- Title
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Oxidative Stress Orchestrates Cell Polarity to Promote Embryonic Wound Healing
- Authors
- Hunter, M.V., Willoughby, P.M., Bruce, A.E.E., Fernandez-Gonzalez, R.
- Source
- Full text @ Dev. Cell
Wound Repair in Zebrafish Embryos Is ROS- and Src-Dependent (A) Volume rendering of wound closure in the enveloping layer (EVL) of a zebrafish embryo expressing non-muscle myosin light-chain 12-GFP (Myl12.1-GFP). (B and B′) Wound healing in the EVL of a zebrafish embryo expressing Myl12.1-GFP (B) and treated with the ROS dye Amplex UltraRed (B′). (C, D, G, and H) Wound closure in the EVL of a zebrafish embryo expressing Myl12.1-GFP and treated with 1% DMSO (C and G), and 250 μM DPI in 1% DMSO (D) or 20 μM PP2 in 1% DMSO (H). Yellow dotted lines outline wounds. (B–D, G, and H) Scale bars, 10 μm. (A–D, G, and H) Time after wounding is shown. Red lines indicate wound sites. (E, F, I, and J) Mean wound area over time (E and I) or mean myosin fold change in the visible segments of the wound margin -quantified 3 min after wounding to avoid the accumulation of apically extruded cellular debris- (F and J) for zebrafish embryos treated with DMSO (blue; E and F, n = 5; I and J, n = 6), DPI (red; E and F, n = 5), or PP2 (red; I and J, n = 5). Wound areas for DPI are underestimated due to wound overexpansion out of the field of view. (E and I) Error bars, SEM. (F and J) Horizontal line, mean; box, SEM; error bars, SD. ∗, p < 0.05. |
Mitochondrial ROS are produced as a result of tissue damage. (a) Wound closure in the epidermis of a Drosophila embryo injected with H2DCFDA, a membrane-permeable ROS dye (a), and expressing myosin:mCherry (a’). Red and blue boxes outline the regions used to quantify epidermal fluorescence in (b). (b) Mean DCF fluorescence over time inside the wound (red, n = 5) and in the surrounding epidermis (blue, n = 5). Note that negative intensities indicate fluorescence values below the background (the image mode). (c) Wound closure in the epidermis of a Drosophila embryo expressing mitoTimer, a mitochondrial ROS biosensor that contains a mitochondrially targeted dsRed variant whose fluorescence shifts from green (c) to red (c’) upon oxidation. (d) Epidermal cells in a Drosophila embryo expressing E-cadherin:tdTomato in embryos injected with DCF and subsequently impaled using the injection microneedle. Images were acquired ~5 minutes after wounding. Yellow dotted lines outline the wound. Scale bars, 5 μm. (e-g) mitoTimer fluorescence (red signal) in the epidermis of a Drosophila embryo injected with DMSO (e), the superoxide dismutase mimetic EUK134 (f), or the NADPH oxidase inhibitor VAS2870 (g). (h) Maximum red:green mitoTimer fluorescence ratio inside the wound for embryos injected with DMSO (blue, n = 6 wounds), EUK134 (green, n = 5), or VAS2870 (yellow, n = 4). Horizontal line, mean; box, SEM; error bars, standard deviation (SD). *, P < 0.05; n.s., not significant. (i) Mean Amplex UltraRed fluorescence inside the wound for zebrafish embryos treated with DMSO (blue, n = 5 wounds) or the ROS inhibitor DPI (red, n = 3). (b, i) Error bars, SEM. (j-k) Amplex Ultrared fluorescence in the enveloping layer of wounded zebrafish embryos treated with DMSO (j) or with the ROS inhibitor DPI (k). Time after wounding is indicated. Red lines indicate wound sites. Scale bars, 10 μm. (l-n) Epidermal cells in Drosophila embryos injected with DPI and expressing mitoTimer (l, red signal shown), or co-injected with H2DCFDA (m) or Amplex Ultrared (n). (a, c, e-g, j-n) Anterior left, dorsal up. Red lines indicate wound sites. Scale bars, 5 μm. |
Reprinted from Developmental Cell, 47, Hunter, M.V., Willoughby, P.M., Bruce, A.E.E., Fernandez-Gonzalez, R., Oxidative Stress Orchestrates Cell Polarity to Promote Embryonic Wound Healing, 377-387.e4, Copyright (2018) with permission from Elsevier. Full text @ Dev. Cell