Fig 2: CD34+ hfDSC progeny repopulate DS and DP cells during an ongoing hair cycle. (A) Outline of lineage-tracing strategy in aSMA:mTmG mice. (B) Experimental design showing fate mapping assessment of CD34+ hfDSCs labeled at P49-53 and harvested at different stages of the hair cycle. (C) Typical images of HFs in aSMA:mTmG mice at different stages. Confocal reconstructions of an anagen HF showed that aSMA+ hfDSCs supplemented DP cells (right-most panel). (D) Representative telogen HFs 45 days post first depilation showing the distribution of mGFP fluorescent protein in hfDSCs (left panel). A higher-magnification view of the left panel (right panel). (E) Fate of CD34-lineage cells (green) at different stages of the hair cycle. Dotted lines: approximate location of DP. (F) ITGA5 immunostaining of an anagen HF from CD34:mTmG mice. (G) An anagen HF from CD34:mTmG mice immunostained for aSMA. aSMA, alpha smooth muscle actin. (H) Schematic summary of the fate of CD34-lineage cells (green) during the second depilation-induced hair cycle. Scale bars, 50 µm.

Fig 3: Ectopic activation of ß-catenin leads to senescent HFs without DP cells. (A) Experimental timeline of RU486 injections and analysis in young CD34:mTmG, old CD34:mTmG, and CD34:mTmG:ß-catact mice. (B) Young CD34:mTmG (left panel), old CD34:mTmG (middle panel), and CD34:mTmG:ß-catact mice (right panel) that had undergone depilation-induced hair cycle twice. (C) Skin sections of 1.5-year-old CD34:mTmG mice. (D,E) CD34:mTmG:ß-catact skin sections showing HFs without DP cells. Note that there was a ring of DS cells at the bottom of the HF, but the HFs did not contain DP. (F) Schematic illustration of the evolution of senescent HFs without DP cells. (G) CD34:mTmG:ß-catact and CD34:mTmG skin immunostained for anti-cleaved caspase-3 antibody (white). Arrowhead highlights cleaved caspase-3 expression in DP cells. (H) CD34:mTmG:ß-catact and CD34:mTmG skin were immunostained for ß-catenin (white). Note elevated ß-catenin expression in hfDSCs. Scale bars, 2 cm (B); 50 µm (C–H).

Fig 4: CD34 is expressed in hfDSCs adjacent to DP and dermal fibroblasts. (A) Immunostaining analysis for CD34 (purple) in skin tissues of C57BL/6 mice 0, 3, 5, 7 and 10 days after depilation. Arrowheads point to DS cells that express CD34. (B) Confocal microscope images of skin sections from CD34-tdTomato (red) mice immunolabeled with CD34 (white). Note the colocalization of tdTomato with CD34 in DS and fibroblasts. (C) Immunofluorescence analysis of a telogen HF from CD34-tdTomato (red) mice immunolabeled with ITGA5 (green). (D) Experimental scheme for RU486 injection and analysis of HFs at different stages of the depilation-induced hair cycle in CD34:mTmG mice. (E) Images of skin sections harvested from CD34:mTmG without RU486 injection. (F) Representative images showing the fate of CD34-lineage cells (green) at each stage of the first depilation-induced hair cycle in 7-week-old CD34:mTmG mice. (G) The schematic illustrating the tracing of CD34-lineage cells (green) during the first depilation-induced hair cycle. Scale bars, 50 μm.

Fig 5: Activation of ß-catenin leads to HF mesenchymal senescence and reduction of hfDSC progenitor pool. (A) Schematic depicting the timeline of RU486/tamoxifen injections and analysis. (B) Hair coat of CD34:mTmG:ß-catact (left panel) and mTmG:ß-catact (right panel) mice photographed at 60 days after RU486 injections. (C) CD34:mTmG:ß-catact mice exhibited significantly grayer hair coats. (D) Ventral view of CD34:mTmG:ß-catact mice (left panel) and control mTmG:ß-catact littermates (right panel). (E) Hair coats of pairs of aSMA:mTmG:ß-catact (left panel) and mTmG:ß-catact (right panel) mice were photographed 60 days after tamoxifen injections. (F,G) Immunostaining for Cyr61 (F), aSMA (G) and ITGA5 (H) in skin tissues from young (upper panel) and old CD34:mTmG mice (middle panel), and CD34:mTmG:ß-catact mice (lower panel). (I) HFs of 18-month-old CD34:mTmG mice illustrating DP cells above Auber’s line replenished by mGFP-labeled cells. (J) Telogen HFs of 18-month-old CD34:mTmG mice showed a significant increase in lineage-labeled mGFP+ cells within DP. (K) Schematic illustration of the CD34 lineage tracing (green) in the HF of CD34:mTmG mice after multiple cycles. Scale bars, 2 cm (B–E); 50 µm (F–K).