Elucidating and bypassing molecular mechanisms that suppress Muller glia-dependent regeneration of cones in two zebrafish models of chronic retinal damage

项目总结： 视网膜退行性疾病是社会面临的重大医学问题。一种潜在的令人兴奋的方法 恢复视力是指从内源性视网膜细胞群中再生丢失的视网膜神经元，即 米勒·格里亚。我们正在研究斑马鱼的这一过程，与哺乳动物不同，斑马鱼表现出天然的Müler神经胶质-- 依赖性视网膜再生反应。然而，我们对此的理解存在两大差距 视网膜再生反应。第一个原因是为什么快速的急性损伤表现出再生反应和 在人类视网膜退行性疾病中经常观察到的慢性慢性损害不会导致 斑马鱼的再生反应。我们理解的第二个差距是小胶质细胞的作用，即 中枢神经系统的免疫细胞，它是损伤引起的炎症的主要来源 以及一种已知的穆勒神经胶质细胞依赖的视网膜再生调节器。我们将在以下方面解决这两个差距 三个具体目标。目标1将确定两种不同的慢性斑马鱼视网膜变性的可能性 突变体(GOSH，早发型快速视锥感光细胞变性突变体和CEP290，晚发型慢变突变体 锥体变性突变体)，以诱导Müler胶质细胞增殖，并使用不同的刺激再生丢失的锥体。 我们将确定继发性急性损伤或引入的分子在多大程度上 刺激Müler神经胶质细胞增殖可诱导慢性损伤鱼类的锥体再生及其完成程度 再生过程是。在目标2中，我们将进行全面和公正的比较分析 应用多体单核RNA技术研究Müler神经胶质细胞和小胶质细胞的基因表达和染色质可及性 这两个慢性变性突变体和两个小鼠慢性视网膜的SEQ和ATAC-SEQ分析 退化突变体。我们将确定基因表达和染色质的异同 急性和慢性视网膜损伤的Müller胶质细胞和小胶质细胞的可及性。这些 生物信息学分析将揭示转录因子和信号(细胞因子、生长因子、配体/受体 对)分子，这些分子对急性损伤后的再生是必不可少的，并阻止了再生 斑马鱼视网膜长期受损。我们还将确定 斑马鱼和小鼠视网膜的慢性损伤，以确定这些调控成分的相似性 在斑马鱼和老鼠之间。然后，目标3将从功能上测试候选监管机构的角色 以前在我们的scRNA-Seq数据集中或在目标2中通过修改它们的表达或活性来识别 在长期和严重受损的斑马鱼视网膜中。这项工作将是第一个分子分析如何 视网膜再生在长期受损的斑马鱼视网膜中受到调节，并将在翻译过程中发挥关键作用 米勒神经胶质依赖的视网膜再生疗法在人类视网膜退行性疾病中的应用。

Project Summary: Retinal degenerative diseases are a major medical issue for society. One potentially exciting approach to restore vision is the regeneration of lost retinal neurons from an endogenous population of retinal cells, the Müller glia. We are studying this process in zebrafish, which unlike mammals, exhibits a natural Müller glia- dependent retinal regeneration response. However, there are two major gaps in our understanding of this retinal regeneration response. The first is why rapid acute damage exhibits a regeneration response and a slow chronic damage, which is what is often observed in human retinal degenerative diseases, does not induce a regeneration response in zebrafish. The second gap in our understanding is the role of the microglia, the immune cells of the central nervous system, which are the major source of inflammation resulting from damage and a known regulator of the Müller glia-dependent retinal regeneration. We will address these two gaps in three Specific Aims. Aim 1 will determine the potential of two different chronic zebrafish retinal degeneration mutants (gosh, an early onset rapid cone photoreceptor degeneration mutant and cep290, a late onset slow cone degeneration mutant) to induce Müller glia proliferation and regenerate lost cones using different stimuli. We will determine to what extent either a secondary acute damage or the introduction of molecules that stimulate Müller glia proliferation can induce cone regeneration in chronically damaged fish and how complete the regeneration process is. In Aim 2, we will conduct a comprehensive and unbiased, comparative analysis of gene expression and chromatin accessibility in Müller glia and microglia using a multiomic single-nuclear RNA- Seq and ATAC-Seq analysis in these two chronic degeneration mutants, along with two mouse chronic retinal degeneration mutants. We will determine the similarities and differences in gene expression and chromatin accessibility in the Müller glia and microglia between the acutely and chronically damaged retinas. These bioinformatic analyses will reveal transcription factors and signaling (cytokine, growth factors, ligand/ receptor pairs) molecules that are essential for regeneration following acute damage and blocking regeneration in the chronically damaged zebrafish retina. We will also determine the differences and similarities between the chronically damaged zebrafish and mouse retinas to determine how similar these regulatory components are between the zebrafish and mouse. Aim 3 will then functionally test the roles of the candidate regulators previously identified in our scRNA-Seq datasets or in Aim 2 by either modifying their expression or their activity in the chronically and acutely damaged zebrafish retina. This work will be the first molecular analysis of how retinal regeneration is regulated in the chronically damaged zebrafish retina and will be critical in the translation of Müller glial-dependent retinal regenerative therapies into human retinal degenerative diseases.