Embryonic Xenopus Transcriptome Analysis

Stock solutions of Bexarotene (Millipore-Sigma), 9-cis retinoic acid (9-cis RA; Millipore-Sigma) GW3965 (Tocris), LG100754 (Tocris), HX531 (Tocris) were prepared in dimethylsulfoxide (DMSO; Millipore-Sigma), aliquoted, and stored in the dark at −20°C. The 9-cis RA was also stored under nitrogen. Embryos were collected, reared, and treated as described previously (Mitchell et al., 2015 (link)) with treatments performed at 28.5°C, beginning at around 52 hpf, and lasting 24 h total. Once the 24-h treatment was complete, embryos were anesthetized, and the heads were surgically separated from the body, anterior to the heart, using dissecting scissors to ensure transcript measurements were from the CNS, and not the yolk sac or region of the developing liver. Any treatment groups showing gross morphology defects and viability affects were noted. At the doses used, the highest dose of LG100754 (2 µM) occasionally affected viability of a few embryos. Heads were immediately transferred to RNA Later (Invitrogen). Heads from three to nine embryos from each treatment group were pooled into RNA Later and stored at −20°C until RNA extraction.