Tissue homeostasis is sustained by stem cell self-renewal and differentiation. How stem cells coordinately differentiate into multiple cell types is largely unclear. Recent studies underline the heterogeneity among stem cells or common progenitors, suggesting that coordination occurs at the stem cell/progenitor level1,2,3,4. Here, by tracking and manipulating the same stem cells and their progeny at the single-cell level in live mice, we uncover an unanticipated flexibility of homeostatic stem cell differentiation in hair follicles. Although stem cells have been shown to be flexible upon injury, we demonstrate that hair germ stem cells at the single-cell level can flexibly establish all of the differentiation lineages even in uninjured conditions. Furthermore, stem cell-derived hair progenitors in the structure called matrix, previously thought to be unipotent, flexibly change differentiation outcomes as a consequence of unexpected dynamic relocation. Finally, the flexible cell fate determination mechanism maintains normal differentiation and tissue architecture against an ectopic differentiation stimulus induced by Wnt activation. This work provides a model of continual fate channelling and late commitment of stem cells to achieve coordinated differentiation and robust tissue architecture.
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Statistics source data for Fig. 3c and Supplementary Fig. 1b have been provided as Supplementary Table 1. Additional images for Fig. 5 have been deposited at Figshare: https://doi.org/10.6084/m9.figshare.7170422. All of the data that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank J. Moore and E. Legué for feedback on the manuscript and other Greco lab members for helpful discussion. We thank E. Fuchs for the K14-H2BGFP, pTRE-H2BGFP, K14-actinGFP and K14-Cre mice, and M.Taketo for the β-cateninflox(Ex3) mice. We thank the Yale Transgenic Facility for generating the pTRE-dNβcatGFP mice. This work is supported by the New York Stem Cell Foundation, The Edward Mallinckrodt Jr Foundation, the Glenn Foundation for Medical Research, the HHMI Scholar award, and the National Institute of Arthritis and Musculoskeletal and Skin Disease (NIAMS), NIH, grants no. 2R01AR063663-06A1, no. 1R01AR072668-01 and no. 5R01AR067755-02. V.G. is a New York Stem Cell Foundation Robertson Investigator and HHMI Scholar. T.X. was supported by The James Hudson Brown–Alexander Brown Coxe Postdoctoral Fellowships.
Integrated supplementary information
Supplementary Figure 1 Inhibiting proliferation in most stem cell by blocking cell cycle does not affect hair follicle growth rate.
(a), Immunofluorescence staining of early Anagen hair follicles for cell cycle markers Ki67 and BrdU showing the H2BGFP+ cells that also express Cdkn1b in the proliferation inhibition experiments (Lgr5-CreER; R26-flox-STOP-tTA; tetO-Cdkn1b; pTRE-H2BGFP; K14-H2BmCherry) have blocked cell cycle. Images representative of 3 mice. Scale bar, 50 µm. (b), No significant growth rate difference between the Cdkn1b-overexpressing hair follicles carrying non-inhibited stem cells only at a certain position. Plot shows the mean±SD from 3 mice (n=10, n=9 and n=15 hair follicles in each group). ns, not significant, p=0.2385, 0.5210 and 0.4856 when comparing each two groups. Two-sided unpaired t-test was used to calculate p value. Statistical source data are provided in Supplementary Table 1.
Supplementary Figure 2 Photo-inducible labeling and tracing show upward lineage shifting of the matrix progenitors.
Representative images showing lower matrix progenitors labeled by photo-activated H2BmCherry (K14-H2BPAmCherry, red) took over the upper matrix over time through either relocation or expansion. Epithelial membranes were marked by K14-actinGFP (green). Hair follicle epithelium is outlined by white dashed lines. Photo-activated areas are indicated by yellow dashed boxes. H2BmCherry signal was diluted through cell proliferation after photo-activation. Images representative of 7 mice. Scale bar, 20 µm.
Supplementary Figure 3 Lineage tracing of the outer hair follicle layer shows progressively progenitor relocation and lineage change.
Lineage tracing of the outer hair follicle layer, by analyzing non-consecutive sections sampling whole mouse back skin at multiple time points, showing outer cells are progressively relocated towards the upper part of the matrix with corresponding lineage changes. To induce outer layer cell labeling, Lgr5-CreER; R26-flox-STOP-tdTomato; K14-H2BGFP mice were injected a single dose of 2 mg tamoxifen between postnatal day 25 and 26. Back skin was then collected 3 days, 5 days and 7 days after injection, which are referred to as Day 0, 2 and 4. The analyses were typically performed from Anagen V to VI. Mesenchymal dermal papilla was labeled by Lef1-RFP (red). Hair follicle epithelium is outlined by dashed lines in the enlarged images. Scale bars, 100 µm for the large field of view, 20 µm for individual hair follicles. (sample and mouse numbers are indicated in the figure).
