Expression of dachb in the zebrafish pancreas. Whole-mount in situ hybridization was performed for dachb (A–K, blue) and insulin (I, red), somatostatin (J, red), and glucagon (K, red) at the stages of zebrafish development shown. Ventral views (A–D;I–K), and lateral views (E–H) are shown with anterior to the left. On all the panels the yolk was manually removed from the embryos. The notochord (n) and the lateral line (ll) have been indicated in panels A, E and C. Black arrowheads: dachb-expressing cell in the pancreatic region. Scale bar, 50 μm.

Inhibition of DachB expression by morpholino antisense oligonucleotides. Expression of insulin (A–C), somatostatin (D–F) and glucagon (G–I) was analyzed by whole-mount in situ hybridization at 30 hpf embryos injected with the morpholino oligonucleotides shown: a standard control (A, D, G), a mismatch control with 4 bases altered from the complimentary dachb sequence (B, E, H), and an oligonucleotide complimentary to a dachb splice junction (C, F, I). All panels present ventral views of yolk-free embryos with anterior to the left. In panel J, the numbers of cells expressing the hormones shown were assessed in morpholino-injected embryos at 30 hpf. Each data point represents the mean ± standard error of at least 40 embryos for each condition. ***p < 0.001 compared with embryos injected with morpholino 4-mismatch control by Student′s t test. Scale bar, 50 μm.

Effect of the dachb morpholino on the splicing pattern of the dachb mRNA and on the exocrine pancreas. A. Schematic representation of the first three exons of the dachb gene. Dachb morpholino targets the junction between exon 2 and intron 2–3.The arrows indicate the position of PCR primers relative to the exons, the introns, and the new splicing pattern. B. RT-PCR with RNA extracted from morpholino-injected embryos. Dachb morpholino induces a knock-down of the fully spliced dachb mRNA. The primers used are as indicated in (A).The presence of a portion on intron 2–3 is indicated by arrowheads. Lower panels: GFP reporter protein expression of Tg(ptfl a::GFP) embryos at 48 hpf in ptfl a::GFP embryos injected with the following morpholino oligonucleotides: standard control (C), mismatch control (D) and dachb morpholino (E). All panels are optical sections of ventrally viewed yolk-free embryos with anterior to the left. Scale bar, 50 μm.

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