This proposal focuses on the cellular and molecular underpinnings of choroid fissure (CF) closure. CF closure is critical for the containment of the retina and RPE within the optic cup. Defects in CF closure result in colobomas, a congenital defect in formation of the eye. Despite the importance of CF closure for normal eye development, we lack a comprehensive mechanistic understanding of the cellular and molecular regulation of the process in the human eye, or in any of the animal model systems utilized for modeling human eye development and disease. Research in our laboratory has focused on identifying the mechanisms underlying CF closure by using the zebrafish embryo as a model system, capitalizing on the strengths of the system for molecular and genetic manipulations, and in vivo imaging. Our preliminary data support a model in which CF closure occurs in three distinct stages. During Stage 1, retinoblast proliferation generates sufficient cells such that, as optic cup morphogenesis proceeds, the lateral edges of the CF are brought into close apposition. During Stage 2, the basement membrane (BM) lining the CF is degraded, enabling adhesion between cells lining the opposing sides of the CF. During Stage 3, cells on opposing sides of the fissure form adhesions and close the CF. While much research has focused on Stage 1 of CF closure, we know virtually nothing about the cellular and molecular mechanisms underlying BM breakdown (Stage 2) and tissue fusion (Stage 3) during CF closure. Indeed, no comprehensive studies to date have directly examined these processes. Experiments in this proposal focus on BM breakdown and tissue fusion during CF closure. We will identify the cellular components required for BM breakdown and tissue fusion to occur, and the cellular and molecular mechanisms that regulate them. Experiments in Aim 1 test the hypothesis that podosome/invadosome-like degradative complexes mediate BM breakdown during CF closure. Experiments in Aim 2 test the hypothesis that Par3/ Par6/aPKC complex activity is required in CF cells for the formation of p190RhoGAP and Rac1-dependent nascent adhesion complexes, which spread and mature to facilitate fusion of the lateral edges of the CF. To test these hypotheses, we utilize a combination of forward and reverse genetics, innovative spatio-temporal transgenic manipulations and in vivo time-lapse imaging. The results of this study will be instrumental in identifying the molecular and cellular regulation of CF closure, and how defects in these processes can result in colobomas. These experiments fit the mission of the NIH and the NEI because they have direct relevance to furthering our understanding of CF closure and colobomas, and more generally, they will facilitate a better understanding of optic cup morphogenesis, a fundamental process underlying formation of the eye.
Experiments in this proposal focus on choroid fissure closure, defects in which result in colobomas, a congenital defect in formation of the eye. The incidence of colobomas ranges from 2.6 in 10,000 births in the U.S. to 7.5 in 10,000 births in China, and colobomas are estimated to be present in 3-10% of all blind children worldwide. Colobomas are also present in over 50 human genetic disorders (OMIM), often associated with other ocular abnormalities. When completed, the results of our studies will have a significant impact on our understanding of colobomas by identifying the molecular and cellular mechanisms underlying choroid fissure closure that, when deficient, result in colobomas and by revealing new candidate coloboma loci.
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