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Wounding induces dedifferentiation of epidermal Gata6+ cells and acquisition of stem cell properties

Nature Cell Biology volume 19, pages 603–613 (2017)Cite this article

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Abstract

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

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Figure 1: Identification of Gata6 as a marker of the sebaceous duct lineage.
Figure 2: Architectural characterization of the junctional zone.
Figure 3: Gata6 controls the identity of the sebaceous duct (SD) lineage and distinguishes the sebocyte and SD lineages.
Figure 4: Fate of Lrig1 and Gata6 lineages during wound healing.
Figure 5: Dedifferentiation of Gata6 lineage SD cells and acquisition of stem cell properties.
Figure 6: Contribution of Gata6+ cells to reconstituted skin.
Figure 7: Live imaging of suprabasal Gata6 GL cells migrating to the basal layer during wound healing.
Figure 8: Wound-induced plasticity of Gata6 lineage linked to loss of JZ identity and acquisition of multi-lineage differentiation potential.

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Acknowledgements

We thank H. Fujiwara, S. Woodhouse, B. M. Lichtenberger, A. Mishra, V. Proserpio, M. Cereda and all the members of the Watt laboratory for helpful discussions and for excellent suggestions; X. Zou (CR-UK Cambridge Research Institute, Cambridge, UK) and the Mary Lyon Centre at MRC Harwell MRC for assistance in generating the Gata6 KI mouse model; D. Cottle and C. Collins for access to animal samples; the core facilities of CRI and KCL and the NIHR GSTT/KCL Biomedical Research Centre for superb technical assistance; the Paterson Institute Microarray Facility for performing arrays; H. Hamada (Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan) for kindly sharing the Gata6 reporter mouse; B. Rosen and F. Amaral (Wellcome Trust Sanger Institute, Cambridge, UK) for the eGFPCreERT2 cassette. This work was funded by the UK Medical Research Council, the Wellcome Trust and the European Union FP7 programme. K.K. was the recipient of an MRC UK PhD studentship; T.H. is the recipient of a EU Marie Curie Fellowship; E.R. is the recipient of an EMBO long-term fellowship (ALTF594-2014).

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Author notes

  1. Emanuel Rognoni, Toru Hiratsuka and Kifayathullah Liakath-Ali: These authors contributed equally to this work.

Authors and Affiliations

  1. King’s College London Centre for Stem Cells and Regenerative Medicine, 28th Floor, Tower Wing, Guy’s Campus, Great Maze Pond, London, SE1 9RT, UK

    Giacomo Donati, Emanuel Rognoni, Toru Hiratsuka, Kifayathullah Liakath-Ali, Esther Hoste, Kai Kretzschmar & Fiona M. Watt

  2. Cancer Research UK Cambridge Research Institute, Cambridge, CB2 0RE, UK

    Giacomo Donati, Roslin Russell & Klaas W. Mulder

  3. Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123, Turin, Italy

    Giacomo Donati

  4. Department of Biomedical Molecular Biology (Ghent University), VIB Center for Inflammation Research, B-9052, Ghent, Belgium

    Esther Hoste

  5. European Bioinformatics Institute and Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SD, UK

    Gozde Kar & Sarah A. Teichmann

  6. MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK

    Melis Kayikci

  7. Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 1QR, UK

    Kai Kretzschmar

  8. Hubrecht Institute, KNAW and UMC Utrecht, 3584CT, Utrecht, The Netherlands

    Kai Kretzschmar

  9. Department of Molecular Developmental Biology, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands

    Klaas W. Mulder

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  1. Giacomo Donati
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Contributions

G.D. and F.M.W. conceived the study and designed the experiments with input from E.R. and K.W.M. G.D., E.R., T.H., K.L.-A., E.H. and K.K. performed experiments. G.D., E.R., T.H., M.K., G.K., R.R., K.W.M., S.A.T. and F.M.W. analysed the data. G.D. and F.M.W. wrote the manuscript, with contributions from all authors.

