Foxc1 reinforces quiescence in self-renewing hair follicle stem cells

Abstract

Stem cell quiescence preserves the cell reservoir by minimizing cell division over extended periods of time. Self-renewal of quiescent stem cells (SCs) requires the reentry into the cell cycle. In this study, we show that murine hair follicle SCs induce the Foxc1 transcription factor when activated. Deleting Foxc1 in activated, but not quiescent, SCs causes failure of the cells to reestablish quiescence and allows premature activation. Deleting Foxc1 in the SC niche of gene-targeted mice leads to loss of the old hair without impairing quiescence. In self-renewing SCs, Foxc1 activates Nfatc1 and bone morphogenetic protein (BMP) signaling, two key mechanisms that govern quiescence. These findings reveal a dynamic, cell-intrinsic mechanism used by hair follicle SCs to reinforce quiescence upon self-renewal and suggest a unique ability of SCs to maintain cell identity.

Fig. 1. Dynamic expression of Foxc1 in the bulge

(A) In telogen, Foxc1 expresses in the Inf and SGP but not in the bulge. (B) Foxc1 is induced in the HFSC, the SBL, and the IRS at anagen III. (C–G) In mature HF of anagen IV, Foxc1 is highly expressed in the bulge and the IRS. Note in (E) Foxc1 is specifically detected in both K6+ SBL and CD34+ HFSCs and in (G) Foxc1 is detected in the IRS. (H–I) There is no Foxc1 expression in the bulge during catagen and telogen. (J) No Foxc1 expression in telogen bulge is confirmed by IF staining. Inf: infundibulum; SGP: sebaceous gland progenitors; Telo: telogen; Ana: anagen; Bu, bulge; HG, hair germ; DP, dermal papillae; HS, hair shaft; Scale bar 20μm in A–C and E–J; 50μm in D.

Fig. 2. Foxc1 is differentially required by the HFSCs and the suprabasal bulge layer

(A) Differential deletion of Foxc1 by different Cre lines at anagen (P26~P30). (B) Premature HF growth of Foxc1 cKO animals, compared to WT animals, during the second telogen. (C–D) Premature HFSC activation in Foxc1 cKO is shown by BrdU incorporation and HF growth and the loss of club hair in the K14-Cre cKO. (E) Different defects in club hair loss and compromised quiescence are observed in Foxn1-Cre and K15-CrePR models. (F) Quantification of BrdU or EdU+ cells in the HFSCs and HG cells before (P47–P50, n=3 for each genotype) and after (P57–P60, n=3 for K14-Cre n=4 for Foxn1-Cre) the premature HFSC activation and quantification of premature HF growth in the K15-CrePR model with pre-anagen deletion, compared to the WT and the K15-CrePR model with post-anagen deletion. *** p<0.001, ns: not significant. Scale bar 20μm in A, C–E unless otherwise labeled. Dotted lines outline the bulge and solid lines outline the DP.

Fig. 3. Foxc1 KO HFSCs fail to return to quiescence

(A) Functional classification of selected differentially expressed gene in P47 KO HFSCs. Red and blue mark up- and downregulated genes in the KO HFSCs, respectively. (B) qPCR validation of down- and upregulated genes in the HFSCs isolated from different cKO models at the second telogen (P47~P60, n≥3 for each genotype, *p<0.05). (C) GSEA comparison of P47 KO transcriptome to telogen bulge and HG signature genes. (D) PCA of P47 Foxc1 KO transcriptome and profiling data from WT HFSCs, HGs and TACs. Linear discriminant analysis groups Foxc1 KO transcriptome to activated HFSCs.

Fig. 4. Foxc1 activates quiescence gene networks

(A) Functional classification of selected differentially expressed gene in P30 KO HFSCs. (B) Comparison of enriched TF motifs against genomic sequences and against the peaks covering the non-differentially expressed (non-DE) genes in P30 KO HFSCs. (C) GSEA of BMP responsive genes and Nfatc1 targets in P30 KO HFSCs. (D) Venn diagram of the differentially expressed genes in P30 KO HFSCs that contain motifs of Foxc1, Smad and Nfatc1 shows a co-regulated gene network. (E) ATAC-seq track of the Nfatc1 locus. Location of Nfatc1, Smad and Foxc1 motifs are shown in the peak region. (F) ChIP-PCR analysis of Foxc1 confirms association of Foxc1 to the predicted binding site in the Nfatc1 locus. (G) Differential expression of Foxc1 targets, Nfatc1, in the WT and KO HFSCs (* p<0.05).