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Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice

Nature Cell Biology volume 19, pages 155163 (2017) | Download Citation

  • An Erratum to this article was published on 01 April 2017

This article has been updated

Abstract

Tissue repair is fundamental to our survival as tissues are challenged by recurrent damage. During mammalian skin repair, cells respond by migrating and proliferating to close the wound. However, the coordination of cellular repair behaviours and their effects on homeostatic functions in a live mammal remains unclear. Here we capture the spatiotemporal dynamics of individual epithelial behaviours by imaging wound re-epithelialization in live mice. Differentiated cells migrate while the rate of differentiation changes depending on local rate of migration and tissue architecture. Cells depart from a highly proliferative zone by directionally dividing towards the wound while collectively migrating. This regional coexistence of proliferation and migration leads to local expansion and elongation of the repairing epithelium. Finally, proliferation functions to pattern and restrict the recruitment of undamaged cells. This study elucidates the interplay of cellular repair behaviours and consequent changes in homeostatic behaviours that support tissue-scale organization of wound re-epithelialization.

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Change history

  • 07 March 2017

    Owing to technical problems, this Article was published online later than the date given in the print version. The published date should read '1 March 2017', and is correct in the online versions.

References

  1. 1.

    , & Wound repair and regeneration: mechanisms, signaling, and translation. Sci. Trans. Med. 6, 265sr6 (2014).

  2. 2.

    , , & Wound repair and regeneration. Nature 453, 314–321 (2008).

  3. 3.

    , & Tissue engineering for cutaneous wounds. J. Invest. Dermatol. 127, 1018–1029 (2007).

  4. 4.

    , & Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis. Nat. Rev. Mol. Cell Biol. 12, 565–580 (2011).

  5. 5.

    & Getting under the skin of epidermal morphogenesis. Nat. Rev. Genet. 3, 199–209 (2002).

  6. 6.

    , & Hardwiring stem cell communication through tissue structure. Cell 164, 1212–1225 (2016).

  7. 7.

    , & The dynamic duo: niche/stem cell interdependency. Stem Cell Rep. 4, 961–966 (2015).

  8. 8.

    , & A murine living skin equivalent amenable to live-cell imaging: analysis of the roles of connexins in the epidermis. J. Invest. Dermatol. 128, 1039–1049 (2008).

  9. 9.

    , & In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2, 329–333 (2007).

  10. 10.

    , , & Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis. J. Clin. Invest. 89, 1892–1901 (1992).

  11. 11.

    et al. Ephrin-Bs drive junctional downregulation and actin stress fiber disassembly to enable wound re-epithelialization. Cell Rep. 13, 1380–1395 (2015).

  12. 12.

    et al. Wound healing revised: a novel reepithelialization mechanism revealed by in vitro and in silico models. J. Cell Biol. 203, 691–709 (2013).

  13. 13.

    in Biology of the Integument 2 Vertebrates Ch. 23, 443–471 (Springer-Verlag, 1986).

  14. 14.

    Some observations on stratum corneum. Curr. Med. Res. Opin. 7, 26–28 (1982).

  15. 15.

    & The cellular mechanisms of keratinocyte migration into a skin wound site: an open question with important implications. Cell Vision 3, 217–223 (1996).

  16. 16.

    & Specific transduction of the leading edge cells of migrating epithelia demonstrates that they are replaced during healing. Exp. Eye Res. 74, 199–204 (2002).

  17. 17.

    , , , & Direct visualization of a stratified epithelium reveals that wounds heal by unified sliding of cell sheets. FASEB J. 17, 397–406 (2003).

  18. 18.

    et al. Morphological evidence for the role of suprabasal keratinocytes in wound reepithelialization. Wound Repair Regen. 13, 468–479 (2005).

  19. 19.

    The spreading of epithelial cells during wound closure in Xenopus larvae. Dev. Biol. 76, 26–46 (1980).

  20. 20.

    & Stem cells in the wild: understanding the world of stem cells through intravital imaging. Cell Stem Cell 15, 683–686 (2014).

