Conditional KO of the PI3K subunit p85 phenocopies the <i>Pdgfrα</i> mutant lens.

(A) The PDGFR recruits the p85 subunit of PI3K, which activates AKT signaling by converting PI-4,5-P to PI-3,4,5-P. This reaction can be reserved by Pten to inhibit AKT signaling. (B) Western blot analysis showed the loss of p85 and pAKT, but not pERK, in E16.5 p85 CKO lens lysates. (C) p85 immunostaining was primarily localized to the transitional zone of the control lens (arrows) but lost in p85 CKO mutants. HE staining revealed the retarded elongation of primary fiber cells at E12.5 and the anterior shift of the transitional zones at E14.5 and P0 (arrowheads). (D) The p85 CKO lens showed normal pERK staining but lost pAKT in the transitional zone (arrows). The anterior lens epithelium displayed extensive TUNEL staining, indicative of apoptosis, and the encroachment of p57 positive cells that suggests the premature differentiation of progenitor cells. Further deletion of Pten in p85 CKO led to the up-regulation of pAKT in the entire lens but a modest increase in pERK. Although the apoptosis defect was not rescued by Pten deletion, the transitional zone in p85;Pten CKO lens marked by p57 staining was reverted back to the equatorial region of the lens. (E–F) The relative fluorescent intensities of pAKT and pERK at the transitional zones of the lenses. One-way ANOVA test, *P n = 3 embryos for each genotype. (G) The percentage of Ki67+ cells in the lens epithelium. One-way ANOVA test, *P P n = 3 embryos for each genotype. The numerical data used in panels E, F, and G are included in S1 Data. AKT, Protein kinase B; E, embryonic day; ERK, Extracellular Signal-Regulated Kinase; HE, hematoxylin–eosin; KO, knockout; pAKT, phospho-AKT; PDGFR, platelet-derived growth factor receptor; pERK, phospho-ERK; PI3K, Phosphoinositide 3-kinase; PI-4,5-P, Phosphatidylinositol 4,5-bisphosphate; Pten, phosphatase and tensin homolog; p85 CKO, Le-Cre;p85αf/f;p85βKO/KO.