Genetic and Chemical Screens for Factors Regulating Retinal Regeneration

项目总结 哺乳动物的视网膜在视网膜细胞丢失后不会自我修复。这一事实导致了这样的假设 哺乳动物的视网膜不能自我修复。然而，最近的研究表明， 用于再生的视网膜是完整的，即使在人类中也是如此；人类M？ler神经胶质细胞培养物能够提供 分化为视网膜神经元，M？ler胶质细胞可作为损伤诱导的视网膜干细胞发挥作用 哺乳动物模型在外源性因素的刺激下，尽管功能修复仍然难以捉摸。 综上所述，这些研究表明：1)M？ler胶质细胞的再生潜力在 人类和；2)了解视网膜干细胞是如何调节的，特别是M？ler 胶质细胞对细胞丢失的反应可能有助于疾病再生疗法的发展 导致视力丧失和失明。 M？ler glia最近作为干细胞出现，负责强健的视网膜再生 斑马鱼，为研究斑马鱼的再生潜力提供了一个很好的模型系统 神经胶质细胞受到调控。到目前为止，斑马鱼的研究只涉及到少数几个分子调控因子 视网膜再生。为了扩大对视网膜修复机制的理解，我们建议使用 无偏见的遗传和化学筛选方法：1)识别再生缺陷斑马鱼 发育正常视网膜但不能在细胞特异性后再生杆状感光细胞的突变体 消融(目标1)，和2)发现促进视网膜再生的化合物加快速度 在突变体中研究杆状细胞替换动力学或促进杆状细胞再生(目标2)。 我们已经建立了一种转基因系，在其中选择性消融视杆感光细胞可以 诱导性。将这一行用于正在进行的试点屏幕，我们已经成功地确定了三个 再生缺陷突变体和大量显示杆状细胞不完整的潜在突变体 替代，证明了基因筛查策略的原则证据。化学筛选将会 使用我们开发的体内高通量筛选(HTS)系统来测量 个体鱼中的荧光报告水平。这一系统使我们能够发现影响 通过量化数千条鱼的细胞丢失和替换的动力学研究杆状细胞的再生 天。确定突变体如何破坏和合成化合物的细胞和分子机制 调节再生过程将有助于加深对视网膜干细胞生物学的理解。 此外，该项目将为研究界产生/验证新的有用资源：1) 新的斑马鱼突变株系，用于定义细胞特异性的再生控制方式 修复调节整个组织再生的机制；2)体内药物HTS平台 适用于广泛研究项目的发现。

PROJECT SUMMARY The mammalian retina does not repair itself following retinal cell loss. This fact led to the assumption that the mammalian retina is incapable of self-repair. However, recent studies suggest the potential for the retina to regenerate is intact, even in humans; human M¿ller glia cell cultures are capable of giving rise to retinal neurons, and M¿ller glia cells can function as injury-induced retinal stem cells in mammalian models when stimulated with exogenous factors, albeit functional repair remains elusive. Together, these studies suggest that: 1) the regenerative potential of M¿ller glia cells is conserved in humans and; 2) an understanding of how retinal stem cells are regulatedin particular, M¿ller glia responses to cell losscould aid development of regenerative therapies for diseases causing vision loss and blindness. M¿ller glia recently emerged as the stem cells responsible for robust retinal regeneration in zebrafish, providing an excellent model system for investigating how the regenerative potential of M¿ller glia cells is regulated. To date, zebrafish studies have implicated only a few molecular regulators of retinal regeneration. To expand mechanistic understanding of retinal repair, we propose to use unbiased genetic and chemical screening approaches to: 1) identify regeneration deficient zebrafish mutants that develop a normal retina but fail to regenerate rod photoreceptors following cell-specific ablation (Aim 1), and 2) discover compounds that promote retinal regenerationincrease the pace of rod cell replacement kinetics or promote rod cell regeneration in mutants (Aim 2). We have established a transgenic line in which selective ablation of rod photoreceptor cells can be induced. Using this line for an ongoing pilot screen, we have succeeded in identifying three regeneration deficient mutants and numerous potential mutants that display incomplete rod cell replacement, demonstrating proof of principle of the genetic screening strategy. Chemical screens will use an in vivo high-throughput screening (HTS) system we developed for measuring changes in fluorescent reporter levels in individual fish. This system allows us to discover compounds that effect rod cell regeneration by quantifying the kinetics of cell loss and replacement in thousands of fish per day. Defining cellular and molecular mechanisms that underlie how mutants disrupt and compounds modulate the regenerative process will serve to further our understanding of retinal stem cell biology. Additionally, this project will generate/validate new and useful resources for the research community: 1) novel mutant zebrafish lines for defining how regeneration is controlledranging from cell-specific repair to mechanisms regulating whole tissue regeneration, and; 2) an in vivo HTS platform for drug discovery that is applicable to a broad range of research programs.