Identification of gene regulatory networks that control proliferative and neurogenic competence in mammalian Müller glia

项目摘要 冷血脊椎动物的Müler胶质细胞可以重新进入细胞周期，并在视网膜后上升为光感受器 而哺乳动物已经失去了这种能力。作为NEI大胆目标倡议的一部分，我们进行了一项 ü 斑马鱼损伤诱导基因表达和染色质可及性变化的综合分析， 鸡和小鼠的M ler胶质细胞，使我们能够识别进化上一致的和物种特有的基因 调节神经胶质细胞重新编程的调节网络。这确定了一套专门的基因调控 小鼠的Tühat网络限制了M ler胶质细胞的增殖和神经源性能力。我们的目标是使用这些 对调节神经源性能力的分子机制有了更全面的了解 在哺乳动物的M ler胶质细胞中，并开发能够最大限度地产生神经胶质细胞来源的治疗方法 光感受器同时不会耗尽现有神经胶质细胞的数量。为了做到这一点，我们建议 产生增殖性和非增殖性基因负调控因子的单个功能缺失突变 ü 利用AAV介导的CRISPR/Cas9基因破坏的神经发生能力。我们将首先验证其疗效 靶向单个转录因子的sgRNAs，全面分析反应性M ler胶质细胞表达Channes的情况 随着这些基因功能的丧失，并表征了M ler胶质细胞的命运。到时候我们会的 进行M ler神经胶质细胞重编程负调控功能的组合性丧失以促进生成 ü 野生型和nfia/b/x缺陷小鼠神经胶质源性视网膜祖细胞的研究。最后，我们将结合CRISPR- 过表达Ascl1、CRX和NRL促进巨噬细胞生成的功能丧失分析 野生型和营养不良视网膜中的神经胶质源性视杆感受器。我们预测这些研究可能 大幅推进基于细胞的再生治疗，旨在恢复因 致盲的疾病。

Project Summary Müller glia of cold-blooded vertebrates can re-enter the cell cycle and rise to photoreceptors following retinal injury, while mammals have lost this ability. As part of the NEI Audacious Goals Initiative, we have conducted a ü comprehensive analysis of injury-induced changes in gene expression and chromatin accessibility in zebrafish, chick and mouse M ller glia, allowing us to identify both evolutionarily consüerved and species-specific gene regulatory networks that regulate glial reprogramming. This has identified a set of dedicated gene regulatory networks in mice tühat restrict proliferative and neurogenic competence in M ller glia. We aim to use these findings to gain a more complete insight into the molecular mechanisms that regulate neurogenic competence in mammalian M ller glia, and to develop treatments that can maximize generation of glial-derived photoreceptors while simultaneously not depleting the number of existing glia. To do this, we propose to generate individual loss of function mutants of the top candidate negative regulators of proliferative and ü neurogenic competence using AAV-mediated CRISPR/Cas9 gene disruption. We will first validate efficacy of sgRNAs targeting individual TFs, comprehensively profile chanüges in geüne expression in reactive M ller glia following loss of function of these genes, and characterize the fate of M ller glia-derived cells. We will then conduct combinatorial loss of function of negative regulators of M ller glia reprogramming to enhance generation ü of glial-derived retinal progenitor cells in wildtype and Nfia/b/x-deficient mice. Finally, we will combine CRISPR- mediated loss of function analysis with overexpression of Ascl1, Crx and Nrl to enhance generation of M ller glia-derived rod photoreceptors in both wildtype and dystrophic retina. We predict that these studies may substantially advance cell-based regenerative treatments aimed at restoring retinal photoreceptors lost due to blinding diseases.