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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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.
This file contains Supplementary Figures S1-S3 with Legends. (PDF 1453 kb)
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. (MOV 2159 kb)
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. (MOV 3041 kb)
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. (MOV 516 kb)
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. (MOV 4317 kb)
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. (MOV 1097 kb)
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. (MOV 1201 kb)
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. (MOV 3038 kb)
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. (MOV 2321 kb)
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Niethammer, P., Grabher, C., Look, A. et al. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459, 996–999 (2009). https://doi.org/10.1038/nature08119
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