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

项目概要 冷血脊椎动物的穆勒胶质细胞可以重新进入细胞周期并继视网膜之后上升为光感受器 受伤，而哺乳动物已经失去了这种能力。作为 NEI 大胆目标计划的一部分，我们开展了一项 ü 综合分析损伤引起的斑马鱼基因表达和染色质可及性变化， 鸡和小鼠 M ller 神经胶质细胞，使我们能够识别进化保守基因和物种特异性基因 调节神经胶质细胞重编程的调节网络。这已经确定了一套专门的基因调控 小鼠中的网络限制了米勒神经胶质细胞的增殖和神经源性能力。我们的目标是利用这些 研究结果可以更全面地了解调节神经源能力的分子机制 哺乳动物米勒神经胶质细胞，并开发可以最大限度地产生神经胶质细胞的治疗方法 光感受器，同时不耗尽现有神经胶质细胞的数量。为此，我们建议 产生增殖和增殖的顶级候选负调节因子的个体功能丧失突变体 ü 使用 AAV 介导的 CRISPR/Cas9 基因破坏的神经发生能力。我们将首先验证功效 靶向单个 TF 的 sgRNA，全面分析反应性 M ller 胶质细胞中基因表达的变化 这些基因功能丧失后，并表征 M ller 胶质细胞衍生细胞的命运。我们随后将 进行 M ller 胶质细胞负调节因子功能的组合丧失重新编程以增强生成 ü 野生型和 Nfia/b/x 缺陷小鼠中神经胶质源性视网膜祖细胞的变化。最后，我们将结合CRISPR- 通过过表达 Ascl1、Crx 和 Nrl 介导的功能丧失分析来增强 M ller 的生成 野生型和营养不良型视网膜中的神经胶质细胞衍生的杆状光感受器。我们预测这些研究可能 大幅推进基于细胞的再生治疗，旨在恢复由于视网膜光感受器而丧失的视网膜光感受器 致盲疾病。

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.