Transcriptomic analysis of embryonic epidermal cell populations.

(A) Diagram depicting the stratification process and different cell populations (basal and K10+ suprabasal layers) existing in the epidermis from E14.5 to E16.5.

(B) Images of EdU staining (white) at E14.5, E15.5 and E16.5. Immunofluorescence of suprabasal cells labeled with K10 in red and the basement membrane, with β4-integrin in green. Scale bars: 20 μm.

(C) Quantification of the percentage of EdU+ cells in the basal and the first two layers of suprabasal cells at E14.5, E15.5 and E16.5. n= 3 embryos/time point. Data are presented as mean ± standard deviation (SD). ns: not significant, *: p < 0.5, **: p < 0.01, ***: p < 0.001, ordinary two-way ANOVA, Tukey’s multiple comparisons test.

(D) Schematic showing basal and suprabasal cell populations collected for RNA sequencing: K14-RFP;K10-rtTA;TRE-H2B-GFP pregnant dams were all fed with doxycycline from E12.5, and then sacrificed at either E14.5 or E16.5. The E14.5 intermediate cells (K10-GFP+;K14-RFP-), E16.5 spinous cells (K10-GFP+;K14-RFP-), and basal cells at E14.5 and E16.5 (K10-GFP-;K14-RFP+) cells were collected separately and sent for RNA sequencing. Granular cells were excluded from E16.5 samples.

(E) PCA score plot of the first two principal components (PC1: 33.5% variance and PC2: 21.1% variance) for gene expression levels from samples of cell populations indicated in (D) (n=3 embryos/cell population).

Identifying markers for intermediate and spinous cells.

(A) The core gene signature of intermediate cells was obtained from the list of genes only upregulated in ICs (log2 fold > 1, p < 0.05; as compared with both basal cells at E14.5 and spinous cells at E16.5). Total genes in the IC signature were 610. From this list, Scara5 and St8sia6 were two of the most highly upregulated genes compared to spinous cells at E16.5.

(B and C) RNAscope of Scara5 (B) and St8sia6 (C) in WT embryos at E14.5 and E16.5. Basement membrane is indicated as a dotted line. Scale bars: 20 μm.

(D and E) Quantification of the percentage of Scara5+ (D) and St8sia6+ (E) basal and suprabasal cells at E14.5 and E16.5. n= 3 embryos/time point. Data are presented as the mean ± SD. ****: p < 0.0001, ordinary two-way ANOVA, Sidak’s multiple comparisons test.

(F) The core gene signature of spinous cells was obtained from the list of genes only upregulated in spinous cells (log2 fold > 1, p < 0.05 compared with both basal cells at E16.5 and intermediate cells at E14.5). Total genes in the spinous cell signature were 163.

(G and H) Immunofluorescence staining of MafB (G) and Cox1 (H) in WT embryos at E14.5 and E16.5. Basement membrane is indicated as a dotted line. Scale bars: 20 μm.

(I) Quantification of the percentage of MafB+ basal and suprabasal cells at E14.5 and E16.5. n= 3 embryos/time point. Data are presented as the mean ± SD. ****: p < 0.0001, ordinary two-way ANOVA, Sidak’s multiple comparisons test.

(J) Quantification of Cox1 puncta/standardized area in intermediate cells at E14.5 was compared to the first two layers of spinous cells at E16.5. n= 3 embryos/time point. Data are presented as the mean± SD. *: p < 0.05, two-tailed unpaired t-test.

Mis-expression of MafB in intermediate cells is sufficient to reduce proliferation but not to induce spinous fate.

(A) Image of EdU staining (magenta) and MafB-expressing ICs immunolabeled with HA (green) in K10-MafB and control mice at E14.5, doxycycline fed since E12. All scale bars in this panel: 20 μm. Dotted lines represent the basement membrane.

