Knockdown of rap1b leads to angiogenesis defects in zebrafish in vivo. (A) Low- (i-ii) and high-power (iii-v) images of the trunk region of 28 hpf flk1:EGFP zebrafish embryos injected at 1- to 2-cell stage with 6 ng of MO, as indicated. Boxed areas (i-ii) indicate the region where intersomitic vessel (ISV) evaluation was performed. Missing ISVs (iv arrows) and “hammerhead” ISVs (v asterisks) are visible in embryos injected with rap1b-MOs but not with control-MOs. All images shown are lateral views with anterior side (A) oriented left and dorsal side (D) oriented up. DLAV indicates dorsolateral anastomotic vessel; DA, dorsal aorta. Scale bars 75 μm. (B-C) Quantitation of ISV (B) and gastrulation (C) defects in MO-injected embryos is expressed as number of affected embryos (numerator) in each group (denominator). No defects in ISV formation were observed in control MO injected embryos, whereas embryos injected with rap1b-MO1 (MO1) and rap1b-MO2 (MO2) displayed inhibition in ISV sprouting. (D-E) Analysis of Rap1b deletion efficacy (D) RT-PCR amplification of F1-R1 fragment (rap1b exon 1 to exon 4; 307bp) and F2-R1 fragment (exon 2 to exon 4; 212bp) of rap1b cDNA is substantially reduced in 6ng rap1b-MO2 injected zebrafish embryos compared with control. Ten to 20 embryos per group were injected at 1-2 cell stage. Total RNA was isolated at 28 hpf. (E) Rap1 expression in rap1b-MO (MO1, MO2) or control-MO (Con) injected embryos at 28 hpf was detected using M90 monoclonal IgG. Relative amount of Rap1 to actin is indicated in parentheses for each group; blot is representative of n = 3 independent experiments.