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Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin

Nature Cell Biology volume 18, pages 619–631 (2016)Cite this article

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Abstract

The interfollicular epidermis regenerates from heterogeneous basal skin cell populations that divide at different rates. It has previously been presumed that infrequently dividing basal cells known as label-retaining cells (LRCs) are stem cells, whereas non-LRCs are short-lived progenitors. Here we employ the H2B–GFP pulse-chase system in adult mouse skin and find that epidermal LRCs and non-LRCs are molecularly distinct and can be differentiated by Dlx1CreER and Slc1a3CreER genetic marking, respectively. Long-term lineage tracing and mathematical modelling of H2B–GFP dilution data show that LRCs and non-LRCs constitute two distinct stem cell populations with different patterns of proliferation, differentiation and upward cellular transport. During homeostasis, these populations are enriched in spatially distinct skin territories and can preferentially produce unique differentiated lineages. On wounding or selective killing, they can temporarily replenish each other’s territory. These two discrete interfollicular stem cell populations are functionally interchangeable and intrinsically well adapted to thrive in distinct skin environments.

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Figure 1: H2B–GFP LRCs and non-LRCs reside in distinct territories of the epidermis.
Figure 2: H2B–GFP LRCs and non-LRCs are molecularly distinct.
Figure 3: H2B–GFP dilution implies two independent cell populations with different proliferation and upward cellular transport rates.
Figure 4: Slc1a3CreER and Dlx1CreER mark long-lived stem cells in the back epidermis.
Figure 5: Dlx1CreER- and Slc1a3CreER-marked cells replenish distinct tail skin territories with distinct differentiated progeny.
Figure 6: The emergence of Dlx1CreER- and Slc1a3CreER-marked cells during skin development.
Figure 7: Dlx1CreER- and Slc1a3CreER-marked cells replenish each other’s territories following selective killing.
Figure 8: Wounding experiments and model of epidermal lineage organization and SC dynamics.

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Acknowledgements

We thank L. G. Sayam for FACS; C. J. Bayles, R. M. Williams and J. M. DelaCruz for imaging; B. Hover and J. Mosher for microarray; the Cornell Center for Animal Resources and Education facility for mouse care; D. J. McDermitt for help with mouse crossings; and K. L. Goodyear for help with mathematical modelling. Support was from: Empire State Stem Cell Foundation, New York State-Department of Health (NYS-DOH), Contract no. C026718 (in part) for Cornell Cytometry core; NIH Grant 1S10RR025502-01 for Cornell Biotechnology Research Center and Imaging Facility. The research was financially supported by NYSTEM Grant C024354, NIH Grant R21AR063278 to T.T., a Long-Term Fellowship from the Human Frontier Science Program to A.S. and a postdoctoral fellowship for research abroad from the Japan Society for the Promotion of Science to A.S.

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Author notes

  1. Fadi Jacob, Eva Leung and Sherry Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA

    Aiko Sada, Fadi Jacob, Eva Leung, Sherry Wang, David Shalloway & Tudorita Tumbar

  2. Division of Oncology, Department of Medicine, Washington University, St Louis, Missouri 63110, USA

    Brian S. White

  3. McDonnell Genome Institute, Washington University, St Louis, Missouri 63110, USA

    Brian S. White

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Contributions

A.S. and T.T. designed the experiments. A.S., D.S. and T.T. wrote the manuscript. A.S., F.J., E.L. and S.W. performed experiments. D.S. and B.S.W. performed mathematical modelling.

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Correspondence to Tudorita Tumbar.

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Integrated supplementary information

Supplementary Figure 1 H2B-GFP tet-off system to count cell division frequency in skin cells in vivo.

