Fig. 2. Design and verification of activin B antisense morpholino oligonucleotides. (A) Sequence of the 5' untranslated region of activin B derived from GenBank (i) and derived from the Xenopus colony at the Wellcome Trust/Cancer Research UK Gurdon Institute (ii). Differences between the two sequences are highlighted and the sequences targeted by the antisense morpholino oligonucleotides used in this paper are boxed. (B) MO1 inhibits in vitro translation of activin B. Arrow indicates activin B. mMO1 was included in one reaction at a final concentration of 5 µM and MO1 was included at 0.5, 2.0 and 5.0 µM. – indicates no addition of morpholino oligonucleotide. (C) MO1 inhibits, in a dose-dependent fashion, translation of RNA encoding HA-tagged activin B following injection into Xenopus embryos. RNA encoding activin B-HA, together with RNA encoding an HA-tagged version of the FYVE and SBD domains of SARA (see Materials and methods), was injected into Xenopus embryos at the one-cell stage in the absence of MO1 or at the indicated concentrations of morpholino. Embryos were cultured to early gastrula stage 10 and subjected to western blotting using an anti-HA antibody. Activin B-HA and FYVE/SBD-HA are indicated by arrows. Inhibition of activin B translation is not observed with mMO1. (D) MO1 prevents activin-induced expression of Xbra in Xenopus animal caps. Xenopus embryos were injected with the indicated combinations of activin and MO1. They were cultured to early gastrula stage 10.5 and assayed for expression of Xbra by real-time RT-PCR. (E-G) MO1 inhibits activin-induced elongation of animal caps. Animal pole regions were derived from uninjected embryos (E) or embryos injected with RNA (5 pg) encoding activin B in the absence (F) or the presence (G) of 40 ng MO1. MO1 inhibits the elongation of animal pole regions (G). (H-M) MO1, but not mMO1, inhibits the function of exogenous activin in intact Xenopus embryos; mMO1 but not MO1 inhibits the function of a mutated form of activin in which the sequence has been mutated to match that of mMO1. Embryos were injected with wild-type activin (H-J; Activin B-HA; 10 pg) or mutated activin (K-M; mActivin B-HA; 10 pg) in the absence of morpholino oligonucleotides or in the presence of MO1 (40 ng) or mMO1 (40 ng).