Fig. S5

Tg(mpeg1:NTR-YFP) fish line affords inducible systemic ablation of macrophages. (A) Schematic of a new inducible macrophage ablation fish line Tg(mpeg1:NTR-eYFP) made using NTR/MTZ technology. Briefly, fish expressing the mpeg1 promoter ubiquitously express a fusion NTR-YFP protein. Addition of metronidazole (MTZ) in fish water results in catalytic electron transfer and activation of Met in cells expressing NTR fusion protein. Activated Met promotes DNA breaks and ultimately cell death. Representative image of mpeg1+ cell (macrophage) in upper panel. Scale bar = 10 μm. (B) Macrophages (mpeg1+) are easily visualized throughout the caudal fin in Tg(mpeg1:NTR-eYFP) fish as green YFP+ fluorescent cells (upper panels). Subsequent incubation in 2.5 mM MTZ for 12h and 36h results in dissipation of this green signal and significant reduction of overall numbers of macrophages throughout the fish, including the caudal fin (lower panels). Scale bar = 200 μm. (C) MTZ-induced cell ablation in Tg(mpeg1:NTR-eYFP) fish as assessed in uninjured +/- MTZ treated fins via flow cytometry shows an 80-90% reduction in eYFP+ cells post-MTZ treatment (2d). Flow cytometry was conducted on pooled samples (NTR+MTZ, N=8-10; NTR-MTZ, N=8-10). Data is representative of 3 separate experiments. Error bars are S.E.M. (D) Quantification of macrophage cell number near the amputation planes for both the proximal and distal cuts (N=7) from uncut to 14 dpa in Tg(mpeg1:NTR-eYFP) fish. Data is representative of 2 separate experiments.