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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012200-22
Application #
9759932
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Araj, Houmam H
Project Start
2000-01-01
Project End
2022-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
22
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
081266487
City
Bronx
State
NY
Country
United States
Zip Code
10461
Martynova, Elena; Bouchard, Maxime; Musil, Linda S et al. (2018) Identification of Novel Gata3 Distal Enhancers Active in Mouse Embryonic Lens. Dev Dyn 247:1186-1198
Zhao, Yilin; Wilmarth, Phillip A; Cheng, Catherine et al. (2018) Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers. Exp Eye Res 179:32-46
Diacou, Raven; Zhao, Yilin; Zheng, Deyou et al. (2018) Six3 and Six6 Are Jointly Required for the Maintenance of Multipotent Retinal Progenitors through Both Positive and Negative Regulation. Cell Rep 25:2510-2523.e4
Esteban-Martínez, Lorena; Sierra-Filardi, Elena; McGreal, Rebecca S et al. (2017) Programmed mitophagy is essential for the glycolytic switch during cell differentiation. EMBO J 36:1688-1706
Cvekl, Ales; Zhao, Yilin; McGreal, Rebecca et al. (2017) Evolutionary Origins of Pax6 Control of Crystallin Genes. Genome Biol Evol 9:2075-2092
Cvekl, Ales; Callaerts, Patrick (2017) PAX6: 25th anniversary and more to learn. Exp Eye Res 156:10-21
Liu, Wei; Cvekl, Ales (2017) Six3 in a small population of progenitors at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice. Dev Biol 428:164-175
Cvekl, Ales; Zhang, Xin (2017) Signaling and Gene Regulatory Networks in Mammalian Lens Development. Trends Genet 33:677-702
Cavalheiro, Gabriel R; Matos-Rodrigues, Gabriel E; Zhao, Yilin et al. (2017) N-myc regulates growth and fiber cell differentiation in lens development. Dev Biol 429:105-117
Sun, Jian; Zhao, Yilin; McGreal, Rebecca et al. (2016) Pax6 associates with H3K4-specific histone methyltransferases Mll1, Mll2, and Set1a and regulates H3K4 methylation at promoters and enhancers. Epigenetics Chromatin 9:37

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