(a) Models of cellular lineage hierarchy in the epidermis. (b) Schematic of microscopic inspection of skin sections or whole-mounts. (c) Adult H2B-GFP tet-off mice subjected to doxy chases. After 1 week of doxy administration, many epidermal cells still have high levels of GFP signal. A chase period of 2 weeks can detect a fraction of bright H2B-GFP clustered cells, indicative of a more infrequent division history. After 3 weeks of doxy chase, rare H2B-GFP bright cells are present (arrowhead). (d) Mice were subjected to 2 weeks of doxy and 2 days of EdU to verify inverse correlation between proliferation and H2B-GFP retention. Data from two mice are shown as individual bars. (e) Unchased control for the tail epidermis whole-mount. Both interscale (K10+) and scale (K10−) show similar level of the original H2B-GFP signal. (f) Whole-mount immunostaining of the tail skin after 6 weeks of doxy chase and 2 days of BrdU, showing basal cells on the scale are more proliferative. The dashed line surrounds non-LRC area (e,f) or represents epidermal-dermal junctions (c). Asterisks indicate HFs. Scale bars, 100 μm (c,e,f). (g) Number of hair follicles within each structure. Data from two mice are shown as individual bars. Experiments are repeated twice with 2 mice for all representative images (c,e,f). (h) Number of blood vessel branch points within each structure detected in back skin. Data from two mice are shown as individual bars.

Supplementary Figure 2 Microarray analysis of isolated epidermal basal LRCs and non-LRCs.

(a) Section immunostaining of the back epidermis after 2 weeks of doxy chase. CD34, a HFSC (bulge) marker used for FACS analysis, is co-localized with bulge LRCs but not epidermal LRCs and is used to exclude the HFSCs along with K14CreER x Rosa-tdTomato induced to mark preferentially the epidermis away from HFs. The dashed line bounds the bulge or represents epidermal-dermal junctions. Scale bar, 100 μm. (b) Expression of indicated epidermis/bulge markers determined by qRT-PCR in FACS sorted populations Error bars show s.e.m. where n > 3, and have been calculated for Cd34, n = 3 mice; tdTomato, n = 3; a6-integrin, n = 3; b4-integrin, n = 3; K1, n = 3; K10, n = 3; Involcrin, n = 4. Ctgf, Ltbp2 and Loricrin, the graph shows the average of two mice. (c) Number of genes determined by microarray analysis that are differentially (≥2-fold) expressed between BL LRCs and non-LRCs. (d,e) BL LRCs versus non-LRCs genes examined in comparison between telogen bulge (quiescent HFSCs)/anagen bulge (proliferative HFSCs) (top) or K14+/Inv+ population (bottom). The top analysis shows that only 30% of BL LRC signatures correlate with quiescence and that 20% of BL non-LRC signatures correlative with active proliferation. The bottom analysis shows that our BL LRC and non-LRC signature genes have minimal overlap with those of the K14CreER with low TM and InvCreER basal populations, indicating their distinct molecular properties. Experiments are repeated twice with 2 mice for images in (a). Microarray are repeated triplicate for BL LRCs and BL non-LRCs and duplicate for SL LRCs, SL non-LRCs, GL, bulge LRCs and bulge non-LRCs (c,d).

Supplementary Figure 3 Dynamical modelling of H2B-GFP dilution experiments.

(a) Schematics of additional dynamical models. The schematic for the single progenitor model (P-D) is shown in Fig. 3c. Δ assesses the best-fit; smaller is better. Red arrows denote division coupled to transport. (b) Cell division number (d)-distributions for the BL, SL, and GL compartments after different chase periods. The left bars (green) are the normalized experimental d-distributions (days 3 and 7, data from 2 mice; day 21, data from 5 mice). The data from individual mice are shown as dots with green bars showing the mean. The other bars are the predictions from the indicated models using the best-fit parameters; they are subdivided to represent individual model component contributions (see Supplementary Note). Left-to-right: single-stem (uncoupled) (SC, blue), single-progenitor (P-D, grey; using the Clayton et al. value fp = 0.22 for the fraction of progenitor cells), single-stem (coupled) (SC, dark blue), two-stem (hybrid coupling) [2 × SC, yellow; dark: SC1 (fast), light: SC2(slow)] models. (c) Doxy-chase scheme for the H2B-GFP fluorescence density calculation in tail. (d) Tail sections from H2B-GFP tet-off mice subjected to doxy chases for indicated periods. Dotted lines delineate scale-interscale boundaries or epidermal-dermal junctions. Asterisks indicate HFs. Scale bars, 100 μm. (e) Decay of H2B-GFP fluorescence density after different chase periods in scale (red) and interscale (blue) tail regions. Experimental values (normalized to day 0) are indicated by (BL) and (total nucleated). The data from 2 mice are shown as individual dots. The solid (BL) and dashed (total nucleated) curves are the predictions for the isolated fast (red) and slow (blue) 2 × SC model subpopulations using the best-fit division rates (see Supplementary Note). The FACS experiments are repeated twice with 2 mice for day 3 and 7; twice with 5 mice for day 2 (b). Experiments are repeated twice with 2 mice for images in (d).

