FIGURE 7

Localization of each 4w_Mg cluster in the retina. (A) Feature plot showing expression of apoeb, rxraa, bzw2, and lipg genes at 4 wpf. (B) Classification strategy of 4 wpf microglial clusters, based on HCR‐FISH with apoeb, rxraa, bzw2, and lipg RNA probes. (C) Color‐coded assignment to eight retinal regions at 4 wpf. (D) HCR‐FISH of 4 wpf microglial clusters, 4wpf_Mg1/2/3/4, in Tg[mpeg1.1:EGFP] transgenic wild‐type and pde6c mutant retinas with apoeb, rxraa, bzw2, and lipg RNA probes. Higher magnification images show microglia located in IPL/INL/OPL area for 4w_Mg1, CMZ/RGC/IPL/INL area for 4w_Mg2, and INL/OPL/ONL/OS area for 4w_Mg3 and 4w_Mg4. Dotted lines demarcate the interface between retinal areas. 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 4 wpf retinal microglia. At 4 wpf, four microglial clusters are identified as homeostatic/stress‐response microglia (4w_Mg1), degeneration‐response/homeostatic microglia (4w_Mg2), degeneration‐response/stress‐response microglia (4w_Mg3), and degeneration‐response microglia (4w_Mg4) in accordance with their transcriptomic profile. Both 4w_Mg3 and 4w_Mg4 are rare in wild‐type siblings, but appear and approach the photoreceptor layer in pde6c mutants, indicating pde6c mutant‐specific microglia clusters. Furthermore, 4w_Mg3 serve as pro‐inflammatory microglia. lipg is enriched in 4w_Mg3; however, all other Mg subtypes in pde6c mutants upregulate lipg expression. 4w_Mg4 upregulates lgals2a in pde6c mutants, suggesting putative anti‐inflammatory microglia. 4w_Mg2 approaches the photoreceptor layer in pde6c mutants.