Dermal sheath contraction powers stem cell niche relocation during hair cycle regression

Abstract

Tissue homeostasis requires the balance of growth by cell production and regression through cell loss. In the hair cycle, during follicle regression, the niche traverses the skin through an unknown mechanism to reach the stem cell reservoir and trigger new growth. Here, we identify the dermal sheath that lines the follicle as the key driver of tissue regression and niche relocation through the smooth muscle contractile machinery that generates centripetal constriction force. We reveal that the calcium-calmodulin-myosin light chain kinase pathway controls sheath contraction. When this pathway is blocked, sheath contraction is inhibited, impeding follicle regression and niche relocation. Thus, our study identifies the dermal sheath as smooth muscle that drives follicle regression for reuniting niche and stem cells in order to regenerate tissue structure during homeostasis.

Fig. 1.. The dermal sheath is required for hair follicle regression.

(A) Schematic of catagen regression during the hair cycle. Hair shaft, inner root sheath (IRS) and matrix progenitors are eliminated by terminal differentiation and extrusion from the skin. The majority of outer root sheath (ORS) progenitors (blue) are eliminated by apoptosis. It is unknown how the surviving dermal papilla (DP) niche relocates to the bulge and germ stem cell reservoir to activate new hair growth after a period of rest. (B) Immunofluorescence (IF) for ACAN, secreted by ITGA8⁺ dermal sheath (DS) cells. (C) Schematic of cytotoxic DS ablation during regression. (D) IF for αSMA in control (R26^LSL-DTA) and DS-ablated (Acan^CreER;R26^LSL-DTA) back skin. (E) Whole mount IF for ORS marker K14 in P20 back skins (viewed from dermis side, anterior = left). Control follicles are in telogen resting phase. Note elongated follicles after DS ablation stalled in the regression phase. (F) Quantification of % stalled follicles at P20 (n = 698 in control and 895 in DS-ablated follicles in 5 mice). **P = 0.003, unpaired two-tailed t-test. (G) Inset from E: hair shaft and DP remain at bulb tip of stalled follicles. (H) Quantification of follicle lengths (n = 11 P13 control, n =14 P13 ablated, n = 80 P20 control, n = 27 P20 stalled; 11 mice). ****P < 10⁻⁴, unpaired two-tailed t-test. (I) Stalled follicles have no DS (αSMA⁻), but retain intact DP (LEF1⁺). K14⁺ ORS progenitor and K6⁺ companion (Cp) layers are present and lack apoptosis (activated CASP3) or proliferation (Ki67) markers. Scale bars, 50 μm (B, E) and 10 μm (D, I).

Fig. 2.. The dermal sheath expresses the molecular machinery of smooth muscles.

(A, B) Flow cell sorting of DS and DP from Sox2^GFP;Lef1-RFP P5 back skin and IF for PDGFRA. Dermal fibroblasts (DF) were sorted for comparison. (C) Venn diagram of gene signatures. (D) Gene ontology analysis of DS signature. (E) Gene set enrichment analysis (GSEA) for genes involved in smooth muscle contraction and regulation are highly enriched in DS. (F) Schematic of Ca²⁺-dependent smooth muscle contraction pathway. (G) Heatmap of smooth muscle contraction gene expression. Ca²⁺ contraction pathway and pan-smooth muscle genes (asterisks) are highly enriched in DS. (H) 3D IF for αSMA fibers arranged in a concentric ring-like network wrapping around the follicle. (I) IF of smooth muscle contraction components in DS. Scale bars, 50 μm.

Fig. 3.. The dermal sheath functionally contracts and is required for regression in vivo.

