Figure 2. Cellular defects of Pten △Epi mutant neuroepithelial cells.

(A) Comparison of WT and mutant cell shape in the E8.5 cephalic neural plate, using X-linked GFP-expression to mark individual cells. Schematic representations of individual cells for each genotype are shown (white box). Red is phalloidin. Scale bar is 10 μm. (B) Comparison of neural plate height in the cephalic region of WT and mutants. WT E7.75 = 23.9 ± 4.5 μm; Pten △Epi E7.75 = 23.6 ± 4.1 μm: WT and mutant are not different, p = 0.86, by standard t-test. WT E8.0 = 32.5 ± 1.7 μm; Pten △Epi E8.0 = 24.6 ± 3.7 μm: WT is significantly taller than the mutant, *p = 0.0164. WT E8.5 = 49.1 ± 9.6 μm; Pten △Epi E8.5 = 32.6 ± 7.4 μm; WT is significantly taller than the mutant, ****p < 0.0001. For this and similar analyses below, >100 measurements were made from >3 embryos. (C) Comparison of apical cell shape in the cephalic neural epithelium of WT and Pten △Epi embryos viewed en face at E7.75, E8.0 and E8.5. Cell borders are marked by expression of ZO1 (white). Scale bar = 20 μm. (D) Apical surface of cephalic neural epithelial cells, taken from images like those shown in (C). WT E7.75 = 29 ± 17 μm²; Pten △Epi E7.75 = 30 ± 16 μm²: WT and mutant are not different, p = 0.79. WT E8.0 = 20 ± 10 μm²; Pten △Epi E8.0 = 29 ± 15 μm². The WT surface area is significantly smaller than in the mutant, ****p < 0.0001. WT E8.5 = 8 ± 4 μm²; Pten △Epi E8.5 = 14 ± 9 μm². The WT surface area is significantly smaller than in the mutant, ****p < 0.0001. (E) Acetylated microtubule arrays in the neural plate in stage-matched WT and mutant embryos. Transverse sections of cephalic regions of WT and Pten △Epi embryos at E8.0 (0– 2 somites), E8.5 (5–7 somites) and E9.0 (11–13 somites). Green is acetylated tubulin; blue is DAPI. Arrows point to the apical surface of neural plate; arrowheads point to the floor plate. The first region of tubulin acetylation in WT is in the floor plate, which is only region of tubulin acetylation in the mutant. Scale bar = 25 μm.

DOI: http://dx.doi.org/10.7554/eLife.12034.007

Figure 2—figure supplement 1. Acetylated microtubules in the wild type cranial neural plate.

(A) α-Tubulin appears to be in apical-basal arrays in E8.5 wild-type cranial neuroepithelial cells but is more diffuse in Pten △Epi embryos. Red is phalloidin. Scale bar = 20 µm. (B) Western blot analysis shows that of acetylated tubulin increases during maturation of the wild-type neural plate. Each lane represents a pool of the cephalic regions of three embryos. (C) Acetylated microtubule arrays increase with time in the cranial neural plate of WT embryos and acetylated tubulin partially colocalizes with PTEN protein beginning at E8.5. Green is PTEN, acetylated tubulin is red; blue is DAPI. Arrows highlight sites of PTEN enrichment and arrowheads show examples of sites of colocalization of PTEN and acetylated tubulin. Scale bar = 25 μm.

DOI: http://dx.doi.org/10.7554/eLife.12034.008