Fig. 5. Tcf-dependent transcription is required prior to midblastula transition (MBT) for dorsal development. (A) To antagonize ß-catenin function and thereby block dorsal development, axin mRNA (2.5 ng) was injected into both dorsal blastomeres at the four-cell stage, as described (Zeng et al., 1997). To rescue dorsal development, hormone-inducible Xtcf3 (TVGR; 0.5 pg) was co-injected and Dexamethasone (dex) was then added at the four-cell, 500-cell stage, MBT (the 4000-cell stage), or late blastula (stage nine). Phenotypes were scored at tailbud stage. (B) Luciferase assays for embryos injected with Lef-fos (as in Fig. 1E) and TVGR (0.5 pg) into the animal pole at the two-cell stage. Injected embryos were cultured in normal medium (no dex), or dex was added at the 128-cell stage (dex, 128cs) or at MBT (dex, MBT). Embryos were harvested every 30 minutes after MBT and luciferase activity was measured. (C) Activation of Xtcf3 induces pre-MBT transcription in ventralized embryos: Axin mRNA was injected into dorsal blastomeres at the four-cell stage alone or with TVGR, as in (A). Injected embryos were cultured in normal medium (no Dex) or dex was added at the four-cell stage (Dex, 4cs). Embryos were harvested at the 1000-cell stage and analyzed for Xnr5 and Xnr6 expression. (EF1- was the loading control). (D) Activation of Xtcf3 rescues expression of Siamois and Xnr3 only if hormone is added before MBT, although activation after MBT can still rescue Xnr6 transcription: injection and manipulation were performed as described in (A). Embryos were harvested at early gastrula stage (stage ten+) and analyzed by RT-PCR for Siamois, Xnr3 and Xnr6 expression (EF1- was a loading control). Lane 1 represents uninjected gastrula-stage embryos.

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