Fig. 5. (A,B) Microinjections of anti-FoxA4a/Pintallavis (A,A') and anti-Xvent2 (B,B') morpholinos elicit short-range posterior expansion of the Xanf1 expression. In the case of the anti-sense to FoxA4a/Pintallavis, the expression of Xanf1 is expanded toward the posterior only along the midline of the neural plate, i.e. just in the region where the endogenous FoxA4a/Pintallavis is expressed (A, red arrows). No expansion of the Xanf1 expression was seen in the morpholino-containing cells occupying lateral parts of the neural plate (the upper rows of embryos in A and A'). By contrast, only lateral expansion of the Xanf1 expression was observed in embryos microinjected with the anti Xvent2 (B,B') morpholino and these embryos demonstrated normal expression of Xanf1 in the midline of the neural plate. (C) The mixture of anti-FoxA4a/Pintallavis morpholino and a synthetic FoxA4a/Pintallavis mRNA can interfere with the posterior expansion of endogenous Xanf1 expression elicited by the morpholino alone. (D) To monitor the effectiveness of CHX on suppression of protein synthesis, we microinjected Xenopus embryos at the two blasomere stage with plasmids expressing luciferase under the control of the CMV (commercial pGL3 vector, Promega) or the `full-length' Xanf1 promoter (Eroshkin et al., 2002). CHX was added to one half of the microinjected embryos at the midblastula transition (CMV-Luc) or at the midgastrula stage (Xanf1-Luc). After 2 hours of incubation, luciferase activity was measured in extracts from three independently collected groups of embryos (10 embryos in each group) treated and not treated with CHX. (E,F) Experiments with the hormone-inducible versions of FoxA4a/Pintallavis and Xvent2 under conditions of inhibition of protein synthesis by cycloheximide demonstrate that Xanf1 is the direct target of these transcription factors. The dexamethasone (DEX) treatment of embryos subsequently elicits activation of VP16-FoxA4a/Pintallavis-BDGR (E,E') or VP16-Xvent2-BDGR (F,F'), which under those conditions can activate transcription of only their own direct genetic targets, in particular, the transcription of Xanf1. Although only short-distance posterior expansion of the Xanf1 expression was observed in the case of VP16-FoxA4a/Pintallavis-BDGR (E, red arrows), much broader spreading of the expression was seen in embryos microinjected with VP16-Xvent2-BDGR mRNA (F, red arrows).