Table_8_The regeneration-responsive element careg monitors activation of Müller glia after MNU-induced damage of photoreceptors in the zebrafish retina.XLSX

In contrast to mammals, zebrafish can regenerate their damaged photoreceptors. This capacity depends on the intrinsic plasticity of Müller glia (MG). Here, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. After methylnitrosourea (MNU) treatment, the retina became deteriorated and contained damaged cell types including rods, UV-sensitive cones and the outer plexiform layer. This phenotype was associated with the induction of careg expression in a subset of MG until the reconstruction of the photoreceptor synaptic layer. Single-cell RNA sequencing (scRNAseq) analysis of regenerating retinas revealed a population of immature rods, defined by high expression of rhodopsin and the ciliogenesis gene meig1, but low expression of phototransduction genes. Furthermore, cones displayed deregulation of metabolic and visual perception genes in response to retina injury. Comparison between careg:EGFP expressing and non-expressing MG demonstrated that these two subpopulations are characterized by distinct molecular signatures, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that TOR signaling became progressively switched from MG to progenitors. Inhibition of TOR with rapamycin reduced the cell cycle activity, but neither affected careg:EGFP expression in MG, nor prevented restoration of the retina structure. This indicates that MG reprogramming, and progenitor cell proliferation might be regulated by distinct mechanisms. In conclusion, the careg reporter detects activated MG, and provides a common marker of regeneration-competent cells in diverse zebrafish organs, including the retina.

与哺乳动物不同，斑马鱼可修复受损的光感受器。这一修复能力依赖于米勒胶质细胞（Müller glia, MG）的内在可塑性。本研究发现，作为鳍与心脏再生标志物的转基因报告基因careg，同样参与斑马鱼视网膜的修复过程。经甲基亚硝基脲（methylnitrosourea, MNU）处理后，斑马鱼视网膜发生退行性病变，受损细胞类型涵盖视杆细胞、紫外敏感视锥细胞及外网丛层。该表型与部分米勒胶质细胞中careg表达的诱导激活相关，这一过程持续至光感受器突触层完成重建。对再生视网膜开展单细胞RNA测序（single-cell RNA sequencing, scRNAseq）分析，结果显示存在一群未成熟视杆细胞，其特征为高表达视紫红质与纤毛发生基因meig1，但光转导基因表达量较低。此外，视锥细胞在视网膜损伤后出现代谢与视觉感知相关基因的表达失调。对表达careg:EGFP与不表达该报告基因的米勒胶质细胞亚群进行比较分析，结果显示二者具有截然不同的分子特征，表明它们对再生程序的响应存在异质性。核糖体蛋白S6磷酸化的动态变化分析显示，TOR信号通路的激活逐渐从米勒胶质细胞转向视网膜祖细胞。使用雷帕霉素抑制TOR通路可降低细胞周期活性，但既不影响米勒胶质细胞中careg:EGFP的表达，也不会阻碍视网膜结构的修复。这表明米勒胶质细胞的重编程与祖细胞增殖可能通过不同的调控机制实现。综上，careg报告基因可标记激活态的米勒胶质细胞，同时可作为斑马鱼包括视网膜在内的多种器官中具备再生能力细胞的通用标志物。