Genetic Analysis of Retinal Development

描述（由申请人提供）：本项目的长期目标是以斑马鱼为模型，了解脊椎动物视网膜发育的分子机制。该应用程序的重点是视网膜神经节细胞的发展，这是重要的正常视力和缺陷，其中涉及许多人类眼睛疾病，包括青光眼。最近的研究结果表明，刺猬（Hh）信号，细胞外信号调节激酶（ERK）的活性，和无调同源物5（ath 5），在视网膜神经节细胞的发育中的重要作用的直接或间接的证据。已知这些途径对蝇眼的发育至关重要。值得注意的是，视网膜神经节细胞的分化以波浪状的方式进行，让人想起蝇眼中R8光感受器的分化的进展。本申请旨在确定Hh，ERK和Ath 5通路在脊椎动物视网膜发生中的作用和相互作用。我们将使用斑马鱼突变体，由于平滑（smo）基因，Hh受体的一个组成部分，和逆转录病毒介导的基因表达系统的破坏，缺乏Hh信号，以操纵这些途径在视网膜中的活动。从这项研究中获得的信息将有重要的意义，在了解脊椎动物视网膜的进化和机制，包括人类在内的脊椎动物视网膜发生。本研究的具体目的是：1）确定Hh信号转导是否以细胞自主或非自主的方式调节相互卵裂球移植和逆转录病毒介导的激活Smo或蛋白激酶A（PKI）特异性抑制剂在smo突变型视网膜中表达的嵌合体中视网膜分化; 2）确定ERK途径是否是视网膜神经节细胞分化所必需的，并通过表达ERK途径的正性和负性调节物以及组成性活性和显性负性组分来鉴定ERK活性的上游调节物; 3）确定在发育的脊椎动物视网膜中Hh信号传导是否调节ERK活性，并且ERK反过来调节ath 5活性，以及ath 5诱导的视网膜神经节细胞分化是否需要Hh和/或ERK信号传导。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular mechanisms governing retinal development in vertebrates using zebrafish as a model. This application focuses on the development of retinal ganglion cells, which are important for normal vision and defects in which are involved in many human eye diseases including glaucoma. Recent results have demonstrated direct or indirect evidence for important roles of hedgehog (Hh) signaling, extracellular signal-regulated kinase (ERK) activity, and atonal homologue 5 (ath5), in retinal ganglion cell development. These pathways are known to be essential for development of the fly eye. Remarkably, the differentiation of retinal ganglion cells proceeds in a wave-like manner, reminiscent of progression of the differentiation of R8 photoreceptors in the fly eye. This application aims to determine the roles of and interactions among Hh, ERK, and Ath5 pathways in vertebrate retinogenesis. We will use zebrafish mutants that lack Hh signaling due to the disruption of smoothened (smo) gene, a component of Hh receptor, and a retrovirus-mediated gene expression system to manipulate the activities of these pathways in the retina. Information gained from this study will have important implications in understanding the evolution of the vertebrate retina and the mechanisms governing retinogenesis in vertebrates, including humans. The specific aims of this study are: 1) to determine whether Hh signal transduction regulates retinal differentiation in a cell-autonomous or nonautonomous manner in mosaics generated by reciprocal blastomere transplantation and by retrovirus-mediated retinal expression of either activated Smo or a specific inhibitor of protein kinase A (PKI) in smo mutant retina; 2) to determine whether the ERK pathway is essential for retinal ganglion cell differentiation and to identify the upstream regulators of ERK activity by expressing positive and negative regulators, and constitutively active and dominant-negative components of the ERK pathway; 3) to determine whether Hh signaling regulates ERK activity, and ERK in turn regulates ath5 activity in the developing vertebrate retina, and whether ath5-induced retinal ganglion cell differentiation requires Hh and/or ERK signaling.