  21. 21.

    et al. Defining the epithelial stem cell niche in skin. Science 303, 359–363 (2004).

  22. 22.

    , & Inhibition of cell migration and cell division correlates with distinct effects of microtubule inhibiting drugs. J. Biol. Chem. 285, 32242–32250 (2010).

  23. 23.

    , , , & The role of microtubules and their dynamics in cell migration. J. Biol. Chem. 287, 43359–43369 (2012).

  24. 24.

    , & Emerging interactions between skin stem cells and their niches. Nat. Med. 20, 847–856 (2014).

  25. 25.

    & Human wound repair I. Epidermal regeneration. J. Cell Biol. 39, 135–151 (1968).

  26. 26.

    & Further observations on epithelialization of small wounds: an autoradiographic study of incorporation and distribution of 3H-thymidine in the epithelium covering skin wounds. J. Invest. Dermatol. 55, 20–25 (1970).

  27. 27.

    et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods 6, 153–159 (2009).

  28. 28.

    et al. Spatiotemporal coordination of stem cell commitment during epidermal homeostasis. Science 352, 1471–1474 (2016).

  29. 29.

    et al. Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1. J. Cell. Sci. 120, 1480–1490 (2007).

  30. 30.

    et al. The function of KGF in morphogenesis of epithelium and reepithelialization of wounds. Science 266, 819–822 (1994).

  31. 31.

    et al. EGFR enhances early healing after cutaneous incisional wounding. J. Invest. Dermatol. 123, 982–989 (2004).

  32. 32.

    , , , & Numerous keratinocyte subtypes involved in wound re-epithelialization. J. Invest. Dermatol. 126, 497–502 (2006).

  33. 33.

    et al. A molecular mechanotransduction pathway regulates collective migration of epithelial cells. Nat. Cell Biol. 17, 276–287 (2015).

  34. 34.

    et al. Unified quantitative characterization of epithelial tissue development. eLife 4, e08519 (2015).

  35. 35.

    & Wound repair at a glance. J. Cell. Sci. 122, 3209–3213 (2009).

  36. 36.

    & Wound repair: a showcase for cell plasticity and migration. Curr. Opin. Cell Biol. 42, 29–37 (2016).

  37. 37.

    et al. Integrin α3β1 inhibits directional migration and wound re-epithelialization in the skin. J. Cell. Sci. 122, 278–288 (2009).

  38. 38.

    , & Recapitulation of morphogenetic cell shape changes enables wound re-epithelialisation. Development 141, 1814–1820 (2014).

  39. 39.

    et al. Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)α and PPARβ mutant mice. J. Cell Biol. 154, 799–814 (2001).

  40. 40.

    et al. A role for skin gammadelta T cells in wound repair. Science 296, 747–749 (2002).

  41. 41.

    et al. Impaired epidermal to dendritic T cell signaling slows wound repair in aged skin. Cell 167, 1323–1338.e14 (2016).

  42. 42.

    , , , & Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors. J. Cell Biol. 166, 261–272 (2004).

  43. 43.

    , , & Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium. Dev. Cell 3, 367–381 (2002).

  44. 44.

    et al. ERBB3-independent activation of the PI3K pathway in EGFR-mutant lung adenocarcinomas. Cancer Res. 75, 1035–1045 (2015).

  45. 45.

    , , , & Conditional expression of the ErbB2 oncogene elicits reversible hyperplasia in stratified epithelia and up-regulation of TGFα expression in transgenic mice. Oncogene 18, 3593–3607 (1999).

  46. 46.

    , , , & Cdkn1b overexpression in adult mice alters the balance between genome and tissue ageing. Nat. Commun. 4, 2626 (2013).

  47. 47.

    et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

  48. 48.

    , , , & A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

  49. 49.

    , , & The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl Acad. Sci. USA 96, 8551–8556 (1999).

  50. 50.

    et al. The hairless mouse ear: an in vivo model for studying wound neovascularization. Wound Repair Regen. 2, 138–143 (1994).

  51. 51.

    et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317, 666–670 (2007).

