Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Cellular and Molecular Medicine Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2025;2848:217-247. doi: 10.1007/978-1-0716-4087-6_14.
Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.
各种替代退行性疾病中丧失的视网膜神经元的策略正在研究中，包括刺激 Muller 胶质细胞（MG）作为损伤诱导的视网膜干细胞的内源性再生能力。固有的再生物种，如斑马鱼，为调节 MG 去分化为干细胞样状态以及 MG 和 MG 衍生祖细胞（MGPC）的增殖的机制提供了关键见解。有趣的是，促进 MG/MGPC 的增殖对于再生来说并不足够，但机制研究通常集中在这一措施上。为了全面描述再生过程，并促进调节细胞再生的因素的筛选，需要一种定量细胞替代的测定方法。因此，我们改编了一种自动化报告基因辅助表型筛选平台，以定量分析幼鱼选择性细胞消融后细胞再生动力学的速度。在这里，我们详细介绍了一种使用这种方法来识别控制选择性视网膜细胞消融后视网膜细胞再生速度的化学物质和基因的方法。
