Pax6 as a key regulator of lens development

描述（由申请人提供）：本项目的长期目标是通过研究DNA结合转录因子Pax 6阐明哺乳动物透镜发育的分子机制。以往的研究表明Pax 6对透镜祖细胞的建立和晶状体蛋白基因表达的调控是必不可少的。然而，Pax 6在透镜形态发生中的其他作用仍有待确定。遗传学研究表明Pax 6调控透镜前体细胞的细胞周期退出。透镜纤维细胞分化的外部调节由BMP和FGF信号传导通过转录因子Gata 3和Prox 1介导。使用染色质免疫沉淀结合DNA测序（ChIP-seq）和Pax 6突变晶状体中的RNA表达谱，我们现在已经确定了一组由Pax 6直接调控的基因，包括Prox 1，FGFR 2和Etv 1/ER 81。Prox 1的表达在透镜囊泡的后部上调，并且Prox 1调节Cdkn 1b/p27和Cdkn 1c/p57的表达，这两种蛋白质是透镜前体细胞退出细胞周期所需的。FGFR 2和Etv 1/ER 81是FGF信号传导的组分。Gata 3的表达局限于透镜囊泡的后部，位于Cdkn 1b/p27和Cdkn 1c/p57的上游。提示Pax 6依赖的细胞周期出口包括FGFR 2、Etv 1/ER 81、Prox 1。BMP信号以Pax 6非依赖性方式调节Gata 3的表达。Gata 3和Prox 1共同调节Cdkn 1b/p27和Cdkn 1c/p57的表达。为了实现这一长期目标，提出了以下具体目标：（1）确定Pax 6依赖的基因调控网络，控制Prox 1，FGFR 2和Etv 1/ER 81的表达，并阐明FGF依赖的Prox 1在胚胎透镜中的上调。(2)目的：建立Gata 3通过BMP和FGF信号在透镜细胞中表达的分子基础。(3)为了证明Cdkn 1b/p27和Cdkn 1c/p57的表达在透镜中通过Gata 3和Prox 1的组合在转录水平上调节。这些目标将通过识别和表征 利用转基因报告基因和细胞培养研究Prox 1、FGFR 2、Etv 1/Er 81和Gata 3基因的远端增强子，在透镜染色质和体外鉴定这些因子的结合位点，并分别鉴定Gata 3中BMP和FGF依赖性增强子以及Prox 1基因中FGF响应性增强子。

DESCRIPTION (provided by applicant): The long-term goal of this program is to elucidate the molecular mechanisms of mammalian lens development through studies of DNA-binding transcription factor Pax6. Previous studies have shown that Pax6 is essential for establishing lens progenitor cells and regulation of crystallin gene expression. However, additional roles of Pax6 in lens morphogenesis remain to be determined. Genetic studies have shown that Pax6 regulates cell cycle exit of lens precursor cells. The external regulation of lens fiber cell differentiation is mediated by BMP and FGF signaling, through transcription factors Gata3 and Prox1. Using chromatin immunoprecipitation in combination with DNA sequencing (ChIP-seq), and RNA expression profiling in Pax6 mutant lenses, we have now identified a group of genes directly regulated by Pax6 including Prox1, FGFR2 and Etv1/ER81. Expression of Prox1 is upregulated in the posterior part of the lens vesicle and Prox1 regulates expression of Cdkn1b/p27 and Cdkn1c/p57, two proteins required for cell cycle exit of lens precursor cells. FGFR2 and Etv1/ER81 are components of FGF signaling. Gata3 expression is restricted to the posterior part of lens vesicle, and is upstream of Cdkn1b/p27 and Cdkn1c/p57. These findings suggest that the Pax6-dependent cell cycle exit includes FGFR2, Etv1/ER81, Prox1. BMP signaling regulates expression of Gata3 in Pax6-independent manner. Gata3 and Prox1 jointly regulate expression of Cdkn1b/p27 and Cdkn1c/p57. In order to carry out this long-term goal, the following specific aims are proposed: (1) To define Pax6-dependent gene regulatory networks governing expression of Prox1, FGFR2, and Etv1/ER81, and to elucidate FGF-dependent up-regulation of Prox1 in the embryonic lens. (2) To establish molecular basis of Gata3 expression in lens cells via BMP and FGF signaling. (3) To demonstrate that expression of Cdkn1b/p27 and Cdkn1c/p57 is regulated in lens by a combination of Gata3 and Prox1 at the level of transcription. These Aims will be achieved through the identification and characterization of distal enhances in Prox1, FGFR2, Etv1/Er81 and Gata3 genes using transgenic gene reporter and cell culture studies, identification of binding sites of these factors in lens chromatn and in vitro, and identification BMP- and FGF-dependent enhancers in Gata3, and FGF- responsive enhances in Prox1 gene, respectively.