Altered differentiation trajectories and Wnt signaling in CerS4-deficient HFSCs. (A) Ceramide levels in FACS-purified CD34⁺ integrin α6⁺ HFSCs and CD34⁻ integrin α6⁺ non-HFSCs determined by quantitative LC-ESI-MS/MS analysis (n = 5 mice/genotype; ratio paired t test). (B) Receptor–ligand interaction analyses (CellPhoneDB) from scRNAseq data of control (Ctr) and CerS4^epi−/− skin. Communication from the OB cells to the IB is illustrated. (C and D) RNA expression levels of selected canonical Wnt target genes in Ctr and CerS4^epi−/− skin from scRNAseq data. (E) Representative images and quantification of P21 Ctr and CerS4^epi−/− back skin sections stained for Lef1 (grey) and keratin-14 (magenta). Note increased Lef1 expression (arrows) in CerS4^epi−/− OB (scale bar 50 µm; n = 4; mean ± SD; unpaired t test). (F) Experimental outline for Wnt activation using Chir99021. (G) FACS-based quantification of HFSCs and non-HFSCs from Ctr and CerS4^epi−/− organoids treated with Chir99021 (1 µM). Note a decrease in HFSCs in Chir99021-treated CerS4^epi−/− organoids but not in Ctr organoids (n = 10 mice/genotype, mean ± SD; unpaired t test). (H) Representative images of Ctr and CerS4^epi−/− organoids treated with Chir99021 and stained for Sox9 (grey) and keratin-14 (magenta). Scale bars 20 µm. (I) Representative images and quantification of Ctr and CerS4^epi−/− organoids treated with Chir99021 and stained for keratin-6 (grey) and keratin-14 (magenta). Note increased keratin-6 expression and loss of cell cohesion in CerS4^epi−/− organoids, indicated by arrows (scale bar 20 µm; n = 8 organoids/genotype; mean ± SD; unpaired t test). LC-ESI-MS/MS, LC coupled to electrospray ionization tandem MS/MS.