Fig. 5. XSOX3 effects on Siamois. (A) The 800bp Siamois/firefly-luciferase reporter together plasmid encoding Renilla luciferase were injected into either the dorsal or ventral side of fertilized eggs. Embryos were homogenized at stage 9 and luciferase activities were measured. Activity was normalized using Renilla luciferase levels and the dorsal activity was set to 1. (B) Fertilized eggs were injected with the Siamois/luciferase reporter plasmid together with 1 ng of XSOX3-V5H₆ RNA. Reporter activity on the dorsal or ventral sides of the embryo in the absence of exogenous XSOX3 RNA (UN) was set independently to 1. Co-injection of XSOX3 wild-type or m7 RNAs activated the Siamois reporter on the dorsal but not on the ventral sides of embryos; the m8 polypeptide had no effect on either side of the embryo. (C) To determine the effect of XSOX3 RNA injection on the endogenous Siamois gene, embryos were ventralized by UV illumination during the first cell cycle. The dorsal axis was rescued by the injection of mutationally stabilized ß-catenin RNA (1 ng). RNA was isolated from wild-type, UV-treated, UV-treated, ß-catenin-rescued and UV-treated, ß-catenin- and XSOX3-RNA-injected embryos at stage 9, and RT-PCR was performed to visualize expression of the dorsalizing gene Siamois; EF-1 was used as a control. Siamois is expressed in intact embryos, greatly reduced in UV-ventralized embryos and returned to nearly wild-type levels in ß-catenin RNA-injected embryos. The co-injection of ß-catenin and either wild type or m7 XSOX3 RNA (1 ng) suppressed the reappearance of Siamois expression; co-injection of m8 RNA (1 ng) did not suppress ß-catenin-induced Siamois expression. (D) Streptavidin-agarose bound biotinylated mutant Siamois promoter fragment (biot. Sia null), biotinylated wild-type Siamois promoter fragment (biot. Sia), biotinylated DC5 (biot. DC5') or biotinylated TCF (biot. TCF) DNAs were incubated with stage-8 embryo lysates (Lys). Bound proteins were eluted and analysed by immunoblot using the anti-XTCF3n and anti-XSOX3c antibodies. Neither XTCF3 nor XSOX3 bound to the mutated Siamois sequence. XTCF3, but not XSOX3, bound to the wild-type Siamois and TCF sequences, and this binding was blocked by incubation with a 10- to 20-fold excess of unbiotinylated TCF oligonucleotide. XSOX3, but not XTCF3, bound to the DC5 sequence and this binding was blocked by incubation with a tenfold excess of unbiotinylated DC5 oligonucleotide. No binding of XTCF3 or XSOX3 was observed when biotinylated DNAs were omitted from the assay (No DNA, SA beads).

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