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: 8719118
• 负责人: JEFFREY MUMM
• 金额: $ 39.69万
• 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/8719118
• 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; Human; Human Development; Individual; Injury; Kinetics; Lead; Libraries; Mammals; Measures; Modeling; Molecular; Molecular Mechanisms of Action; Natural regeneration; Neuroglia; Neurons; Nitroreductases; Outcome; Photoreceptors; Process; Property; 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; public health relevance; 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无法自我修复，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，最近的研究表明视网膜再生的潜力是完整的。 ，即使在人类中；神经胶质细胞培养物能够产生视网膜神经元，并且 M¿当受到外源因素刺激时，小胶质细胞可以作为哺乳动物模型中损伤诱导的视网膜干细胞发挥作用，但功能修复仍然难以捉摸，这些研究表明：1）M¿胶质细胞在人类中是保守的；2）了解视网膜干细胞是如何调节的？特别是 M¿神经胶质细胞对细胞损失的反应？可以帮助开发针对导致视力丧失和失明的疾病的再生疗法。勒胶质细胞最近出现作为负责斑马鱼视网膜稳健再生的干细胞，为研究 M 的再生潜力提供了一个优秀的模型系统迄今为止，斑马鱼研究仅涉及视网膜再生的少数分子调节因子，为了扩大对视网膜修复机制的理解，我们建议使用公正的遗传和化学筛选方法来：1）识别再生缺陷的斑马鱼突变体。发育出正常的视网膜，但在细胞特异性消融后无法再生杆状光感受器（目标 1），以及 2）发现促进视网膜再生？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（目标2）我们已经建立了一种转基因系，其中可以诱导视杆细胞感光细胞的选择性消融，使用该系进行正在进行的试点筛选。我们成功地鉴定了三种再生缺陷突变体和许多表现出不完全视杆细胞替换的潜在突变体，证明了化学筛选策略将使用体内高通量筛选（HTS）系统的原理证明。我们开发的这个系统是为了测量个体鱼中荧光报告基因水平的变化，通过量化每条鱼的细胞损失和替换的动力学，我们可以发现影响视杆细胞再生的化合物。 定义突变体如何破坏和化合物调节再生过程的细胞和分子机制将有助于进一步了解视网膜干细胞生物学。此外，该项目将为研究界生成/验证新的有用资源：1）新颖。用于定义如何控制再生的突变斑马鱼系？范围从细胞特异性修复到调节整个组织再生的机制；2) 用于药物发现的体内 HTS 平台，适用于广泛的研究项目。