Fig. S7

Knockdown of insb results in severe developmental defects. (A) Diagram illustrating insb pre-mRNA structure in wild type and insbMO-injected cells. insbMO targets the exon 4-intron 4 boundary of preproinsulin-b mRNA and promotes the deletion of exon 4. Primers complementary to exon 3 and exon 5 amplify the differentially spliced cDNA products. (B) Agarose gel showing amplified insb PCR product from 2 dpf control and insbMO-injected embryos. Note that a PCR product with a size of 427 bp was detected in control embryos (arrowhead) but not insbMO injected embryos. A weak product of 210 bp can be detected in insbMO-injected embryos (arrow). β actin was used as an internal control. (C) Dose dependent phenotypes at 48 hpf after insbMO injection. Arrow indicates head defect first observed at 2 ng insbMO dose. (D,E) RT-PCR analysis of cDNA in 30 hpf insbMO-injected (D) and insaMOinjected (E) embryos. Neither insbMO nor insaMO affected the splicing or expression of the mRNA of the respective paralog. (F-G′) Merged and single channel confocal planes of 24 hpf control (F,F′) and insbMO-injected (G,G′) islets of Tg(ins:CFP-NTR) embryos stained for CFP (β cells, green) and insulin (red). (H) Quantification of ins:CFP-NTR+ β cells in 24 hpf control (n=5) and insbMO-injected embryos (n=6). Student′s t-test was used to determine significance in H.