Fig. 2

Tissue-specific damage to the heart upon exposure of Tg(cmlc2:CFP-NTR)^s890 larvae to Metronidazole (Mtz). A,B: Brightfield images of a control Tg(cmlc2:GFP) larva (A) and Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larva (B) exposed to 10 mM Mtz (in 0.2% dimethyl sulfoxide [DMSO]) at 48 hours postfertilization (hpf) for 24 hr. A′,B′: Fluorescence images of the larvae in A,B, showing Tg(cmlc2:GFP) expression (green). C-J: Confocal images of the heart taken after Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae were exposed to 0.2% DMSO alone (C,E,H), or 10 mM Mtz in 0.2% DMSO (D,F,G,I,J), from 58 to 76 hpf. C,D: Ventricles of DMSO (C) and Mtz-treated (D) Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae stained for F-actin (red), Tg(cmlc2:GFP) expression shown in green. E-G: Heart sections of DMSO (E) and Mtz-treated (F,G) Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae immunostained for activated Caspase-3 (blue) and stained with rhodamine phalloidin (red), Tg(cmlc2:GFP) in green. G: Same larva as in F at a higher magnification and thicker optical section. H-J: Heart sections of DMSO (H) and Mtz-treated (I,J) Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae processed for terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection (red), Tg(cmlc2:GFP) in green. Scale bars = 20 μm. Exposure of 48 hpf embryos to 10 mM Mtz for 24 hr did not affect control larvae (A,A′), but resulted in a severe functional cardiac phenotype in the CFP-NTR⁺ larvae (B,B′): collapsed atrium and ventricle, shown by Tg(cmlc2:GFP) expression (B′), and consequent failure in blood circulation with blood pooling (arrow in B). Confocal imaging of the CFP-NTR/Mtz-mediated damaged ventricle shows a significant loss of actin filaments as well as changes in the overall heart ventricle shape and dimensions (D), compared with ventricles of Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae exposed to DMSO only (C). Cell death was detected in hearts of Tg(cmlc2:CFP-NTR)^s890; Tg(cmlc2:GFP) larvae exposed to 10 mM Mtz - both by immunostaining for activated Caspase-3 (F,G) and TUNEL assay, in the ventricle (I) as well as in the atrium (J) - whereas in control Tg(cmlc2:CFP-NTR)^s890 larvae treated with DMSO alone, no Caspase-3 expression (E) or TUNEL (H) was detected. TUNEL and Caspase-3 expression was detected exclusively in Tg(cmlc2:GFP)-expressing cells (F,G,I,J), indicating that cell death occurred specifically in cardiomyocytes and without affecting the neighboring CFP-NTR-negative cells.