Fig. S4

Control experiments to validate the specificity and efficacy of the morpholino. Schematic diagrams show the relative positions of the designed MOs and reporter constructs for either foxi3a (A) or foxi3b (B). The 52UTR and partial exon 1 sequences of foxi3a or foxi3b were PCR-amplified from zebrafish cDNA and in-frame-fused with the green fluorescent protein (GFP) reporter gene. The resulting chimeric vectors of pCS2+3aUTR:GFP or pCS2+3bUTR:GFP contained the complementary sequences for testing the specificity and efficacy of both MO1 and MO2. (C) When five mismatched controls of 3a MO1^mis (0.5 mM) were co-injected with 3aUTR:GFP mRNA (250 pg), all embryos (100%, n = 94) showed strong GFP expression. This result suggests that the 3a MO1mis is unable to target endogenous foxi3a mRNA. (D) When the 3a MO1 (0.5 mM) was co-injected with 3aUTR:GFP mRNA (250 pg), GFP expression was completely abolished in 78% of the injected embryos (n = 83). (E) When 3a MO2 (0.5 mM) was co-injected with 3aUTR:GFP mRNA (250 pg), GFP expression was completely abolished in all injected embryos (n = 119). This result suggests that both 3a MO1 and 3a MO2 can target endogenous foxi3a mRNA. However, the efficacy of 3a MO2 was superior to 3a MO1. (F) When five mismatched controls of 3b MO1^mis (0.5 mM) were co-injected with 3bUTR:GFP mRNA (250 pg), all embryos (100%, n = 94) showed strong GFP expression. This result suggests that the 3b MO1^mis is unable to target endogenous foxi3b mRNA. (G) When the 3b MO1 (0.5 mM) was co-injected with 3bUTR:GFP mRNA (250 pg), it was only sufficient to abolish GFP expression in 58% of injected embryos (n = 71). (E) When the 3b MO2 (0.5 mM) was co-injected with 3bUTR:GFP mRNA (250 pg), it was sufficient to abolish GFP expression in all injected embryos (n = 145). This result suggests that both 3b MO1 and 3b MO2 can target endogenous foxi3b mRNA. However, the efficacy of 3b MO2 was superior to 3b MO1. The number and percentage on the right bottom corner of C-H refer to the ratios of embryos that express GFP. ORF, open reading frame; UTR, untranslated region.