Figure 4

Loss of Mll1 in retinal progenitor cells results in proliferation and cell cycle defects. (A-C) Ki67 immunostaining (green) for proliferative cells with DAPI nuclear stain (blue) of retinal cross-sections in CreNeg (A), Mll1Het (B) and Mll1KO (C) mice at P0. (D) Quantification of Ki67 + cells in three 100 μm² regions per cross-section, three cross-sections per sample and three samples per genotype. Results are displayed as mean + SEM (n = 3). (E–G) Phospho-histone H3 (PH3) immunostaining (green) with DAPI (blue) of the same samples for cells in mitotic (M-) phase of the cell cycle. (H) Quantification of the number of Ki67+ cells positive for PH3 per cross-section, normalized to the number in CreNeg sections. The results are displayed as mean + SEM. (I) To determine S-phase cells in P0 retinas, EdU was injected IP into P0 mouse pups, and retinas harvested 4 hours later. (J–L) EdU labeled cells (green) with DAPI (blue) in retinal cross-sections of harvested retinas. (M) Quantification of the number of Ki67+ cells reactive for EdU in three 100 μm² regions per cross-section, relative to the number in CreNeg sections. The results are displayed as mean + SEM. *p < 0.05, n.s. = not significant by two-way ANOVA with Tukey’s multiple comparisons (n = 3). Antibody information is given in Supplementary Table S2. Reductions in proliferating cells were also detected in Mll1KO retinas at P3 and P5 using EdU pulse-labeling (Supplementary Fig. S3A).