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Fig. S3
Extent of wound angiogenesis is proportional to number of pro-angiogenic macrophages versus anti-angiogenic neutrophils present at wound site.
A Representative fluorescent stereomicroscope images taken from Tg(mpeg:KalTA4); Tg(UAS:nfsB-mCherry) transgenic zebrafish, either treated with control DMSO or metronidazole as indicated. Metronidazole treatment resulted in > 90% macrophage ablation using this genetic model. N = 10 independent fish per condition. B Representative fluorescent stereomicroscope images taken from Tg(mpeg:mCherry) transgenic zebrafish, injected with either control liposomes or clodronate liposomes, showing similar levels of macrophage ablation to metronidazole treatment. N = 10 independent fish per condition. C Two metronidazole treatment protocols were used to ablate macrophages prior to injury, or throughout injury. Wounded Tg(mpeg:KalTA4); Tg(UAS:nfsB-mCherry); Tg (fli:GFP) transgenic zebrafish larvae were treated with DMSO (control) or metronidazole as indicated. Boxed area denotes wound site. D Quantification of number of macrophages at the wound site of DMSO-treated control fish versus fish treated under the two ablation protocols, measured from images represented in (C) and Fig 2. Under protocol 1, fish recovered into fresh water post-injury saw a partial rescue of macrophage numbers at the wound site by 4 DPF. N = 12 independent fish per condition per timepoint. E Quantification of total blood vessel length, measured from images represented in (C), using Angioanalyser. Under protocol 1, fish with partially rescued macrophages also showed a trend towards increased wound angiogenesis compared to full macrophage ablation. Treatment of Tg(fli:GFP) transgenic zebrafish with metronidazole showed no difference in extent of wound angiogenesis compared to untreated injury. N = 12 independent fish per condition per timepoint. Statistical significance, as determined by one-way ANOVA, is P ≤ 0.0001. Subsequent Bonferonni multiple comparison test, determines level of significance, as indicated. Significance values: ***P ≤ 0.0001. F 1% agarose gel, showing that both mflt1 and sflt1 products are present in whole zebrafish cDNA, but only sflt1 cDNA is present in FAC-sorted Tg(mpx:GFP) neutrophils in early zebrafish wounds, as outlined in Fig 3F.