Fig. 4. Pre-midblastula transition (MBT) transcription of Xnr5 and Xnr6 is regulated by ß-catenin and Xtcf3 in dorsal blastomeres. (A) Zygotic expression of Xnr5 and Xnr6 is detectable by RT-PCR as early as the 256-cell stage whereas siamois expression begins at the 4000-cell stage. Ornithine decarboxylase (ODC), a maternally expressed gene that does not increase significantly at MBT, was used as a loading control. `No-RT' indicates control lacking reverse transcriptase. (B) Regulation of pre-MBT transcription by ß-catenin: embryos were injected ventrally with ß-catenin mRNA (500 pg), treated at the 32-cell stage with LiCl (0.3 M for 10 minutes), or injected dorsally with either dnXtcf3 mRNA (500 pg) or morpholino antisense oligonucleotide against ß-catenin (10 ng). Embryos were harvested at the 1000-cell stage and analyzed by RT-PCR for Xnr5 and Xnr6 expression (FGFR: FGF receptor was a loading control). (C) Pre-MBT transcription of Xnr5 and Xnr6 is localized to dorsal blastomeres. Embryos were dissected into dorsal and ventral halves at the 500-cell stage, RNA was isolated from each half, and Xnr5 and Xnr6 expression was assessed by RT-PCR as in panels (A) and (B). Control whole embryo RNA is shown in lane 1. (D) RNA polymerase II-dependent pre-MBT transcription of Xnr5 and Xnr6: embryos were treated with actinomycin D (ActD) from the two-cell stage, injected with the RNA polymerase II-specific inhibitors -amanitin or 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) at the one-cell stage, or treated with LiCl as in (B). Embryos were harvested at the 1000-cell stage for analysis of Xnr5 and Xnr6 expression as above.