Fig. S3

ip6k2 mRNA rescues the Hh-related phenotypes in ip6k2^SPMO-injected embryos. (A) Schematic of ip6k2 genomic organization with exons (rectangular boxes) and introns (lines). Arrows indicate relative positions of ip6k2^SPMO and oligonucleotide primers used to amplify ip6k2 cDNA. (B) ip6k2^SPMO blocks splicing at exon2-intron2 junction. Total RNA prepared from uninjected (U) and ip6k2^SPMO injected (M) embryos at different stage (5–20 hpf) were analyzed by twostep RT-PCR using ip6k2-F and -R primers (shown above). Reduced sized, aberrantly spliced ip6k2 product was detectable at 5 hpf and later time points in ip6k2^SPMO-injected embryos (Upper). PCRs with β-actin-specific primers served as controls (Lower). (C–N) ip6k2 mRNA injection reverses inhibitory effects of ip6k2SPMO on somite development (C–F), NCC migration (G–J), and Hh target gene expression (K–N). Anterior is toward the left and dorsal toward the top. (C) Lateral view of chevron-shaped somites of a live uninjected embryo at 24 hpf. (D and E) Rescue of U-shaped somites in embryos injected with ip6k2^SPMO (D) by coinjecting ip6k2 mRNA (E). (F) ip6k2 mRNA injection alone did not have any effect. Dotted lines indicate somite boundaries. (G–J) Whole- mount in situ hybridizations showing crestin expression in trunk NCC in uninjected (G), ip6k2^SPMO-injected (H), ip6k2^SPMO + ip6k2 mRNA-coinjected (I), and ip6k2 mRNA injected (J) embryos at 24 hpf. Expression of crestin in ip6k2^SPMO-injected embryos was restored by ip6k2 mRNA coinjection (H and I). ip6k2 mRNA injection alone did not alter crestin expression (J). (K–N) Whole-mount in situ hybridizations showing ptc1 expression in uninjected (K), ip6k2^SPMO-injected (L), ip6k2^SPMO + ip6k2 mRNA-coinjected (M), and ip6k2 mRNA-injected (N) embryos at 24 hpf. It is apparent that ip6k2 mRNA coinjection rescued ptc1 expression in embryos injected with ip6k2^SPMO (L and M), whereas ip6k2 mRNA injection alone had no effect (N).