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Correspondence to Giacomo Donati or Fiona M. Watt.

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Integrated supplementary information

Supplementary Figure 1 K14ΔNLef1 phenotypes, Gata6 expression in wild type adult skin. Gata6-tdTomato reporter analysis and Gata6 ChIP-Seq.

(a) Late K14ΔNLef1 phenotypes: ectopic sebaceous glands (SG) (middle panel) and cysts (right panel). Haematoxylin and Eosin stained dorsal skin sections from wild type (WT) and K14ΔNLef1 mice at 22 weeks. (b) Heatmap of genes that are differentially expressed between WT and K14ΔNLef1 epidermal cells. (c) RT-qPCR of RNA from microdissected WT and K14ΔNLef1 hair follicles (HF) compared to heart (positive control for Gata4 and Gata6 expression). Data are means ± s.d. from n = 3 independent biological samples. (d) Tamoxifen treated (lower panel) and untreated (top panel) Lgr6EGFPCreERT2 Rosa26-fl/STOP/fl-tdTomato back skin sections stained with antibodies to Gata6. Representative images of 2 independent experiments. (e) Adult WT dorsal skin sections (bottom panels) or tail epidermal whole mounts (top panels) were stained for Gata6 and markers for different hair follicle stem cell populations. Insets are higher magnification views of boxed areas. Representative images of 3 independent experiments. (f) Adult ear skin sections from Gata6 reporter mouse showing co-localization of endogenous Gata6 and tdTomato. Representative images of 4 mice. (g,h) Gata6-tdTomato adult ear (g) and tail (h) epidermal whole mounts showing endogenous tdTomato and Krt14 antibody labelling. White arrows indicate the absence of Gata6 expression in lower growing HF during anagen. Yellow brackets indicate the bulge areas. Representative images of 4 mice. (i) A Z stack image (left panel) of a HF from whole mount tail Gata6-tdTomato epidermis stained with anti-Krt14. Orthogonal images from the 3D reconstruction (right panels), at the plane indicated by the white lines, show basal cell Gata6 expression confined to the junctional zone in proximity to the sebaceous glands (upper right panel). Flow cytometry analysis of Gata6-tdTomato dorsal epidermis shows absence of Gata6 + CD34 + cells in adult telogen HF (right panel). Representative image/plot of n = 3 mice j, Gata6 and Gapdh western blot of control (empty vector) and Gata6-overexpressing cultured primary mouse keratinocytes. Unprocessed scan of the blot are shown in Supplementary Fig. 9. (k) Average signal intensity of Gata6 binding sites detected by two Gata6 antibodies (blue and light blue). (l) Representative plots of ChIP-Seq reads aligned to the Lpl and Cldn4 gene loci. (m) Motif enrichment analysis on Gata6 target regions performed by Meme Suite identified Gata6 expected DNA consensus and putative co-regulators. Scale bars, 50 μm (a,d–f,i); 150 μm (g,h).

Supplementary Figure 2 Structural complexity of the junctional zone (JZ) revealed by gene expression profiling and antibody labelling.

(a) Expression profile analysis (RT–qPCR) of sorted basal JZ cells (green), non-basal SD cells (violet), bulge cells (yellow) and all remaining basal cells (grey) from telogen mouse back skin. Data are means ± s.d. from n = 3 mice for Lgr6, Gata6, tdTomato, Cd34 and Tnc; n = 7 mice for Plet1 and Lrig1; n = 5 mice for Blimp1. (b,c) Gata6 and Blimp1 co-localize in the outermost differentiated layers of the JZ/SD. Wild type tail epidermal section stained for Gata6 and Blimp1 (b, top panels). Masks after color threshold analysis with ImageJ software and single fluorescent channel images of the magnified white insert are shown (b, bottom panels). Quantification of Gata6 expression in Blimp1 positive cells in JZ/SD and IFE (c). n = 35 images from 4 mice. Data are means ± s.e.m. (d–j) Wild type back (d,f,g) or tail (e,h,i,j) skin sections stained with the indicated antibodies. Representative images of 3 mice Dashed lines demarcate epidermal-dermal boundaries. Scale bars, 50 μm.

