Fig. S3

Zonal Variations in Tissue [H₂O₂] Related to the Path of an Individual Neutrophil Migrating towards the Wound Zone, Related to Figure 4, and a Model of the H₂O₂/Myeloperoxidase Interaction after Wounding
(A) Still images over 1 h showing path of a single lyz:DsRED2-marked neutrophil (upper row) and associated HyPer ratiometic data reflecting the [H₂O₂] data for the wound zone. Note the asymmetrical regional reduction on the dorsal (upper) half of the fin where the DsRED2-marked neutrophil is located, most evident by the loss of extent of yellow signal on the heat map. Stills are from Movie S5. Scale bar, 100 μm.
(B) (i) Merged transmission and red fluorescence image at 98 min after injury, overlayed with the migratory paths of lyz:DsRED2 neutrophils (4 green paths, only one of which is relevant to regions 1 and 2) and lyz:DsRED2-negative macrophages (purple, blue and cyan paths, all relevant to region 3). Regions 1-4 are: (1) and (2), regions where the single DsRED2-marked neutrophil passes in sequence; (3) region where three DsRED2-negative macrophages pass; (4) control region, distal to wound. Scale bar, 100 μm. (ii) HyPer ratiometic data reflecting the temporal [H₂O₂] data for the 4 regions of the injured zebrafish larvae detailed in (i); data are median HyPer ratio for each of the regions shown. Note that the median [H₂O₂] decreases sequentially in region 1 then 2 as the neutrophil passes through them, but remains elevated in region 3 where only macrophages pass. The [H₂O₂] remains at background in the control region (4).
(C) Representative HyPer heat maps depicting [H₂O₂] across the tail fin of a WT embryo 30 and 60 min after embedding only (i.e. without wounding by tail transection) showing resolution of small focal H₂O₂ bursts at minor nick injuries that were sustained during embedding (white arrowheads). Adjacent overlaid transmission (Trans) and DsRED images document that this resolution occurred without the arrival of any lyz:DsRED2-marked neutrophils. Scale bar, 200 m.
(D) Table showing the extent of overlap of leukocyte marker transgene expression in compound transgenic zebrafish embryos at 2-3 dpf. * = actual genotype Tg(mpeg1:GAL4)x(10xUAS:Kaede) na = not applicable ¶ = Wound margin (WM) corresponds to cell within 100 μm of the wound edge 2 h after amputation.
(E) Model of the H₂O₂/myeloperoxidase interaction in the wound zone. Schematic diagrams at three timepoints before (i), just after (ii) and 60-90 min after (iii) wounding, showing for each time point the stage in the post-wounding H₂O₂ burst (upper panels), a whole tail overview (middle panels), and a detail of a neutrophil (lower panels). Prior to wounding (i), intra-neutrophil and surrounding tissue [H₂O₂] is low. Neutrophils carry the Src-family kinase Lyn capable of H₂O₂ sensing (green), and myeloperoxidase (Mpx, blue) packaged in primary granules.
Immediately after wounding (ii), a tissue-scale [H₂O₂] gradient (red, low-high [H₂O₂] as indicated in scale) is generated by Dual oxidase (Duox, yellow). H₂O₂ is sensed by Lyn in neutrophils, initiating their migration. Initially, within migrating neutrophils the sum of their own endogenous H2O2 production (possibly generated by NADPH oxidase 2 (Nox2) and superoxide dismutase (SOD), and/or as a by-product of mitochondrial respiration) plus the diffusion of tissue-sourced H₂O₂ results in the net outcome of higher intracellular [H₂O₂] than surrounds.
The decline from peak tissue gradient H₂O₂ levels (iii) coincides with neutrophil arrival and is dependent on their delivery of enzymatically-active Mpx which interacts intracellularly with H₂O₂. Some migrating neutrophils approaching the wound develop and sustain an intracellular [H₂O₂] that is lower than their surrounds – they are persistent net consumers of endogenous and tissue-sourced H₂O₂. These neutrophils function as intracellular H₂O₂ sumps. At the same time, some other wound-proximate neutrophils display higher internal [H₂O₂] than their surrounds. However, the net effect of Mpx-dependent intracellular enzymatic HH₂O₂ consumption is dampening of the overall wound-stimulated tissue H₂O₂ burst.