The pattern of mitosis in p110α knockout lenses was altered on P0. The smaller lenses observed in p110α knockout mice could have resulted from abnormal epithelial cell mitosis in the embryonic or postnatal period, as has been observed for other mouse knockout models with microphthalmia,
38,39 or was due to an increased level of apoptosis. TUNEL staining, or immunostaining for cleaved caspase 3, showed that any apoptosis was very difficult to detect in both the wild-type and the p110α-deficient lenses, with no increase observed in knockout animals (data not shown). To examine whether loss of p110α influenced the magnitude or pattern of mitosis, lenses were labeled with EdU between embryonic day 14 (E14) and P2. From E14 through E17, both the wild-type (
Fig. 5A–D) and the knockout (
Fig. 5G–J) lenses displayed similar patterns of robust EdU labeling, with the highest level of fluorescence in the germinative zone near the lens equator as previously reported.
40,41 On P0, wild-type lenses continued to show the greatest level of proliferation at the equator (
Fig. 5E), whereas the p110α knockout lenses lacked the ring of increased labeling at the equator and showed a more homogeneous pattern of EdU incorporation (
Fig. 5K). By P2, both the wild-type (
Fig. 5F) and the knockout (
Fig. 5L) lenses once again displayed the greatest levels of proliferation in the equatorial germinative zone. To quantify this transient change in mitotic pattern, line scans were performed using EdU-labeled images from wild-type and p110α knockout lenses on P0 (
Fig. 6A) and P2 (
Fig. 6B), and the mean (±SE) values were plotted against the position along the lens diameter. On P0, wild-type lenses had clear peaks of fluorescence intensity near the equator. In contrast, p110α knockout lenses lacked the equatorial peaks and had maximum fluorescence values 41% lower than those in wild-type lenses (
P < 0.05) lenses. On P2, both the wild-type and the knockout lenses displayed clear peaks of EdU signal in the equatorial germinative zone, and the mean value of maximum fluorescence in p110α-deficient lenses was only 12% lower than those in wild-type lenses (
P > 0.05). These data suggested that the significant reduction in lens size following p110α deletion could be caused by a transient loss of proliferating epithelial cells in the germinative zone on P0.