Supplementary Figure 4 Dlx1CreER or Slc1a3CreER without Tamoxifen injection.

(a) Dlx1- or Slc1a3CreER/Rosa-tdTomato double-transgenic mice without TM injection, showing no leakiness of Cre. (b) Dlx1CreER or Slc1a3CreER/Rosa-tdTomato double-transgenic mice subjected different doses of TM injection, showing Cre is induced in a dose-dependent manner. The dashed line represents the boundary of the tail epidermis scale structure (the remaining marked area is interscale) or, in section images, represents epidermal-dermal junctions. Asterisks indicate HFs. Scale bars, 100 μm (a,b). Experiments are repeated twice with 2 mice for all representative images.

Supplementary Figure 5 Lineage tracing of Dlx1CreER- or Slc1a3CreER-marked cells in tail skin.

(a) Schematic view of the tail epidermis structures and quantification of the relative area of each structure. Data from two mice are shown as individual bars. (b) Immunostaining of tail skin with K10 (interscale marker) or K31 (scale marker) showing distinct localization of Dlx1CreER- versus Slc1a3CreER-marked cells. (c) Low magnification image of lineage tracing data described in Fig. 5e to show multiple scale/interscale structures in one field of view. The dashed lines delineate the boundaries of tail epidermis structures: scales (white circles) and interscale (yellow for line and white for non-line). Asterisks indicate HFs. Scale bars, 100 μm (b–d). (d) Fluorescence images of tail skin sections from Dlx1/ or Slc1a3CreER/Rosa-tdTomato double-transgenic mice. Arrowheads indicate tdTomato+ basal cells. The dashed line represents epidermal-dermal junctions. (e) Quantification of tdTomato+ area/clone at 1-year chase. Data from two mice are shown as individual bars. Dlx1CreER-marked cells in the interscale non-line region produce larger clones than those in the scale region, suggesting that the Dlx1 lineages not only show biased localization within the interscale, but also thrive more in regenerating the tissue in this territory. (f) Number of tdTomato+ clones per structure. Data from 4, 4, 2, and 2 mice for the 2-week, 3-month, 6-month, and 1-year chases, respectively, are shown, with individual mice plotted as dots; the lines show the means. (g) Persistence of relative clone number in non-line (blue) and line (green) interscale and scale (red) tail regions. Each dot represents a mouse data-point (4, 2, and 2 for the 3, 6 and 12 month time points respectively). The solid curves are the (neutral-drift) best-fit predictions (Supplementary Note) for the LRC SC subpopulation with the division rate determined by the H2B-GFP experiments (λ = 0.14/day) and partially coupled to transport with uncoupling (symmetry) parameter u = 0.18 (interscale, non-line), 0.12 (interscale, line), or 0.10 (scale). The dotted curves are the best-fits if it is assumed that division is completely uncoupled (that is, symmetric, u = 1) with much slower rates: λ = 0.030/day (interscale, non-line), 0.021/day (interscale, line), or 0.019/day (scale). Experiments are repeated twice with 2 mice for representative images in (b); and twice with 4 mice (Slc1a3 1 day, Slc1a3 2 weeks), 3 mice (Dlx1 3 days and Dlx1 3 months) and 2 mice (Dlx1 2 weeks, Dlx1 1 year, Slc1a3 3 months and Slc1a3 1 year) for images in (c,d).

Supplementary Figure 6 Attempt to deplete Dlx1CreER+ cells.

