Chemically induced genetic cell ablation of zebrafish larval cardiac valves. (A) Outline of chemical treatment of UAS-E1b:NfsB-mCherry transgenic embryos with Metronidazole (MTZ). Metronidazole was applied at 3.5 dpf embryos and washed-off at 4 dpf. Embryos were then left for recovery in order to observe regeneration process. (B,C) Gfp and mCherry Gal4 driven expression in 4 dpf cardiac valves (Arrows). AV: atrioventricular canal, (BA): bulbus arteriosus. Scale bars: 100 μm. (D) Confocal z stack projection of a double transgenic Tg(hspGFFDMC73A/UAS-E1b:NfsB-mCherry) embryonic heart stained with cell-cell adhesion marker Dm:grasp (blue). Scale bar: 20 μm (F,G) Gfp and mCherry Gal4 driven expression in 4 dpf cardiac valves of MTZ treated embryos. Injury is observed at AV canal and BA nfsb-mCherry+ cells that have been ablated. Scale bars: 100 μm. (H) Confocal z-stack projection of a double transgenic Tg(hspGFFDMC73A/UAS-E1b:NfsB-mCherry) heart treated with MTZ and stained with cell-cell adhesion marker Dm-grasp (blue). Arrowheads depict the position where AV canal and BA differentiated cells should be. Scale bar: 20 μm. (E–I) Snapshots of high frame live videos of a DMSO treated and a MTZ-treated Tg(gSAIzGFFD703A/UAS-E1b:NfsB-mCherry) embryo 4 dpf, respectively (S. Movies 3 and 4). Bars show the haemodynamic flow patterns at the atriovantricular canal of uninjured and injured valves per heartbeat. The number of frames per total frames of a heartbeat was measured for forward flow (+) no flow (0) and reverse flow (−). The reverse flow fraction is increased 4,91 times (quantified from n = 8 ctrl embryos and 8 MTZ-treated embryos. p < 0.001 using paired t-test) upon valve ablation. +: forward fraction. 0: null fraction. −: reverse fraction.
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