Regulation of Retinal Progenitor Cell Properties

描述(申请人提供)：视网膜疾病的细胞替代和再生疗法面临的一个主要挑战是产生具有治疗意义所必需的质量的视网膜细胞或组织。为了应对这一挑战，我们需要能够控制祖细胞/干细胞的生长和发展潜力，以便能够选择某些命运而不是其他命运，并且分化计划足够强大，以赋予所需的功能。对这一点的关键见解来自于使用从果蝇到小鼠的各种遗传易驯化的动物模型系统研究视网膜是如何发育的。在这个项目中，我们从促进神经发生、维持神经发生环境中的祖细胞和产生神经元多样性的角度，研究了关键的发育转录因子如何调节视网膜前体细胞(RPC)的神经发生特性。在第一个目的中，我们测试了这样的假设，即同源结构域蛋白Vsx2(以前称为Chx10)通过一个细胞自主回路来控制胚胎视网膜中神经发生的开始时间，该回路设置了前神经源性RPC中至少四个其他转录因子的适当表达水平/活性：MITF、p27Kip1、Pax6和Sox2。我们还测试了一种假设，即依赖Notch信号作为祖细胞维持机制的获得与这一时机有关。在第二个目标中，我们提出了一种假设，即同源结构域蛋白LHX2是视网膜神经发生过程中前体细胞维持、神经源性输出和前体能力的关键调节因子。利用条件失活遗传学绕过LHX2的S在视泡图案化和视杯形态发生中的早期和重要作用，我们将检验LHX2维持高神经发生潜能的RPC的模型，并通过这样做，与其他RPC建立动态平衡，使它们持续存在于视网膜发育的整个阶段。第二，我们将测试 模型认为，在选定的RPC中需要LHX2来响应来自有丝分裂后、分化的视网膜前体的负反馈信号，并且通过这样做，限制了特定RPC的产生 视网膜中的细胞类型和对神经元多样性的产生有贡献。第三，我们已经确定了视网膜发育早期的一个关键窗口，LHX2是RPC转换到另一种能力状态所必需的，这是产生后来的细胞类型所必需的。只有几个其他因素被提出来调节早期和晚期的能力，并将测试与LHX2的潜在关系。我们还将采用新描述的HA标签方法与转录组分析相结合，以发现由LHX2调控的促进能力转变的新基因。这些研究的完成将为在胚胎发生过程中如何控制RPC的维持和神经生成潜力提供新的见解，这些信息可以应用于干细胞工程和再生医学。

DESCRIPTION (provided by applicant): A major challenge facing cell replacement and regenerative therapies for retinal diseases is generating retinal cells or tissue at the quality an scale necessary to be therapeutically relevant. To meet this challenge, we need to be able to control the growth and developmental potential of progenitor/stem cells such that certain fates can be selected over others and differentiation programs are robust enough to impart desired functions. Key insights into this come from studying how the retina develops using various genetically tractable animal model systems ranging from Drosophila to mouse. In this project, we examine the question of how key developmental transcription factors regulate the neurogenic properties of retinal progenitor cells (RPCs), from the perspectives of promoting neurogenesis, maintaining progenitors in a neurogenic environment, and generating neuronal diversity. In the first aim, we test the hypothesis that the homeodomain protein Vsx2 (formerly Chx10) controls the timing of onset of neurogenesis in the embryonic retina through a cell-autonomous circuit that sets the proper expression levels/activities of a minimum of four other transcription factors in pre-neurogenic RPCs; Mitf, p27Kip1, Pax6, and Sox2. We also test the hypothesis that the acquisition of a dependence on Notch signaling as a mechanism for progenitor cell maintenance is linked to this timing. In the second aim, we address the hypothesis that the homeodomain protein Lhx2 is a key regulator of progenitor cell maintenance, neurogenic output, and progenitor competence during retinal neurogenesis. Using conditional inactivation genetics to bypass Lhx2's early and essential role in optic vesicle patterning and optic cup morphogenesis, we will test the model that Lhx2 maintains RPCs with high neurogenic potential, and by doing so, establishes a dynamic balance with other RPCs such that they persist through the entire interval of retinal development. Second, we will test the model that Lhx2 is required in select RPCs to respond to negative feedback signals from postmitotic, differentiating retinal precursors, and by doing so, limits the production of specific cell types and contributes to the generation of neuronal diversity in the retina. Third, we've identified a critical window during early retinal development Lhx2 is required for RPCs to transition into another competence state necessary for production of later cell types. Only a couple of other factors have been proposed to regulate early versus late competence, and a potential relationship with Lhx2 will be tested. We will also adapt the newly described HA-tagging method with transcriptome profiling to discover new genes regulated by Lhx2 that promote the transition in competence. Completion of these studies will provide new insight into how RPC maintenance and neurogenic potential is controlled during embryogenesis, information that can be applied to stem cell engineering and regenerative medicine.