The long-term goal of this program is to define the requirements for mammalian eye development by elucidating the mechanisms employed by three DNA-binding transcription factors, including Pax6, Prox1, and Gata3, in ocular lens morphogenesis. These factors form a regulatory network that controls key steps of mammalian lens formation. This network is governed by multiple tissue-specific and developmentally controlled enhancers regulated by BMP and FGF signaling through their signal-regulated transcription factors (SRTFs) such as AP-1, Ets, and Smads. Pax6 regulates all essential steps of lens development from the birth of lens progenitor cells, followed by the expansion and organization of the progenitor cells to form the lens placode, reciprocal invagination of the lens placode with the optic vesicle to form the lens vesicle and optic cup, and finally, the formation of posterior lens fibers and anterior lens epitelium that constitute the mature ocular lens. To elicidate the molecular mechanisms underlying this intricate developmental program, we employed unbiased genomic studies to generate a comprehensive map of Pax6-determinate chromatin-regulatory landmarks that revealed novel lens-specific Pax6-bound 5'- and 3'- distal enhancers of the Pax6 locus. We discovered that Pax6 directly regulates the actions of a second critical lens transcription factor, Prox1, and that differentiation of primary lens fibers also requires transcription factor Gata3. We also identified a lens-specific distal enhancer of the Gata3 locus that is enriched for cis-motifs recognized by the BMP- and FGF-regulated SRTFs. Collectivelly, our new data provide the basis for the central premise of this proposal: Pax6, Prox1, Gata3, and SRTFs orchestrate lens development as integrators of BMP and FGF signaling, employ Pax6 autoregulation to maintain lens-cell type, employ genetic cascade including Pax6 ? Prox1 and SRTFs/Prox1 ? Gata3 modules, and include direct regulation of Cdk inhibitors of cell cycle progression, Cdkn1b/p27 and Cdkn1c/p57, by Gata3, Prox1, and SRTFs. To reveal these regulatory mechanisms, we propose (1) To establish the molecular mechanisms required for lens cell formation during early embryogenesis, and (2) To elucidate the molecular mechanisms by which Pax6, Prox1, Gata3, and SRTFs control cell cycle exit-coupled differentiation of lens cells.The proposed studies are supported by strong preliminary data identifying two novel lens-specific Pax6 ?super-enhancers?, requirements of BMP and FGF factors for lens induction, direct regulation of Prox1 by Pax6 via two novel enhancers, and disrupted lens differentiation from the stage of lens vesicle in Gata3 conditional lens mutants. These studies are expected to define for the first time those sequential events required for the onset and propagation of Pax6 expression in the prospective lens ectoderm, identify molecular phenotype of lens cells, and establish gene regulatory networks (GRN) regulating cell cycle exit- coupled differentiation in the lens vesicle and of the primary lens fiber cells.
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 placode formation and invagination, cell cycle exit of the lens precursor cells, and terminal differentiation in lens fiber cells. Mutations in PAX6 and its downstream target genes including MAF, FOXE3, PITX3, DNase II?, and crystallins, are known to cause human congenital eye diseases. 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 (T2D). GATA3 haplo- insufficiency has been linked to cataract. Disrupted regulation of lens cell cycle exit is found through depletion of the retinoblastoma protein pRb (Rb1), E2F, Cdkn1b/p27, Cdkn1c/p57, Gata3, Smarca5, and other proteins. Studies of these proteins are critical for understanding malignant transformation and cancer.
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