Fig 2: Hyperproliferation of Plexin-B1/Plexin-B2 double-deficient epidermal stem cells induces overcrowding, cell shape anisotropy and differentiation.a Upper row: Confocal images of whole-mount immunostainings (top view) of murine epidermis at E15.5 using an anti-E-cadherin antibody (green). Shown are representative images of the basal cell layer. Lower row: Segmentation analyses of the images depicted in the upper row. Scale bar, 25 µm. b Quantification of the number of epidermal stem cells (basal layer) per area (mean ± s.d.; n = 3 mice per genotype; p = 0.0199; two-sided unpaired t-test). c Analysis of epidermal stem cell areas (violin plot with first quartile, median, and third quartile; control: n = 415 from three mice, PlexDKO: n = 509 from three mice; F-test of equality of variances: p < 0.0001, two-sided Mann–Whitney U test: p = 0.0011). d, e Quantification of cell shape anisotropy (box plot with minimum, first quartile, median, third quartile and maximum; control: n = 415 from three mice, PlexDKO: n = 509 from three mice; two-sided Mann–Whitney U test: p = 0.0003). f Immunohistochemistry on murine embryonic epidermis using an anti-NICD antibody (brown). Dashed lines indicate the basement membrane. Scale bar, 25 µm. g, h Quantification of the data in f (mean ± s.d.; E15.5 control: n = 5 mice, E15.5 PlexDKO: n = 4 mice, E16.5: n = 6 mice per genotype; p = 0.0917 at E15.5, p = 0.0391 for E16.5; two-sided unpaired t-test). i Confocal images of immunostainings of murine epidermis at E18.5 using anti-K14 (green) and anti-K10 (red) antibodies. Scale bar, 25 µm. j, k Quantification of the data in i (mean ± s.e.m.; n = 3 mice per genotype; j p = 0.0041, k p = 0.0569, two-sided unpaired t-test).

Fig 3: B-plexins suppress YAP activity in human basal cell carcinoma cells.a Human primary epidermal keratinocytes (hPEKS) and human primary basal cell carcinoma cells (hPBCCs) were lysed, and proteins were detected using the indicated antibodies. b, c hPBCCs engineered to express GFP (“control”) or myc-tagged Plexin-B2 (“Plexin-B2”) and cultured at dense conditions were immunostained for YAP (red) and myc (green). Blue: DAPI. Representative confocal images are shown in b with dashed boxed areas depicted in the right panel. A quantification of YAP nuclear intensity is shown in c (mean ± s.d.; n = 3 independent experiments; p = 0.0495; two-sided unpaired t-test). d hPBCCs were engineered to express GFP (“control”) or myc-tagged Plexin-B2 (“Plexin-B2”), and cultured at dense conditions. Relative mRNA expression levels of the YAP target genes ctgf and cyr61 were determined by quantitative RT-PCR (mean ± s.d.; n = 5 independent experiments; p = 0.0123 for ctgf, p = 0.0002 for cyr61; two-sided unpaired t-test).

Fig 4: Plexin-B1/Plexin-B2 suppress epidermal stem cell proliferation during embryonic development.a Confocal images of immunostainings of murine skin at embryonic day 15.5 (E15.5), 16.5 (E16.5), 18.5 (E18.5) and at an adult stage using anti-phospho-histone H3 (pHH3; red) and anti-keratin 14 (K14; green) antibodies. Dashed lines indicate the basement membrane. Scale bar, 25 µm. b Quantification of pHH3-positive cells at different time points of embryonic (E) development and in the adult (mean ± s.e.m.; n = 3 mice per time point). c mRNA expression levels of genes encoding B-plexins (plxnb) in the murine skin at the indicated time points as determined by quantitative RT-PCR (mean ± s.e.m.; n = 3 mice per time point). d Confocal images of immunostainings of murine epidermis at embryonic day 16.5 (E16.5) using anti-Plexin-B1 (green) and anti-Plexin-B2 antibodies (red). Dashed lines indicate the basement membrane. Scale bar, 10 µm. e Confocal images of immunostainings of murine epidermis at E16.5 using anti-Plexin-B2 (red), and anti-keratin 14 (K14) or anti-keratin 10 (K10) antibodies (green). Scale bar, 10 µm. f Immunohistochemistry on murine embryonic epidermis using an anti-BrdU antibody (brown). “contr.”: control mice (genotype plxnb1flox/flox;plxnb2flox/flox), “PlexDKO”: epidermis-specific Plexin-B1/Plexin-B2 double-knockout mice (genotype K14-Cre;plxnb1flox/flox;plxnb2flox/flox). Blue: Hematoxylin. Scale bar, 50 µm. g Quantification of the data in f (mean ± s.e.m.; E15.5: n = 5 mice per genotype, E16.5: n = 4 mice per genotype; p = 0.0429 for E15.5, p = 0.0175 for E16.5; two-sided unpaired t-test). h H&E-stained histological sections of murine epidermis at the indicated time points of embryonic development. Brackets indicate epidermal thickness. Scale bar, 50 µm. i Quantification of the data in h (mean ± s.e.m.; E14.5: n = 6 mice per genotype, E15.5 and E16.5: n = 7 mice per genotype and time point; p = 0.625 for E14.5, p = 0.494 for E15.5, p = 0.005 for E16.5; two-sided unpaired t-test). j Confocal images of immunostainings of murine embryonic epidermis using anti-K14 (green) and anti-K10 (red) antibodies. Scale bar, 25 µm. k Quantification of K14-positive cells (mean ± s.e.m.; n = 7 mice per genotype and time point; p = 0.38 for E14.5, p = 0.0009 for E15.5, p = 0.0497 for E16.5; two-sided unpaired t-test).

Fig 5: Plexin-B1/Plexin-B2 control YAP activity and cell proliferation in basal cell carcinoma.a Schematic illustration of the generation of epidermis-specific tamoxifen-inducible Gas/Plexin-B1/Plexin-B2 triple-deficient mice. b Kaplan–Meier curves representing the percentage of lesion-free mice. Time point “0” indicates the start of tamoxifen treatment (wildtype mice: n = 3, Gas KOind mice: n = 16, Gas KOind;PlexDKOind mice: n = 16; p < 0.0001; Mantel–Cox test for Gas KOind compared to Gas KOind;PlexDKOind). c–h Histological/immunofluorescence stainings (left panels) and respective quantifications (right panels) of adult murine skin of mice with the indicated genotypes 30 days after treatment with tamoxifen. Boxed areas are magnified in the lower rows. Tumors are marked by dashed lines. c Goldner-Elastica stain of histological sections. Scale bar, 100 µm (upper row), 25 µm (lower row). d Quantification of tumor areas (mean ± s.d.; Gas KOind mice: n = 8, Gas KOind;PlexDKOind mice: n = 12; p = 0.0049; two-sided unpaired t-test). e Confocal images of immunostainings using an anti-Ki67 antibody (red). Scale bar, 100 µm (upper row), 25 µm (lower row). f Quantification of Ki67-positive cells (mean ± s.d.; Gas KOind mice: n = 7, Gas KOind;PlexDKOind mice: n = 7; p = 0.0049; two-sided unpaired t-test). g Confocal images of immunostainings using anti-active YAP (red) and anti-K14 antibodies (green). Scale bar, 100 µm (upper row), 25 µm (lower row). h Quantification of cells positive for active YAP (mean ± s.d.; Gas KOind mice: n = 7, Gas KOind;PlexDKOind mice: n = 10; p = 0.0143; two-sided unpaired t-test).