  52. 52.

    , & Spatial organization within a niche as a determinant of stem-cell fate. Nature 502, 513–518 (2013).

  53. 53.

    et al. Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 487, 496–499 (2012).

  54. 54.

    et al. Intravital imaging of hair follicle regeneration in the mouse. Nat. Protoc. 10, 1116–1130 (2015).

  55. 55.

    Mitomycin C: small, fast and deadly (but very selective). Chem. Biol. 2, 575–579 (1995).

  56. 56.

    & Colcemid and the mitotic cycle. J. Cell Sci. 102, 387–392 (1992).

  57. 57.

    & Inability of colchicine to inhibit newt epidermal cell migration or prevent concanavalin A-mediated inhibition of migration. Studies in vivo. Exp. Cell Res. 116, 15–19 (1978).

  58. 58.

    et al. Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool. Nature 522, 94–97 (2015).

  59. 59.

    et al. Spontaneous tumour regression in keratoacanthomas is driven by Wnt/retinoic acid signalling cross-talk. Nat. Commun. 5, 3543 (2014).

  60. 60.

    et al. β-catenin activation regulates tissue growth non-cell autonomously in the hair stem cell niche. Science 343, 1353–1356 (2014).

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Acknowledgements

We thank E. Fuchs (Rockefeller University, USA) for the K14-H2BGFP, K14-actinGFP, K14-rtTA mice; K. Politi (Yale, USA) for the TetO-Cre mice; and V. V. Verkhusha (Albert Einstein, USA) for the PAmCherry construct. We thank the Marine Biological Laboratory (MBL, USA) for their support while writing this manuscript; S. Williams (UNC, USA), M. Schober (NYU, USA) and M. Lee for critical feedback; and S. Werner (ETH, Switzerland), R. Clark (Stony Brook, USA) and P. Martin (Bristol University, UK) for their constructive criticism of this work. V.G. is supported by the National Institute of Arthritis and Musculoskeletal and Skin Disease (NIAMS), NIH, grants no. 5R01AR063663-04 and 1R01AR067755-01A1 and by a Mallinckrodt Scholar Award. V.G. is a New York Stem Cell Foundation Robertson Investigator and a HHMI Scholar. S.P. was supported by the James Hudson Brown-Alexander Brown Coxe Postdoctoral Fellowship and is currently supported by the CT Stem Cell Grant 14-SCA-YALE-05. D.G.G. and A.M.H. are supported by the Yale Rheumatic Disease Research Core Center (P30 AR053495-08). K.R.M. was and S.B. is supported by the NIH Predoctoral Program in Cellular and Molecular Biology, grant no. T32GM007223. K.R.M. is currently supported by the NSF Graduate Research Fellow. E.D.M. is supported by the National Institutes of Health, grant no. T32 GM007499. P.R. was a New York Stem Cell Foundation-Druckenmiller Fellow and was supported by the CT Stem Cell Grant 13-SCA-YALE-20. K.C. is supported by a Canadian Institutes of Health Research Postdoctoral Fellowship. Y.B. is supported by CNRS, INSERM, Institut Curie, ERC Advanced (TiMoprh, 340784), ARC (SL220130607097), ANR Labex DEEP (11-LBX-0044, ANR-10-IDEX-0001-02) and PSL grants.

Author information

Affiliations

  1. Department of Genetics, Yale School of Medicine, New Haven, Connecticut 06510, USA

    • Sangbum Park
    • , David G. Gonzalez
    • , Jonathan D. Boucher
    • , Katie Cockburn
    • , Edward D. Marsh
    • , Kailin R. Mesa
    • , Samara Brown
    • , Panteleimon Rompolas
    •  & Valentina Greco
  2. Department of Laboratory Medicine, Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut 06510, USA

    • David G. Gonzalez
    •  & Ann M. Haberman
  3. Polarity, Division and Morphogenesis Team, Genetics and Developmental Biology Unit (CNRS UMR3215/Inserm U934), Institut Curie, 75248 Paris Cedex 05, France

