Transcriptional Regulation of Retinal Development

描述(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，在各种内在和外在因素的作用下，有7类细胞是由多能祖细胞特异性产生的。最近涉及功能丧失和功能获得的分子遗传学研究发现，许多转录因子是视网膜发生的关键内在调节因子。这些因素被发现在不同的发育过程中起作用，以建立祖细胞的多能性，定义祖细胞的能力，决定细胞的命运，和/或指定细胞类型和亚型。因此，在视网膜形成过程中，转录因子在控制细胞规格和分化方面起着至关重要的作用。然而，尽管有这些重要的进展，许多参与视网膜发育的转录因子的分子机制和调控基因网络仍然知之甚少。在这一应用中，建议进行实验，重点研究Ptf1a和Tcfap2转录因子调控的分子和发育事件，这些转录因子导致无长突细胞和水平细胞分化。我们的初步数据和以前的工作使我们提出，Ptf1a可能与Rbpj辅因子形成一个关键的蛋白质复合体，激活Tcfap2a和2b的表达，而Tcfap2a和2b可能是无长突细胞和水平细胞分化所必需的。该项目主要通过追求三个具体目标来验证这一假说：i)研究Ptf1a-Rbpj复合体在指定无长突细胞和水平细胞命运中的需求。通过分析几种不同的Ptf1a和Rbpj功能丧失小鼠模型的视网膜表型，我们旨在验证Ptf1a可能与Rbpj形成Notch非依赖蛋白复合体的工作假设，以指定无长突细胞和水平细胞；ii)测试Tcfap2a和2b作为Ptf1a在无长突细胞和水平细胞发育中的靶点。通过抑制、染色质免疫沉淀和表型挽救实验，我们旨在研究Ptf1a是否能够直接激活Tcfap2a和2b的表达，进而介导Ptf1a在无长突细胞和水平细胞发育中的作用；以及iii)分析Tcfap2a和2b在无长突细胞和水平细胞分化中的作用。通过在视网膜前体细胞中错误表达Tcfap2a和2b，以及在发育中的视网膜中同时敲除这两个基因，我们旨在检验Tcfap2a和2b可能作为Ptf1a效应基因发挥作用的假设，这些基因是无长突细胞和水平细胞分化所多余的。这项拟议的研究有望阐明Ptf1a调控的分子机制，该分子机制控制着视网膜前体细胞的无长突细胞和水平细胞规格，从而为不同类型视网膜细胞分化的调控基因网络以及 基于干细胞的治疗视网膜变性的未来改进框架。 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和构建调控基因网络，从而从祖细胞中指定和分化不同类型的视网膜细胞。由于干细胞的分化通常概括了胚胎发生过程中发生的事件，因此更好地了解视网膜前体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，拟议的视网膜发育研究可能为未来基于干细胞的治疗人类视网膜变性的改进提供一个框架。

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.