Supporting Information

Sacilotto et al. 10.1073/pnas.1300805110 SI Materials and Methods The placenta was removed for use in genotyping. DNA was Generation of Tol2-Mediated Mosaic Transgenic Fish. F0 transient extracted by digestion in tail lysis buffer (100 mM NaCl, 25 mM mosaic transgenic zebrafish embryos were generated by the Tol2 EDTA, 1% SDS, 10 mM Tris·Cl, 200 g/mL proteinase K, pH 8.0) at system (1). Briefly, 0.5 nL of 50 ng/μL Tol2 transposase capped 55 °C overnight. Digested samples were extracted once with phe- mRNA (mMESSAGE mMACHINE SP6 Kit; Ambion) and 60 nol–chloroform and ethanol precipitated. Genotyping was con- ng/μL pE1b/enhancer/GFP expression vector were injected into ducted by PCR using LacZ genotyping primers (detailed below). one-cell embryos obtained by natural spawning of wild type (WT) adult zebrafish raised and maintained at 28.5 °C in system Generation of Mutant Dll4 Enhancer Constructs. All of the mutated water. Embryos were maintained in E3 medium (5 mM NaCl, versions of the Dll4 intron 3 (Dll4in3) and Dll4-12 enhancers were generated by PCR (Phusion Taq; Thermo Scientific), am- 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) at 28.5 °C. plifying the pCR8/GW/TOPO TA entry vectors containing the Generation of Dll4in3-GFP Stable Transgenic Fish Line. The Dll4in3: Dll4 enhancer with mutated forward (F) and reverse (R) oligo- GFP stable line tg(Dll4in3:GFP) was generated from an initial nucleotides (full primer sequences are detailed below). These outcross of adult F0 carriers generated by the Tol2 system de- mutated entry vectors were then used to generate expression scribed above. GFP positive embryos from these initial out- vectors by LR recombination. The specific mutations are de- crosses (F1 generation) were analyzed for expression, and one scribed here, with lowercase denoting mutated nucleotides. representative line selected. For the Dll4in3 enhancer, the TBX site TTACACCTT was mutated to TTACgtaTTT, the RBPJ-a site CGTGGGAA was Analysis of Transgenic Fish. All embryos were anesthetized using mutated to CcccGGgt, the RBPJ-b site GGTGAGAA was mu- 0.01% tricaine and imaged at 28.5 °C with the Zeiss LSM 710 tated to GcTGcagA, the MEF2C site TTATTTTTGG was mu- confocal microscope. Where necessary, embryos were also em- tated to TcccgggTTGG, the SOX-a site CACAATTG was bedded in 0.5% low-melting point agarose. For the time course of mutated to CcCgggTG, the FOXC2 site TGTATTTT was mu- GFP expression pattern in the tg(Dll4in3:GFP) stable line, a single tated to cGggTTTT, the ETS-a site GTTTCCTGC was mutated zebrafish embryo was imaged at 1–4 days post-fertilization (dpf) to GTcTgCTGC, the ETS-b site TCTTCCTGT was mutated whereas imaging of the tg(Dll4in3:GFP) fish after morpholino to TCTaaCTca, and the ETS-g/h site GGAAAAGGAT was (MO) injection used a different embryo for each time point. mutated to GctAAAGctT. To delete SOX-b, a 20-bp region GFP expressioninTol2-mediatedmosaictransienttransgenic fish (CACAACAATTCTCACTTTGA) was deleted by inverse PCR. was examined at 24 hours post-fertilization (hpf), and each embryo For the Dll4-12 enhancer, the SOX-b site CATTGTG was was scored positive or negative for GFP expression in the vascu- mutated to CccgGgG, the SOX-d site AACAATG was mutated lature. GFP expression in the vasculature was scored as positive to ccCgggG, and a 30-bp region containing the RBPJ site when at least one intersegmental vessel, or the equivalent expres- (GGTGGGAATAAGGGTCCCCAGGACAAAGCC) was deleted sion in either of the axial vessels or head vessels, was observed. by inverse PCR.

