CerS4 is required for the establishment of the HFSC compartment. (A) Representative images and quantification of hair follicle stage of H/E-stained back skin sections of control (Ctr) and CerS4^epi−/− back skin at P16, 18, and 19 showing a developmental delay in hair follicle morphogenesis and enlarged SGs (scale bars 100 µm; n = 4–6 mice/genotype; unpaired t test). (B) Representative FACS plots of P47 murine epidermal cells stained for CD34 and integrin α6. (C) FACS-mediated quantification of CD34⁺ integrin α6⁺ HFSCs at P21 (n = 13 Ctr, 9 CerS4^epi−/− mice), P47 (n = 9 Ctr, 11 CerS4^epi−/− mice), P128 (n = 8 Ctr, 9 CerS4^epi−/− mice), and P365 (n = 12 Ctr, 8 CerS4^epi−/− mice). Note progressive loss of stem cells in CerS4^epi−/− mice (mean ± SD; unpaired t test). (D) Cell counts of cell clusters determined by scRNAseq. (E) Schematic illustration of the IFE and the pilosebacous unit containing the SG and the hair follicle and its bulge stem cell compartment. Keratin-14-Cre is expressed in basal cells throughout the tissue, allowing keratin-14-Cre–mediated deletion of CerS4 (CerS4^epi−/−) in all stem cell compartments. Schematic and UMAP projections of cells identified using scRNAseq. Note comparable presence of all epidermal cell states but increased OB population in CerS4^epi−/− mice (n = 2 mice/genotype, pooled). (F) Gene expression of indicated stem and progenitor cell marker genes in the OB stem cell compartment in Ctr and CerS4^epi−/− skin. IFE = interfollucular epidermis, IB = inner bulge, OB = outer bulge, uHF = upper hair follicle, SG = sebaceous gland, TC = T cells, LH = Langerhans cells.