Targeted knockdown of zebrafish pegasus. (A) Targeting of pegasus with anti-sense morpholinos. Portion of the zebrafish pegasus gene showing encoded amino acids below the corresponding nucleotide sequence, with numbering at the left (nucleotide—black; amino acid—pink). Exons are shown in upper-case and introns in lower-case. The relative positions of the morpholinos targeting pegasus are indicated: PegUTRMO, targeting the 5′ untranslated region; PegATGMO, targeting the start codon (bold and red), and PegSSBMO, targeting a splice-site. Arrows (green) indicate the location of primers used to quantify the extent of pegasus knockdown with PegSSBMO. (B–C) Confirmation of knockdown by splice-site morpholino. Total RNA was extracted from 24 hpf embryos injected with a scrambled morpholino (ScrambleMO) or splice-site morpholino (PegSSBMO), and incubated in the presence (+) or absence () of reverse transcriptase (RT), as indicated. PCR was performed on these samples using the pegasus primer set indicated in panel A, with β-actin gene-specific primers as a control (B). The extent of knockdown was quantified by densitometric analysis of these bands across three independent experiments (C). (D–T) Molecular analysis of the developing gut and pancreas in pegasus morphants. Embryos injected with scrambled morpholino (ScrambleMO: D, N, J), pegasus ATG morpholino alone (PegATGMO: E, F, K, O, R), or with p53 morpholino (PegATGMO+p53MO: L) or morpholino-resistant mRNA (PegATGMO+mRNA: S), or pegasus splice-site blocking morpholino (PegSSBMO: G, H) were subjected to WISH at or 3 dpf with the gut-specific ifabp (D–H, J–L) and pancreas-specific insulin (N–O, Q–S) genes. Embryos are of dorsal view, with anterior to the left, with purple staining evident. Embryos were quantified with respect to the percentage expressing ifabp (I, M) or insulin (O,T) on the left, right or middle following injection with scrambled (Scr), PegATG (Peg), PegSSB (Peg-SSB) and p53 (p53) morpholino or morpholino-resistant mRNA (mRNA), as indicated.
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