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

Abstract

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 (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor. This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm 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 H(2)O(2) pattern, and the first evidence that H(2)O(2) signals to leukocytes in tissues, in addition to its known antiseptic role.

Figure 1

Wound margin H₂O₂ production in zebrafish larvae. (a) Experimental procedure. (b) HyPer imaging in an injured zebrafish larva. [H₂O₂] is inferred from the YFP₅₀₀/YFP₄₂₀ excitation ratio of HyPer. Greyscale scaling is adjusted to improve contrast. (c) Temporal [H₂O₂] profile in a ∼10−30 μm broad region of interest along the wound margin. Arrival of first leukocyte at wound (solid red line) ± SD (dashed red line). (d) [H₂O₂] line profile normal to the wound margin. (e) Imaging of leukocyte recruitment and [H₂O₂] in a lysC::DsRED2 fish line. Coloured lines: superimposed leukocyte tracks. Scale bars: 100 μm.

Figure 2

Nox/Duox activity is required for wound margin H₂O₂ production and leukocyte recruitment. (a) Scheme of mammalian NADPH oxidases also found in zebrafish,. (b) Wound margin [H₂O₂] ± DPI or VAS2870 (VAS), or carrier (1% DMSO) imaged 17 min pw. (c) Statistical quantification of wound margin [H₂O₂]. (d) Injured tail fins of mpo::GFP larvae ± DPI (42 min pw). Coloured lines: leukocyte tracks derived from the corresponding time-lapse movies. (e) Statistical quantification of leukocyte recruitment to wound margin. Error bars: SEM of indicated number of larvae (brackets). *** P < 0.001 (vs. control). Scale bars: 100 μm.

Figure 3

Duox activity is required for wound margin H₂O₂ production and leukocyte recruitment. (a) Wound margin H₂O₂ after morpholino mediated duox knockdown (MO1-duox) or injection of a corresponding 5-misprime morpholino (5-MP) imaged 17 min pw. Inset: RT-PCR of a duox mRNA region flanking the targeted splice site. (b) Quantification of wound margin [H₂O₂]. (c) Injured tail fins of mpo::GFP larvae injected with MO1-duox, or 5-MP (42 min pw). Coloured lines: leukocyte tracks. (d) Quantification of leukocyte recruitment. Error bars: SEM of indicated number of larvae (brackets). ** P < 0.01, *** P < 0.001 (vs. control). Scale bars: 100 μm.