(A) Flrt2 is expressed by SACs, a second amacrine population, and a subset of RGCs. In situ hybridization for Flrt2 RNA (magenta) was combined with immunostaining for calbindin (green), a selective SAC marker at the ages shown (P1 and P6). Yellow arrows indicate Flrt2+ SACs. Cells in the inner nuclear layer (INL) expressing Flrt2 but not calbindin (purple arrows) define a non-SAC Flrt2+ amacrine population. Non-SACs in the ganglion cell layer (GCL) are likely RGCs, based on their large soma size (purple arrows). Among SACs, Flrt2 is detected predominantly in ON SACs (which reside in the GCL) at P1 whereas it is detected more readily in OFF SACs (which reside in the INL) at P6. However, ON SACs positive for Flrt2 are observed at P6 (yellow arrow in GCL), suggesting that Flrt2 is not selective for one SAC population over the other. (B) RGCs expressing Flrt2 include direction-selective ganglion cells (DSGCs). Double staining for Flrt2 and CART, an ooDSGC marker, at P1 and P6. Double-labeled cells (yellow arrows) are observed in the GCL. Not all ooDSGCs express Flrt2, however, as CART+ Flrt2– cells are also apparent (green arrows). Purple arrows indicate Flrt2+ cells that are not ooDSGCs; this group likely includes SACs. Scale bar, 10 µm. (C-E) SACs express FLRT2 protein. Dissociated SACs from P2 Chat-Cre::RosaLSL-tdTomato mice that specifically express tdTomato (magenta) in SACs. Neurons were co-stained with an antibody against Tuj1 (green) and (C) FLRT1, (D) FLRT2, (E) FLRT3 (cyan). Only FLRT2 co-localized with tdTomato-positive SACs. SACs were also negative for Unc5s as shown in Figure 7—figure supplement 1. (F-G) tdTomato SACs (magenta) grown on Unc5C (F) or FLRT2 (G) stripes (green). Stripes were visualized by addition of PLL-FITC to the purified Unc5C or FLRT2 protein patterned. Unc5C (F) but not FLRT2 (G) repelled SACs. (H-I) Dissociated Drd4-GFP ooDSGCs (green) in culture harvested from P3 mice that specifically express GFP in ooDSGCs. (H) Drd4-GFP neurons on Unc5C stripes co-stained with an antibody against Tuj1 (green) and FLRT2 (cyan). (I) Drd4-GFP neurons on FLRT2 stripes stained with an antibody against Tuj1 (green). Neurons cultured 8 DIV. Scale bar, 30 µm.