Letter | Published:

A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish

Nature volume 459, pages 996999 (18 June 2009) | Download Citation



Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H2O2), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H2O2 during the early events of wound responses in zebrafish larvae expressing a genetically encoded H2O2 sensor1. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting 3 min after wounding and peaking at 20 min, which extended 100–200 μm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role.

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  1. 1.

    et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nature Methods 3, 281–286 (2006)

  2. 2.

    , & Membrane transport of hydrogen peroxide. Biochim. Biophys. Acta 1758, 994–1003 (2006)

  3. 3.

    & Redox regulation of cellular functions. Biochemistry 72, 132–145 (2007)

  4. 4.

    , , , & Wound healing and inflammation: embryos reveal the way to perfect repair. Phil. Trans. R. Soc. Lond. B 359, 777–784 (2004)

  5. 5.

    , , , & Modeling inflammation in the zebrafish: how a fish can help us understand lung disease. Exp. Lung Res. 33, 549–554 (2007)

  6. 6.

    et al. Birth and life of tissue macrophages and their migration in embryogenesis and inflammation in medaka. J. Leukoc. Biol. 81, 263–271 (2007)

  7. 7.

    & Reverse leukocyte migration can be attractive or repulsive. Trends Cell Biol. 18, 298–306 (2008)

  8. 8.

    et al. Fluorescent probes for hydrogen peroxide based on a non-oxidative mechanism. Angew. Chem. Int. Edn Engl. 43, 2389–2391 (2004)

  9. 9.

    et al. Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J. Leukoc. Biol. 80, 1281–1288 (2006)

  10. 10.

    , , , & The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev. Biol. 7, 42 (2007)

  11. 11.

    & Redox signals in wound healing. Biochim. Biophys. Acta 1780, 1348–1361 (2008)

  12. 12.

    & The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007)

  13. 13.

    , & Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol. Biol. 7, 109 (2007)

  14. 14.

    et al. Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity. J. Biol. Chem. 280, 30046–30054 (2005)

  15. 15.

    et al. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. Cardiovasc. Res. 71, 331–341 (2006)

  16. 16.

    et al. Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cells. Biochem. Biophys. Res. Commun. 344, 200–205 (2006)

  17. 17.

    et al. Platelet-derived growth factor BB stimulates vasculogenesis of embryonic stem-cell-derived endothelial cells by calcium-mediated generation of reactive oxygen species. Cardiovasc. Res. 81, 159–168 (2009)

  18. 18.

    et al. Compounds containing a N-heteroaryl moiety linked to fused ring moieties for the inhibition of NAD(P)H oxidases and platelet activation. Patent WO/2005/111041. (2005)

  19. 19.

    , , , & Dual oxidases. Phil. Trans. R. Soc. Lond. B 360, 2301–2308 (2005)

  20. 20.

    , , , & Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J. 17, 1502–1504 (2003)

  21. 21.

    , , & A direct role for dual oxidase in Drosophila gut immunity. Science 310, 847–850 (2005)

  22. 22.

    , , , & Regulated hydrogen peroxide production by Duox in human airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 32, 462–469 (2005)

  23. 23.

    , , , & Airway epithelial cell migration and wound repair by ATP-mediated activation of dual oxidase 1. J. Biol. Chem. 282, 3213–3220 (2007)

  24. 24.

    , & Hydrogen peroxide-induced chemotaxis of mouse peritoneal neutrophils. Eur. J. Cell Biol. 70, 347–351 (1996)

  25. 25.

    , , & Hydrogen peroxide-mediated, lysyl oxidase-dependent chemotaxis of vascular smooth muscle cells. J. Cell. Biochem. 78, 550–557 (2000)

  26. 26.

    et al. Redox modulation of T-type calcium channels in rat peripheral nociceptors. Neuron 31, 75–85 (2001)

  27. 27.

    et al. Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors. Proc. Natl Acad. Sci. USA 101, 16419–16424 (2004)

  28. 28.

    et al. Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-γ and PTEN. Nature 442, 457–460 (2006)

  29. 29.

    et al. Tumor suppressor PTEN is a physiologic suppressor of chemoattractant-mediated neutrophil functions. Blood 109, 4028–4037 (2007)

  30. 30.

    et al. Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1. Nature Cell Biol. 9, 36–44 (2007)

  31. 31.

