FIGURE 2

Microglia upregulate expression of homeostatic and inflammatory genes when Müller glia commence regeneration. (A) Timeline of the onset of Müller glia‐mediated neuronal regeneration in pde6c mutants. (B) qPCR of retinal regeneration genes in the eye cup at 3, 4, and 5 wpf. Statistical significance was evaluated by unpaired t‐test: means ± SD. ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) Four wpf wild‐type sibling and pde6c mutant retinas labeled with anti‐PCNA (green) and zrf‐1 (magenta) antibodies. Dotted lines indicate the OPL. Bottom panels are higher magnification images of rectangles shown in the upper panels. Yellow arrows indicate zrf‐1 signals associated with PCNA‐positive cells, indicating proliferative Müller glia. Scale bars: 20 μm. (D) Five wpf wild‐type sibling and pde6c mutant retinas labeled with anti‐PCNA (green) and anti‐Sox2 (red) antibodies. Nuclei are counterstained with Hoechst (blue). Bottom panels are higher magnification images of rectangles shown in upper panels. Yellow arrows and arrowheads indicate Sox2 and PCNA‐double positive cells in the INL and ONL, respectively. ONL, outer nuclear layer; INL, inner nuclear layer; RGCL, retinal ganglion cell layer. Scale bars: 30 μm. (E) Graph of the number of Sox2⁺ Müller glia in the ONL, INL, and both nuclear layers (total) per retinal section of wild‐type siblings and pde6c mutants. Each dot indicates the average number of 2–3 sections from one fish. Two‐way ANOVA with Sidak multiple comparison test: means ± SD, ns, p > 0.05; ****p ≤ 0.0001. (F) Flat‐mount retinas of wild‐type siblings and pde6c mutants at 4 wpf. Microglia are visualized with Tg[mpeg1.1:EGFP] (green). Double cone‐type photoreceptors are labeled with zpr1 antibody (magenta). Top panels indicate confocal z‐axis images of wild‐type sibling and pde6c mutant retinas. Three optical slice positions corresponding to (1) the subretinal area (rod OS and RPE), (2) the zpr1⁺ cone layer, and (3) the inner nuclear layer (INL) are indicated by arrows. Bottom panels show three optical slices. Many microglia are observed only in the subretinal area and the cone layer in pde6c mutant retinas (yellow asterisks). Scale bar: 70 μm. (G) Microglial density is calculated as the number of microglia per mm². Each dot indicates an individual fish. Nested t‐tests (two tailed): means ±SD, *p ≤ 0.05, ***p ≤ 0.001; ****p ≤ 0.0001. (H) Magnified images of 4 wpf wild‐type sibling and pde6c mutant Tg[mpeg1.1:EGFP] transgenic retinas labeled with zpr1 antibody. Top and bottom panels indicate the cone layer and INL of flat‐mounted retinas, respectively. Two higher magnification images indicating microglial cells phagocytosing zpr1⁺ cone material in cone layers of pde6c mutants are presented. Scale bars: 20 μm. (I) Sphericity of microglia in the inner retinal layer and cone layer of wild‐type siblings and pde6c mutants. Sphericity of each microglial cell was calculated using surface‐rendered objects prepared with IMARIS software. Each point represents one object. Brown‐Forsythe and Welch ANOVA with Dunnett's T3 multiple comparisons test: Box‐and‐whisker plot, ns, p > 0.05; *p ≤ 0.05, ****p ≤ 0.0001. (J) PCR of microglia‐related genes in wild‐type sibling and pde6c mutant eyes at 3, 4, and 5 wpf. Four‐six biological replicates of 2–4 eyes each were tested. ND: not detected. Unpaired t‐tests: means ± SD. ns, p > 0.05; *p ≤ 0.05, ***p ≤ 0.001; ****p ≤ 0.0001.