The role of Insm1a in photoreceptor differentiation

描述(申请人提供)：视网膜色素变性(RP)和相关遗传性视网膜营养不良是导致失明的主要原因，目前还没有治愈的方法。视网膜退行性疾病的一种潜在治疗方法是基于细胞的移植疗法，即将祖细胞移植到患病的眼睛中，以取代丢失的光感受器。虽然这是一种令人兴奋的可能性，但移植治疗的实施必须克服几个挑战，包括视网膜前体细胞(RPC)与受体视网膜的整合效率低下，以及难以获得足够数量的光感受器前体用于临床应用。必须制定体外培养RPC的方案，以产生大量的光感受器前体，并在移植后促进其存活和分化。确定促进光感受器前体规范的转录网络是治疗策略进化的必要的下一步。我们实验室的长期目标之一是通过研究斑马鱼的光感受器发育和再生来为这些努力做出贡献。斑马鱼对研究光感受器生物学特别有用，因为除了杆状细胞外，它的视网膜还包含许多锥形细胞亚型。此外，与哺乳动物不同，斑马鱼的视网膜能够在实验损伤时再生神经元。我们最近发现转录抑制因子Insm1a是光感受器分化和再生的候选调节因子。这项提案中描述的实验将通过应用遗传学和分子遗传学方法来确定Insm1a在视网膜神经发生中的作用。我们的具体目标如下：特定目标一：在细胞水平上确定Insm1a在视网膜发育过程中调节光感受器分化中的作用，使用功能丧失和功能获得实验将Insm1置于已知光感受器发育基因的遗传层次中，并确定它是否是视网膜前体细胞周期退出所必需的；特定目标II：确定视网膜中Insm1调节的分子靶点。 采用基因表达谱、数字基因表达分析、体外报告分析和体内染色质免疫沉淀相结合的方法。我们的建议的完成将弥合我们对脊椎动物光感受器分化的分子机制的理解的重要空白，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理。

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.