(B) Percentage of dividing suprabasal cells (EdU+) in control, and HA+ and HA-cells in K10-MafB mice at E14.5 (n= 3 embryos/genotype). Data are presented as the mean ± SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ordinary one-way ANOVA, Tukey’s multiple comparisons test.

(C) Immunofluorescence staining of Tgm1 in K10-MafB vs control mice at E14.5.

(D) Quantification of Tgm1 average fluorescence in suprabasal cells of control vs K10-MafB at E14.5 (n= 3 embryos/genotype). Data are presented as the mean ± SD. *: p < 0.05, two-tailed unpaired t-test.

(E) RNAScope of Scara5 in red and immunostaining with MafB in white.

(F) Quantification of the average number of Scara5 RNAScope puncta per cell in suprabasal layers of control, and MafB+ and MafB-cells of K10-MafB mice at E14.5 (n= 3 embryos/genotype). Data are presented as the mean. ns: not significant, repeated-measures one-way ANOVA, Tukey’s multiple comparisons test.

(G) Immunofluorescence staining of Cox1 in K10-MafB vs control mice at E14.5.

(H) Quantification of number of Cox1 puncta per standardized area of suprabasal layer in K10-MafB vs control mice at E14.5. n= 3 embryos/genotype. Data are presented as the mean. ns: not significant, two-tailed unpaired t-test.

Intermediate cells are transcriptionally similar to granular cells and express granular markers.

(A) Gene Ontology (GO) term enrichment analysis of genes in the intermediate cell signature.

(B) Venn diagram illustrating the number of genes in the intermediate cell signature compared with the granular signature (log2 fold > 1, p < 0.05) for each cell population. A total of 209 genes were commonly enriched in ICs and granular cells, which represents 34% of the total IC signature.

(C and E) Immunofluorescence staining of Tgm1 (C) and Abca12 (E) in green and K14 (red) to mark basal cells in WT embryos at E14.5 vs E16.5. Scale bars: 20 μm.

(D) Quantification of Tgm1 average fluorescence intensity at suprabasal cells at E14.5 (IC) vs the first two layers of suprabasal cells at E16.5 (spinous cells). n = 3 embryos/time point. Bar represents the mean. *: p < 0.05, two-tailed paired t-test.

(F) Quantification of Abca12 average fluorescence intensity at suprabasal cells at E14.5 (ICs) vs the first two layers of suprabasal cells at E16.5 (spinous cells). n= 3 embryos/time point. Data are presented as the mean ± SD. *: p < 0.05, two-tailed unpaired t-test.

Intermediate cells do not differentiate into spinous cells.

(A, B and C) Immunofluorescence staining of spinous cells with MafB in red and labeled cells with H2B-GFP from K10-rtTA;TRE-H2B-GFP mice that were injected with a low dose of doxycycline at E14.5 to label only ICs and sacrificed at E15.5 (A), E16.5 (B) or E18.5 (C).

Basement membrane is indicated as a dotted line. Scale bars: 20 μm.

(A’ and B’) Percentage of MafB+ and MafB-suprabasal cells expressing H2B-GFP at E15.5 (n= 3 embryos) (A) and E16.5 (n= 4 embryos) (B). Data are presented as the mean ± SD. ****: p < 0.0001, two-tailed unpaired t-test.

(D) Immunofluorescence staining of spinous cells with MafB in red and labeled cells with H2B-GFP in K14-rtTA;TRE-H2B-GFP mice that were injected with a low dose of doxycycline at E14.5 and sacrificed at E15.5. Scale bar: 20 μm.

(D’) Percentage of basal cells and MafB+ suprabasal cells expressing H2B-GFP. Paired samples are quantifications from the same mouse (n= 3 K14-rtTA;TRE-H2B-GFP embryos).

Intermediate cells show upregulation of contractility markers as compared to spinous cells.

(A) GO term enrichment analysis of genes upregulated in intermediate cells vs spinous cells at E16.5, as shown in the above diagram, regardless of their expression in basal cells (log2 fold > 1, p < 0.05) reveals upregulation of biological processes related to cytoskeleton organization.

