Fig 1: Runx1 and Fst label RGC subtypes. a Runx1 expression is enriched in the RGC subtype 27 (mean ± SEM shown). b–g The t-SNE graph with V1,V2 2D view and color-code as in Fig. 1d, shows clusters distribution in (b) and just the expression of Runx1 in (c) and Fst in (d). An alternative V1,V3 2D view t-SNE graph shows clusters distribution in (e) and just the expression of Runx1 in (f) and Fst in (g), demonstrating clearer separation between the clusters 27 and 3, as well as their markers (Runx1 and Fst), in the 3rd dimension (which is color-coded in the V1,V2 2D view). h Representative image of purified P5 RGCs at 12 h in culture probed by FISH for Runx1, an RGC marker RBPMS, and DAPI, as marked. i, j Insets of two Runx1+ RGCs outlined with dashed box in the upper panels (in the middle i and on the bottom j) are shown enlarged below (cell soma outlined with dashed line). Merged image in the upper panel also includes brightfield, which was used to outline cell soma shape in the insets. Granularized signal (arrowheads) in the insets is a property of the single-molecule sensitivity FISH kit. k Interpolation line shows a higher distribution peak over RGCs with fewer Runx1 puncta compared to RGCs with more puncta. l Fst expression is highly enriched in the RGC subtype 3 (mean ± SEM shown). m Representative image of purified P5 RGCs at 12 h in culture immunostained for Fst, an RGC marker RBPMS, and DAPI, as marked. n, o Insets of Fst+ and Fst− RGCs outlined with dashed box in the upper panels (in the middle n and on the bottom o) are shown enlarged below (cell soma outlined with dashed line). p–s Representative images of purified P5 RGCs at 12 h in culture probed by FISH for Runx1, and then immunostained for Fst and DAPI, as marked. Insets of Runx1−/Fst+ (p) and Runx1+/Fst− (r) RGCs outlined with dashed box in the upper panels are shown enlarged below (q and s, respectively; cell soma outlined with dashed line). (Scale bars: upper panels, 100 µm; lower panels, 5 µm)