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Mechanical waves identify the amputation position during wound healing in the amputated zebrafish tailfin

Nature Physics volume 19, pages 1362–1370 (2023)Cite this article

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Abstract

Highly regenerative animals can regrow lost appendages and the rate of regrowth is proportional to the amount of appendage loss. This century-old phenomenon prompted us to investigate whether the mechanism of wound healing, as the first stage of regeneration, is responsible for discerning the amputation position. In vitro studies have revealed great insights into the mechanics of the wound-healing process, including the identification of mechanical waves in collective epithelial cell expansion. It has been suggested that these mechanical waves may also be involved in positional sensing. Here we perform live-cell imaging on adult zebrafish tailfins to monitor the collective migration of basal epithelial cells on tailfin amputation. We observed a cell density wave propagating away from the amputation edge, with the maximum travelling distance proportional to the amputation level and cell proliferation at later stages. We developed a mechanical model to explain this wave behaviour, including the tension-dependent wave speed and amputation-dependent travelling distance. Together, our findings point to an in vivo positional sensing mechanism in regenerative tissues based on a coupling of mechanical signals manifested as a travelling density wave.

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Fig. 1: In toto image showing the distribution of BECs in the zebrafish tailfin.
Fig. 2: Illustration of the re-epithelization process after amputation.
Fig. 3: Amputation-level-dependent responses of BEC migration.
Fig. 4: The wave is transmitted through mechanical tension.
Fig. 5: A one-dimensional active spring model depicts the wave response of collective BEC migration.
Fig. 6: H2O2 manipulation influences cell motility, Lstretch levels, cell proliferation and regeneration.

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Data availability

The plasmids, generated zebrafish lines, raw data and all other data supporting the plots and graphs in this paper are available from the corresponding authors upon request. Source data are provided with this paper.

Code availability

Customized scripts and usage instructions are available via GitHub at https://github.com/mpdeleonTIGPMBAS/Mechanical-wave.git.

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Acknowledgements

We thank the Taiwan Zebrafish Core Facility at Academia Sinica (108-2319-B-400-002) for zebrafish care; principal investigators at the Institute of Cellular and Organismic Biology for discussions; Chen and Lin laboratory members, S. Q. Schneider, T. B. Saw and Y.-L. Wang, for comments on the manuscript; and M. J. Calkins and K. Hatch for English editing and comments. We thank M.-W. Hsueh for helping with some of the codes and the schematic of the three-dimensional drawing. We acknowledge the Data Science Statistical Cooperation Center of Academia Sinica (AS-CFII-111-215) for statistical support, and intramural funding support from the Institute of Cellular and Organismic Biology and the Institute of Physics, Academia Sinica, to C.-H.C. and K.-H.L.; grants from Academia Sinica to C.-H.C. (AS-CDA-109-L03) and the Academia Sinica Innovative Materials and Analytical Technology Exploration Program to K.-H.L. (AS-iMATE-109-12 and AS-iMATE-111-14); and grants from the Ministry of Science and Technology, Taiwan, to C.-H.C. (MOST 106-2628-B-001-001-MY4 and MOST 110-2628-B-001-016), K.-H.L. (MOST 109-2122-M-001-001-MY2) and F.-L.W. (MOST 109-2112-M006-028-MY3).

