Regulation of Melanocyte Development and Differentiation

在过去的一年里，我们集中在三个主题的调节黑素细胞的发展和分化：（1）了解神经纤维蛋白在黑素细胞的发展和分化的作用，（2）检查生存的耳黑素细胞在小眼白杂合子小鼠，人类Waardenburg和Tietz综合征的模型，和（3）开发一个诱导系统的基因表达在体内的小鼠黑素细胞。我们的成果如下：（1）我们今年致力于更好地确定黑素细胞发育和黑素细胞分化期间神经纤维蛋白对Kit信号通路的活性之间的差异。为了证实我们的概念，神经纤维蛋白调节黑素基因的表达，通过不同的机制，其作为一个负调节Ras信号的影响，我们一直致力于优化小鼠原代黑素细胞培养系统，这将使我们能够最充分地研究这个问题。为此，我们利用细胞表面标记物，使我们能够分析原代培养物中黑素细胞的存在，并开始分选原代黑素细胞以获得纯的黑素细胞培养物。这些技术将有助于检查Ras信号通路扰动对黑素基因表达的影响，而不会因污染细胞群的存在而引起歧义。(2)我们的观察，耳黑素细胞的生存，而不是皮肤黑素细胞，是选择性损害小眼白杂合子小鼠促使我们调查皮肤衍生的因素，如试剂盒配体，内皮素-1，碱性成纤维细胞生长因子，可能会促进耳黑素细胞的生存在这种遗传背景的影响。使用耳蜗器官培养物的一组体外实验表明，多种因素的组合可以促进这些耳黑素细胞的存活，从而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见解。(3)我们已经产生了一套转基因创始人小鼠表达四环素反式激活剂tTA和rtTA的多巴色素互变异构酶（Dct）启动子。我们计划表征这些转基因株系的高效和诱导表达，并将其与现有的酪氨酸酶-rtTA小鼠进行比较，以确定胚胎发生期间表达开始的时间和在黑素细胞干细胞中的持续表达。在表征之后，我们计划使用小鼠在小鼠中诱导表达活化的Ras和活化的Ras信号通路组分，以了解Ras如何调节黑素细胞的发育和分化，并评估这些小鼠在小鼠黑色素瘤模型中的使用。

During the past year, we have focused on three topics in the regulation of melanocyte development and differentiation: (1) understanding the role of neurofibromin in melanocyte development and differentiation, (2) examining survival of otic melanocytes in the Microphthalmia-white heterozygous mouse, a model for human Waardenburg and Tietz syndromes, and (3) developing an inducible system for gene expression in vivo in murine melanocytes. Our progess is as follows:(1) We have worked this year to define better differences between the activity of neurofibromin on the Kit signalling pathway during melanocyte development and during melanocyte differentiation. To confirm our notion that neurofibromin regulates melanogenic gene expression via mechanisms distinct from its effects as a negative regulator of Ras signalling, we have devoted effort to optimizing mouse primary melanocyte culture systems that will permit us to examine this question most fully. To this end, we have utilized cell surface markers that permit us to analyze primary cultures for the presence of melanocytes and to begin sorting primary melanocytes to obtain pure melanocyte cultures. These techniques will be helpful for examining the effects of Ras signalling pathway perturbations on melanogenic gene expression without ambiguities introduced by the presence of contaminating cell populations.(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 Kit ligand, endothelin-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.(3) We have generated sets of transgenic founder mice expressing the tetracycline transactivators tTA and rtTA from the dopachrome tautomerase (Dct) promoter. We plan to characterize these transgenic lines for efficient and inducible expression and compare them to existing tyrosinase-rtTA mice for the timing of the onset of expression during embryogenesis and for persistent expression in melanocyte stem cells. Following characterization, we plan to use the mice to express activated Ras and activated Ras-signalling pathway components inducibly in mice to understand how Ras modulates melanocyte development and differentiation and to evaluate the use of these mice in murine melanoma models.