Supplementary Figure 4 Outer root sheath cells (ORS) expand and move downwards during hair follicle growth.
(a), Representative images showing lower bulge cells of the resting hair follicle give rise to upper ORS cells which expand and move downwards during hair follicle growth. Images representative of 5 mice. (b), Representative images showing both upper and lower ORS cells expand and move downwards during hair follicle growth. Images representative of 8 mice. Epithelial nuclei were marked by K14-H2BGFP (green). Lgr5-CreER and R26-flox-STOP-tdTomato (red) were used to induce bulge cell or ORS cell labeling. The tracking was performed either from Telogen to Anagen VI (a) or from Anagen IIIc to VI (b). Hair follicle epithelium is outlined by white dashed lines. Scale bar, 20 µm. (c), Frequency of relocation or concurrent multi-lineage differentiation without relocation of the matrix progenitors in total hair follicles analyzed or hair follicles with single progenitor labeled.
Supplementary Figure 5 βCatGOF mutant matrix cells are integrated into the normal differentiation process.
(a), Revisits of a hair follicle where βCatGOF mutation had been induced in both the stem cell compartment and the matrix by Hopx-CreER. While an outgrowth (arrowhead) emerged from the stem cell compartment, the mutant cell in the matrix were gradually relocated into the top matrix area without causing aberrancy. Epithelial nuclei were marked by K14-H2BGFP (green). R26-flox-STOP-tdTomato (red) was used to identify the mutant cells. Asterisk and arrow indicate the original and current progenitor cell position respectively. Images representative of 5 mice. (b), Representative image of hair follicles having GFP-tagged βCatGOF mutation (dNβCatGFP) induced in the stem cells showing ectopic outgrowths (arrowheads and outlined by dashed lines) emerged in the upper part of the hair follicles. Epithelial membrane was marked by K14-ActinmCherry (red). Lgr5-CreER and R26-flox-STOP-tTA were used to induce the dNβCatGFP expression under the Doxycycline-inducible promoter (pTRE-dNβCatGFP) in the stem cells. (c), Revisits of hair follicles where dNβCatGFP had been induced in the matrix. The mutant cells were gradually relocated into the top matrix area and sent progeny upwards to differentiate normally without causing aberrancy. Images representative of 5 mice. Epithelial nuclei were marked by K14-H2BmCherry (red). Shh-CreER and R26-flox-STOP-tTA were used to induce the dNβCatGFP expression. Note that dermal papillae are out of the planes in Day 0 hair follicle images, thus the dashed lines inside the hair follicles do not outline the actual dermal papillae surfaces. Arrows indicate the positions of the individual progenitors labeled. Yellow dashed lines separate different lineages. For a and c, the tracking was performed from Anagen IV to VI and hair follicle epithelium is outlined by white dashed lines. Scale bar, 20 µm.
The βCat exon2-4 primers amplify a 363 bp fragment of the wild type β-catenin gene and a 135 bp fragment of the recombined βCatGOF allele.
Supplementary Figures 1–6, Supplementary Table and Supplementary Video legends
Statistics source data
3D view and surface rendering of hair follicles from the K14-Cre;mTmG (R26-flox-membrane tdTomato-STOP-membrane GFP) mice showing progressive encapsulation of mesenchymal dermal papilla (red) by epithelium (green) during hair follicle growth
3D views of the same hair follicle in Telogen and Anagen I from the K14-H2BGFP (green) mice showing there are no cells in the suprabasal space (purple circle) of the Telogen hair germ (white, left), while suprabasal cells (pink highlights one such cell) emerge in the Anagen I hair germ (white, right)
Time-lapses showing cell death of lower hair germ cells (arrow). Epithelial nuclei were marked by K14-H2BmCherry (left) and K14-H2BGFP (right)
3D view and surface rendering of a hair follicle during lineage tracing showing suprabasal displacement (blue) of cells deriving from lower stem cell (left) and basal expansion of cells deriving from upper stem cell (right). Epithelial nuclei were marked by K14-H2BGFP (green). Lgr5-CreER and R26-flox-STOP-tdTomato (red) were used to induce stem cell labelling
3D view and surface rendering of a hair follicle from the Lgr5-CreER; R26-flox-STOP-tTA; tetO-Cdkn1b; pTRE-H2BGFP; K14-H2BmCherry mice showing induction of Cdkn1b and H2BGFP (green, proliferation-impaired cells) expression in all but one (red, proliferation-competent cell) hair germ stem cells