(B) RNA-Seq heatmap depicting differential expression of contractome genes in intermediate and spinous cells at E16.5. Gene expression by FPKM was log2-transformed.

(C, D and E) Immunofluorescence staining of Phalloidin (C), MyosinIIA (D) and α18 (E) in green at E14.5 vs E16.5. Basement membrane is indicated as a dotted line. Scale bars: 20 μm.

(C’, D’ and E’) Fluorescence intensity of suprabasal-suprabasal cell boundaries in the first two layers of suprabasal cells. Line scans were performed in WT embryos at E14.5 (between intermediate cells) and E16.5 (between spinous cells). Line scan graphs show centered measurements across six cell-cell boundaries at each time point. Data are presented as the mean ± SD (shown as colored shadows in green at E14.5 and pink at E16.5). Scale bars: 20 μm.

(C’’, D’’ and E’’) Quantification of average fluorescence intensity from the line scan maximum values at suprabasal-suprabasal cell boundaries in WT E14.5 and E16.5. For bar plots, bars represent the mean, joined dots are paired samples from each time point. n= 3 embryos/time point, average maximum values of at least 15 line scans at suprabasal cell boundaries in each embryo were calculated. *: p < 0.05, two-tailed paired t-test.

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.

Live imaging of mitotic suprabasal cells during early epidermal stratification.

(A) Diagram depicting alleles used for live imaging of suprabasal cells, and treatment timeline: Pregnant dams were doxycycline fed 48 hrs before embryos were collected and their tissue harvested for explant imaging.

(B) Snapshots from live imaging of back skin explants from E14.5, E15.5 and E16.5 K10-rtTA;TRE-H2B-GFP embryos, which express H2B-GFP in suprabasal cells. Images are from the first plane of H2B-GFP+ cells, corresponding to the suprabasal layer immediately on top of basal cells from E14.5, E15.5 and E16.5 explants. Time interval between snapshots: 15 min. Scale bars: 20 μm.

(C) Percentage of mitotic events counted during 2 hrs of live imaging. Only suprabasal cells from layers on top of basal cells were quantified. n= 5 different fields for E14.5 and E15.5, and n= 4 different fields for E16.5 from at least 2 embryos for each time point were analyzed. ****: p < 0.001, ordinary one-way ANOVA, Tukey’s multiple comparisons test.

Intermediate cells do not express some granular markers at the protein level.

(A and B) Immunofluorescence staining of Loricrin(A) or St8sia6 (B) in green labeling granular cells at E16.5 but not in intermediate cells at E14.5. Basement membrane is marked with β4-integrin in red. Scale bars: 20 μm.

The overall rate of suprabasal cell proliferation in K10-MafB is not significantly different from controls.

(A) Diagram depicting alleles in K10-MafB used to induce MafB expression in intermediate cells. Pregnant dams were fed with doxycycline chow from E12.5 and sacrificed at E14.5.

(B) Percentage of suprabasal cells expressing MafB (HA+) in K10-MafB mice at E14.5.

(C) Percentage of dividing cells among all suprabasal cells at E14.5 in K10-MafB vs controls is not significantly different. n= 3 embryos/genotype. Data are presented as the mean. ns: not significant, two-tailed unpaired t-test.

Intermediate and granular cells shared pathways related to lipid biosynthetic processes and endomembrane systems.

GO analysis from commonly enriched genes in intermediate and granular cell signatures.

Intermediate cells differentiate into granular cells

(A) Image showing H2B-GFP expression in intermediate cells in a E14.5 K10-rtTA;TRE-H2B-GFP mouse fed with doxycycline from E12.5. Basement membrane is indicated as a dotted line. Scale bars: 20 μm.

(B) Immunofluorescence staining of Loricrin in red colocalizing with H2B-GFP labeled cells in K10-rtTA;TRE-H2B-GFP mice injected with a low dose of doxycycline at E14.5 and sacrificed at E16.5. Scale bars: 20 μm.

