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. 2016 Jan 26;5:e12034. doi: 10.7554/eLife.12034

Figure 3. Expression of an activated form of PI3 Kinase mimics the Pten mutant neural plate phenotype.

(A) Loss of Pten (Pten △Epi) or expression of the activating mutation Pik3caH1047R-Epi in the epiblast leads to phosphorylation of AKT in E8.5 embryos. Representative Western blots (n = 3) show the two phosphorylated forms of AKT in WT, Pten △Epi and Pik3caH1047R–Epi embryos. Numbers indicate approximate MW. (B) Pik3caH1047R–Epi embryos phenocopy Pten △Epi embryos. Whole embryos (inset) and expanded view of the cephalic region of E8.5 WT and Pik3caH1047R-Epi embryos; dorsal view. Scale bar = 120 μm. (C) The apical surface of the neural plate, viewed en face; cell borders marked by expression of ZO1 (white) (top row), and acetylated tubulin (green) in transverse sections of the cephalic neural epithelium of E8.5 WT and Pik3caH1047R-Epi embryos. Blue is DAPI. Scale bar = 20 μm. (D) Comparison of apical surface area of cephalic neural epithelial cells at E8.5. WT = 8 ± 4 μm2; Pten △Epi = 14 ± 9 μm2; Pik3caH1047R-Epi = 15 ± 10 μm2. The surface areas of both mutants are significantly larger than wild type, ****p < 0.0001. (E) Cephalic neural plate height at E8.5. WT = 49.1 ± 9.6 μm; Pten △Epi = 32.6 ± 7.4 μm; Pik3caH1047R-Epi = 31.5 ± 7.2 μm. Cells in both mutants are significantly shorter than in wild type, ****p < 0.0001.

DOI: http://dx.doi.org/10.7554/eLife.12034.009

Figure 3.

Figure 3—figure supplement 1. Inhibition of PI3 kinase restores pseudostratification in the Pten △Epi neural plate.

Figure 3—figure supplement 1.

(A) Mothers of Pten △Epi mutant embryos were injected with 25 mg/kg of LY294002 24 hr prior to E8.5 embryo dissection. This treatment leads to elevation of the neural folds and prevents the formation of abnormal folds in the Pten △Epi neural plate phenotype without affecting wild-type development. (B) Immunostaining for acetylated tubulin (green) of transverse sections of the cephalic neuroepithelium of treated E8.5 WT and Pten △Epi embryos. Blue is DAPI. (C) Western blot analysis of untreated and LY294002-treated Pten mutant embryos shows an increase in tubulin acetylation and inhibition of phosphorylation of AKT at Ser473, indicating that the treatment effectively inhibited PtdIns(3,4,5)P3 production. Scale bar in A = 120 μm; in B = 20 μm.
DOI: http://dx.doi.org/10.7554/eLife.12034.010