Cell Fate Regulation by Transcriptional Repression

描述(由申请人提供)：随着发育的进行，细胞获得新的命运，以便它们能够在生物体内执行其适当的功能。传统上，细胞命运的指定被认为是根据转录因子的激活，然后激活细胞类型特定的基因。在这项研究中，我们研究了有翼螺旋转录抑制因子foxd3在斑马鱼神经脊细胞命运选择中的作用。我们假设foxd3参与细胞命运选择，不是通过激活特定细胞类型的基因，而是通过阻止参与获得其他命运的基因的表达。我们认为foxd3特异性地抑制bHLH转录因子mitfa，这是黑素母细胞规范所必需的。我们将从以下几个方面探讨这一假说：1.我们将检验这一假说，即MITFA在神经胶质细胞前体细胞中瞬时转录，随后在非黑素细胞中被抑制。我们将比较转基因动物中色素细胞标志物的表达和MITFA启动子驱动的GFP mRNA的表达，并将使用荧光报告程序作为计时器来区分转录和翻译发生的时间。2.我们将检验foxd3抑制mitfa表达的假设。我们建议：鉴定mitfa启动子中的foxd3结合位点，并测试foxd3和mitfa是否共表达。3.我们将验证foxd3通过调节mitfa来调节神经嵴细胞命运的假设。我们将通过在整个胚胎和单个神经脊细胞中过度表达或反义吗啉敲除来测试foxd3对神经脊细胞命运的影响，并在mitfa和c-kit突变背景下测试foxd3功能的丧失/获得。这些实验将阐明一种可能的常见胶质/黑素母细胞前体细胞的分化，并有助于理解这种前体细胞的异常发育可能是先天性肿瘤的基础。

DESCRIPTION (provided by applicant): As development proceeds, cells acquire new fates so that they can carry out their proper function within the organism. Conventionally, cell fate specification has been thought of in terms of the activation of transcription factors that subsequently activate cell type specific genes. In this proposal we examine a role for a winged-helix transcriptional repressor, foxd3, in zebrafish neural crest cell fate choice. We hypothesize that foxd3 is involved in cell fate choice not by activating genes for particular cell types but instead by preventing expression of genes involved in acquisition of other fates. We propose that foxd3 specifically represses the bHLH transcription factor mitfa, which is necessary for melanoblast specification. We will address this hypothesis with the following aims: 1. We will test the hypothesis that mitfa is transiently transcribed in glial-pigment cell progenitors, and subsequently repressed in non-melanogenic cells. We will compare the expression of pigment cell markers to the expression of GFP mRNA driven by the mitfa promoter in transgenic animals, and will construct a transgenic line using a fluorescent reporter that acts as a timer to distinguish when transcription and translation occur. 2. We will test the hypothesis that foxd3 represses mitfa expression. We propose to: identify foxd3 binding sites in mitfa promoter, and test whether foxd3 and mitfa are co-expressed. 3. We will test the hypothesis that foxd3 regulates neural crest cell fate decisions by regulating mitfa. We will test the effects of foxd3 on neural crest cell fates by overexpression or antisense morpholino knockdown within the whole embryo and within individual neural crest cells, and test foxd3 loss/gain of function in the context of the mitfa and c-kit mutant backgrounds. These experiments will shed light on the differentiation of a possible common glial/melanoblast precursor and lead to understanding how abnormal development of this precursor might underlie congenital tumors.