Supplementary Figure 3 Additional data confirming the role of Gata6 in specifying the SD lineage.

(a) RT-qPCR of mRNA from dorsal epidermis of wild type (WT) and Gata6 knockout (cKO) mice confirming loss of Gata6 expression in the Gata6 cKO. Data are means ± s.d. from n = 4 mice. (b) Quantification of basal Itga6 + Cd34- keratinocytes by flow cytometry (see Fig. 1h). Data are means ± s.d. from n = 3 mice. c, Tail skin sections from WT, Gata6 epidermal knockout (cKO), K14ΔNLef1 and K14ΔNLef1 x Gata6 cKO (K14ΔNLef1 cKO) stained for the duct marker Plet1. Inserts are higher magnification views. (d,e) K14ΔNLEF1 cysts express Plet1 and Atp6v1c2 and expression is reduced in the absence of Gata6. RT–qPCR analysis of mRNA from WT, cKO, K14ΔNLef1 and K14ΔNLef1 cKO epidermis (d). K14ΔNLef1 and K14ΔNLef1 cKO back skin sections were stained for the SD marker Atp6v1c2 and the SG marker Scd1 (e). Data are means ± s.d. from n = 4 WT, n = 5 cKO, n = 3 K14ΔNLef1 and n = 4 K14ΔNLef1 cKO mice. ∗P < 0.05; ∗∗P < 0.005, by unpaired Student’s t-test. Scale bars, 50 μm. (f) Gene Ontology enrichment analysis of Gata6 direct target genes highly expressed in Gata6 + Itga6 + cells (see Fig. 2a). g, Plots of ChIP-Seq reads aligned to the Cep55, Ncaph and Spc24 (representative genes involved in mitosis) loci are shown. h, RT–qPCR of RNA from wild type (WT), Gata6 knockout (cKO), K14ΔNLef1 and K14ΔNLef1 x Gata6 cKO (K14ΔNLef1 cKO) epidermal cells. (i–k) Validation of gene expression profiles from manual microdissected tail interfollicular epidermis (IFE), hair follicles (HF) and sebaceous glands (SG). Bright field image of tail epidermal whole mount showing schematic of microdissection strategy (i). Z score heat maps representing array results for differentially expressed genes (j) validated by RT-qPCR (k). Data are means ± s.d. from n = 4 mice for IFE; n = 6 mice for SG; n = 2 for HF; n = 6 for whole tail epidermis (EPID). l, Sections of wild type (WT), Gata6 epidermal knockout (cKO), K14ΔNLef1 and K14ΔNLef1 x Gata6 cKO (K14ΔNLef1 cKO) dorsal skin stained for Gata6 and Blimp1 showing reduction in Blimp1 + cells in HF but not IFE in absence of Gata6. Inserts are higher magnification views of the JZ (red channel only). Asterisk indicates nonspecific staining. Dashed lines indicate epidermal-dermal boundary. (m) Nuclear Androgen Receptor, Ar (black arrows), normally present in WT SG (insert panel), is reduced in K14ΔNLef1 ectopic SG (top panel) but restored in K14ΔNLef1 epidermis when Gata6 is ablated (bottom panel). Back skin sections were labeled with Ar antibody. o, RT–qPCR analysis of RNA from WT, cKO, K14ΔNLef1 and K14ΔNLef1 cKO adult back skin dermis. Data are means ± s.d. from n = 4 WT, n = 5 cKO, n = 3 K14ΔNLef1 and n = 4 K14ΔNLef1 cKO mice ∗P < 0.05 by unpaired Student’s t-test. (p) Schematic representation of Gata6 regulation of Ar and Blimp1. Representative images of n = 3 mice per genotype. Scale bars, 50 μm.

