-螺旋-亮氨酸拉链转录因子MITF。KIT和MITF表现出复杂的相互作用，即MITF是黑素母细胞中Kit表达维持所必需的，而KIT信号传导调节黑素细胞系中MITF的活性和稳定性。利用来自纯合Kit无效等位基因（由插入的LacZ基因标记，Kit(W-LacZ)）胚胎的原代神经嵴细胞培养物，我们发现黑素母细胞中Mitf表达的起始并不需要KIT。事实上，只要存在黑素细胞生长因子内皮素-3，在Kit(W-LacZ)/Kit(W-LacZ)培养物中就可以将少量MITF/β-半乳糖苷酶阳性细胞维持至少2周。这些细胞表达几种色素细胞特异性基因，这些基因被认为或已被证明可被MITF激活，包括二羟苯丙氨酸互变异构酶、pMel 17/银和酪氨酸酶相关蛋白-1，但缺乏MITF靶基因酪氨酸酶的表达，酪氨酸酶编码黑色素合成中的限速酶。因此，这些细胞仍无色素沉着。添加霍乱毒素可提高cAMP水平并模拟KIT信号通路的一部分，增加Kit(W-LacZ)/Kit(W-LacZ)培养物中MITF阳性细胞的数量，导致酪氨酸酶表达，并诱导黑素母细胞分化为成熟的、有色素的黑素细胞。即使在培养的第5-6天添加，霍乱毒素仍能挽救酪氨酸酶的表达和分化。因此，结果表明MITF的存在不足以使黑素母细胞中表达酪氨酸酶，并且KIT信号传导以基因选择性方式影响黑素细胞发育过程中的基因表达。
