Transcriptional Regulation of Retinal Development

描述（由申请人提供）：本提案的长期目标是了解导致不同视网膜细胞类型的确定、分化和维持的分子事件。在哺乳动物视网膜发生过程中，通过各种内在和外在因素的作用，从多能祖细胞中指定了七类细胞。最近的分子遗传学研究涉及功能丧失和功能获得的方法已经发现了一些转录因子作为视网膜发生的关键内在调节因子。发现这些因子在不同的发育过程中起作用，以建立祖细胞多能性，定义祖细胞能力，决定细胞命运和/或指定细胞类型和亚型。因此，转录因子在视网膜形成过程中控制细胞的特化和分化中起着重要作用。尽管有这些重要的进展，然而，参与视网膜发育的许多转录因子的分子机制和调控基因网络仍然知之甚少。 在本申请中，提出的实验将集中在Ptf 1a和Tcfap 2转录因子，导致无长突和水平细胞分化的分子和发育事件的调节。我们的初步数据和以前的工作使我们提出Ptf 1a可能与Rbpj辅因子形成一个关键的蛋白质复合物，以激活Tcfap 2a和2b的表达，这反过来又可能是无长突细胞和水平细胞分化所必需的。该项目主要旨在通过追求三个具体目标来测试这一假设：i）研究Ptf 1a-Rbpj复合物在指定无长突和水平细胞命运中的需求。通过分析几种不同的Ptf 1a和Rbpj功能丧失小鼠模型中的视网膜表型，我们的目的是测试Ptf 1a可能与Rbpj形成Notch非依赖性蛋白复合物以在视网膜发生期间指定无长突和水平细胞的工作假设; ii）测试Tcfap 2a和2b作为无长突和水平细胞发育中的Ptf 1a靶标。通过错表达、染色质免疫沉淀和表型拯救实验，我们旨在研究Ptf 1a是否能够直接激活Tcfap 2a和2b的表达，进而介导Ptf 1a在无长突细胞和水平细胞发育中的功能; iii）分析Tcfap 2a和2b在无长突细胞和水平细胞分化中的作用。通过在视网膜祖细胞中错误表达Tcfap 2a和2b，并在发育中的视网膜中同时敲低这两个基因，我们的目的是测试工作假设，即Tcfap 2a和2b可能作为Ptf 1a效应子基因，这些基因是无长突和水平细胞分化所必需的。这些研究有望阐明控制视网膜祖细胞无长突和水平细胞特化的Ptf 1a调节的分子机制，从而为不同视网膜细胞类型分化的调控基因网络提供重要的见解，并为进一步研究Ptf 1a调节的视网膜祖细胞分化机制提供参考。 为将来改善基于干细胞的治疗视网膜变性的框架。 公共卫生相关性：拟议的研究旨在了解视网膜发育的分子基础，特别是识别转录因子并建立调控基因网络，从而导致不同视网膜细胞类型从祖细胞的特化和分化。由于干细胞的分化通常重演胚胎发生过程中发生的事件，更好地了解视网膜祖细胞及其发育将使我们能够更好地操纵，维持和利用视网膜干细胞用于治疗目的。因此，对视网膜发育的拟议研究可能为未来改善基于干细胞的治疗人类视网膜变性提供框架。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the molecular events that lead to the determination, differentiation and maintenance of different retinal cell types. During mammalian retinogenesis, seven classes of cells are specified from multipotent progenitors by the action of various intrinsic and extrinsic factors. Recent molecular genetic studies involving loss-of-function and gain-of-function approaches have uncovered a number of transcription factors as pivotal intrinsic regulators of retinogenesis. These factors are found to act at different developmental processes to establish progenitor multipotency, define progenitor competence, determine cell fates, and/or specify cell types and subtypes. Therefore, transcription factors play essential roles in controlling cell specification ad differentiation during retinogenesis. Despite these important advances, however, the molecular machinery and regulatory gene networks of many transcription factors involved in retinal development still remain poorly understood. In this application, experiments are proposed that will focus on the molecular and developmental events regulated by the Ptf1a and Tcfap2 transcription factors that lead to amacrine and horizontal cell differentiation. Our preliminary data and previous work have led us to propose that Ptf1a may form a crucial protein complex with the Rbpj cofactor to activate the expression of Tcfap2a and 2b, which in turn may be redundantly required for the differentiation of amacrine and horizontal cells. This project primarily aims to test this hypothesis by pursuing three specific aims: i) to investigate the requirement for a Ptf1a-Rbpj complex in specifying amacrine and horizontal cell fates. By analyzing retinal phenotypes in several different Ptf1a and Rbpj loss- of-function mouse models, we aim to test the working hypothesis that Ptf1a may form a Notch-independent protein complex with Rbpj to specify amacrine and horizontal cells during retinogenesis; ii) to test Tcfap2a and 2b as Ptf1a targets in amacrine and horizontal cell development. By misimpression, chromatin immunoprecipitation and phenotype rescue experiments, we aim to investigate whether Ptf1a is be able to directly activate Tcfap2a and 2b expression which in turn may mediate the Ptf1a function in amacrine and horizontal cell development; and iii) to analyze the role of Tcfap2a and 2b in differentiation of amacrine and horizontal cells. By misexpressing Tcfap2a and 2b in retinal progenitors and by simultaneous knockdown of both genes in the developing retina, we aim to test the working hypothesis that Tcfap2a and 2b may function as Ptf1a effectors genes that are redundantly required for amacrine and horizontal cell differentiation. The proposed studies are expected to elucidate the Ptf1a-modulated molecular machinery that controls amacrine and horizontal cell specification from retinal progenitors, thereby providing important insights into te regulatory gene network underlying the differentiation of different retinal cell types as well as a framework for future improvement of stem cell-based therapy to treat retinal degeneration. PUBLIC HEALTH RELEVANCE: The proposed studies aim to understand the molecular basis of retinal development, in particular, to identify transcription factors and build regulatory gene networks leading to the specification and differentiation of different retinal cell types from progenitors. Because differentiation of stem cells normally recapitulates the events that occur during embryogenesis, a better understanding of retinal progenitors and their development will enable us to better manipulate, maintain and exploit retinal stem cells for therapeutic purposes. Thus, the proposed research on retinal development may provide a framework for future improvement of stem cell-based therapy to treat human retinal degeneration.