FIGURE 4

Localization of each 5d_Mg cluster in the retina. (A) Feature plot showing expression of apoeb, rxraa, and snx33 genes at 5 dpf. (B) Classification strategy of 5 dpf microglial clusters, based on HCR‐FISH with apoeb, rxraa, and snx33 RNA probes. (C) Color‐code assignment to seven retinal regions at 5 dpf. (D) HCR‐FISH of 5 dpf microglial clusters, 5d_Mg1/2/3, in Tg[mpeg1.1:EGFP] transgenic wild‐type sibling and pde6c mutant retinas with apoeb, rxraa, and snx33 RNA probes. Higher magnification images show microglia located in the CMZ/RGCL/IPL/INL area for wild‐type siblings and the INL/OPL/ONL/OS area for pde6c mutants. Dotted lines demarcate the interface between each retinal area. Yellow line indicates the outline of microglia defined by Tg[mpeg1.1:EGFP] expression. Scale bars: 10 μm. (E) Bar charts showing the distribution of each cluster across retinal areas in wild‐type siblings and pde6c mutants, using color‐code assignment defined in (C). Microglia were sampled from four fish for each group using 3–4 sections per fish. (F) Graphical summary of scRNA‐seq analysis of zebrafish 5 dpf retinal microglia. At 5 dpf, three microglial clusters, 5d_Mg1, 5d_Mg2, and 5d_Mg3, are identified as degeneration‐response microglia, stress‐response microglia, and homeostatic microglia, respectively, in accordance with their transcriptomic profiles. In wild‐type sibling retinas, 5d_Mg1 is broadly localized in the CMZ, RGCL, INL, and ONL, whereas 5d_Mg2 and 5d_Mg3 are preferentially localized in the CMZ and RGCL. In pde6c mutant retinas, all three Mg clusters move toward the OPL, ONL, OS/RPE, and upregulate lgals2a/lgasl9l1. In addition, 5d_Mg1 in pde6c mutants upregulate oxidative phosphorylation (OXPHOS)‐related genes, suggesting a neuroprotective role.