Retinal Epithelial Polarity and Cellular Patterning

描述(申请人提供)：视网膜工程很可能是治疗由视网膜细胞退化引起的失明疾病的最终方法。视网膜工程的前提是了解脊椎动物视网膜细胞模式形成的机制。我们的长期研究兴趣是定义这些机制。最近的研究表明，视网膜上皮细胞的极性在神经性视网膜模式的形成中起着至关重要的作用，但其涉及的机制在很大程度上尚不清楚。我们已经证明了三个斑马鱼极性基因(NOK、PARD3和zLinT)是建立成熟视网膜的细胞结构所必需的。这些蛋白质都含有成熟的蛋白质-蛋白质相互作用结构域。我们的数据表明，zLin7可能与NOK相互作用，NOK是神经视网膜和并列的视网膜色素上皮(RPE)之间相互作用所必需的。我们的中心假设是，NOK、PARD3和zLin7以发育调节的方式与其他蛋白质结合，共同建立和/或维持视网膜上皮细胞的适当极性，从而确保神经视网膜和RPE之间的准确相互作用。这些相互作用可能使视网膜模式形成所需的细胞迁移线索得到适当的分布。本项目要进行的研究的主要目标包括：1)通过制作和分析在眼睛中显示镶嵌NOK表达的转基因胚胎，确定NOK在控制神经视网膜细胞模式形成方面的功能在多大程度上需要NOK在RPE中的活动；2)确定NOK是否与zLin7‘S的一个或多个异构体发生物理作用以及参与相互作用的蛋白质结构域；3)通过亲和纯化、酵母双杂交和候选基因方法鉴定和鉴定在视网膜发育过程中与NOK、PARD3和zLin7复合体相互作用的蛋白质。

DESCRIPTION (provided by applicant): Retinal engineering is likely to be the ultimate cure for blinding disorders caused by degeneration of retinal cells. A prerequisite for retinal engineering is understanding the mechanisms of cellular pattern formation in the vertebrate retina. Our long-term research interest is to define these mechanisms. Recent studies suggest that the polarity of the retinal epithelia plays an essential role in neural retinal pattern formation; but the mechanisms involved are largely unknown. We have shown that three zebrafish polarity genes (nok, Pard3, and zLinT) are required to establish the cellular architecture of the mature retina. These proteins all contain well-established protein-protein interaction domains. Our data suggest that zLin7 likely interacts with Nok and that Nok is required for the interactions between the neural retina and the juxtaposed retinal pigmented epithelium (RPE). Our central hypothesis is that Nok, Pard3, and zLin7 associate with other proteins in a developmentally regulated fashion to collectively establish and/or maintain the proper polarity of retinal epithelia and consequently ensure accurate interactions between the neural retina and RPE. These interactions likely enable the proper distribution of cell migration cues required for retinal pattern formation. The major aims of the research to be pursued in this project include: 1) Determine to what extent Nok's function in controlling cellular pattern formation in the neural retina requires Nok's activity in the RPE by making and analyzing transgenic embryos that display mosaic Nok expressions in the eye; 2) Determine whether Nok physically interacts with one or more isoforms of zLin7's and what protein domains are involved in the interaction; 3) Identify and characterize the proteins that interact with Nok, Pard3, and zLin7 complexes during retinal development by using affinity purification, yeast two-hybrid, and candidate genes approaches.