Table_3_The Regenerating Adult Zebrafish Retina Recapitulates Developmental Fate Specification Programs.DOCX

Adult zebrafish possess the remarkable capacity to regenerate neurons. In the damaged zebrafish retina, Müller glia reprogram and divide to produce neuronal progenitor cells (NPCs) that proliferate and differentiate into both lost neuronal cell types and those unaffected by the damage stimulus, which suggests that developmental specification/differentiation programs might be recapitulated during regeneration. Quantitative real-time polymerase chain reaction revealed that developmental competence factors are expressed following photoreceptor damage induced by intense light or in a genetic rod photoreceptor cell ablation model. In both light- and N-Methyl-D-aspartic acid (NMDA)-damaged adult zebrafish retinas, NPCs, but not proliferating Müller glia, expressed fluorescent reporters controlled by promoters of ganglion (atoh7), amacrine (ptf1a), bipolar (vsx1), or red cone photoreceptor cell competence factors (thrb) in a temporal expression sequence. In both damage paradigms, atoh7:GFP was expressed first, followed by ptf1a:EGFP and lastly, vsx1:GFP, whereas thrb:Tomato was observed in NPCs at the same time as ptf1a:GFP following light damage but shifted alongside vsx1:GFP in the NMDA-damaged retina. Moreover, HuC/D, indicative of ganglion and amacrine cell differentiation, colocalized with atoh7:GFP prior to ptf1a:GFP expression in the ganglion cell layer, which was followed by Zpr-1 expression (red/green cone photoreceptors) in thrb:Tomato-positive cells in the outer nuclear layer in both damage paradigms, mimicking the developmental differentiation sequence. However, comparing NMDA- to light-damaged retinas, the fraction of PCNA-positive cells expressing atoh7:GFP increased, that of thrb:Tomato and vsx1:GFP decreased, and that of ptf1a:GFP remained similar. To summarize, developmental cell specification programs were recapitulated during retinal regeneration, which adapted to account for the cell type lost.

成年斑马鱼具备令人瞩目的再生神经元的潜能。在受损的斑马鱼视网膜中，穆勒细胞重编程并分裂，产生神经元祖细胞（NPCs），这些细胞增殖并分化为丧失的神经元细胞类型以及未受损伤刺激影响的细胞类型，这表明在再生过程中可能重现了发育的特化/分化程序。定量实时聚合酶链反应揭示了在强光诱导的感光细胞损伤或遗传性棒状感光细胞消融模型中，发育能力因子在感光细胞损伤后表达。在光损伤和N-甲基-D-天冬氨酸（NMDA）损伤的成年斑马鱼视网膜中，NPCs表达荧光报告基因，这些基因受神经节（atoh7）、无长突（ptf1a）、双极（vsx1）或红色圆锥形感光细胞能力因子（thrb）启动子的调控，其表达顺序呈现时间性。在两种损伤模型中，atoh7:GFP首先表达，随后是ptf1a:EGFP，最后是vsx1:GFP，而thrb:Tomato在光损伤后与ptf1a:GFP同时表达于NPCs中，但在NMDA损伤的视网膜中与vsx1:GFP的表达时间发生位移。此外，HuC/D，作为神经节和神经节细胞分化的标志，在ptf1a:GFP表达之前与atoh7:GFP共定位，随后在两种损伤模型中，Zpr-1（红/绿圆锥形感光细胞）的表达出现在thrb:Tomato阳性细胞的外核层中，模拟了发育分化顺序。然而，比较NMDA损伤和光损伤的视网膜，表达atoh7:GFP的PCNA阳性细胞比例增加，而thrb:Tomato和vsx1:GFP的比例减少，ptf1a:GFP的比例保持相似。总之，在视网膜再生过程中，发育细胞特化程序得以重现，并适应了细胞类型丧失的补偿机制。