Effects of morpholinos (MOs) against dhps, eif5a1, and eif5a2 on pancreas length in zebrafish. A, model of the first three exons of dhps and the binding site of the dhps MO. PCR products amplified by primers between exon 1 (forward) and exon 3 (reverse) are shown: the normally spliced product and the disrupted-splicing products resulting from dhps MO. B, representative 2% agarose gel of RT–PCR products showing disrupted dhps transcript splicing with dhps MO in 24 h post fertilization (hpf) embryos. C, representative immunoblot analysis of total eIF5A protein levels in the absence or the presence of dhps MO at 3 days post fertilization (dpf). D, representative images and quantification of embryonic Ptf1a;GFP zebrafish pancreas length at 3 dpf following control, dhps, or eif5a1/2 MO injection. Control MO was injected at 8 ng/embryo. dhps MO was injected at 4 ng/embryo alongside 4 ng/embryo control MO. eif5a1 MO was injected at 4 ng/embryo alongside 4 ng/embryo eif5a2 MO. Scale bar represents 50 μm. Each point on the plot represents one embryo (N = 28–56 embryos). E, model of the first three exons and the binding site of the eif5a1 and eif5a2 MOs. F, representative immunoblots of total eIF5A protein in zebrafish embryos at 3 dpf following combinations of eif5a1 and eif5a2 MOs. Each MO was injected at 4 ng/embryo. G, representative images and quantification of embryonic Ptf1a;GFP zebrafish pancreas length at 3 dpf following control, dhps, or combined dhps and eif5a1/2 MO (triple MO) injection. Scale bar represents 50 microns. Each point on the plot represents one embryo (N = 28–51 embryos). Control MO was injected at 12 ng/embryo. dhps MO was injected at 4 ng/embryo alongside 8 ng/embryo control MO. dhps, eif5a1, and eif5a2 MOs were each injected at 4 ng/embryo together in triple MO embryos. H, representative 2% agarose gel of RT–PCR products showing disrupted dhps transcript splicing with triple MO in 24 hpf embryos. Data are presented as mean ± SEM, and statistical significance was determined by a one-way ANOVA with Tukey’s post hoc test. eIF5A, eukaryotic translation initiation factor 5A.
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