Regulation of Melanocyte Development and Differentiation

在过去的一年里，我们专注于黑素细胞发育和分化调控的三个主题：(1)了解神经纤维蛋白在黑素细胞发育和分化中的作用，(2)检测小眼球-白色杂合子小鼠黑素细胞的存活率，人类Waardenburg和Tietz综合征的模型，以及(3)建立小鼠黑素细胞体内基因表达的诱导系统。我们的进展如下：(1)今年我们致力于确定在黑素细胞发育和分化过程中Kit信号通路上神经纤维蛋白活性的更好差异。为了证实我们的观点，即神经纤维素通过与其作为RAS信号负调节因子的作用不同的机制来调节黑素生成基因的表达，我们致力于优化小鼠原代黑素细胞培养系统，使我们能够最充分地研究这一问题。为此，我们利用了细胞表面标记，使我们能够分析原代培养的黑素细胞的存在，并开始对原代黑素细胞进行分选，以获得纯净的黑素细胞培养。这些技术将有助于检测RAS信号通路的扰动对黑素生成基因表达的影响，而不会因为污染细胞群体的存在而产生模棱两可的结果。(2)我们观察到，在小眼球白色杂合子小鼠中，黑素细胞(而不是皮肤黑素细胞)的生存受到选择性地损害，促使我们研究皮肤衍生因子，如Kit配体、内皮素-1和碱性成纤维细胞生长因子，这些因素可能促进黑素细胞在这种遗传背景下的生存。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新的见解。(3)我们已经产生了几组表达来自DOPAChrome互换酶(DCT)启动子的四环素反式激活剂TTA和RTTA的转基因创始小鼠。我们计划鉴定这些转基因品系的高效和可诱导表达，并将它们与现有的酪氨酸酶-RTTA小鼠在胚胎发育期间开始表达的时间和黑素细胞干细胞中持续表达的时间进行比较。在鉴定之后，我们计划在小鼠中诱导表达激活的RAS和激活的RAS信号通路组件，以了解RAS如何调控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应用。

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.