Genetic and Chemical Screens for Factors Regulating Retinal Regeneration

描述（由申请人提供）：哺乳动物视网膜在视网膜细胞丢失后不会自我修复。这一事实导致了哺乳动物视网膜无法自我修复的假设。然而，最近的研究表明，即使在人类中，视网膜再生的潜力也是完整的;人类M？ller胶质细胞培养物能够产生视网膜神经元，并且M？ller胶质细胞在受到外源性因素刺激时，可以在哺乳动物模型中充当损伤诱导的视网膜干细胞，尽管功能修复仍然难以捉摸。总之，这些研究表明：1）Müller胶质细胞的再生潜力在人类中是保守的; 2）了解视网膜干细胞是如何调节的？特别是，米勒神经胶质细胞对细胞损失的反应？可以帮助开发再生疗法，治疗导致视力丧失和失明的疾病。Müller胶质细胞最近成为负责斑马鱼强大视网膜再生的干细胞，为研究Müller胶质细胞的再生潜力如何调节提供了一个很好的模型系统。迄今为止，斑马鱼的研究只涉及视网膜再生的几个分子调节剂。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）鉴定再生缺陷的斑马鱼突变体，这些突变体发育正常的视网膜，但在细胞特异性消融后不能再生视杆细胞（目的1），2）发现促进视网膜再生的化合物？增加视杆细胞替换动力学的速度或促进突变体中的视杆细胞再生（目的2）。我们已经建立了一个转基因株系，其中可以诱导视杆细胞的选择性消融。使用这个线进行中试筛选，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，显示不完全的视杆细胞替代，证明遗传筛选策略的原则。化学筛选将使用我们开发的体内高通量筛选（HTS）系统，用于测量个体鱼中荧光报告水平的变化。这个系统使我们能够发现化合物，通过量化细胞损失和更换的动力学在成千上万的鱼，每 天确定突变体如何破坏和化合物如何调节再生过程的细胞和分子机制将有助于我们进一步理解视网膜干细胞生物学。此外，该项目将产生/验证新的和有用的资源，为研究界：1）新的突变斑马鱼线，以确定如何再生控制？从细胞特异性修复到调节整个组织再生的机制，以及; 2）适用于广泛研究计划的用于药物发现的体内HTS平台。

DESCRIPTION (provided by applicant): 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 regulated?in particular, M�ller glia responses to cell loss?could 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 implicate 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 regeneration?increase 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 controlled?ranging 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.