Genetic Regulation of Eye Development

描述(由申请人提供)：本提案侧重于脉络膜裂隙闭合的细胞和分子基础。Cf闭合对于将视网膜和RPE封闭在视杯内是至关重要的。CF闭合缺陷会导致眼球缺陷症，这是一种先天性的眼球形成缺陷。尽管闭合对眼睛的正常发育很重要，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的了解。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，利用该系统在分子和遗传操作方面的优势，以及活体成像来确定CF关闭的机制。我们的初步数据支持一个模型，在该模型中，CF闭合发生在三个不同的阶段。在第一阶段，视网膜母细胞增殖产生足够的细胞，例如 视杯形态发生过程中，视神经纤维束的侧缘紧密对接。在第二阶段，衬里CF的基底膜(BM)被降解，使得衬里CF相对两侧的细胞之间能够粘连。在第三阶段，裂隙相对两侧的细胞形成粘连并闭合CF。虽然许多研究都集中在CF关闭的第一阶段，但我们几乎对CF关闭过程中骨髓破裂(阶段2)和组织融合(阶段3)的细胞和分子机制知之甚少。事实上，到目前为止，还没有任何全面的研究直接审查这些过程。本方案中的实验重点是在CF闭合过程中BM的分解和组织融合。我们将确定发生骨髓分解和组织融合所需的细胞成分，以及调节它们的细胞和分子机制。目标1中的实验验证了足体/侵袭体样降解复合体在CF关闭过程中介导BM分解的假设。Aim 2中的实验验证了这一假设，即在CF细胞中需要Par3/Par6/aPKC复合体的活性，以形成依赖于p190RhoGAP和rac1的新生黏附复合体，这些复合体扩散和成熟以促进CF侧缘的融合。为了验证这些假说，我们利用了正向和反向遗传学、创新的时空转基因操作和活体时间推移成像的组合。这项研究的结果将有助于确定CF闭合的分子和细胞调节，以及这些过程中的缺陷如何导致缺损。这些实验符合NIH和NEI的使命，因为它们与加深我们对CF闭合和缺损的理解有直接关系，更广泛地说，它们将有助于更好地理解视杯形态发生，这是眼睛形成的基本过程。

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.