Table_4_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，产生神经元祖细胞（NPCs），这些细胞增殖并分化为受损神经元细胞类型以及未受损伤刺激影响的神经元细胞类型，这表明发育过程中的特异性/分化程序可能在再生过程中得以重现。定量实时聚合酶链反应揭示了在由强光诱导的视觉感受器损伤或在遗传性棒状视觉感受器细胞消融模型中，发育能力因子在损伤后表达。在由光或N-甲基-D-天冬氨酸（NMDA）损伤的成年斑马鱼视网膜中，NPCs而非增殖的Müller胶质细胞，在神经元细胞能力因子（atoh7）、无长突细胞能力因子（ptf1a）、双极细胞能力因子（vsx1）或红锥体视觉感受器细胞能力因子（thrb）的启动子控制下表达荧光报告基因，呈现时间表达顺序。在两种损伤模式中，首先表达atoh7:GFP，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，但在NMDA损伤的视网膜中与vsx1:GFP同步移动。此外，HuC/D，作为神经节细胞和神经节细胞分化的标志，在ptf1a:GFP表达之前与atoh7:GFP共定位，随后在两种损伤模式的外核层中thrb:Tomato阳性细胞中表达Zpr-1（红/绿锥体视觉感受器），模拟了发育分化顺序。然而，与光损伤视网膜相比，NMDA损伤视网膜中表达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。总结而言，在视网膜再生过程中，发育细胞特异性程序得以重现，并据此适应细胞类型损失的情况。