Increased contractility in spinous cells is sufficient to induce many granular genes.
(A and B) GO term analysis of the list of genes commonly upregulated in suprabasal K10-Spastin (vs control spinous cells at E16.5, log 2-fold > 1, p < 0.05) and granular cell signature (A) or intermediate cell signature (B).
(C and D) Immunofluorescence staining of MafB (C) or Tgm1 (D) in red, and suprabasal cells expressing Spastin marked by HA (green) in K10-Spastin and controls. Images of E16.5 embryos, doxycycline fed starting at E14.5. Insets to the left of all images in this panel show close-ups of HA+ cells in suprabasal layers adjacent to basal cells. Scale bars: 20 μm.
(E and F) Immunofluorescence staining of MafB (F) or Tgm1 (H) in red, and suprabasal cells expressing Arhgef11CA marked by HA (green) in K10-Arhgef11 and controls. Images of E17.5 embryos, doxycycline fed from E10.5. Scale bars: 20 μm.
(C’ and E’) Percentage of MafB+ suprabasal cells in controls, and in HA+ and HA-cells in K10-Spastin at E16.5 (C’) or K10-Arhgef11CA at E17.5 (E’). Total number of mice analyzed was three embryos per genotype. Data are presented as the mean ± SD. *: p < 0.05, ****: p < 0.0001, repeated-measures one-way ANOVA, Tukey’s multiple comparisons test. For all plots in this panel, cells located in the first two layers of suprabasal cells immediately above the basal layer were considered for quantification.
(D’ and F’) Quantification of average fluorescence intensity of Tgm1 in suprabasal cells of control and HA+ cells in K10-Spastin at E16.5 (D’) or K10-Arhgef at E17.5 (F’). Total number of mice analyzed was three embryos per genotype. *: p < 0.05, two-tailed paired t-test.
(G) RNAScope of Scara5 in red and immunostaining with HA in green in control and K10-Arhgef11. Images of E16.5 embryos, doxycycline fed starting at E14.5. Scale bars: 20 μm. (G’) Violin plot of number of Scara5 puncta per suprabasal cell in control, and HA+ cells and neighbor HA-cells in K10-Spastin at E16.5. n= 224 HA+ cells and 160 WT cells from 2 embryos per genotype. ns: not significant, two-tailed unpaired t-test.