2.1. Zebrafish Husbandry and Chemical Treatment

Zebrafish (Danio rerio) and embryos were raised and maintained in a 28.5 °C fish room based on the Zebrafish Book guidelines [39]. The regulations were approved from the National Sun Yat-Sen University Animal Care Committee (approval reference #10231). Wild-type fish and transgenic fish lines Tg(flk:eGFP), Tg(fli1a:negfp)^y7, Tg(gata1:dsRed)^sd2, and Tg(kdrl:mCherry)^ci5 [13,40,41,42] were used in this study from the Taiwan Zebrafish Core Facility at Academia Sinica. Embryos were cultured in E3 medium and dechorionation by incubation was performed in 2 mg/mL pronase (Sigma, St. Louis, MO, USA). 0.003% 1-Phenyl-2-Thiourea (PTU) (Sigma), which was added into the medium at 6 h post fertilization (hpf) to inhibit pigmentation. To investigate signal pathways, the embryos were treated with 15 μM of the VEGF inhibitor SU5416 (Calbiochem, Nottingham, UK) and 75 μM of the Notch signal inhibitor DAPT (Sigma) at a working concentration in E3 fish water at 6 hpf. Concentrations of 10 μM Dorsomorphin (DM, Calbiochem) or 40 μM DMH1 (Calbiochem) were used to inhibit BMP signals at 20 hpf. The control embryos were cultured in 0.3% DMSO medium.