Author information

Author notes

  1. These authors contributed equally: Marco P. De Leon, Fu-Lai Wen.

Authors and Affiliations

  1. Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan

    Marco P. De Leon, Ying-Ting Wang, Hsiao-Yuh Roan, Chung-Han Wang & Chen-Hui Chen

  2. Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan

    Marco P. De Leon

  3. Institute of Physics, Academia Sinica, Taipei, Taiwan

    Fu-Lai Wen, Giovanni J. Paylaga & Keng-Hui Lin

  4. International Center for Wound Repair and Regeneration, National Cheng Kung University, Tainan, Taiwan

    Fu-Lai Wen

  5. Department of Science Education, National Taipei University of Education, Taipei, Taiwan

    Fu-Lai Wen

  6. Department of Physics, National Central University, Chung-Li, Taiwan

    Giovanni J. Paylaga & Keng-Hui Lin

  7. Department of Physics, Mindanao State University, Iligan Institute of Technology, Iligan City, Philippines

    Giovanni J. Paylaga

  8. Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taiwan

    Chung-Der Hsiao

  9. Department of Physics, National Taiwan University, Taipei, Taiwan

    Keng-Hui Lin

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Contributions

M.P.D.L., F.-L.W., K.-H.L. and C.-H.C. designed the experiments, analysed the data and prepared the manuscript. Y.-T.W. and H.-Y.R. performed the RT-qPCR and histology measurements. C.-H.C., H.-Y.R. and C.-H.W. designed and generated the transgenic zebrafish lines. M.P.D.L. performed the imaging experiments. F.-L.W. conducted the theoretical modelling. M.P.D.L., F.-L.W., G.J.P. and K.-H.L. performed the quantitative analyses. G.J.P. assisted in creating the macros and scripts for image analysis. C.-D.H. contributed Tg(krt4:NLS-EGFP)cy34. All authors reviewed and edited the paper.

Corresponding authors

Correspondence to Keng-Hui Lin or Chen-Hui Chen.

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Nature Physics thanks Yutaka Matsubayashi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of the inter-ray anatomy.

(a) Sagittal view of an inter-ray of a Tg(krt4:NLS-EGFP;krt19:H2A-mCherry) zebrafish (stitched). SECs (green) cover the entire inter-ray, and BECs (red) sandwich the mesenchyme. Piecewise fitted curves based on the centroids of the SECs/BECs represent the SEC/BEC layer. The distal–proximal axial distance is calculated from the arc length of one of the SEC/BEC curves, starting from the most distal BEC. The inter-ray/mesenchyme thickness is the normal distance from one layer to the next layer (green/magenta double-headed arrow). The z-axis was rescaled to 20x to provide a clear visualization. Scale bar: 20 µm. (b–e) Inter-ray thickness (b), mesenchyme thickness (c), inter-ray width (d), and average inter-nuclear distance <ℓnuc> (e) from the distal to the proximal end. The inset in (e) presents a representative image of <ℓnuc> based on Delaunay triangulation (n = 4). Scale bar: 10 µm.

Source data

Extended Data Fig. 2 Individual velocity profiles of BECs after amputation.

(a) Velocity profiles of single cells over time determined by manual tracking in one inter-ray. The blue line indicates the average velocity of cells approximately 90 µm from the amputation plane (near, n = 4), and the red line indicates the average velocity of cells approximately 250 µm from the amputation plane (distant, n = 4). The dot and shaded region indicate the mean ± SD of the cell velocity. (b) Montage of time-lapse stitched images of Tg(krt19:LifeAct-mScarlet) cells after tailfin amputation. A BEC marked by an asterisk is near the amputation plane, while a BEC marked by a cross is further away. White arrows point to a lamellipodium protrusion. The red arrow points to the amputation plane. The lamellipodium of the distant cell is activated at a later time point after the cell is stretched. Each image frame is denoted in minutes post-amputation (mpa). Scale bar: 5 µm. (c) Overlay of the spatial distribution of <ℓnuc> (green), BEC velocity (red), and ‘white wave’ (blue) over time. Units in the y-axis are based on Fig. 3e–g. (d) Schematics of ‘white wave’ analysis. The red arrow points to the amputation plane. Raw images for time 1 (T1) and time 2 (T2) are shown in cyan and magenta, respectively. In the overlay image, regions containing cyan and magenta indicate locations in which cells are moving while regions containing white indicate regions in which cells are not moving. In the difference image, that is, the ‘white wave’ image, white regions indicate locations in which cells are moving, and black regions indicate locations in which cells are not moving. (e) ‘White wave’ kymograph and corresponding quantitative analysis. Each column is the average value of a ‘white wave’ image of an inter-ray stacked over time. The parameters for extracting the CMZ speed (cyan), back CMZ (green), CMZ length (magenta), and Lstretch (yellow) are denoted in the image.

Source data

Extended Data Fig. 3 Amputation-level-dependent responses.

