Fig. S6

UBIAD1 Regulates a Blood Flow-Dependent NO Signaling and NO-dependent Oxidative Stress, Related to Figure 7 (A) Embryos from bar^t31131 heterozygote intercrosses were injected at one-cell stage with tnnt2 morpholino (tnnt2 MO) or Kruppel-like factor 2a morpholino (klf2a MO) or control morpholino (ctrl MO). Histogram shows the percentage of larvae showing the bar mutant phenotype at 72 hpf after injection. Compared to control, impairment of the blood flow-Klf2a pathway by morpholino injection significantly delayed the penetrance of the bar phenotype, evaluated as trunk vessels disintegration. Results are shown as a mean of n = 2 independent experiments for each condition.(B) Fluorescent micrographs of DLAV and Se of Tg(kdrl:GFP)^s843bar mutants at 72 hpf after injection of control morpholino (ctrl MO), tnnt2 morpholino (tnnt2 MO), klf2a morpholino (klf2a MO). Knockdown of tnnt2 and klf2a delayed Se and DLAV disintegration in bar mutants. Se, intersegmental vessels; DLAV: dorsal longitudinal anastomotic vessel. Scale bar, 75 �m.(C) Histograms show oxidative stress levels in endothelial cells (EC) derived from Tg(kdrl:GFP)^s843bar^t31131 after klf2a morpholino injection. barolo mutants were injected with control morpholino (ctrl MO) or klf2a morpholino (klf2a MO). Compared to control-injected embryos, klf2a MO-injected barolo (bar) mutants show reduced EC oxidative stress levels. Results are shown as a mean of n = 2 independent experiments for each condition.(D) Histograms show oxidative stress levels in endothelial cells derived from Tg(kdrl:GFP)^s843 and bar^t31131 (bar) mutant at 65 hpf after treatment with the NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) or DMSO as control. ROS levels in endothelial cells of zebrafish embryos were measured by flow cytometric analyses using the specific CellROX probe on Kdrl:GFP+ cells. NO overload in barolo cardiovascular tissues significantly enhances oxidative stress level. Results are shown as a mean of n = 2 independent experiments for each condition.(E) Representative confocal 3D projections of trunk vasculature between 10^th and 18^th somites of Tg(Kdrl:mCherry)^uto2barolo^s847(bar) mutant at 65 hpf. Embryos were treated from 48 hpf with the NO inhibitor, N- Nitro-L-arginine methyl ester hydrochloride (L-NAME; 500 �M) or NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 100 �M) or equivalent volume of DMSO as control.Compared to DMSO-treated embryos, L-NAME treatment efficiently prevents cardiovascular failure as indicated by intact dorsal aorta (DA), posterior cardinal vein (PCV) and intersegmental vessels integrity (Se) (arrows). On the other hand, SNAP treatment accelerates oxidative stress and cardiovascular failure in bar mutant embryos. Scale bar, 150 �M.(F) Confocal transverse sections of Tg(kdrl:GFP)^s843bar^t31131 trunk vasculature at the level of 10^th somite and stained for S-nitroso-cysteine, a biomarker of oxidative damage at 65 hpf. Confocal acquisitions are showed as single channel images and relative merged image: DNA (DAPI, blue), S-nitroso-cysteine (SNO-Cys, red), endothelium (Kdrl; green). bar^t31131 were previously treated with the NO inhibitor, L-NAME (500 �M) or NO donor, SNAP (100 �M) or DMSO as relative control. Oxidative stress-positive cells are detectable in dorsal aorta (DA) and posterior cardinal vein (PCV) in bar^t31131 (arrows) and SNAP-treated bar embryos. L-NAME treatment blocks oxidative stress in cardiovascular cells but not in pronephros suggesting a eNOS-dependent mechanism in oxidative stress caused by the loss of Ubiad1. DA, dorsal aorta; PCV posterior cardinal vein. Scale bar, 20 �m.(G) Histograms show numbers of endothelial cells in dorsal aorta (DA) and posterior caudal vein (PCV) positive for S-nitroso-cysteine (SNO-Cys) in bar mutant embryos treated with DMSO or the NO donor, SNAP (100 �M). Comparative confocal 3D projections were analyzed for SNO-Cys-positive endothelial cells in dorsal aorta and posterior cardinal vein. Results are shown as a mean of n = 3 independent experiments for each condition.(H) Histograms show NOS activity in protein extracts from barolo mutants (bar) and siblings (sib) at 72 hpf. Evaluation of NOS activity was based on conversion of [³H]-L-arginine to [³H]-L-citrulline. bar^t31131 and bar^s843 mutants show significant reduction in NOS activity compared to controls. These results indicate that loss of Ubiad1 impairs NOS enzymatic activity and therefore NO production. Results are shown as a mean of n = 2 independent experiments for each condition.(I) Representative micrographs of bar mutant embryos (bar) and siblings (sib) stained for nitric oxide (NO) with the green fluorescent probe 4,5 Diaminofluorescein Diacetate (DAF-2DA) at 72 hpf. NO production in the notochord (NC; arrows) and bulbus arteriousus (BuA; arrowheads) are evident in sibling but not in bar mutants. Scale bar, 300 �m. BuA, bulbus arteriosus; NC, notochord.All data are means � SEM; p < 0.05, p < 0.01, p < 0.001.