    & Zebrafish (Oxford Univ. Press, 2002)

  32. 32.

    et al. Loss of function of def selectively up-regulates Delta113p53 expression to arrest expansion growth of digestive organs in zebrafish. Genes Dev. 19, 2900–2911 (2005)

  33. 33.

    et al. Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo. Development 134, 4147–4156 (2007)

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P.N. was supported by a Human Frontiers Science Program long-term fellowship. This work was supported by the National Institutes of Health grant GM023928. We would like to thank A. Huttenlocher and P. Crosier for providing us with the mpo::GFP and lysC::DsRED2 transgenic zebrafish lines, respectively.

Author Contributions P.N. and T.J.M. conceived the project. P.N. and C.G. designed and executed the experiments. C.G. and A.T.L. contributed expertise in the zebrafish system. P.N. and T.J.M. contributed expertise in imaging and pharmacology. T.J.M. and A.T.L. provided guidance and institutional support. P.N., C.G. and T.J.M. wrote the text.

Author information

Author notes

    • Philipp Niethammer
    •  & Clemens Grabher

    These authors contributed equally to this work.

    • Clemens Grabher

    Present address: Karlsruhe Institute of Technology, Forschungszentrum Karlsruhe GmbH, Institute of Toxicology and Genetics, 76344 Eggenstein-Leopoldshafen, Germany.


  1. Department of Systems Biology, Harvard Medical School, Boston,

    • Philipp Niethammer
    •  & Timothy J. Mitchison
  2. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Clemens Grabher
    •  & A. Thomas Look
  3. Division of Hematology/Oncology, Department of Pediatrics, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

    • A. Thomas Look


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Corresponding author

Correspondence to Philipp Niethammer.

Supplementary information

PDF files

  1. 1.

    Supplementary Figures

    This file contains Supplementary Figures S1-S3 with Legends.


  1. 1.

    Supplementary Movie 1

    This movie shows time-lapse imaging of H2O2 production in response to tail fin wounding. Imaging starts ˜3 min pw (2 min/frame). Same colour scheme as in Figure 1b (HyPer ratio scale: 2-7). Scaling of individual fluorescence channels is adjusted to improve greyscale contrast. Scale bars: 100 µm.

  2. 2.

    Supplementary Movie 2

    This movie shows H2O2 production and leukocyte movements imaged simultaneously in a lysC::DsRED2 leukocyte reporter larva (3 dpf). Imaging starts ˜3 min pw (2 min/frame). Same colour scheme as in Figure 1e (HyPer ratio scale: 0.5-3.5). Scale bar: 100 µm.

  3. 3.

    Supplementary Movie 3

    This movie shows H2O2 production in response to pharmacological NADPH oxidase inhibition (100 µM DPI). Imaging starts ˜3 min pw (2 min/frame). Larvae different from those depicted in Figure 2b are shown. Same colour scheme as in Figure 2b (HyPer ratio scale: 0.5-4.5). Scale bar: 100 µm.

  4. 4.

    Supplementary Movie 4

    This movie shows the effect of NADPH oxidase inhibition (100 µM DPI) on leukocyte recruitment to the wound (4 dpf mpo::GFP larvae). Imaging starts ˜3 min pw (30 sec/frame). Scale bar: 100 µm.

  5. 5.

    Supplementary Movie 5

    This movie shows H2O2 production in MO-cyba injected larva (Cyba MO) compared to control. Imaging starts ˜3 min pw (2 min/frame). Larvae different from those depicted in Supplementary Figure S2b are shown. Same colour scheme as in Supplementary Figure S2b (HyPer ratio scale: 0.5-8.0). Scale bar: 100 µm.

  6. 6.

    Supplementary Movie 6

    This moves shows H2O2 production in MO1-duox (DUOX MO) injected larvae vs. 5-MP control. Larvae different from those depicted in Figure 3a are shown. Same colour scheme as in Figure 3a (HyPer ratio scale: 0.5-8.0). Imaging starts ˜3 min pw (2 min/frame). Scaling of fluorescence channels is adjusted to improve greyscale contrast. Scale bar: 100 µm.

  7. 7.

    Supplementary Movie 7

    This movie shows Leukocyte recruitment in MO1-duox (DUOX MO) injected 3 dpf mpo::GFP larvae vs. 5-MP control. Imaging starts ˜3 min pw (30 sec/frame). Scale bar: 100 µm.

  8. 8.

    Supplementary Movie 8

    This movie shows Leukocyte recruitment in MO1-duox (DUOX MO) injected 3 dpf mpo::GFP larvae vs. 5-MP control. Imaging starts ˜3 min pw (30 sec/frame). Scale bar: 100 µm.

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