Figure 4. Cell cycle analysis in Pten^Δ/Δ, Fgfr2^Δ/Δ, and (Pten/R2)^Δ/Δ lenses.

BrdU incorporation assay was used on Cre-negative controls (A, A’, E), Pten^Δ/Δ (B, B’, F) Fgfr2^Δ/Δ (C, C’, G), and (Pten/R2)^Δ/Δ (D, D’, H) to assess the impact of deleting Pten, Fgfr2, and the combination of the two on lens epithelial cell proliferation and cell cycle withdrawal. By E12.5, control lenses failed to exhibit any BrdU incorporation in the differentiating fiber cells (A, A’). E12.5 Pten^Δ/Δ (B, B’) ,Fgfr2^{Δ/ Δ (}C, C’), and (Pten/R2)^Δ/Δ (D, D’) lenses displayed BrdU incorporation of nuclei in the fiber cell mass, although Fgfr2^Δ/Δ lenses contained the most proliferation in posterior lens cells (compare C’ to B’ and D’). Furthermore, overall proliferation was increased in E12.5 Fgfr2^Δ/Δ lenses, yet was not significantly increased in Pten^Δ/Δ or (Pten/R2)^Δ/Δ lenses at this stage (I). By E15.5 (E–H, J) lens epithelial proliferation was not altered in Pten^Δ/Δ (F, J) Fgfr2^Δ/Δ (G, J), or (Pten/R2)^Δ/Δ (H, J). A’–D’ represent boxed regions of A–D selected for higher magnification. Dashed white lines outline the edges of the lens and white arrows point towards posterior lens cells remaining prolific (A’–D’). Errors bars on the graphs represent s.e.m, with each bar representing a minimum of 9 measurements (3 sections from the lens center of 3 different embryos). Scale bars: 100 μm in A–D; 50 μm in A’–D’; 200 μm in E–H.