Regulation of Melanocyte Development and Differentiation

在过去的一年里，随着实验室向黑色素瘤研究的过渡，我们努力完成了对黑素细胞发育和分化调控的两个主题的研究：(1)了解神经纤维蛋白在黑素细胞发育和分化中的作用，以及(2)检测小眼球-白色杂合小鼠(人类Waardenburg和Tietz综合征的模型)黑素细胞的存活率。我们去年在这一节中描述的在小鼠黑素细胞中开发可诱导的基因表达系统的工作，在今年的项目2中进行了描述，因为它与我们在恶性黑色素瘤方面的工作更相关，而不是我们正在完成的黑素细胞分化方面的工作。我们的进展如下：(1)今年我们进一步确定了神经纤维蛋白在黑素细胞发育过程中和黑素细胞分化过程中Kit信号通路上的活性差异。为了更直接地验证神经纤维素通过其对RAS信号的影响来调节黑素生成基因表达的假设，我们成功地优化了用流式细胞术纯化原代小鼠黑素细胞的系统。这种方法使我们能够直接比较神经纤维蛋白单倍体不足对黑素生成基因表达的影响，而不考虑培养中存在其他类型细胞所引入的变量。这些实验的结果表明，与培养的黑素细胞中ERK激活相关的神经纤维蛋白单倍体不足，增加了原代小鼠黑素细胞中黑素生成基因的表达。与这一发现一致的是，通过抑制上游激酶MEK而使ERK激活的药物失活减少了野生型和神经纤维蛋白单倍体缺乏的黑素细胞中黑素生成基因的表达。这些结果，除了从混合原代培养的原代黑素细胞获得的结果外，还支持RAS依赖的信号在黑素生成基因表达中的作用。NF1基因缺陷小鼠与小鼠毛色Kit和MITF基因突变的遗传杂交的其他结果支持神经纤维蛋白在体内调节黑素细胞发育中的作用。(2)我们观察到，在小眼白杂合子小鼠中，眼球黑素细胞而不是皮肤黑素细胞的存活受到选择性的影响，这促使我们研究了皮肤来源的因子，如干细胞因子(SCF)/Kit配体、内皮素-1(ET-1)和碱性成纤维细胞生长因子(Basic FGF)在这种遗传背景下可能促进黑素细胞存活的作用。一系列使用耳蜗器培养的体外实验表明，多种因素的组合可以促进这些黑素细胞的存活，为部分缺乏黑素细胞转录因子MITF的细胞中黑素细胞存活的决定因素提供了额外的洞察力。此外，用分离的新生小鼠血管纹进行器官培养实验表明，ET-1和SCF的结合可以促进这些MITF缺陷的黑素细胞在工业环境中的存活。这些结果表明，小鼠毛囊内或附近的环境因素可能对促进MITF缺陷黑素细胞在该位置的存活起着重要作用。这一发现可能与先天性色素沉着瓦登堡综合征和蒂茨综合征患者的黑素细胞存活率有关。

During the past year, we have worked to complete research on two topics in the regulation of melanocyte development and differentiation as the lab undergoes a transition to focus upon melanoma research: (1) understanding the role of neurofibromin in melanocyte development and differentiation, and (2) examining survival of otic melanocytes in the Microphthalmia-white heterozygous mouse, a model for human Waardenburg and Tietz syndromes. Our work on developing an inducible system for gene expression in vivo in murine melanocytes, described in this section last year, is described under Project 2 this year because it is more relevant to our work on malignant melanoma than to work we are completing on melanocyte differentiation.Our progess is as follows:(1) We have worked further this year to define differences between the activity of neurofibromin on the Kit signalling pathway during melanocyte development and during melanocyte differentiation. To test in a more direct way the hypothesis that neurofibromin regulates melanogenic gene expression via its effects upon Ras signalling, we have successfully optimized a system to purify primary mouse melanocytes by flow cytometry. This approach has permitted us to compare directly the effect of neurofibromin haploinsufficiency upon melanogenic gene expression without accounting for variables introduced by the presence of other types of cells in culture. Results from these experiments show that neurofibromin haploinsufficiency, which is associated with Erk activation in cultured melanocytes, increases melanogenic gene expression in primary murine melanocytes. Consistent with this finding, pharmacologic inactivation of Erk activation by inhibition of the upstream kinase Mek decreases melanogenic gene expression in both wild-type and neurofibromin-haploinsufficient melanocytes. These results, in addition to those obtained from primary melanocytes in mixed primary cultures, support a role for Ras-dependent signaling in melanogenic gene expression. Additional results from the genetic crossing of Nf1-deficient mice with murine coat color mutations in the Kit and Mitf genes support a role for neurofibromin in the regulation of melanocyte development in vivo. (2) Our observation that the survival of otic melanocytes, as opposed to cutaneous melanocytes, is selectively compromised in Microphthalmia-white heterozygous mice prompted us to investigate the effect of skin-derived factors, such as stem cell factor (SCF)/Kit ligand, endothelin-1 (ET-1), and basic FGF that might promote otic melanocyte survival in this genetic background. A set of in vitro experiments using cochlear organ culture suggest that a combination of factors can promote survival of these otic melanocytes, providing additional insight into the determinants of melanocyte survival in cells partially deficient in the melanocyte transcription factor Mitf. Going further, organ culture experiments with isolated stria vascularis that has been isolated from neonatal mice show that the combination of ET-1 and SCF can promote survival of these Mitf-deficient melanocytes in the strial environment. These results show that environmental factors in or near the murine follicle are likely to be important for promoting the survival of Mitf-deficient melanocytes in this location. This finding may be relevant to the survival of melanocytes in patients with the congenital disorders of pigmentation Waardenburg syndrome and Tietz syndrome.