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Fig. S3 Testing efficacy of fzd gene morpholinos. A) 10 ng of fzd3a-MO can substantially suppress the normal splicing of fzd3a mRNA. RT-PCR analysis was carried out as previously reported (See Materials and Methods). We injected 10 ng of morpholinos into wild-type embryos, and extracted total RNA at 24 hpf. Whilst control random oligo-injected (lane 2) and uninjected (lane 3) samples showed a clear fzd3a transcript band of the expected size (465 bp, arrowhead)., this band was barely detectable after 10 ng of fzd3a-MO injection due to the inhibition of correct splicing (lane 1). Primers against β-actin cDNA were used as a positive control for RNA extraction and RT-PCR (lanes 4, 5, 6.). B) Schematic drawing of fusion constructs to test ability of fzd9b and fzd10 morpholinos to bind to the target sites and suppress translation in vivo. Target sites for fzd9b- and fzd10-MO (red) are fused to cDNA of egfp (green). Actual sequences of target site and the first 3 bps of egfp sequence are shown below the scheme. Original ATG for egfp gene is modified into ATC, which is next to GTG in green, in order to avoid translation from this. C–F, C′–F′) Our fzd9b- and fzd10-MOs can efficiently suppress GFP fluorescence derived from injected fusion constructs. In all cases, 100 pg of mRNA of the respective fusion construct was injected, and embryos were observed at 10 hpf stage. GFP fluorescence was clearly detected in embryos injected with 5 ng of control random morpholinos (C, C′, E, E′). In contrast, injection of even just 1 ng of the respective experimental morpholinos suppressed fluorescence completely (D, D′, F, F′). C–F are bright field images of C′–F′, respectively. Numbers in C′–F′ are ratios of GFP-positive embryos out of surviving injected embryos. Scale bar: 500 μm.