Cardiomogen 1

To perform ventricular CM ablation, Tg(vmhc:mCherry-NTR) larvae were treated with 6 mM MTZ (metronidazole, Sigma-Aldrich) in E3 water for 4 h at 3 dpf as previously described [11 (link)]. To modulate signaling pathways, larvae were incubated with the following chemicals for the indicated time period: 100 μM DAPT (Sigma-Aldrich), 12 μM AG1478 (Sigma-Aldrich), 5 μM cardiomogen-1 (Sigma-Aldrich), 7.5 μM dorsomorphin (Sigma-Aldrich), 5 μM LDN193189 (Selleck), or 10 μM rapamycin (Cell Signaling Technology). To stop blood flow, larvae were treated with 1.8 mM tricaine (3-aminobenzoic acid ethyl ester, Sigma-Aldrich) or 10 mM BDM (2,3-butanedione monoxime, Sigma-Aldrich) in E3 water for the indicated time period, and then washed with fresh E3 water.

To influence blood flow, control or ablated Tg(vmhc:mCherry-NTR) larvae were incubated in E3 water with 1.8 mM Tricaine (3-aminobenzoic acid ethyl ester, Sigma, St. Louis, MO, USA) or 10 mM BDM (2,3-butanedione monoxime, Sigma, St. Louis, MO, USA) directly after ablation for 15 h [43 (link)]. For signaling pathway studies, zebrafish larvae were incubated in E3 water with different drugs or solvent right after ablation for 20 h. A quantity of 100 μM of DAPT (Sigma, St. Louis, MO, USA) was used to inhibit Notch signaling [5 (link)]; 5 μM Cardiomogen-1 (Sigma, St. Louis, MO, USA) was used to inhibit Wnt signaling [51 (link)]; 7.5 μM K02288 (Selleck, Houston, TX, USA) or 5 μM LDN193189 (Selleck, Houston, TX, USA) was used to inhibit Bmp signaling [53 (link),55 (link)]; 1 μM RA (Sigma, St. Louis, MO, USA) was used to activate RA signaling [69 (link)]; 5 μM PD153035 (Selleck, Houston, TX, USA) was used to inhibit EGF signaling [70 (link)].