Figure 4.

Proliferation is upregulated in the RPE layer in the Nf2^CKO optic cup. (A–D: E11.5; E–J: E11.0) A, B: Phospho-histone H3 (pHH3) labeling (red) of control (A) and Nf2^CKO (B) detects ocular cells in G2/M phases of the cell cycle (red). Dapi is used as a nuclear label (blue). Quantification shows that the number of phospho-histone H3-labeled cells is not changed in the ventral optic cup in Nf2^CKO eyes (C); however, there is a small significant increase in the ventral-most area that harbors the OF (V2; D). E-J: EdU labeling and quantification. EdU-labeled cells in control (E; green), with nuclear Dapi co-labeling (blue). In the RPE of the Nf2^CKO ventral optic cup, the number of EdU-labeled cells is increased (F; arrowheads), in comparison with controls (E, arrowheads). Quantitative analysis of EdU incorporation reveals that the number of cells in S-phase is significantly upregulated specifically in the RPE but not in the retina (G, H). (RPE: control: 19.91% ± 1.74 SD; Nf2^CKO: 29.07% ± 4.60 SD) The increase in EdU-labeled cell number is significant for the ventral RPE (I; control: 27.82% ± 5.66 SD; Nf2^CKO: 49.86% ± 7.10 SD), while the dorsal RPE shows no change. Significant stimulation of proliferation in the ventral RPE applies to all regions in the ventral optic cup, including the V2 region harboring the OF (J). Data are shown as means ± SD, and P-values are indicated above the horizontal lines in each graph (significant < 0.05). Statistical analysis was performed using unpaired, two-tailed Student T-test or 2-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Images show sagittal view with nasal orientation to the left. Scale bar: 100 μm in A, E.