Fat3 RNA is enriched in OFF-cone bipolar cells. (A) Schematic representation of the retina highlighting the position of bipolar cell bodies, as stained by VSX2. (B)In situ hybridization for Fat3 RNA in WT P22 retinas. (C)In situ hybridization for Fat3 RNA in Fat3^∆TM/∆TM P22 retinal tissue. (B′ and C′) In B and C the RNA puncta are shown in white and the yellow brackets indicate the area of VSX2⁺ cell bodies (cyan, B′ and C′). Yellow dashed lines demark the inner nuclear layer (INL) and the outer misplaced plexiform layer (OMPL) in Fat3^∆TM/∆TM tissue. The squares demark the insets seen in B′ and C′ at higher magnification. VSX2 protein is seen in cyan. (D) Hybridization chain reaction-immunohistochemistry (HCR-IHC) of FAT3 in wild type retinas. (D′) Inset demarked in a yellow box in D is shown at higher magnification in D′. (E) HCR-IHC of FAT3 in Fat3^∆TM/∆TM mutant retinas. (E′) Inset demarked in a yellow box in E is shown at higher magnification in E′. (F) Schematic representation of Grik1 and Grm6 RNA enrichment in bipolar cells, according to data in Fig. S2. (G and G′)in situ hybridization of Grik1 RNA (magenta) and Grm6 RNA (yellow, G′) with immunostaining for VSX2 (cyan). (G″ and G‴) The insets in G and G′ are shown at a higher magnification in G″ and G‴. (H) Triple in situ hybridization of Fat3 (white), Grik1 (magenta), and Grm6 (yellow) RNA. (I–I‴) Inset in H is seen at higher magnification in I–I‴. (I) Higher magnification of inset shown in H. Yellow dashed lines in I″ and I‴ demark Grik1 RNA⁺ cell bodies. Fat3 RNA (white) is shown together with Grm6 (yellow) RNA in I′, with Grik1 (magenta) RNA in I″ and alone in I‴. Scale bars: 20 µm.