Like granular cells, more intermediate cells have nuclear YAP than spinous cells.

(A) Immunofluorescence staining of YAP/TAZ at E14.5 and E16.5. Scale bars: 20 μm.

(B) Percentage of nuclear active YAP in basal cells and in the first two suprabasal layers at E14.5 and E16.5. Active YAP was also quantified in granular cells at E16.5. *:p < 0.05, multiple unpaired t-test. n= 3 embryos/time point.

Inducing constitutively active YAP in spinous cells does not increase their proliferation.

(A) Image of EdU staining in magenta and immunofluorescence of Loricrin in red from control and K10-YAPCA embryos at E16.5. Cells expressing constitutively active nuclear YAP are labeled with H2B-GFP in green. Mice were fed with doxycycline from E13.5. Scale bars: 20 μm.

(B) Percentage of dividing suprabasal cells in control vs K10-YAPCA. n= 3 embryos/genotype. 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. *: p < 0.05, ns: not significant, two-tailed unpaired t-test.

K10-Spastin cells are more granular-like than intermediate-like, and lipid metabolic processes are commonly upregulated among K10-Spastin, IC and granular cells.

(A and B) Size proportional-Venn Diagrams show that K10-Spastin cells are more granular-like (A: 46.6% similarity) than intermediate-like (B: 11.7% similarity). Green: genes enriched in K10-Spastin vs WT embryos at E16.5; blue: genes in intermediate cell signature, pink: genes in granular signature.

(C) GO term analysis of genes commonly upregulated in ICs, granular cells and K10-Spastin (total: 50 genes) are lipid metabolic processes.

Increased contractility in spinous cells of adult epidermis induces some granular markers in adult epidermis.

(A and B) Immunofluorescence staining of MafB in back skin (A) or glabrous skin (B) in red, and suprabasal cells expressing Arhgef11CA marked by HA (green) in adult K10-Arhgef11 and controls. Mice were fed with doxycycline since adulthood. Insets show close-ups of HA+ cells in suprabasal layers adjacent to basal cells. Scale bars: 20 μm.

(C and D) Immunofluorescence staining of Tgm1 in back skin (C) or glabrous skin (D) in red, and suprabasal cells expressing Arhgef11CA marked by HA (green) in adult K10-Arhgef11 and controls. Insets show close-ups of HA+ cells in suprabasal layers adjacent to basal cells. Mice were fed with doxycycline since adulthood. Scale bars: 20 μm.

Increased contractility in spinous cells accelerates acquisition of granular markers.

(A and B) Immunofluorescence staining of MafB (A) or Tgm1 (B) in red, and suprabasal cells expressing Arhgef11CA marked by HA (green) in adult K10-Arhgef11 and controls. Images from E17.5 embryos, fed with doxycycline starting at E15.5. Insets show close-ups of HA+ cells in suprabasal layers adjacent to basal cells. Scale bars: 20

Inducing constitutively active YAP in spinous cells does not induce granular markers

(A and C) Immunofluorescence of MafB (A) or Tgm1 (C) in red, active YAP in green (YAP-H2B-GFP) in control, and K10-YAPCA at E16.5. Scale bars: 20 μm.

(B) Average MafB fluorescence intensity per cell in control and K10-YAPCA. n=113 suprabasal cells from 3 control embryos, and 103 from 3 K10-YAPCA embryos. ns: not significant, two-tailed unpaired t-test.

(D) Average fluorescence intensity of suprabasal cells, area above basal cells in control and K10-YAPCA embryos at E16.5 were measured. n= 3 embryos/genotype. ns: not significant, two-tailed unpaired t-test.

Increased contractility does not alter H3K27me3 levels.

Quantifications of nuclear H3K27me3 levels in control spinous and K10-Arhgef-expressing spinous cells.