Genetic and Chemical Screens for Factors Regulating Retinal Regeneration

遗传和化学筛选调节视网膜再生的因素

• 批准号: 9127241
• 负责人: JEFFREY MUMM
• 金额: $ 40.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-06 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9127241
• 关键词: Ablation; Biological Models; Blindness; Cell Culture Techniques; Cell Cycle Kinetics; Cells; Chemicals; Cicatrix; Communities; Development; Disease; Exogenous Factors; Eye; Fishes; Genes; Genetic; Genetic Screening; Hair Cells; Health; Human; Individual; Information Resources; Injury; Kinetics; Lead; Libraries; Mammals; Measures; Modeling; Molecular; Molecular Mechanisms of Action; Natural regeneration; Neuroglia; Neurons; Nitroreductases; Outcome; Photoreceptors; Process; Property; Regenerative response; Reporter; Research; Resources; Retina; Retinal; Retinal Photoreceptors; Series; Shapes; Source; Specificity; Stem cells; System; Testing; Therapeutic; Tissues; Toxic effect; Transgenic Organisms; Vision; Vision Disorders; Zebrafish; cell type; chemical genetics; dosage; drug discovery; high throughput screening; in vivo; mutant; novel; programs; regenerative; regenerative therapy; repaired; response; retinal neuron; retinal progenitor cell; retinal regeneration; retinal rods; rho; screening; small molecule; stem; stem cell biology; therapeutic development; tissue regeneration

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.

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能自我修复。这一事实导致了一种假设，即哺乳动物的视网膜不能自我修复。然而，最近的研究表明，即使在人类中，视网膜的再生潜力也是完整的；人类莫勒神经胶质细胞培养物能够产生视网膜神经元，并且莫勒神经胶质细胞在受到外源因素刺激时可以在哺乳动物模型中作为损伤诱导的视网膜干细胞发挥作用，尽管功能修复仍然难以捉摸。综上所述，这些研究表明：1)莫勒神经胶质细胞的再生潜能在人体内是保守的；2)了解视网膜干细胞是如何被调控的？特别是，莫勒胶质细胞对细胞丢失的反应？可以帮助开发再生疗法来治疗导致视力丧失和失明的疾病。最近，科学家发现了一种能够促进斑马鱼视网膜再生的干细胞，这为研究神经胶质细胞的再生潜能是如何调控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：1)鉴定再生缺陷斑马鱼突变体，这些突变体发育正常视网膜，但在细胞特异性消融后不能再生杆状光感受器（目的1）；2)发现促进视网膜再生的化合物。增加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（目的2）。我们已经建立了一个转基因系，其中可以诱导杆状光感受器细胞的选择性消融。使用该细胞系进行正在进行的试点筛选，我们已经成功地确定了三种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，用于测量单个鱼类荧光报告蛋白水平的变化。该系统使我们能够通过量化每个数千条鱼的细胞损失和替换动力学来发现影响杆状细胞再生的化合物