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. Author manuscript; available in PMC: 2014 Sep 9.
Published in final edited form as: Nat Cell Biol. 2014 Jul 13;16(8):758–769. doi: 10.1038/ncb3001

Figure 3.

Figure 3

Loss- or gain-of-function in spindle orientation genes does not alter early stratification behaviour. (a) Lentiviral overexpression of EYFP–mInsc in E15.5 (left, centre) and E17.5 (right) mitotic basal cells. Although EYFP–mInsc relocalizes LGN to the apical domain in ~90% of cells at E17.5, it is markedly less efficient at E15.5. (b) Quantification of LGN localization in control (YFP−) and EYFP–mInsc+ mitotic basal cells at E15.5, showing no significant difference between groups. (c) Quantification of LGN crescent orientation from lateral (0°) to apical (90°), similar to Fig. 1e. Although EYFP–mInsc polarizes infrequently at E15.5, it can promote apical LGN localization when present. n = 36 cells for control, n = 45 for EYFP–mInsc from 4 to 5 independent animals. Box boundaries indicate the 25% and 75% quartiles, the middle bar the median, and the plus symbol the mean. (d,e) Radial histograms of division orientation at E15.5 (d) and E17.5 (e). At E15.5, division angles are essentially randomized in controls (Scramble shRNA), as in wild-type embryos (Fig. 1f). Neither knockdown of LGN nor overexpression of EYFP–mInsc has any effect at E15.5. However, strong division reorientation phenotypes are observed at E17.5, with EYFP–mInsc promoting perpendicular divisions and loss of LGN inducing planar divisions. Chi-square P values can be found in Supplementary Table 1. n indicates cells from 4 to 6 independent animals. (f) Ectopic expression of EYFP–mInsc (green) does not accelerate differentiation at early ages. The differentiation marker Keratin 1 (K1, red) is expressed at low levels at E13.5 (left), and by E15.5 (right), K1 becomes restricted to spinous layer cells, while K14 (blue) marks basal cells. Epidermal thickness is indicated by the double arrow. (g) Quantification of spinous (K10+) layer thickness in skin transduced with EYFP–mInsc, normalized to age-matched littermates. There is no statistically significant difference between EYFP–mInsc and controls at E15.5, whereas at E17.5, EYFP–mInsc promotes spinous thickening. n indicates sections analysed from >3 animals per age. (h) Quantification of spinous layer thickness at E15.5, normalized to littermates. Knockdown of LGN, Numa1 or the LGN homologue AGS3/Gpsm1 does not affect early stratification. n indicates sections analysed from n≥4 embryos for knockdown and littermate controls. Scale bars, 10 μm (a), 50 μm (f). Error bars (g,h): s.d. P values determined by Mann–Whitney non-parametric test in c and two-tailed Student’s t-tests in g,h; *** P < 0.0001; NS, not significant.