Transcriptional regulation of retinal cell differentiation and function

项目摘要 Müller神经胶质细胞在形态和基因上与视网膜祖细胞（RPC）非常相似， 表达谱，是发育中的视网膜中产生的最后一种细胞类型。然而，哺乳动物的缪勒神经胶质， 快速丧失增殖和生成神经元的能力。的分子机制 使RPC成为胶质细胞，并导致Müller胶质细胞失去神经原性能力仍然很差 明白我们最近已经确定LIM同源域因子Lhx 2作为MG的中枢调节因子 分化和功能，现在试图描述其作用机制。根据我们初步的 研究发现，我们假设Lhx 2与NFI和SoxE因子合作启动胶质细胞生成。我们提出 NFI和SoxE因子与控制神经胶质生成和神经胶质特异性基因表达的靶序列结合 在晚期RPC中，然后暴露Lhx 2的结合位点。Lhx 2然后诱导这些区域的染色质开放， 位点，这反过来又导致这些基因的稳定激活。相反，在有丝分裂后的神经胶质前体中， Rnf 12水平抑制Ldb 1/Lhx 2依赖的神经原性bHLH基因转录激活，最终 导致成熟的Müller神经胶质细胞中神经原性能力的永久丧失。我们的目标是确定NFI， SoxE和Lhx 2起驱动神经胶质分化和限制神经原性能力的作用。我们将首先确定 NFI因子是否促进视网膜中的胶质细胞生成能力和胶质细胞分化。接下来我们将测试 NFI和SoxE直接调节胶质细胞生成和胶质细胞特异性基因的表达，并决定是否 Lhx 2在分化胶质细胞中的选择性缺失降低了染色质在顺式调节元件处的可接近性， 控制神经胶质特异性基因。我们还将测试NFI和SoxE是否直接通过Lhx 2在晚期进行靶位点选择。 阶段RPC和神经胶质前体。除了这些关于神经胶质分化是如何控制的研究，我们还打算 以确定Lhx 2依赖的神经原性能力是如何在分化Müller胶质细胞中丢失的。我们将 确定Rnf 12是否以Lhx 2依赖的方式作用，以驱动神经原性能力的丧失， 分化成熟的Müller胶质细胞。最后，我们将鉴定介导Lhx 2依赖性 出生后视网膜的转录抑制。我们预计，通过以下方式充分了解该机制， Lhx 2调节胶质细胞生成的机制将确定药物和基因治疗的关键靶点， 通过控制Müller胶质细胞的去分化来实现视觉。

Project Summary Müller glia, which closely resemble retinal progenitor cells (RPCs) in their morphology and gene expression profile, are the last cell type generated in the developing retina. Mammalian Müller glia, however, rapidly lose the ability to proliferate and generate neurons following specification. The molecular mechanisms that allow RPCs to become gliogenic, and lead Müller glia to lose neurogenic competence remain poorly understood. We have recently identified the LIM homeodomain factor Lhx2 as a central regulator of MG differentiation and function, and now seek to characterize its mechanism of action. Based on our preliminary findings, we hypothesize that Lhx2 cooperates with NFI and SoxE factors to initiate gliogenesis. We propose that NFI and SoxE factors bind to target sequences that control expression of gliogenic and glial-specific genes in late-stage RPCs, which then exposes binding site for Lhx2. Lhx2 then induces chromatin opening at these sites, which in turn leads to stable activation of these genes. In postmitotic glial precursors, in contrast, rising levels of Rnf12 inhibit Ldb1/Lhx2-dependent transcriptional activation of neurogenic bHLH genes, eventually leading to a permanent loss of neurogenic competence in mature Müller glia. We aim to determine how NFI, SoxE, and Lhx2 act to drive glial differentiation and restrict neurogenic competence. We will first determine whether NFI factors promote gliogenic competence and glial differentiation in retina. We will next test whether NFI and SoxE directly regulate expression of gliogenic and glial-specific genes, and determine whether selective loss of Lhx2 in differentiating glia reduces chromatin accessibility at cis-regulatory elements that control glial-specific genes. We will also test whether NFI and SoxE direct target site selection by Lhx2 in late- stage RPCs and glial precursors. In addition to these studies of how glial differentiation is controlled, we intend to determine how Lhx2-dependent neurogenic competence is lost in differentiating Müller glia. We will determine whether Rnf12 acts in an Lhx2-dependent manner to drive loss of neurogenic competence in differentiating and mature Müller glia. Finally, we will identify molecules that mediate Lhx2-dependent transcriptional repression in postnatal retina. We anticipate that a full understanding of the mechanism by which Lhx2 regulates gliogenesis will identify key targets for drug and gene-based therapies aimed at restoring vision through controlled dedifferentiation of Müller glia.