    • Boris Guirao
    •  & Yohanns Bellaïche
  4. Departments of Cell Biology and Dermatology, Yale Stem Cell Center, Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut 06510, USA

    • Valentina Greco

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Contributions

S.P. and V.G. designed experiments and wrote the manuscript. S.P. performed the experiments and analysed the data. D.G.G. and A.M.H. performed three-dimensional imaging analysis and quantifications. J.D.B. and E.D.M. performed immunofluorescence. B.G. and Y.B. performed characterization of tissue deformation. Y.B., K.R.M., J.D.B., A.M.H., D.G.G., K.C. and S.B. assisted with critical feedback on the research and manuscript. P.R. generated the K14-H2BPAmCherry mouse line.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Valentina Greco.

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Videos

  1. 1.

    Tracking of epithelial migration over 12 hours shows a gradient migration pattern from the wound.

    Imaris track analysis was done using K14-H2BGFP (h:mm:ss.sss) (representative analysis from 3 mice). Bright cells located closer to the wound edge (left) are presumably dead and are not observed to move on the top of the epidermis. Each nucleus is marked by a dragon tail track, which highlights its migration path. The color of the tracks change from blue to red indicating passage of time. Also see Figure 1b. Scale bar, 100 μm.

  2. 2.

    Tracking of leading edge cells shows upward movement in vivo.

    Tracking analysis from Imaris of a time-lapse recording of the x-z view of the leading edge region using K14-H2BGFP (hh:mm:ss.sss) (representative analysis from 3 mice). Also see Figure 1e. Scale bar, 10 μm.

  3. 3.

    Cell migration behind leading edge.

    Epidermal nuclei from K14-H2BGFP are represented by dots which have different colors depending on their distance from the basement membrane. All the dots are shown in x-y, x-z, and y-z views (t = 12 hours; representative analysis from 2 mice). Also see Figure 1g.

  4. 4.

    Identification of cell shape changes in the different layers of the epidermis in vivo.

    Time-lapse recording of epithelial cells using membrane-fluorescent mice (K14-CreER;mTmG) shows that lamellipodia are formed during migration in basal and spinous layer cells but not in granular layer cells. The asterisk indicates a migrating spinous layer cell (h.hh) (representative analysis from 3 mice). The movie was looped 3 times to facilitate identification of the cellular behaviors. Scale bar, 10 μm.

  5. 5.

    Identification of distinct regions of cellular behavior at varying distances from the wound.

    Time-lapse recording of epithelial cells show 1) cells migrating but not proliferating closer to the wound edge (migration- left); 2) cells both proliferating and migrating beyond the wound edge (mixed- center); 3) cells only proliferating at the distance from the wound (proliferation- right) using K14-H2BGFP (h.hh) (representative analysis from 3 mice). The movie was looped 3 times to facilitate identification of the cellular behaviors. Also see Figure 3b. Scale bar, 10 μm.

  6. 6.

    Lamellipodia formation before cell division in the mixed zone.

    Time-lapse recording of an epithelial cell in the mixed zone in K14-CreER;mTmG mice shows that lamellipodia are formed during migration. However, all lamellipodia disappear just before cell division (representative analysis from 3 mice). Arrowheads indicate lamellipodia. The movie was looped 3 times to facilitate identification of the cellular behaviors. Scale bar, 10 μm.

  7. 7.

    Quantification of local tissue deformations.

    First, nuclei positions are used as seed to generate a Voronoi tessellation defining a region for each nucleus (grey lines) and a set of neighboring nuclei. Then, a grid made of regions of about 80 × 80 μm2 is used to select nuclei that will define the regions of the tissue over which deformations will be determined (black contours). The evolution of the links connecting the centroids of neighboring Voronoi “cells” is used to calculate the deformation of each tissue patch. Between two successive images: blue links are conserved and are used in the calculation of the patch deformation; magenta links disappear (dashed lines) and appear (full lines), and are not used in the calculation (t = 12 hours; representative analysis from 2 mice). Scale bar, 50 μm.

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https://doi.org/10.1038/ncb3472

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