Genetic and Chemical Screens for Factors Regulating Retinal Regeneration

项目摘要 哺乳动物视网膜在视网膜细胞损失后不会自我修复。这一事实导致了一种假设， 哺乳动物的视网膜无法自我修复然而，最近的研究表明， 即使在人类中，再生的视网膜也是完整的;人类M？ller神经胶质细胞培养物能够提供 视网膜神经元，M？ller胶质细胞可以作为损伤诱导的视网膜干细胞， 在哺乳动物模型中，当用外源因子刺激时，尽管功能性修复仍然难以捉摸。 总之，这些研究表明：1）M？ller神经胶质细胞的再生潜力是保守的， 人类; 2）了解视网膜干细胞是如何调节的特别是，M？ller 胶质细胞对细胞损失的反应可以帮助发展再生疗法， 导致视力丧失和失明。 米勒神经胶质细胞最近被认为是负责视网膜再生的干细胞， 斑马鱼，提供了一个很好的模型系统，用于研究M？ller的再生潜力 神经胶质细胞受到调节。到目前为止，斑马鱼的研究只涉及到一些分子调节剂， 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 无偏遗传和化学筛选方法：1）鉴定再生缺陷的斑马鱼 突变体发育正常的视网膜，但不能再生杆光感受器后，细胞特异性 消融术（目标1），以及2）发现促进视网膜再生的化合物加快步伐 的视杆细胞替代动力学或促进视杆细胞再生的突变体（目的2）。 我们已经建立了一个转基因株系，在该株系中可以选择性地切除视杆细胞。 诱导。使用这条线进行试点屏幕，我们已经成功地确定了三个 再生缺陷突变体和许多潜在的突变体显示不完整的视杆细胞 替代，证明遗传筛选策略的原理。化学筛将 使用我们开发的体内高通量筛选（HTS）系统来测量 个体鱼的荧光报告水平。这个系统使我们能够发现化合物， 通过量化成千上万条鱼的细胞损失和替换的动力学， 天定义突变体如何破坏和复合的细胞和分子机制 调节再生过程将有助于我们进一步了解视网膜干细胞生物学。 此外，该项目将为研究社区生成/验证新的有用资源：1） 新的突变斑马鱼系用于定义再生是如何控制的从细胞特异性 修复调节整个组织再生的机制，以及2）用于药物治疗的体内HTS平台 这一发现适用于广泛的研究计划。

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.