zfGrx2 is essential for development of the vasculature. (A) Confocal microscopy of fli:EGFP transgenic embryos with or without zfGrx2 at 35 and 48 h postfertilization (hpf). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; PC, parachordal sprouts. (Scale bars, 100 μm.) (B) Statistical analysis of A shows mean ± SEM, significance (***P < 0.01) was calculated by two-tailed Student’s t test, 50 pg WT or mutated zfGrx2 mRNA were injected per single egg for rescue experiments. (C) Statistical analysis of intersegmental vessel formation in fli:EGFP embryos, zfGrx2 knockdown embryos, and zfGrx2 knockdown embryos rescued with 15 ng/embryo zfSIRT1 mRNA (48 hpf) (Fig. S2), mean ± SEM, significance (*P < 0.05) was calculated by two-tailed Student’s t test. (D) Quantitative real-time PCR of zfSirt1 transcripts in WT and zfGrx2 knockdown embryos 48 hpf. Presented are mean ± SEM of triplicates of two independent experiments.

Knockdown of zfGrx2 does not induce gross changes in morphology of zebrafish embryos. (A) At 48 hpf, fli:EGFP transgenic embryos displayed malformation (arrows) of the fluorescent vasculature after morpholino induced knockdown of zfGrx2. The overall morphology was unaffected by the loss of zfGrx2. (Scale bar, 200 μm.) (B) Densitometric analyses of three Western blots revealed significant down-regulation of zfGrx2 at 48 hpf after morpholino injection. (C) Visualization of cell death by Acridine Orange staining in fli:EGFP control embryos and zfGrx2 knockdown embryos with confirmed defects in vessel formation 48 hpf. (Scale bars, 100 μm.)

Phenotype of zfGrx2 knockdown is connected to zfSIRT1. Intersegmental vessels in fli:EGFP control embryos, zfGrx2 knockdown embryos, and zfGrx2 knockdown embryos rescued with 15 ng/embryo zfSIRT1 mRNA (48 hpf). (Scale bars, 100 μm.)