Zebrafish Embryo Chemical Screen

Zebrafish wild-type (WT) embryos were arrayed and treated with small molecules using the ICCB Known Bioactives Library as described (Poureetezadi et al., 2014 (link)). Zebrafish embryos were staged at 2 hpf, then at least 30 fertilized embryos were arrayed into the chambers of 24-well plates and incubated at 28°С in E3 media until just prior to 4 hpf. Working stocks of small molecules were stored at −80°С, then dissolved in 100% DMSO to make 10 mM concentrations (Lengerke et al., 2011 (link)). For drug exposure, the E3 media was completely drawn off the embryos using a glass transfer pipet and the appropriate solution of DMSO, PGB₂, dmPGE₂, indomethacin, SC-560, NS-398, AH6809, or PF04418948 was applied at a discrete development time point (eg 4 hpf or 12 ss) (American Bioanalytical, Enzo Life Sciences, Santa Cruz) (North et al., 2007 (link); Eisinger et al., 2007 (link); Jin et al., 2014 (link)). Embryos were raised to the 20 ss or 24 hpf, washed three times with E3, then fixed in 4% paraformaldehyde. For rescue of prostaglandin activity, ptgs1 or ptgs2a deficient embryos were generated, and cohorts of approximately 30 embryos were arrayed in 24-well plates with E3, then placed in a 28°С incubator until 4 hpf. E3 was then completely drawn off the wells using a glass transfer pipet and a 50 µM concentration of dmPGE₂ was applied. The embryos were placed into a 28°С incubator, raised until 24 hpf, washed three times with E3, and fixed as previously described.