TRANSCRIPTIONAL CONTROL OF DEVELOPING CILIARY EPITHELIUM

描述(摘自申请者的摘要)：眼睛由三个人发育而成 细胞群、外胚层、神经上皮和神经脊细胞。一位伟大的 Deal知道眼睛发育的早期阶段，眼睛的分化 晶状体和神经视网膜的形成。然而，这种差异化 视杯前部进入睫状体和虹膜 结构仍然模糊不清。调查员的长期目标是确定 控制纤毛特性的转录因子 视杯前部的上皮细胞和调节发育的 这座建筑的。为了实现这一目标，转录机制控制 纤毛组织中长亚型胶原α1(IX)基因的表达 将对上皮组织进行研究。这种基因只在未来的 发育早期的纤毛上皮。因此，知道它的 转录调控将提供有关调控的信息 睫状体上皮细胞分化。在初步研究中，近端 分析了胶原α1(IX)基因的启动子。捆绑在一起的两个片段 鉴定了转录因子。两种蛋白质，cZic2和cDtx2，可能 与近端启动子的结合在单杂交筛选中被鉴定。至 标识其他顺式元素，此应用程序将使用一种新方法， 这使得人们能够研究活着的基因表达的调节 胚胎。携带不同启动子片段的载体与报告基因融合， GFP，将通过体内微电穿孔的方法导入发育中的眼睛。 在初步研究中，胶原α1(IX)基因的一个片段是 使用此方法标识的足以驱动 分化中的睫状体上皮中的报告基因。的具体目标 这一应用是为了(1)鉴定胶原中的顺式元件 转录调控所必需的α1(IX)启动子 (2)分析cZic2和cDtx2的结合特异性 α-胶原(IX)基因近端启动子的体外蛋白和 在体内，以及(3)鉴定与纤毛结合的转录因子 上皮特异性顺式作用元件(在第一个特定目的中确定) 使用单混合屏幕。最后，老鼠和人类的纤毛同源物 通过搜索DNA和DNA，将获得上皮特异性转录因子 蛋白质数据库及其与人类遗传性眼病的相关性将是 已评估。

DESCRIPTION (Adapted from applicant's abstract): The eye develops from three cell populations, ectoderm, neuroepithelium, and neural crest cells. A great deal is known about the early stages of eye development, the differentiation of the lens and the formation of the neural retina. However, the differentiation of the anterior part of the optic cup into the ciliary body and the iris structures remains obscure. The long-term goal of investigator is to determine the transcription factors that control the specification of the ciliary epithelium at the anterior of the optic cup and that regulate the development of this structure. To achieve this goal transcriptional mechanism controlling the long-isoform of collagen alpha1 (IX) gene expression in the ciliary epithelium will be investigated. This gene is expressed only in the prospective ciliary epithelium in the early stages of development. Therefore, knowing its transcriptional regulation will provide information on the regulation of ciliary epithelium differentiation. In preliminary studies, the proximal promoter of the collagen alpha1 (IX) gene was analyzed. Two fragments that bind transcription factors were identified. Two proteins, cZic2 and cDtx2 that may bind to the proximal promoter were identified in a one-hybrid screen. To identify additional cis-elements, this application will use a new approach, which permits one to study the regulation of gene expression in the living embryo. Vectors carrying different promoter fragments fused with reporter gene, GFP, will be introduced into the developing eye by in ovo microelectroporation. In preliminary studies, a fragment of the collagen alpha1(IX) gene was identified using this method that is sufficient to drive the expression of a reporter gene in the differentiating ciliary epithelium. The Specific Aims of this application are to (1) identify the cis-elements in the collagen alpha1(IX) promoter that are essential for transcriptional regulation in the ciliary epithelium, (2) analyze the binding specificity of cZic2 and cDtx2 proteins to the proximal promoter of the collagen alphal(IX) gene in vitro and in vivo, and (3) identify the transcription factors that bind to the ciliary epithelium-specific cis-acting elements (identified in the first specific aim) using a one-hybrid screen. Finally, mouse and human homologs of the ciliary epithelium-specific transcription factors will be obtained by searching DNA and protein databases and their relevance to human hereditary eye diseases will be evaluated.