Clonal reduction of p15 ⁺ RPCs in WT retinae is not due to apoptosis. (A) Representative image showing TUNEL signals (magenta) in 28-hpf p15 overexpression (p15⁺) retina (n = 11). The schematic insert shows TUNEL signals (magenta) in p15⁺ retinae (gray). Yellow arrowheads indicate TUNEL signals. (B) Whole mount GFP immunostaining (green) and TUNEL assay (magenta with yellow arrowheads) of 28-hpf p15⁺ host retinae (black box on the left, n = 11) and WT host retinae (black box on the right, n = 10) transplanted with gfp-expressing p15⁺ donor RPCs. Schematic inserts show TUNEL signals (magenta) in p15⁺ donor RPCs (green) in p15⁺ and WT host retinae (gray). In each group, zoom-in images of retinal areas (dashed yellow rectangles) at the merged and three separate channels are shown in the right panels. (C) The level of TUNEL signals. In the p15 mRNA group, the TUNEL level was determined by the ratio of TUNEL+ nuclear fragments to total nucleus number (DAPI+) in single retina sections (TUNEL+/DAPI+). In transplantation groups, the TUNEL level was determined by the ratio of the number of GFP+TUNEL+ (GFP and TUNEL double positive) nuclear fragments in p15⁺ donor RPCs to the total number of GFP+ (GFP positive) p15⁺ donor RPCs (GFP+TUNEL+/GFP+) in either p15⁺ or WT host retinae. (D) Whole mount GFP immunostaining (green) and TUNEL assay (magenta with yellow arrowheads) of 28-hpf WT host retinae transplanted with gfp-expressing p15⁺ (black box on the left, n = 10) or p15⁺/bcl2a⁺ (black box on the right, n = 13) donor RPCs. In each group, zoom-in images of retinal areas (dashed yellow rectangles) at the merged and three separate channels are shown in the right panels. Schematic inserts show TUNEL signals (magenta) in p15⁺ and p15⁺/bcl2a⁺ donor RPCs (green) in WT host retinae (gray). (E) The ratio of GFP+TUNEL+ nuclear fragments in p15⁺ and p15⁺/bcl2a⁺ donor RPCs to the total number of GFP+ donor RPCs in WT host retinae. (F and G) Representative images (F) and quantification of clone size (G) of 72-hpf clones derived from single 12-hpf p15⁺ (n = 35) and p15⁺/bcl2a⁺ (n = 41) donor RPCs in WT retinae. OPL, the outer plexiform layer; IPL, the inner plexiform layer. Areas within white dashed contour lines are retinae in A, B, and D. Data in C, E, and G represent mean ± SEM. Scale bars, 10 µm.