Pax6 as a key regulator of lens development

描述(申请人提供)：该项目的长期目标是通过研究DNA结合转录因子Pax6来阐明哺乳动物晶状体发育的分子机制。以往的研究表明，Pax6在晶状体祖细胞的建立和晶体蛋白基因表达的调控中起着至关重要的作用。然而，Pax6在晶状体形态发生中的其他作用仍有待确定。遗传学研究表明，Pax6调节晶状体前体细胞的细胞周期退出。BMP和成纤维细胞生长因子通过转录因子GATA3和Prox1介导晶状体纤维细胞分化的外在调节。利用染色质免疫沉淀结合DNA测序(CHIP-SEQ)，以及Pax6突变晶状体中的RNA表达谱，我们现在已经鉴定了一组由Pax6直接调控的基因，包括Prox1，FGFR2和ETV1/Er81。Prox1在晶状体囊泡的后部表达上调，并调节晶状体前体细胞周期退出所需的两种蛋白--CDKn1b/p27和CDKN1c/p57的表达。FGFR2和ETV1/Er81是成纤维细胞生长因子信号转导系统的组成部分。GATA3的表达局限于晶状体囊泡的后部，位于CDKn1b/p27和CDKN1c/p57的上游。这些发现表明，依赖于Pax6的细胞周期出口包括FGFR2、Etw1/Er81、Prox1。BMP信号以不依赖于Pax6的方式调节GATA3的表达。GATA3和Prox1共同调节CDKN1b/p27和CDKN1c/p57的表达。为了实现这一长期目标，提出了以下具体目标：(1)确定依赖于Pax6的基因调控网络，调控Prox1、FGFR2和ETV1/Er81的表达，并阐明依赖于FGF的Prox1在胚胎晶状体中的上调。(2)通过骨形态发生蛋白和成纤维细胞生长因子信号转导，建立晶状体细胞表达GATA3的分子基础。(3)证实GATA3和Prox1在转录水平上共同调控晶状体中CDKN1b/p27和CDKN1c/p57的表达。这些目标将通过识别和表征来实现 利用转基因报告基因和细胞培养技术对Prox1、FGFR2、Etw1/Er81和GATA3基因的远端增强进行了研究，鉴定了这些因子在晶状体色素和体外的结合部位，并分别在GATA3和Prox1基因中鉴定了BMP和成纤维细胞生长因子依赖的增强子，以及在Prox1基因中对成纤维细胞生长因子的反应增强。

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.