Supplementary Figure 4 Gata6 genetic labeling of epidermis under homeostatic conditions and Gata6 regulation by Vitamin A and Myc.

(a) Untreated (top left panel) and tamoxifen (4OHT) treated adult back skin sections from Gata6EGFPCreERT2 x Rosa26-fl/STOP/fl-tdTomato reporter mice. Dotted white lines indicate sebaceous glands. Samples were collected at the time points indicated. Gata6 genetically labeled cells are restricted to the junctional zone and sebaceous ducts (white arrow) and are not present in the lower HF. Representative images of 2 independent experiments. (b) Schematic of known upstream regulators of Gata6 expression in multiple cell contexts11,12. (c) The transcription factor Myc induces Gata6 expression in the junctional zone, in the sebaceous glands and in the interfollicular epidermis (white arrows); in contrast, in K14ΔNLef1 skin, Gata6 expression is only induced in the epidermal cysts (see Fig. 1c). Sections of tamoxifen treated dorsal skin from wild type (WT) and K14MycER mice10 stained for Gata6 (red) and Krt14 (green). Representative images of 3 mice. (d,e) Quantification of Gata6 expressing cells per hair follicle (d) and per length of interfollicular epidermis (IFE) (e) in the indicated mouse strains. Box-and-whisker plots: mid-line, median; box, 25th to 75th percentiles; and whiskers, minimum and maximum (n = 12 hair follicles). (f,g) Gata6 expression in the interfollicular epidermis correlates with the presence of ectopic SG (white and black arrowheads) in Tamoxifen treated skin from K14MycER mice. Dorsal skin sections were stained with Haematoxylin and Eosin (f) or Gata6 and Scd1 (g). Representative images of n = 3 mice. (h,i) Treatment with Vitamin A (Retinoid Acid – RA) leads to the presence of Gata6 expressing suprabasal cells in the interfollicular epidermis and lower hair follicle (white arrows) of adult dorsal skin, where normally Gata6 is not expressed. Acetone- (vehicle) and RA-treated skin sections were stained for Gata6, Ki67, and Fabp5. Insert with white lines is magnified in right panel h. Representative images of 3 mice. Dashed lines demarcate epidermal-dermal boundaries. Scale bars, 25 μm (a), 50 μm (c,f–i).

Supplementary Figure 5 Characterization and quantification of Lrig1 and Gata6 genetically labelled (GL) cells prior to wounding and during re-epithelialization.

(a) Quantification of Gata6 and Lrig1 genetically labeled (GL) cells after tamoxifen treatment, 48 h after wounding in tail skin (n = 5 mice, 116 HF for Gata6; 27 HF for Lrig1). Data are means ± s.d. (b) Flow cytometry analysis showing lack of EdU incorporation by GL Gata6 tail keratinocytes in unwounded skin. Representative flow cytometry plots of epidermal Lrig1 (dark grey) and Gata6 (red) GL cells from back and tail skin (top left panels). Gates in upper panels mark EdU positive cells analyzed in bottom left density plots (dashed green lines demarcate tdTomato negative controls). Quantification of EdU incorporation in all epidermal cells (top right panels) and tdTomato Gata6 (red) or Lrig1 (dark grey) GL cells (bottom right panels) is shown. (n = 6 mice for Gata6; n = 3 mice for Lrig1). Data are means ± s.d. (c) Histological analysis confirming the absence of EdU incorporation into GL Gata6 in unwounded tail skin. Tail skin sections showing tdTomato GL Gata6 (left) or Lrig1 cells (right)) and EdU localization. EdU masks after color threshold analysis with ImageJ software together with tdTomato mask (top panel) or single tdTomato channel (bottom panel) are shown. Representative images of 2 independent experiments. Quantification of EdU incorporation by GL (Lrgi1 or Gata6) cells in JZ/SD (n = 3 mice each; 49HF) is shown. Scale bars, 50 μm (d) Suprabasal Gata6 GL cells acquire a basement membrane position during wound re-epithelialisation. Position in the epidermal layers of Gata6 GL cells at the indicated day after wounding tail skin is shown. Measurements are position relative to the basement membrane of the lowest Gata6 GL cell of a column of labelled cells in the wound bed (“0” corresponds to the basal cell layer). From n = 3 mice for day 5, 9, 12 and n = 4 mice for day 7 (average of 70 columnar cell units per mouse). The yellow bars in the violin plots represent median and black lines the 25th and 75th percentiles. (e) Quantification of flow cytometry analysis of High-Itga6 and Mid/Low-Itga6 expressing Gata6 and Lrig1 GL cells in the tail wound bed at the indicated days after wounding. Data are means ± s.d. from n = 4 mice for Gata6 GL 5d, 12d and Lrig1 GL 5d; n = 3 for Lrig1 GL 12d. Representative flow cytometric plots (right panels) are shown. Light blue lines represent gating strategy used for quantification in left hand panel13. ∗P < 0.05; ∗∗P < 0.005, by unpaired Student’s t-test. (f) Quantification of Ki67 + Lrig1 and Gata6 tdTomato GL cells at day 6 after wounding. Data are means ± s.e.m. from n = 25 cell clusters.

