CerS4 controls HFSC lineage specification. (A) Linage trajectories as determined by the SlingShot of Ctr and CerS4^epi−/− cells from scRNAseq data (n = 2 mice/genotype, pooled). Note abnormal trajectory from OB cells into SG. (B) Lineage trajectories as determined by Paga of Ctr and CerS4^epi−/− cells from scRNAseq data (n = 2 mice/genotype, pooled). Note trajectories from the uHF I cell cluster and OB cells to the SG population in CerS4^epi−/− skin. (C) Pseudotime analyses of Ctr and CerS4^epi−/− cells from scRNAseq data (n = 2 mice/genotype, pooled) confirming altered linage progression with expression of Plet1 remaining high, Lhx2 low, and keratin-6 high in CerS4^epi−/− OB stem cells. (D) Representative images and quantification of P21 back skin sections stained for Lhx2 (grey) and keratin-14 (magenta) show reduced expression of Lhx2 (arrows) in bulge stem cells (scale bars 50 µm; n = 4 mice/genotype; mean ± SD; unpaired t test). (E) Representative images and quantification of P21 control and CerS4^epi−/− back skin sections stained for keratin-6 (grey), keratin-14 (magenta) show ectopic keratin-6 expression (arrows) in the OB and uHF of CerS4^epi−/− mice (scale bars 25 µm; n = 4 mice/genotype; mean ± SD; unpaired t test). (F) Representative images and quantification of lineage-tracing analysis of β-galactosidase⁺ Lgr5 progeny from Ctr and CerS4^fl/fl-Lgr5eGFP-CreERT2 (Lgr5Cre⁺) mice. Quantification of hair follicles containing β-galactosidase⁺ in the SG, uHF, and IFE are shown (scale bars 80 µm; n = 3 Ctr and 7 = CerS4^fl/fl Lgr5Cre⁺ mice).