(a) Representative kymographs depicting CMZ propagation after DA, MA, and PA. The red arrow points to the amputation position. (b, c) Maximum CMZ length (n = 16 [DA], 14 [MA], 19 [PA]) and average BEC velocity (20 to 30 mpa; n = 11 [DA], 7 [MA], 13 [PA]) for different amputation levels. The value of n indicates the number of inter-rays analyzed. Statistical analyses were performed via one-way ANOVA. The midlines and boxes show the mean ± SD, whereas the whiskers indicate the minimum and maximum values. (d) Representative time-lapse PIV images of BEC movement after DA, MA, and PA. Each image frame is denoted in minutes post-amputation (mpa). The red arrow points to the amputation position.

Source data

Extended Data Fig. 4 Tissue tension governs the CMZ speed.

(a) Illustration of tailfin laser ablation. The white box indicates the imaging area. (b) Image sequence of the tailfin laser ablation experiment. The cyan box indicates the area chosen for the kymograph. Scale bar: 25 µm. (c) Vrecoil is calculated from the rate of change of the edges (green lines). Vertical scale bar: 3 µm. Horizontal scale bar: 10 s. (d) Illustration of experimental conditions of the laser ablation position and tailfin tension. Left to right: Laser ablation at a sub-DA location on a complete intact, non-stretched tailfin (NS@DA) and after a DA tailfin has healed (low stretch, LS@DA) and at a sub-MA location on a complete intact tailfin (NS@MA) and after an MA tailfin has healed (high stretch, HS@MA). (e) Representative kymographs of the incision experiments illustrated in (d). Vertical scale bar: 3 µm. Horizontal scale bar: 10 s. Green lines indicate fitted lines for calculating the recoil speed. (f) Quantification of Vrecoil for control NS@DA (n = 5, water), LS@DA (low stretch; n = 7, water), control NS@MA (n = 15, control; water), HS@MA (high stretch; n = 9, water), Control@MA (n = 17, DMSO), Yoda1@MA (n = 17, DMSO), and Gd@MA (n = 15, water). The value of n indicates the number of inter-rays analyzed. Statistical analyses were performed via Kruskal–Wallis ANOVA. The midlines and boxes show the mean ± SD, whereas whiskers indicate the minimum and maximum values. (g) Scatter plot of Vrecoil and <ℓnuc> for stretched tissue. The points and lines indicate the mean ± SD. Pearson’s correlation coefficient is 0.999 (p=0.006). Two-tailed test of significance is used. Vrecoil (n = 15 [NS@MA], n = 9 [HS@MA], n = 5 [NS@DA], n = 7 [LS@DA]) and <ℓnuc> (n = 6 [NS@MA], n = 6 [HS@MA], n = 5 [NS@DA], n = 5 [LS@DA]). The value of n indicates the number of inter-rays analyzed. (h) Representative kymographs of CMZ propagation in epidermal tissue at different stretch levels. Top: Illustration of the tailfin amputation conditions. Bottom (left to right): Kymographs showing the CMZ of an NS and LS tailfin after DA and of an NS and HS tailfin after MA. The red arrow indicates the amputation position. Colored lines are fitted lines used to calculate the CMZ speed. Lines from non-stretched conditions are overlaid on plots for the stretched conditions. (i) Quantification of VCMZ after amputation for control NS@DA (n = 12, water), LS@DA (low stretch; n = 16, water), NS@MA (n = 46, control; water), HS@MA (high stretch; n = 18, water), Control@MA (n = 9, DMSO), Yoda1@MA (n = 8, DMSO), and Gd@MA (n = 10, water). The value of n indicates the number of inter-rays analyzed. Statistical analyses were performed via one-way ANOVA. The midlines and boxes show the mean ± SD, whereas whiskers indicate the minimum and maximum values. (j) Scatter plot of VCMZ and Vrecoil that includes ablation at the DA position under NS and LS conditions. The points and lines indicate the mean ± SD. Pearson’s correlation coefficient is 0.891 (p=0.007). Two-tailed test of significance is used. VCMZ (n = 12 [NS@DA], n = 16 [LS@DA], n = 46 [NS@MA], n = 18 [HS@MA], n = 9 [Control@MA], n = 8 [Yoda1@MA], n = 10 [Gd@MA]) and Vrecoil (n = 5 [NS@DA], n = 7 [LS@DA], n = 15 [NS@MA], n = 9 [HS@MA], n = 17 [Control@MA], n = 17 [Yoda1@MA], n = 15 [Gd@MA]). The value of n indicates the number of inter-rays analyzed.

