FIGURE 1

Microglia phagocytose dying photoreceptors in zebrafish pde6c mutants at 5 dpf. (A) Schematic drawing of zebrafish retinal neural circuit. RGCL, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, photoreceptor outer segment; RPE, retinal pigmented epithelium. (B) Pathological process of photoreceptor degeneration and Müller glia‐mediated rod regeneration in zebrafish pde6c mutants. (C) Retinal sections of wild‐type sibling and pde6c mutant embryos at 3–5 dpf. Microglia and nuclei are visualized with Tg[mpeg1.1:EGFP] (green) and Hoechst staining (gray), respectively. Apoptotic cells are labeled with TUNEL (magenta). Upper panels indicate only images of green and magenta channels. Dotted lines indicate the OPL. Scale bars: 20 μm. (D) Number of TUNEL‐positive ONL cells per section. Each dot represents the average of 4 sections from one fish. Nested t‐tests (two tailed): means ± SD, ns, p > 0.05; ***p ≤ 0.001. (E) Number of microglia in the ONL per section. Each dot indicates the average of 4 sections from one fish. Nested t‐tests (two tailed): means ± SD, ns, p > 0.05; **p ≤ 0.01. (F) Projection view of 3D confocal scanning of 5 dpf wild‐type sibling and pde6c mutant retinas. Microglia are visualized with Tg[mpeg1.1:EGFP] (green). Dotted lines indicate the outline of the retina. The right‐hand graph indicates the number of microglia per eye. Unpaired t‐tests (two tailed): means ± SD, **p ≤ 0.01. Scale bars: 30 μm. (G) Live imaging of 5 dpf wild‐type sibling and pde6c mutant retinas carrying the Tg[mfap4:tdTomato‐CAAX] transgene (green). Dying cells are stained with acridine orange (magenta). Inset panels show higher magnification images of the ONL shown in the top panels (dotted rectangle). The third row represents IMARIS rendering of acridine orange signals as spots and mfap4:tdTomato signal as surface objects. The bottom panel shows the object‐rendered images filtered to show only acridine orange‐derived spots engulfed by microglia (yellow arrowheads). Dotted lines indicate the OPL. Scale bars: 30 μm. (H) qPCR of microglia‐related genes in wild‐type sibling and pde6c mutant eyes at 5 dpf. Three biological replicates of 20 eyes each were tested. Unpaired t‐test: means ± SD. ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.