Pax6 as a key regulator of lens development

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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 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 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 achived through the identification and characterization of distal enhancres 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 chromatin and in vitro, and identification BMP- and FGF-dependent enhancers in Gata3, and FGF- responsive enhancres in Prox1 gene, respectively.