Supplementary Figure 6 Fate of Blimp1 genetically labelled cells during wound healing.

(a) Blimp1-eGfp reporter mouse shows Blimp1 promoter activity in differentiated epidermal layers in undamaged epidermis and in the wound bed. Section of re-epithelialised wound from Blimp1-eGfp mouse stained for Gfp (green) and Itga6 (red). White arrow indicates newly formed dermal condensate in the magnified insert (Gfp channel only). Representative images of n = 3 mice. (b,c) Sections of back skin showing Blimp1 genetically labelled cells (green) in Blimp1Cre cag-cat-eGfp mice stained for Itga6 (red) in homeostatic (b) and hyperproliferative (c) skin. Hyperproliferation was induced by irradiation with 1,000 J m−2 UVB. White arrows indicate eGfp labelled Blimp1 expressing cells in the differentiated epidermal layers. Representative images of 3 mice. (d) Section of re-epithelialized wounded back skin stained with antibodies to Blimp1 and Krt14. Blimp1 expression is restricted to terminally differentiated epidermal cells in undamaged epidermis and in the wound bed. Inserts with white lines are higher magnification views. Representative images of 3 mice. (e) Differentiated, Blimp1 positive, epidermal cells dedifferentiate during re-epithelialization. Section of back skin showing Blimp1 lineage cells (red) stained with Itga6. White arrows (left hand insert) indicate tdTomato expressing Blimp1 GL cells in the differentiated epidermal layers far from the wound area. In the middle insert tdTomato labeled Blimp1 cells in the dermal papilla are shown. Right insert shows wound edge. The yellow boxed area is further magnified in the right hand panels. Single fluorescent channel images (left to right: dapi, Itga6, tdTomato) of two Gata6 GL cells indicated by the yellow arrows are shown. Representative images of 2 independent experiments. (f–h) Sections of tail (f,g) or back skin (h) at the indicated time points after wounding showing columns of IFE cells derived from dedifferentiation of Blimp1 GL cells (tdTomato). Sections are stained with Krt14 or Itga6. Nuclear expression of Blimp1 (red) is restricted to the suprabasal layers in the wound bed (g, lower panel). Representative images of n = 2 independent experiments. Inserts with white lines are higher magnification views. Dashed lines indicate dermal-epidermal junction. Scale bars, 50 μm (a–f); 25 μm (g,h).

Supplementary Figure 7 Additional evidence for the plasticity of differentiated Gata6 + cells following skin reconstitution.

