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.
Our application combines the study of lens cataract, a major cause of worldwide blindness, with the continued investigation of Pax6, a gene governing the formation of lens progenitor cells, lens induction, cell cycle exit of the lens precursor cells, an terminal differentiation in lens fiber cells. Mutations in PAX6 and its downstream target genes including MAF, PITX3, DNase IIb, and crystallins, are known to cause human congenital eye diseases. Disrupted regulation of lens cell cycle exit is found through depletion of the retinoblastoma protein (Rb1), E2F, Cdkn1b/p27, Cdkn1c/p57, and other proteins that are otherwise critical for understanding malignant transformation and cancer. Mutations in PAX6 also cause a variety of neurological disorders including autism, cognitive disorders, epilepsy and mental retardation. PAX6 has also been implicated in type II diabetes. Mutations in FGFR2 cause Apert-Crouzon syndrome characterized by craniofacial abnormalities. Although no eye abnormalities were reported in GATA3 haplo-insufficiency, heterozygous mutations in this gene are linked to hypoparathyroidism, sensori-neural deafness and renal dysplasia syndrome.
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