Fig. 2

Knockdown of ftr82 causes vascular defects by using splicing block morpholino. (A,B) Bright field images of uninjected control and ftr82^e1i1 morpholino injection at 26 hpf; (C–H) At 26 hpf, loss of ftr82 shows intersegmental vessel (ISV) growth defect (hollow arrowheads in (F)) and less or no angiogenic sprouting from the caudal vein compared to uninjected controls (arrowheads in (E) and arrows in (G)) at 30 hpf; (E,G) are enlarged figures from (C), and (F,H) are enlarged figures from (D). At 30 hpf, in uninjected control embryos, ISV has reached the dorsal longitudinal anastomotic vessel (DLAV) at the dorsal aspect of the embryo (K, arrowheads), and the CVP formed honeycomb-like structures at the tail (M, arrows). At the same stage, ISVs are stalled at mid-somite in ftr82^e1i1 morphants (L, hollow arrowheads), and less honeycomb structure in the CVP (N); At 48 hpf, less or no CVP capillary loop can be observed (P) compared to the control (O, arrows); (Q) Quantification of angiogenic sprouting from the caudal vein shows a five-fold decrease in ftr82 morphants at 26 hpf (n = 29 in control; n = 26 in ftr82 MO); (R) Quantification of percentage of completed ISV shows a ~60% increase compared to ftr82 morphants (n = 32 in control and n = 28 in ftr82 MO) at 30 hpf; (S) Quantification of the deformation of CVP in ftr82 morphants by different morpholino dosage injections (1, 3 and 8 ng) shows MO in a dose-dependent manner at 48 hpf; (T,U) The efficiency of ftr82 splicing morpholino knockdown in embryos; (T) Schematic drawing showing the exon-intron structure of the ftr82 gene, the targeting area of the ftr82 splicing morpholino (ftr82^e1i1 MO), and the suggested loss of the expression fragment can be detected by an ftr82mo_f and ftr82mo_r primer set; (U) cDNA from uninjected controls or 3.4 ng morpholino-injected ftr82 morphants were subjected to PCR with primers for the loading control GAPDH or for the ftr82 gene. In ftr82^e1i1 morpholino-injected embryos, GAPDH levels at 283 bp are unchanged, while the amount of ftr82 product at 487 bp is diminished, indicating the loss of ftr82 expression caused by the morpholino inhibition. (*** refers to p < 0.0001 by an unpaired Student’s t-test.) Scale bars are 200 μm for A–D,I–L and 100 μm for E–H,M–P.