Extended Data Fig. 8: Catagen HFSCs produce and respond to retinoic acid to upregulate the phagocytic program.
From: Stem cells tightly regulate dead cell clearance to maintain tissue fitness
a, Left, Schematic to expose Retinoic Acid Response Element (RARE)-driven RFP; constitutive H2B-GFP expressing HFSCs to signals in vitro. Middle left, Quantification of RARE+ HFSCs to RA isoforms 9-cis retinoic acid (9cRA) or all-trans retinoic acid (ATRA) with or without the RAR-inhibitor (+AGN; AGN193109). Middle right, Quantifications of RARE+ HFSCs exposed to corpses with or without lysophosphatidylcholine (LPC)/free fatty acids (+BEL; bromoenol lactone) (left) or to recombinant arachidonic acid (AA) and LPC with or without retinoic acid (RA) (right). n = 6 experimental replicates per condition. b, Top, Strategy to express RARE-driven RFP with constitutive H2B-GFP reporter in skin. Bottom, Sagittal immunofluorescence of RARE-infected HFSCs across the hair cycle, asterisk denotes hair shaft keratinization; quantified in Fig. 4f. Images and quantification are representative of 10-15 HFs per mouse, n = 2 mice per hair cycle stage. Scale bar, 20 μm. c, Left, Dot plot summarizing retinoic acid synthesis and catabolism enzyme expression across the hair cycle from single cell transcriptomic data. Middle, Sagittal wild type skin sections from mid-late catagen and early telogen stages of the hair cycle stained for retinoic acid rate-limiting enzyme Aldh1a2 (arrowheads). Dashed line, dermo-epithelial border. Scale bar, 20 μm. Right, FACS plots showing normalized counts of Aldh1a activity on the AldeFluor substrate in late catagen HFSCs (top) quantified across the hair cycle (bottom). n = 6 mice per hair cycle stage. d, Left, Experimental strategy to deplete RA from HFs in vivo. TMX, Tamoxifen. Right, Sagittal imunofluorescence of transduced (left and middle) versus untransduced catagen HFs. Boxed regions are magnified at right. Cyp26b1 over-expressing (OE) cells are YFP+RFP+ while virus control are RFP+. Scale bar, 20 um. Data are quantified in Fig. 4f, n = 3 mice per hair cycle stage. Quantifications, pairwise independent Student’s t-tests (2-sided), p-values indicated. n.s. not significant (p > 0.05). Data as box-and-whisker plots; box: first-to-third quartiles and median, whiskers 1.5× inter-quartile range. More details on statistics and reproducibility can be found in the Methods section.
