Genetic and Chemical Screens for Factors Regulating Retinal Regeneration

描述(申请人提供)：哺乳动物的视网膜在视网膜细胞丢失后不会自我修复。这一事实导致了哺乳动物视网膜不能自我修复的假设。然而，最近的研究表明，视网膜再生的潜力是完整的，即使在人类中也是如此；人类M�ler胶质细胞培养能够产生视网膜神经元，在哺乳动物模型中，M�ler胶质细胞在外源因素刺激下可以作为损伤诱导的视网膜干细胞，尽管功能修复仍然难以实现。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中是保守的；2)了解视网膜干细胞是如何调节的，尤其是M�ler胶质细胞对细胞丢失的反应？可能有助于开发针对导致视力丧失和失明的疾病的再生疗法。最近，M�ler胶质细胞作为负责斑马鱼视网膜再生的干细胞出现，为研究M�ler胶质细胞的再生潜力是如何调控的提供了一个很好的模型系统。到目前为止，斑马鱼的研究只涉及到少数几个视网膜再生的分子调节因子。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来：1)鉴定再生缺陷斑马鱼突变体，这些突变体发育正常的视网膜，但在细胞特异性消融后未能再生杆状光感受器(目标1)，以及2)发现促进视网膜再生的化合物？加快杆状细胞替换动力学的速度或促进突变体中的杆状细胞再生(目标2)。我们已经建立了一个转基因细胞系，在其中可以诱导选择性消融视杆细胞。将该品系用于正在进行的中试筛选，我们已经成功地鉴定了三个再生缺陷突变体和许多显示不完全杆状细胞替换的潜在突变体，证明了遗传筛选策略的原理。化学筛选将使用我们开发的体内高通量筛选(HTS)系统，以测量单个鱼类中荧光报告水平的变化。这个系统使我们能够通过量化数以千计的鱼的细胞丢失和替换的动力学来发现影响杆状细胞再生的化合物 天。明确突变体如何扰乱再生过程以及化合物如何调节再生过程的细胞和分子机制将有助于加深我们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)用于定义再生如何控制的新型突变斑马鱼品系--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体内HTS药物发现平台。

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.