Fig. 2

Cytokine genes are dynamically expressed following light damage. (A) qRT-PCR analysis of three cytokine genes, which are commonly associated with inflammatory effects: il-1? (red line, yellow dot), il-6 (red line, green dot), and ifng1 (red line, red dot) and three cytokine genes, which are usually associated with inhibiting inflammation: il-10 (blue line, blue dot), il-13 (blue line, yellow dot), il-4 (blue line, green dot), across light treatment (LT) time course (0, 6, 12, 24, 36, and 72 h LT) and 1-day (120 h) and 7-days (264 h) recovery (after completing 96 h constant LT). All values are normalized to 18 s rRNA reference gene. The mRNA expression levels are displayed as log2-fold change relative to 0 h LT from three independent replicates with a pool of 6-7 dorsal retinas for each replicate. (B) qRT-PCR analysis of il-1? expression at 0 h and 24 h LT in Standard Control morphants (S.C. MO, blue circles) and tnfa morphants (red circles). Data was normalized to 18 s rRNA reference gene and displayed as log2-fold change relative to the S.C. MO control group. For the qRT-PCR, three independent replicates were performed with a pool of 6-7 dorsal retinas for each replicate. (C,D) Previously published single-cell RNA-Seq data (Hoang et al., 2020) were analyzed by violin plots for il-1? expression in microglia (C) and Müller glia (D). The time points included in the snRNA-Seq dataset correspond to 0, 4, 10, 20, and 36 h LT. (E?J?) Confocal images of dark-adapted albino;Tg(gfap:EGFP)nt11 zebrafish retinas that were isolated at 0 (E?E???), 6 (F?F???), 12 (G?G???), 24 (H?H???), 36 (I?I???), and 72 h LT (J?J???). Retinal sections were labeled by in situ hybridization with a probe for il-1? magenta: (E?J,E???J???), grayscale: (E??J?). Sections were also immunostained to detect GFP Müller glia, green: (E?J,E??-J???) and Lcp1 microglia, grayscale: (E?J, E???J???), with DAPI counterstain nuclei, blue: (E?J, E???J?). Orange boxes in (E?J) were magnified to better portray il-1? signal in microglia (E???J??) and yellow boxes in E-J were magnified to better portray il-1? signal in Müller glia (E????J???). The magnified images in (E???J???) represent single z-plane images. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar in E is 20 µm and is the same for F-J? and scale bars in E?? and E??? are 5 µm and are the same for (F???J??) and (F????J???), respectively. Graphs represent the Mean ± SEM and n = 3. *, p < 0.05, ****, p < 0.0001.