(a) Whole-mount immunostaining of Caspase3+ (apoptotic cells) in the tail epidermis. The dashed lines delineate the boundary of the tail epidermis scale structure (the remaining marked area is interscale). Asterisks indicate HFs. Scale bars, 100 μm. (b) Number of caspase3+ cells within each structure. Data from two mice are plotted as individual dots. A small number of cells were ablated in Dlx1CreER/Rosa-DTA mice, while massive apoptosis was induced in Slc1a3CreER/Rosa-DTA mice used here as a control. IS, interscale. (c) Expression of GFP in tail whole-mounts. Scale bars, 100 μm. (d,e) Two days of BrdU administration marks proliferating cells. Quantification of BrdU+ cells per unit area is shown; data from two mice are shown as individual bars. Scale bars, 100 μm (d). (f) Immunostaining of K10 (interscale lineage marker) in the tail epidermis. No differences in the expressions of GFP, BrdU, or K10 were observed after depleting Dlx1CreER+ cells. This was likely due to inefficient marking by Dlx1CreER induction. Scale bars, 100 μm. Experiments are repeated twice with 2 mice for all representative images.

Supplementary Figure 7 Depletion of Slc1a3CreER+ cells in tail skin.

(a) Lineage tracing of surviving Slc1a3CreER+ cells after DTA-mediated cell killing. Whole-mount immunostaining of the tail epidermis in Slc1a3CreERRosa-DTA;GFP/Rosa-tdTomato triple-transgenic mice shows increased apoptosis, as indicated by caspase3+ cells, and the loss of tdTomato+ cells and GFP upon TM induction. (b) Expression of tdTomato and GFP in tail whole-mounts. Small numbers of tdTomato+ cells survived in some scales and showed moderate expansion of clones by 2 weeks. (c,d) Two days of BrdU administration marked proliferating cells. The average number of BrdU+ cells per tdTomato+ cells is shown; data from two mice are shown as individual bars. (e) One week of BrdU administration marks proliferating cells in the BL of the epidermis. Following 5 weeks of chase, infrequently dividing cells were detected as BrdU LRCs and were located preferentially within interscales. The non-LRC (scale) area is marked in the Slc1a3CreER/Rosa-tdTomato mice. (f) Experimental scheme of BrdU and TM treatment in Slc1a3CreER/Rosa-DTA mice. (g,h) Whole-mount (g) or section staining (h) of Slc1a3CreER/Rosa-DTA mice with BrdU pulse-chase, showing that LRCs became more proliferative (Ki67+; arrowheads) by 1 week after the loss of Slc1a3+ cells. Controls were either single-transgenic Slc1a3CreER or Rosa-DTA mice. (i) Quantification of Ki67+ LRCs in scale and interscale regions at 1-week post-induction shows activation of proliferation in all areas (n = 3 mice). Statistical analyses were performed using a two-tailed Student’s t-test. Error bars show s.e.m.; ∗: P < 0.01; ∗: P < 0.05. (Interscale, P = 0.02; scale, P = 0.0006.) Significantly increased LRC proliferation was observed in both scales and interscales indicating that LRCs are activated to migrate and proliferate upon the loss of Slc1a3CreER+ cells. Ki67, cell proliferation. The dashed lines represents the boundaries of the tail epidermis structures (circle is scale, while the remaining marked area is interscale). Asterisks indicate HFs. Scale bars, 100 μm (a,b,c,e,g,h). Experiments are repeated twice with 2 mice for all representative images (a,b,c,e,g,h).

Supplementary Figure 8 Phenotypes of Slc1a3CreER/Rosa-DTA mice in back skin.

(a) Staining of back skin section for caspase3 showing the induction of apoptosis in the BL of the back epidermis. Arrowhead indicates caspase3+ basal cells. Control mice were either single-transgenic Slc1a3CreER or Rosa-DTA. (b) BrdU was administered for 2 days to mark proliferative cells. Similar to the tail epidermis, the proliferation of back skin basal cells was increased by 1-week post-induction. (c) BrdU+ LRCs co-localized with Ki67 expression in experimental mice, indicating that these cells became more proliferative upon the loss of non-LRCs. (d,e) Wounding markers, Tenascin and K6 were up-regulated in Slc1a3CreER/Rosa-DTA mice after TM induction. (f,g) LRCs co-localized with wounding markers. This was unexpected since killing was targeted to the non-LRC areas and might indicate migration of LRCs into the area where Slc1a3CreER+ cells were located before they were killed. The dashed line represents epidermal-dermal junctions. Asterisks indicate HFs. Scale bar, 100 μm (a–g). Experiments are repeated twice with 2 mice for all representative images (a–g).

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Sada, A., Jacob, F., Leung, E. et al. Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin. Nat Cell Biol 18, 619–631 (2016). https://doi.org/10.1038/ncb3359

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