Fig. 4

The overexpression of the fsta gene causes defects in vascular development. In uninjected control embryos, intersegmental vessels (ISV) reached the DLAV at the dorsal region from 24–30 hpf ((A), arrowheads) and the caudal vein plexus (CVP) formed honeycomb-like structures at the tail region from about 36–48 hpf ((A′), arrows). At the same stage, the overexpression of fsta driven by the fli promoter caused ISVs’ stalling at the mid-somite ((B), hollow arrowheads) and less/no honeycomb structure formation at the CVP (B′) Knockdown of fsta by morpholino had no obvious defect in the vasculature (C,C′), but rescued the defect of ISV stalling (solid arrowhead) (D) and restored the honeycomb structure at the CVP (D′). (E) The quantification of the percentage of completed ISV at 30 hpf in wild-type embryos (97 ± 2, n = 31), (fli:fsta)-overexpressing embryos (54 ± 8, n = 24), fsta MO (97 ± 3, n = 15), and rescued embryos (84 ± 6, n = 13). (F) Quantification of loop formation (loops and sprouts) at the CVP at 36 hpf showed an 8-fold increase in the rescued embryos (23 ± 3, n = 16) when compared to (fli:fsta) embryos (3 ± 1, n = 21); fsta MO (28 ± 4, n = 14) has no difference when compared to wild-type embryos (30 ± 2, n = 15). (G,H) Imaging of endothelial nuclei in green and vessels in red at 30 hpf in wild-type controls and (fli:fsta)-overexpressing embryos using Tg(kdrl:mCherry^ci5; fli1a:nEGFP ^y7) double transgenic line. (fli:fsta) embryos showed reduced ISV nuclei numbers (H). (I) Quantification of ISV nuclei number in (fli:fsta) embryos (n = 25) when compared to wild-type controls (n = 18). Data are represented as means ± S.E. *** refers to p< 0.0001 by an unpaired Student’s t-test. Scale bars are 100 μm for (A–D,A′–D′,G,H).