Fig. 4

Knockdown of gas6 induces the inhibition of angiogenesis in intersegmental vessels.

(A) The red arrows (Ex5:In5 and Ex7:In7) indicate morpholino target sites for splicing blocks. Primers (blue arrows, exon 1, forward primer; exon 8, reverse primer) were designed for the testing of morpholino efficacy, as described in the Materials and Methods. (B, C) Testing and quantification of morpholino nucleotide efficacy by RT-PCR in standard control MO (Ctrl-MO) and gas6 MO (EX5-MO and EX7-MO) treated embryos at 26 hpf. In control morphants, gas6 mRNA (768 bp, black arrow) is detectable by RT-PCR (B, first line). In gas6 EX5 morphants, the wild-type gas6 mRNA is undetectable by RT-PCR at doses of 2 and 5 ng/embryo. The morphant mRNA encodes a truncated form of the Gas6 protein. (C) In gas6 EX7 morphants, RT-PCR products reveal Ctrl-MO embryos expressing wild-type gas6 transcript, while EX7-MO embryos express two transcript variants at doses of 1 or 2 ng/embryo. The black arrow shows reduced expression of wild-type gas6 mRNA. The red arrow indicates results from aberrant splicing, resulting in a gain of ~250 base pairs of intron 7, which encodes a premature stop codon that occurs in the Gas6. (D) Angiogenesis defects in gas6 morphants. The flk:GFP transgenic zebrafish embryos were microinjected with Ctrl-MO (n = 30, 4 ng/embryo) and gas6 MO (EX5-MO, n = 35, 4 ng/embryo; EX7-MO, n = 34, 2 ng/embryo), and their blood vessel formation was examined at a cellular level in living embryos at 30 hpf. Normal formation of intersegmental vessels, as shown by GFP-positive endothelial cells, is observed in Ctrl-MO embryos, but severe vascular defects is observed in gas6 MO-injected embryos. The experiment was repeated two times.