TRANSCRIPTIONAL CONTROL OF DEVELOPING CILIARY EPITHELIUM

描述（改编自申请人的摘要）：眼睛从三个 细胞群、外胚层、神经上皮和神经嵴细胞。一个伟大 我们知道眼睛发育的早期阶段， 透镜和神经视网膜的形成。然而，分化 进入睫状体和虹膜 结构仍然模糊。研究者的长期目标是确定 控制纤毛特异性的转录因子 视杯前部的上皮，调节视杯的发育 这个结构。为了实现这一目标，转录机制控制 睫状体中胶原α 1（IX）基因的长型表达 将研究上皮。该基因仅在预期的 睫状上皮在早期发展阶段。所以，知道自己的 转录调控将提供有关调控的信息， 睫状上皮分化在初步研究中，近端 分析胶原α 1（IX）基因的启动子。结合的两个片段 鉴定了转录因子。两种蛋白质，cZic 2和cDtx 2， 在单杂交筛选中鉴定了与近端启动子的结合。到 为了鉴定另外的顺式元件，本申请将使用新的方法， 这使得人们可以研究生物体中基因表达的调节， 胚胎携带与报告基因融合的不同启动子片段的载体， GFP将通过卵内微电穿孔引入发育中的眼睛。 在初步研究中，胶原蛋白α 1（IX）基因的一个片段， 使用这种方法识别的，足以驱动 报告基因在睫状上皮分化。的具体目标 该应用是（1）鉴定胶原中的顺式元件 alpha1（IX）启动子，这些启动子对于转录调控是必需的， （2）分析cZic 2和cDtx 2的结合特异性 在体外将蛋白质与胶原蛋白α 1（IX）基因的近端启动子连接， 在体内，和（3）确定转录因子，结合到纤毛 上皮特异性顺式作用元件（在第一个特定目标中鉴定） 使用一个单混合屏幕。最后，小鼠和人类的纤毛同源物 上皮特异性转录因子将通过搜索DNA获得， 蛋白质数据库及其与人类遗传性眼病的相关性， 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.