Sb505124

For RNA-sequencing experiments, SB505124 from Sigma-Aldrich was used at a concentration of 100 μM in 12 mL embryo media in a 15 mm × 60 mm Petri dish (DaCosta Byfield et al., 2004 (link); Hagos et al., 2007 (link)). For RT-PCR and whole mount in situ hybridization, SB505124 from Tocris was used at a concentration of 20 μM, which caused a similar phenotype to 100 μM of the Sigma drug, in a total of 30 mL embryo media in a 100 mm × 20 mm Petri dish. For both SB505124 stocks, Dimethyl Sulfoxide (DMSO) was used to make the 10 mM stock solution of SB505124. SB505124 treatment/solution (12 mL for Sigma-Aldrich and 30 mL for Tocris, with a final concentration of 1% DMSO) was added to pooled groups of ZDR embryos at either sphere (4.0 hpf) or 30% epiboly (4.7 hpf) stages and left on until the embryos were snap frozen in liquid nitrogen or fixed in 4% paraformaldehyde. Control embryos were treated with 1% DMSO in embryo media and raised in parallel to inhibitor treated embryos. In addition, at least 20 embryos from each treatment group were left in SB505124 until 24 hpf and scored for pineal phenotype by whole mount in situ hybridization (WISH) to ensure that the embryos had the expected neural tube phenotype. Phenotypes at 24 hpf between both SB50124 stocks were similar at the concentrations used.