Transcriptional regulation of retinal cell differentiation and function

项目摘要 在形态和基因上与视网膜前体细胞(RPC)极为相似的Müler胶质细胞 表达谱，是发育中的视网膜产生的最后一种细胞类型。然而，哺乳动物的Müler胶质细胞， 按照规定迅速失去增殖和生成神经元的能力。分子机制 允许RPC变得胶质生成，并导致Müler胶质细胞失去神经生成能力的情况仍然很差 明白了。我们最近发现LIM同源结构域因子LHX2是MG的中枢调节因子 分化和功能，现在试图表征其作用机制。根据我们的初步调查 发现，我们假设LHX2与NFI和SoxE因子协同启动神经胶质形成。我们建议 NFI和SoxE因子与控制胶质形成和胶质特异性基因表达的靶序列结合 在晚期RPC中，然后暴露LHX2的结合位点。然后LHX2在这些区域诱导染色质开放 这反过来又导致这些基因的稳定激活。相比之下，在有丝分裂后的神经胶质前体细胞中， Rnf12水平抑制LDB1/LHX2依赖的神经源性bHLH基因的转录激活，最终 导致成熟Müler神经胶质细胞神经源性能力的永久性丧失。我们的目标是确定NFI， SOXE和LHX2可促进神经胶质细胞分化，限制神经发生能力。我们将首先确定 NFI因子是否促进视网膜的神经胶质形成能力和神经胶质分化。接下来我们将测试一下 NFI和SoxE直接调节胶质形成基因和胶质特异性基因的表达，并决定是否 胶质细胞分化过程中LHX2的选择性丢失降低了染色质在顺式调控元件上的可及性 控制神经胶质细胞特异性基因。我们还将测试NFI和SoxE是否指导LHX2在晚些时候选择目标地点- 分期RPC和神经胶质前体细胞。除了这些如何控制神经胶质分化的研究外，我们还打算 为了确定LHX2依赖的神经源性能力是如何在分化Müller胶质细胞时丢失的。我们会 确定RNF12是否以LHX2依赖的方式作用于 分化和成熟的穆勒胶质细胞。最后，我们将确定介导LHX2依赖的分子 出生后视网膜中的转录抑制。我们预计，通过以下方式充分了解这一机制 哪个LHX2调节神经胶质形成将确定药物和基于基因的治疗的关键靶点 通过控制Müler神经胶质细胞的去分化而获得视觉。

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.