Source data

Extended Data Fig. 5 A simple one-dimensional active spring model captures both CMZ and BEC dynamics.

(a) In silico simulation of the effects of chemical signals (that is, different propagation rates) in the generation of CMZ under different tension conditions. Black dashed lines indicate the CMZ speed. (b) Illustration of the middle cut-through incision experiment (a is the width of the cut [approximately 40–60 µm], b is the length of the cut [approximately 2 mm]), showing the propagation direction of the p-CMZ/d-CMZ at the proximal/distal side of the tailfin. ‘Near cells’ refer to BECs near the incision site. ‘Distant cells’ refer to BECs distal to the incision site. The blue and pink arrows mark the direction of each wave. Black arrows indicate the direction of cell migration. Red dashed lines mark the incision site. Gray shades highlight a gradient of mechanical factors. (c, d) Simulation results depicting spatiotemporal changes in cell velocity. When the CMZ propagates along a proximal region, the maximum cell velocity is predicted to decrease. In contrast, when the CMZ propagates along a distal region, the maximum cell velocity is predicted to increase. (e, f) Cell velocity determined by single-cell tracking during p-CMZ and d-CMZ propagation. The ‘near cells’ and ‘distant cells’ are located approximately 200 and 300 µm from the incision site (n = 3 each), respectively. Data are shown as the mean and standard error of the mean (SEM) (n = 13 [p-CMZ], 13 [d-CMZ] from 5 animals). The dots and shaded regions indicate the mean ± SEM of the cell velocity. (g) A second CMZ arises when the constraint force is reduced.

Source data

Extended Data Fig. 6 Endogenous hydrogen peroxide regulates CMZ dynamics.

(a) Snap-shot CellROX images overlaid with DA/MA/PA tailfins at 1, 15, 30, 60, and 120 minutes post-amputation (mpa). Scale bar: 100 µm. The red arrow indicates the amputation plane. (b) RNA ISH assay on a krt19:cat tissue section. Catalase over-expression (indicated by blue) is restricted to the BEC layer. The black box indicates the magnified area (shown below), and the red dashed line marks the basement membrane. Scale bars: 200 µm. (c) RT-qPCR assay results for measuring catalase expression levels in adult WT and krt19:cat zebrafish. Uninjured adult tailfin tissues were collected for analysis (n = 4; mean±SEM). (d) Representative CellROX images overlaid with bright-field images of amputated tailfins of WT and krt19:cat zebrafish at 60 mpa. Scale bar: 100 µm. The red arrow indicates the amputation position. (e) The CellROX intensity is much higher in WT than in krt19:cat zebrafish at 60 mpa (n = 8 [WT], 8 [krt19:cat]; two-sample t-test). (f) PIV image of cell velocity in WT and krt19:cat zebrafish at 30 mpa (n = 5 [WT], 5 [krt19:cat]; two-sample t-test). (g) Representative kymographs depicting CMZ propagation in WT and krt19:cat zebrafish. Adult double-transgenic animals, Tg(krt19:H2A-mCherry; krt19:cat), were used to visualize CMZs.

Source data

Extended Data Fig. 7 Hydrogen peroxide levels influence the wound closure rate and cell proliferation.

(a) Confocal images of wound edges on the amputation plane of WT and krt19:cat tailfins at 4 and 26 minutes post-amputation (mpa). The wound edges are highlighted by cyan dashed lines. Scale bar: 20 µm. (b) Wound closure occurs more rapidly in WT than in krt19:cat zebrafish (n = 5 [WT], 5 [krt19:cat]). The dots and shaded regions indicate the mean ± SD of the wound opening length. (c) Bright-field images of a punched-hole injury in WT and krt19:cat zebrafish at 5 and 60 mpa. The cyan dashed lines highlight the advancing wound edges. Scale bar: 100 µm. (d) Wound closure of a punched-hole injury occurs more rapidly in WT than in krt19:cat zebrafish. The plots show the mean and SD of the ratio of the apparent wound size at a particular time point and the original size of the punched hole (n = 5 [WT], 5 [krt19:cat]). The dots and shaded regions indicate the mean ± SD of the wound area. (e) Illustration of EdU quantification in the blastema (that is, the region distal to the lateral third bony ray) and inter-ray (that is, the region between the lateral second and third bony rays). The red dashed line highlights the amputation plane. Green dots represent EdU+ cells, and violet boxes indicate regions in which EdU+ cells were quantified. (f, g) Representative images of MA, PA, WT, and krt19:cat inter-ray and blastema sections from fixed EdU-stained tailfins at 2 days post-amputation (dpa). The red dashed line represents the amputation plane. The left panel presents a stitched maximum-projected image of the inter-ray, where the violet box (length: 1500 µm) indicates the region of EdU+ cell quantification. The right panel presents a magnified image slice of the blastema, where the violet box (120 x 120 µm) indicates the region of EdU+ cell quantification. EdU+ cells in the blastema were obtained from a 10-µm z-section.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Live image of wound closure in an amputated Tg(krt19:H2A-mCherry) tailfin. A three-dimensional rendered image highlighting wound closure and CCM propagation in the inter-ray. Scale bar, 80 µm.

