Genetic Regulation of Eye Development

描述（由申请人提供）：本提案侧重于脉络膜裂隙（CF）闭合的细胞和分子基础。CF闭合对于视网膜和视杯内RPE的控制至关重要。CF闭合缺陷导致结肠瘤，这是一种先天性的眼睛形成缺陷。尽管CF闭合对正常眼睛发育很重要，但我们缺乏对人眼或任何用于模拟人眼发育和疾病的动物模型系统中这一过程的细胞和分子调控的全面机制理解。我们实验室的研究重点是通过使用斑马鱼胚胎作为模型系统，利用该系统的优势进行分子和遗传操作，以及体内成像，来确定CF关闭的机制。我们的初步数据支持一个模型，其中CF关闭发生在三个不同的阶段。在第1阶段，视网膜母细胞增殖产生足够的细胞，如

DESCRIPTION (provided by applicant): 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.