Table_1_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能力。在受损的斑马鱼视网膜中，Müller胶质细胞重编程并分裂，产生神经祖细胞（NPCs），这些细胞增殖并分化为丢失的神经元细胞类型以及未受损伤刺激影响的细胞类型，这表明发育的特异性/分化程序可能在再生过程中得以重演。定量实时聚合酶链反应揭示了在由强光或遗传性棒状光感受器细胞消融模型诱导的光感受器损伤后，发育能力因子得以表达。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表达由神经节（atoh7）、无长突（ptf1a）、双极（vsx1）或红锥体光感受器细胞能力因子（thrb）启动子控制的荧光报告基因，其表达时间序列为：首先为atoh7:GFP，随后为ptf1a:EGFP，最后为vsx1:GFP，而在光损伤后，thrb:Tomato与ptf1a:GFP同时表达于NPCs，但在NMDA损伤的视网膜中，其表达随vsx1:GFP的表达而转移。此外，指示神经节细胞和无长突细胞分化的HuC/D与atoh7:GFP在神经节细胞层中表达前共定位，随后由Zpr-1表达（红色/绿色锥体光感受器）在outer nuclear layer中thrb:Tomato阳性细胞，在两种损伤模型中均模仿了发育分化序列。然而，在NMDA损伤与光损伤的视网膜比较中，表达atoh7:GFP的PCNA阳性细胞比例增加，thrb:Tomato和vsx1:GFP的比例减少，而ptf1a:GFP的比例保持相似。总之，发育细胞特异性程序在视网膜再生过程中得以重演，并据此调整以适应细胞类型的丢失。