(A) Schematic of live imaging microdissected follicles pre-incubated with or without MLCK inhibitor ML7 and after high K⁺ depolarization. (B, C) Still images from brightfield movie at start (black) and end (pink) of high K⁺ incubation. Overlays highlight reduction of follicle width, blocked by ML7. (D) Quantification of follicle widths during live imaging. n = 7 follicles for ML7 and no inhibitor pre-incubation. Data points are mean ± s.d. **P < 0.01, unpaired two-tailed t-test. (E-G) Topical inhibition of MLCK by ML7 blocks hair follicle regression in vivo. Schematic of ML7 or vehicle application during catagen (E). Whole mount IF of P20 back skins show normal regression of follicles into telogen rest in control, but stalled follicles in contraction-inhibited ML7-treated regions (F). (G) Quantification of % stalled follicles (n = 1071 control, n = 1019 ML7-treated; 10 mice). Data bars are mean ± s.d. **P = 0.001, unpaired two-tailed t-test. (H) IF for LEF1, Ki67, αSMA and K14. Stalled follicles have intact DS (αSMA) and DP (LEF1) that are no longer engulfed. Epithelial cells (K14) of stalled follicles are not proliferative (Ki67). Scale bars, 50 μm (B, C, F) and 10 μm (H).

Fig. 4.. Dermal sheath contraction is required for hair shaft and niche relocation.

(A) Triple-fluorescent reporter follicles for intravital 3D time-lapse imaging of catagen regression in live mice (5 hours). Acan^tdT and Tbx18^H2BGFP marks DS cytoplasm and nuclei, respectively. K14-H2BCer highlights all epithelial nuclei. The DP was recognized by low level Tbx18^H2BGFP expression and surrounding epithelial and DS cells. (B) Upward movement of hair shaft and DP during regression. Tracking of individual ORS, shaft and DS cells and of upper and lower bounds of DP during 5-hour imaging. (C) Quantifications of live cell tracking relative to ORS movement (7.5 minute intervals). Shaft and DP move upward relative to ORS and DS. Solid lines are average; shaded areas are s.d. n = 13 HS, n = 8 DP, n = 26 ORS, and n = 17 DS measurements (7 follicles, 3 imaging sessions). (D) In vivo contraction blocking during intravital imaging in K14-H2BCer mice. Inverted grayscale still images at beginning and end of vehicle control followed by ML7 application. Quantification of live tracking of shaft cells (black) relative to ORS (blue). n = 9 follicles from 2 independent imaging sessions. **P < 0.01 and ***P < 0.001, paired two-tailed t-test. (E) Schematic of two historically hypothesized mechanisms for DP niche relocation during regression. (F, G) Fluorescence images from time-lapse movie and quantification of the length of regressing epithelial strand. Strand lengths remain unchanged and stable (n = 10 follicles). P = 0.572, unpaired two-tailed t-test (0 hr v. 5 hr). (H) DS cross-sectional diameter at DP bottom (n = 4 follicles). P = 0.994, unpaired two-tailed t-test (0 hr v. 5 hr). (I) Quantification of DS diameter that remains unchanged over time. Data bars are mean ± s.d. Scale bars, 10 μm.

Fig. 5.. Dermal sheath contraction pushes the hair shaft and indirectly pulls the niche.

(A) Schematic of alternative hypothesis for DP niche relocation by DS contraction at the bottleneck between the shaft-containing club hair and narrower regressing epithelial strand. (B) Live cell tracking of DS centripetal constriction movement at club-epithelial strand bottleneck and of hair shaft upward movement. Arrows are starting and ending positions of 5-hour tracking. (7 follicles, 3 imaging sessions). (C) High magnification of fluorescence time-lapse images at club-epithelial strand bottleneck. (D) DS cross-sectional diameter at club hair-epithelial strand bottleneck. The diameter of the follicle-wrapping DS cell ring decreased over time. (E) Quantification of DS diameter at bottleneck decreases over time (n = 3 follicles). *P = 0.021, **P = 0.009 and P = 0.010, unpaired two-tailed t-test (0 hr v. 5 hr). (F) Model for DP niche relocation during regression. DS contraction forces centripetally constrict the follicle at the bottleneck to move the hair shaft upward, which pulls the DP upward via the epithelial strand. Data bars are mean ± s.d. Scale bars, 10 μm.