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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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.
Integrated supplementary information
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.
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.
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.
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.
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.
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.
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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In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing
Communications Biology (2018)