tom tom 24hpf tom tom 48hpf tom 60hpf tom tom 72hpf tom

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1 a 24hpf c 48hpf d e 60hpf f g 72hpf h

2 i j k ISV ISV Figure 1. Vascular integrity defects and endothelial regression in mutant emryos. (a,c,e,g,i) Bright-field and (,d,f,h,j) corresponding fluorescent micrographs of developing vasculature in wild-types () and mutants () as oserved in the Tg() s843 line at 24 (a,), 48 (c,d), 60 (e,f,i,j) and 72 (g,h) hpf. (a-h) mutant emryos develop normally forming a wild-type-like vascular network. Starting etween 54 and 60 hpf, vascular regression takes place as indicated y the downregulation of expression mainly in the rain and trunk region, and at 72 hpf all the vascular network appears to e affected (arrows). By 72hpf, pericardial edema is evident and circulation is reduced. (i-j) mutants at 60 hpf show vascular regression as assessed y in the intersomitic vessels (ISV), dorsal aorta () and posterior cardinal vein () (arrows). (k) Transverse confocal sections of 60 hpf Tg() s843 and mutant emryos., and ISV regression are evident.

3 a irc2 MO irc2 MO Figure 2. Knock-down of irc2 y MO injection phenocopies the mutation. (a) Bright-field and () corresponding fluorescent micrographs of Tg() s843 larvae at 72 hpf injected with 4 ng of irc2 MO. Knock-down of irc2 led to hemorrhage (arrows) and vascular regression as seen in mutants.

4 a c irc2 48hpf β-catenin d NT ISV e NC Birc2 f irc2 48hpf * * * * β-catenin Birc2 Figure 3. Birc2 is expressed in the cytosol of endothelial cells. In situ analysis of irc2 expression in whole mount (a) and crosssection () at 48 hpf. Intersomitic vessels, dorsal aorta and posterior cardinal vein are indicated (arrows). (c-f) Confocal transverse sections of Tg() s843 emryos at 54 hpf showing the two lateral dorsal aortae (arrows) and cardinal veins (arrowheads) and PHBC (primordial hindrain channel; dashed arrows). Sections were stained for Birc2 (red) and β-catenin (lue) expression. Birc2 is excluded from the nuclei of endothelial cells (green) (asterisks).sections shown are at the level of the 1st somite. Scale ars, 20µm.

5 % input ztr AF2 htr AF2 ztraf2 htraf2 zbirc2 zbirc2-e66a/r67a pcdna3 KDa 64 ztraf2 htraf2 zbirc 2 zbirc 2-E66 A/R6 7A zbirc 2-H61 7A pcdna3 a IP:FLAG IB:TRAF2 IB: FLAG c zbirc2 zbirc2-e66a/r67a zbirc2-h617a pcdna3 ztraf2 (IVT) U-zTRAF2 TRAF2 KDa ztraf Figure 4. A conserved mechanism for BIRC2-TRAF2 interaction. (a) Zerafish Birc2 inds to zerafish Traf2. Zerafish (z) and human (h) Traf2 were translated in vitro (right panel) and incuated with lysates from cells transfected with FLAG-tagged zbirc2 or zbirc2-e66a/r67a (left panel). Protein interactions were immunopurified with anti-flag antiodies. zbirc2 inds h and ztraf2 while the E66A/R67A mutation arogates this inding. () E66A/R67A mutation aolishes zbirc2-traf2 association in vivo. FLAG-tagged zbirc2 constructs were transfected into cells and association with endogenous htraf2 was examined y immunoprecipitation with anti-flag antiodies. zbirc2 and zbirc2-h617a ind endogenous Traf2 while the E66A/R67A mutation arogates the inding. (c) Uiquitin ligase activity of zbirc2 against ztraf2 is impaired y the H617A mutation. In vitro translated, ztraf2 was added to cell lysates of zbirc2, zbirc2-e66a/r67a or zbirc2h617a transfected cells. Ex vivo uiquitination assay was performed y adding E1 and E2 fractions, ATP-generating system, and uiquitin. Polyuiquitination is seen as high molecular weight ladder formation. Birc2-H617A uiquitin activity is reduced compared to zbirc2.

6 % of mutant emryos treatment Figure 5. Caspase-8 inhiitor treatment rescues endothelial apoptosis in mutants Emryos from heterozygote intercrosses were treated with 0.1% DMSO or 300µM of caspase-8 inhiitor IETD-fmk starting at the 15-somite stage. Emryos were genotyped using SfiI as indicated in the Methods section. Histograms indicate the percentage of mutants at 60 hpf after treatment.

7 a PBS c PBS d TNFα TNFα Figure 6. HUVEC apoptosis in response to loss of Birc2/3 expression. BIRC2/3 sirna-transfected HUVECs were treated with PBS (a-) or TNFα for 4 h (c-d). Cells were then fixed and stained with phalloidin (a-c) and active caspase-3 antiody (-d). Reduced expression of Birc2/3 induced apoptosis in endothelial cells after TNFα stimulation. Scale ars, 50µm.

8 a NT NC β-cat g DMSO DMSO DMSO c NT β-cat d h NC NAI NAI NAI e f i NT NC β-cat Figure 7. NF-κB regulates vascular integrity in zerafish emryos. (a-f) Confocal transverse sections of Tg() s843 DMSOtreated (a-), NAI-treated (c-d) and mutant (e-f) emryos at 54 hpf visualized for endothelial cells (, green), β-catenin (lue) and the tight junction protein (red). Vessel morphology is affected in NAI-treated emryos and mutants as shown y Tg() s843 expression and ZO1 staining. NAI-treated and mutants display a normal emryonic morphology as shown y β- catenin staining. (g-i) Schematic drawings of endothelial cells and tight junctions of, d and f, respectively. Sections shown are at the level of the 10th somite. NT, neural tue; NC, notochord;, dorsal aorta;, posterior cardinal vein. Scale ars, 20µm.

9 Movie 1. Vascular regression and apoptosis in mutant larvae Confocal projection movies of Tg() s843 larvae visualized for DNA (lue) at 72 hpf. Wild-type (a-c) and mutant (-d) larvae. The movies were taken with a 20x (A-B) or 40x ojective (C-D) lens on a Zeiss LSM5 confocal microscope. mutants show disintegration of endothelial cells within the dorsal aorta and posterior cardinal vein. Movie 2. Vascular integrity defects in NAI-treated zerafish emryos Confocal projection movies of doule transgenic Tg() s843 ;Tg(gata1:DsRed) sd2 emryos treated with 0.1% DMSO (a) or 100 nm NAI (NF-kB activator inhiitor) () at 54 hpf. NAI-treated emryos showed vascular integrity defects and vascular leakage. The movies were taken with a 20x ojective lens on a Zeiss LSM5 confocal microscope.

10 Methods. s805 mutants were genotyped y polymerase chain reaction (PCR) amplification using the primers 5 -TTGATAAAGGTTGAGCTTCCACAG-3 and 5 -GTGGCAGGTAAGCTCTGGATGAAGCTGCAG-3 followed y restriction enzyme digestion with SfiI. We used a morpholino oligonucleotide targeted against irc2 with the following sequence: 5 - TGTCCGAGGCCCTTACCAACATAAT-3.