The role of Insm1a in photoreceptor differentiation

描述（由申请人提供）：视网膜色素变性（RP）和相关的遗传性视网膜营养不良是目前无法治愈的失明的主要原因。视网膜退行性疾病的一种潜在治疗是基于细胞的移植疗法，其中祖细胞被移植到患病的眼睛中以替代丢失的光感受器。虽然这是一个令人兴奋的可能性，但必须克服实施移植疗法的几个挑战，包括视网膜祖细胞（RPC）整合到受体视网膜中的效率低下，以及难以获得足够数量的光感受器前体用于临床应用。必须开发用于RPC的体外培养的方案，其将产生大量的光感受器前体，并且一旦移植将促进它们的存活和分化。定义促进感光细胞前体特异性的转录网络是治疗策略进化的必要下一步。我们实验室的长期目标之一是通过研究斑马鱼感光器的发育和再生来为这些努力做出贡献。斑马鱼对于研究光感受器生物学特别有用，因为它的视网膜除了视杆细胞外还含有许多视锥细胞亚型。此外，与哺乳动物不同，斑马鱼视网膜能够再生神经元以应对实验性损伤。我们最近已经确定了转录抑制因子Insm 1a作为一个候选人的光感受器的分化和再生的调节。本提案中描述的实验将通过应用遗传和分子遗传学方法来定义Insm 1a在视网膜神经发生过程中的作用。我们的具体目标如下：具体目标I：确定Insm 1a在视网膜发育过程中在细胞水平上调节感光细胞分化的作用，使用功能丧失和获得实验将Insm 1置于已知感光细胞发育基因的遗传层次中，并确定它是否是视网膜祖细胞周期退出所需的;具体目标II：确定视网膜中Insm 1调节的分子靶点， 使用基因表达谱分析、数字基因表达分析、体外报告基因测定和体内染色质免疫沉淀的组合。我们的提案的完成将弥合我们对脊椎动物感光细胞分化的分子机制的理解中的重要差距，并揭示与开发有效治疗人类视网膜疾病的方法相关的基本原则。

DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) and allied inherited retinal dystrophies are a leading cause of blindness for which there is currently no cure. One potential treatment for retinal degenerative diseases is cell-based transplantation therapy, whereby progenitor cells are transplanted into the diseased eye to replace the lost photoreceptors. While this is an exciting possibility, several challenges to the implementation of transplantation therapies must be overcome, including the inefficient integration of retinal progenitor cells (RPCs) into the recipient retina, and the difficulty of obtaining sufficient numbers of photoreceptor precursors for clinical application. Protocols for the in vitro culture of RPCs must be developed that will produce large numbers of photoreceptor precursors and will promote their survival and differentiation once transplanted. Defining the transcriptional networks that promote specification of photoreceptor precursors is a necessary next step for the evolution of therapeutic strategies. One of the long-term goals of our laboratory is to contribute to these efforts by studying photoreceptor development and regeneration in the zebrafish. The zebrafish is especially useful for studying photoreceptor biology, because its retina contains numerous cone subtypes in addition to rods. Furthermore, unlike mammals, the zebrafish retina is able to regenerate neurons in response to experimental damage. We have recently identified the transcriptional repressor Insm1a as a candidate regulator of photoreceptor differentiation and regeneration. The experiments described in this proposal will define the role of Insm1a during retinal neurogenesis through the application of genetic and molecular genetics approaches. Our specific aims are as follows: Specific Aim I: Determine the role of Insm1a in regulating photoreceptor differentiation during retinal development at the cellular level, using both loss- and gain-of-function experiments to place Insm1 within the genetic hierarchy of known photoreceptor development genes and to determine whether it is required for retinal progenitor cell cycle exit; Specific Aim II: Identify the molecular targets of Insm1 regulation in the retina, using a combination of gene expression profiling, digital gene expression analysis, in vitro reporter assays and in vivo chromatin immunoprecipitation. Completion of our proposal will bridge important gaps in our understanding of the molecular mechanisms of vertebrate photoreceptor differentiation, and reveal underlying principles relevant to the development of approaches for the effective treatment of human retinal disease.