Fig. S1

MO-mediated loss of function of Wnt3a and Cdx1a. (A) wnt3aMO inhibits the splicing of the first intron. Embryos injected with wnt3aMO (lanes 3 and 4), wnt3amisMO (lanes 5 and 6), or pCS2+wnt3a plasmid (lane 8) were harvested (lanes 1 and 2, non-injected control embryos) at dome stage, and RNA were isolated from these embryos. cDNAs were synthesized (lanes 1, 3, 5, 7, and 8; lanes 2, 4, and 6 are control without reverse transcriptase) and subjected to PCR using the primers 5′-TGTGGAGTTCGGCAGCATGGT-3′ and 5′-ACCAGCAGGTTTTCACCTCGC-3′ surrounding the first intron. Lane 7 control without template. The correctly spliced wnt3a RNA generates 182 bp PCR products, whereas the RNA deficient in the splicing should produce 814 bp products. Injection of wnt3aMO but not wnt3amisMO inhibited the splicing and generates 814 bp PCR products. (B) wnt3aatgMO inhibits translation of GFP-tagged Wnt3a, in which GFP is tagged at the carboxy terminal of Wnt3a. Expression of Wnt3a-GPF in the embryos that received injection of 50 pg of wnt3a-gfp RNA (a), wnt3a-gfp RNA and wnt3aatgMO (2.1 ng) (b), or wnt3a-gfp RNA and the control MO (c), at 4.5 hpf. wnt3aatgMO but not the control MO inhibited generation (translation) of Wnt3a-GFP. (C) cdx1aMO inhibits translation of GFP-tagged Cdx1a, in which GFP is tagged at the carboxy terminal of Cdx1a. Expression of Cdx1a-GFP in the embryos that received the injection of 100 pg of cdx1a-gfp RNA (a), cdx1a-gfp RNA and cdx1aMO (b), cdx1a-gfp RNA andcdx1amisMO, cdx1a-gfp RNA and the control MO, and expression of GFP in the embryos injected with gfp RNA and cdx1aMO. cdx1aMO inhibited the generation of Cdx1a-GFP protein but not GFP. Neither cdx1amisMO nor the control MO inhibited the translation of Cdx1a-GFP.