Fig. 3

Knockdown of Anxa4 reduces 2F11 staining. (a) Diagram of anxa4 transcripts to depict targets of MOs and RT-PCR primers (green arrows). MO1 is targeted to block translation initiation in Exon 3 and MO2 is targeted to block the splice donor site at the Exon 4/Intron 4 boundary, predicted to cause skipping of Exon 4, leading translation from Exon 3 directly to Exon 5. (b) RT-PCR (with primers indicated in (a)) of control and MO2 morphant embryos showing the predicted 157 bp PCR product from the mis-spliced transcript (red arrow) and a reduction of the 245 bp PCR product from the normally-spliced transcripts (blue arrow) in morphants. (c) The sequence of the mis-spliced transcript confirms Exon 4 skipping and indicates a premature stop codon in Exon 5. (d) Both MOs lead to reduced 2F11 staining in a western blot of whole embryo lysate. (e)-(g′) Ventral views of 48 hpf Tg(sox17:GFP) showing that Anxa4 morphants have reduced 2F11 labeling in the entire hepatopancreas area compared to the control. L, liver; P, pancreas; dP, dorsal pancreas.