Supplementary Video 2

Live images of Tg(krt19:LifeAct-mScarlet) after tailfin amputation. Both nearby and distant BECs show lamellipodium formation during migration. The BEC marked with an asterisk represents a nearby cell, whereas a distant cell is marked with a cross. The images were acquired every 15 s for 1 h. Scale bar, 10 µm.

Supplementary Video 3

Time-lapse images of BECs at PA. Tailfin BECs are labelled by Tg(krt19:H2A-mCherry) (right) and white-wave analysis (left), where the CMZ is highlighted in a red box. The images were acquired from 4 to 80 mpa at 2 min intervals. Scale bar, 100 μm.

Supplementary Video 4

Time-lapse images of BECs after either DA, MA or PA. Tailfin BECs are labelled by Tg(krt19:H2A-mCherry). For each panel, time-lapse images without processing are shown on the left, whereas white-wave images are shown on the right. The red boxes highlight the CMZ. The images were acquired from 10 to 60 mpa at 2 min intervals. Scale bar, 100 μm.

Supplementary Video 5

Representative time-lapse images of the stretched epidermis on a laser-assisted incision. Tailfin BECs are labelled with H2A-mCherry. First row: NS@DA, no stretch tissue; LS@DA, low stretch tissue. Second row: NS@MA, no stretch tissue; HS@MA, high stretch tissue. Third row: WT and krt19:cat zebrafish. Fourth row: control (water) and gadolinium-treated zebrafish. Fifth row: vehicle dimethyl-sulfoxide-treated and Yoda1-treated zebrafish. The images were acquired every 2 s after the incision. End-point wound edge distances are highlighted by a green arrow. Scale bar, 20 µm.

Supplementary Video 6

Raw time-lapse images and PIV heat map of the two-wound-edge experiment. The images were acquired every 2 min for 120 min. Scale bar, 50 µm.

Supplementary Video 7

Stepwise simulation of a one-dimensional active spring on a kymograph. Cell migration in one-dimensional space (left). The positions of cells migrating at speeds larger than a threshold are highlighted (middle). A dynamic plot of CCM over time (right).

Supplementary Video 8

Time-lapse images of BECs after a middle incision. Tailfin BECs are labelled by Tg(krt19:H2A-mCherry). A single cut-through incision in the inter-ray region generates both p-CMZ and d-CMZ, which travel in opposite directions. Time-lapse images without processing (left). Time-lapse images after processing via white-wave analysis (right). The red boxes highlight the CMZ. The images were acquired from 10 to 60 mpa in 2 min intervals. Scale bar, 50 μm.

Supplementary Video 9

Time-lapse images of BECs after tailfin amputation in WT and krt19:cat zebrafish amputated at the middle tailfin region. A WT sibling (left). The catalase overexpression line (right). For each panel, time-lapse images without processing are shown on the left, whereas white-wave images are shown on the right. The red boxes highlight the CMZ. The images were acquired from 10 to 60 mpa at 2 min intervals. Scale bar, 50 μm.

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De Leon, M.P., Wen, FL., Paylaga, G.J. et al. Mechanical waves identify the amputation position during wound healing in the amputated zebrafish tailfin. Nat. Phys. 19, 1362–1370 (2023). https://doi.org/10.1038/s41567-023-02103-6

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