Epidermal cells isolated from the back skin of Gata6-tdTomato reporter/GFP-expressing adult mice in telogen were sorted into differentiated Gata6 Itga6 low/tdTomato positive and Itga6 high cells. These two populations were compared in a skin reconstitution assay (Fig. 6). (a,b). Graft skin sections stained for Gfp (green) and Krt14 (red). Single fluorescent channel images (a) and magnification of boxed inserts (b, left panels) are shown. Yellow arrows indicate Gfp positive basal cells derived from Gata6 + Itga6 low cells in the sebaceous gland (a). Dashed lines demarcate epidermal-dermal boundaries. Representative images from 3 to 6 chamber graft assays (Itga6 high and Itga6 low/tdTomato positive cells respectively). Scale bars, 25 μm.

Supplementary Figure 8 Time lapse imaging of migration of suprabasal Gata6 GL cells during wound healing in adult ear skin.

(a) A single stack image at 0 h indicating the wound edge (magenta dotted line), HFs (white dotted circles), nuclei and collagen (green) and Gata6 genetically labeled (GL) cells (red). The thickness used to reconstruct the orthogonal images to be able to follow the entire group of Gata6 GL cells over time shown in Fig. 7a is indicated by the yellow rectangle. Representative image of 6 independent experiments. Scale bars, 100 μm. (b) Quantification of the speed of cell migration according to whether cells moved upward, downward or straight. (c,d) Representative examples of upward (c) and straight (d) and migration of Gata6 GL cells (white arrows) are shown. Orthogonal images at the indicated time points from the beginning of image acquisition showing tdTomato labeled cells (red), nuclei and collagen (green). White lines indicate the basal membrane. Cartoons summarising the cell movements are shown in the top left panels. Scale bars, 25 μm. (e,f) Cell shape and size analysis of Gata6 GL cells during wound healing. Snapshots from live imaging during wound healing (Fig. 7) were analyzed to assess size and shape of Gata6 GL cells that attached to the basement membrane compared to Gata6 GL cells that maintained a suprabasal position and unlabelled keratinocytes. Heat-mapped XY -view of the shapes of suprabasal Gata6 GL cells (left panel, n = 18 cells) and Gata6 GL cells that attached to the basement membrane (BM) (right panel, n = 9 cells) (e). Individual cells were aligned to the same XY centroid position and rotated to align wound direction horizontally to the right. Note that newly BM-attached GL cells were elongated toward the axis of wound direction as quantified in (f) (Right panel). Quantification of Gata6 GL cell size with reference to control keratinocytes in mouse ear skin (f). The reference cell size was obtained by in vivo labelling of keratinocytes by injection of Isolectin into WT mouse ear skin. Note that Gata6 GL cell that newly attached to the basement membrane had a similar size to normal basal keratinocytes (left panel). Quantification of cell elongation toward the axis of wound direction through the quantification of the migration shape index (MSI), defined as the ratio (α/β) of cell length projected to the axis of wound direction (α) over cell length perpendicular to the axis (β). Blue dotted line indicates MSI = 1, no elongation (right panel). Data are means ± s.d. from n = 21 Basal cells (reference); n = 20 Suprabasal cells (reference); n = 18 Suprabasal Gata6 GL cells; n = 9 newly BM-attached Gata6 GL cells. ∗P < 0.05; ∗∗P < 0.005 by unpaired Student’s t-test.

Supplementary Figure 9 Uncropped images from the western-blots in Supplementary Fig. 1j.

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41556_2017_BFncb3532_MOESM5_ESM.mov

3D representtation of Gata6 positive cells in the JZ/SD visualised with the Gata6-tdTomato reporter. Confocal Z stack projections of tail epidermal whole mount rendered using Volocity software. (MOV 2226 kb)

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Donati, G., Rognoni, E., Hiratsuka, T. et al. Wounding induces dedifferentiation of epidermal Gata6+ cells and acquisition of stem cell properties. Nat Cell Biol 19, 603–613 (2017). https://doi.org/10.1038/ncb3532

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