Analysis of Transgenic Mice. Transgenic mouse embryos were col- Zebrafish Whole-Mount in Situ Hybridization. In situ hybridization lected at the indicated times for F0 analysis along with the yolk sac as procedures used the following probes: dll4 (3); flt4 (4); notch1b previously described (2). Care was taken to ensure that all embryos (5), dlC (6); efnB2a (6); /grl (7) flt1 (8); and notch3 (6). were treated and stained using identical conditions. Briefly, each In situ hybridization was conducted as described (9). Briefly, embryo and associated yolk sac were dissected away from the pla- embryos were collected at the desired stages and fixed overnight at centa, rinsed brieflyinicecold1× PBS, then placed in fix solution 4 °C in 4% paraformaldehyde. Fixed embryos were stored in 100% [2% (vol/vol) paraformaldehyde, 0.2% glutaraldehyde, 1× PBS] at ethanol and rehydrated in PBST. Embryos were treated with 10 μg/ 4 °C. Embryonic day (E) 9 embryos were fixed for 30 min, E11 mL proteinase K (Sigma) for 8 min, followed by two PBST washes, embryos were fixed for 60 min, and E15 embryos and postnatal fixed with 4% PFA for 20 min, and washed five times with PBST. organs were fixed for 2.5 h. After fixation, embryos were rinsed Embryos were transferred into hybridization solution (50% twice for 20 min in rinse solution (0.1% sodium deoxycholate, 0.2% formamide, 5× SSC, 0.1% Tween 20, 50 μg/mL heparin, 500 μg/mL Nonidet P-40, 2 mM MgCl2,1× PBS) at 4 °C, then stained overnight of tRNA adjusted, 10 mM citric acid) for 2 h at 65 °C, then at room temperature in 1 mg/mL 5-bromo-4-chloro-3-indolyl β-D- transferred into diluted antisense riboprobe/hybridization solution galactoside solution (X-Gal) containing 5 mM potassium ferrocy- and incubated overnight at 65 °C. Probes were removed and em- anide, 5 mM ferricyanide, 0.1% sodium deoxycholate, 0.2% Non- bryos transferred to a Biolane HT-1 in situ machine (Intavis). idet P-40, 2 mM MgCl2,and1× PBS. After staining, embryos were Embryos were washed through a dilution series into 2× SSC then rinsed through a series of 1× PBS washes and then fixed overnight 0.2× SSC at 65 °C, followed by room temperature dilution washes in 4% (vol/vol) paraformaldehyde at 4 °C. into 100% MABT (0.1M Maleic Acid, 0.15 M NaCl, pH 7.5). Every embryo was imaged as a whole mount using a Leica Nonspecific sites were blocked with MAB block (MABT with 2% binocular dissecting microscope, and the staining pattern was Boehringer block reagent) at room temperature and incubated for scored for intensity in blood vessels, ectopic expression, and arterial 10 h with anti-DIG antibody (Roche) at 1:5,000 at 4 °C, before specificity/absence of staining in veins. For each enhancer con- washing in MABT. Before staining, embryos were washed in AP struct, multiple embryos were also sectioned for histological buffer and the in situ signal developed at 4–37 °C with BM Purple analysis to confirm these conclusions, as were any embryos where (Roche). Staining was stopped as appropriate by fixation in 4% the specificity of staining in arterial endothelial cells was not clear. paraformaldehyde. Embryos were transferred to 50% glycerol for For histological analysis, embryos were dehydrated through a series imaging and storage. of ethanol washes, cleared by xylene and paraffin wax-embedded. Six-micrometer sections were prepared, dewaxed, and counter- Chromatin Immunoprecipitation Assay. Chromatin immunoprecip- stained with nuclear fast red (Electron Microscopy Sciences). itation (ChIP) assays were performed as described in ref. 10, with

