Figure 5. The Pten neural plate phenotype is independent of AKT.

(A) Effect of the AKT inhibitor MK-2206 treatment on targets of the PI3 kinase pathway in E8.5 embryos. Western blot of the two phosphorylated forms of AKT and pS6 S240/4 in WT and Pten △Epi at E8.5 in control embryos (vehicle) and after 24 or 48 hr of MK-2206 treatment in utero prior to embryo dissection. Numbers indicate approximate MW. (B) Dorsal view (inset) and enlarged image of the cephalic region of E8.5 wild-type and Pten △Epi embryos. There is no change in the morphology of the mutant heads after 24 or 48 hr of MK-2206 treatment in utero. 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); acetylated tubulin (green) in transverse sections of cephalic neural epithelium in wild type and Pten △Epi at E8.5 after 24 or 48 hr of MK-2206 treatment in utero. Blue is DAPI. Scale bar = 10 μm. (D) Height of the E8.5 cephalic neural plate. Wild type, untreated (control) = 44.9 ± 5.7 μm; WT 48 hr treatment = 46.5 ± 9.9 μm; MK-2206 treatment had no significant effect. Pten △Epi untreated (control) = 32.4 ± 7.3 μm; Pten △Epi 24 hr = 33.9 ± 6.8 μm²; Pten △Epi 48 hr = 30.3 ± 7.5 μm. Treated and untreated mutants were all significantly shorter than wild type, but MK-2206 treatment did not significantly rescue cell elongation in the mutant. (E) Apical surface area of E8.5 cephalic neuroepithelial cells. Control = 8 ± 5 μm²; WT 48 hr = 7 ± 4 μm²; Pten △Epi Control = 14 ± 9 μm²; Pten △Epi 24 hr = 13 ± 9 μm²; Pten △Epi 48 hr = 14 ± 10 μm². Treated and untreated mutant cells all had significantly larger surface area than wild type, but MK-2206 treatment did not significantly decrease cell surface area in the mutant.

DOI: http://dx.doi.org/10.7554/eLife.12034.014

Figure 5—figure supplement 1. Inhibition of mTORC1 by rapamycin does not rescue the Pten neural plate phenotype.

(A) Western blot of extracts from control embryos and embryos treated in utero with rapamycin for 48 hr shows that the treatment effectively blocked the expression of pS6 in both WT and Pten △Epi embryos. (B) Transverse sections (acetylated tubulin in green) and dorsal view of cephalic region of E8.5 WT and Pten △Epi embryos after a 48 hr treatment with rapamycin. Scale bar = 10 and 200 μm.

DOI: http://dx.doi.org/10.7554/eLife.12034.015

Figure 5—figure supplement 2. Downstream targets of PDPK1.

(A) Western blots for pNRDG1 and p-aPKCζ/λ in E8.5 WT Pten △Epi, Pdpk1 △Epi and Pten △Epi Pdpk1 △Epi embryos. (B) Western blot analysis of pNRDG1 in WT and Pten △Epi embryos treated with MK2206 for 48 hr.

DOI: http://dx.doi.org/10.7554/eLife.12034.016

Figure 5—figure supplement 3. Myosin-II distribution and levels appear normal in the Pten neural plate.

(A–D) En face images of the distribution of MHC-IIB (green). (A–B) E8.0 embryos. Myosin was anisotropically distributed in the neural plate of all genotypes (WT, Pten, Pdpk1 and Pten Pdpk1) and no preferential enrichment at long or short cell edges was noted. (C–D) E8.5 embryos. Apical myosin is enriched in all genotypes at E8.5. This is especially prominent in WT, and presumably reflects the formation of the actomyosin rings that mediate apical constriction in the next phase of neural tube closure. Phalloidin is red. Scale bar = 20 μm. (E) Western blot detection of phospho-myosin light chain (pMLC) and total MLC in E8.5 Pten △Epi, Pdpk1 △Epi and Pten △Epi Pdpk1 △Epi embryos. No striking differences between genotypes were apparent.

DOI: http://dx.doi.org/10.7554/eLife.12034.017