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 1of15 the following modifications: confluent human umbilical vein Immunohistochemistry on Dll4in3 and Dll4-12 transgenic endothelial cells (HUVECs) were cross-linked with 1% formal- mouse tissue was performed on 4% PFA fixed, paraffin-embedded dehyde at room temperature for 7 min, stopping the reaction samples. Antigen retrieval was used in conjunction with anti-Bgal, with glycine 0.125 M, final concentration. Cells were scraped, Nrp1, and Efnb2 antibodies (described in Materials and Methods). recovered, and lysed in ice-cold cell lysis buffer (10 mM NaCl, 3 In brief, sections were boiled for 10 min in 10 mM citric acid mM MgCl2, 30 mM sucrose, 10 mM EDTA, 0.5% Nonidet P-40, buffer (pH 6.0) followed by 20 min in room temperature 10 mM 10 mM Tris·HCl, pH 7) supplemented with protease inhibitor citric acid buffer (pH 6.0). After two 10-min washes in 1× PBS, all mixture (Sigma), and the intact nuclei were further lysed in samples were blocked in 3% normal goat serum 1× PBS for 1 h at · nuclei lysis buffer (10 mM EDTA, 1% SDS, 50 mM Tris HCl, pH room temperature before the addition of primary antibody. All 8.1) to release the chromatin. antibodies were diluted in PBS plus 0.025% Triton-X-100. Pri- Harvested chromatin was sonicated using the Bioruptor son- mary antibodies were incubated overnight at 4 °C in a humidified icator (Diagenode) at 4 °C to obtain a population of fragments chamber. After three 10-min washes in 1× PBS, secondary anti- with an average size of 500 bp. bodies were incubated for 1 h at room temperature. For each independent sonication, the size of the resulting Whole-mount caspase-3/GFP double immunostaining was con- chromatin fragments was analyzed by reversing the formaldehyde ducted on manually dechorionated zebrafish at the indicated time cross-links at 65 °C overnight and purifying the DNA by phenol: points and fixed in 4% PFA for 1 h at room temperature, followed by chloroform extraction and ethanol precipitation, followed by two 5-min washes in PBS-Tx (1× PBS with 0.25% Triton X-100). resolution of the purified naked DNA by agarose electrophoresis. Diluted chromatin (equivalent to 50 μg of DNA) was incubated Embryos were then blocked in 5% goat serum in PBS-Tx for 1 h at under rotation for 2 h at 4 °C with Dynabeads- G (In- room temperature and incubated overnight at 4 °C with the pri- vitrogen) and 2 μg of the corresponding antibody. An aliquot was mary antibodies in 2% goat serum in PBS-Tx (1:250 dilution for α α treated as above, but in the presence of IgG as negative control, and both rabbit -caspase-3 and chicken -GFP antibodies). Embryos another sample was immunoprecipitated with α-Polymerase II were then washed at room temperature six times for 15 min in PBS- antibody as positive control. Tx and incubated for 2 h at room temperature with the secondary Immunocomplexes were recovered with a magnet, extensively antibodies in 2% goat serum in PBS-Tx (1:500 dilution). After six washed, and eluted from the Dynabeads with elution buffer 15-min washes in PBS-Tx at room temperature, embryos were (EDTA 10 mM, SDS 1%, 50 mM Tris·HCl) at 65 °C for 10 min, mounted in Vectashield mounting medium with DAPI (Vector and incubated at 65 °C overnight to reverse formaldehyde cross- Labs) and imaged in the Zeiss LSM 710 confocal. links, with proteinase K (0.4 mg/mL), together with Input sam- ples, obtained from the first supernatant after IgG incubation. Primers for PCR. Lowercase indicates nucleotides mutated to ab- The DNA (from immunoprecipitation (IP), IgG, and Input late binding sites (as described in Generation of Mutant Dll4 samples) was purified by PCR purification kit (Qiagen) and used Enhancer Constructs): for PCR analysis. mouse Dll4in3 F:CTGCTGATATCGCTATCTCTAATGTCCC Electrophoretic Mobility Shift Assay. Electrophoretic mobility shift mouse Dll4in3 R:AGAGTTTCCTGGCGAAGTCTCTGC assays (EMSAs) were performed as described previously (2). zebrafish Dll4in3 F:CACTCACCTTACTCGGATCTACCT- were made using the TNT Quick Coupled Transcrip- GTAG tion/Translation System (Promega) as described in the manu- facturer’s directions. zebrafish Dll4in3 R:CTTATCTGCATGTTAGGGTTGTAT- The truncated version of ETS1 (ETS1DBD) and full-length GCGT ERG, ELF1, ELF2, SOX7, SOX18, MEF2C, and TBX2 were in mouse dll4in3ΔTBX F:CTGACGGGCCTCTTCCTGTATTT- the pCITE2 plasmid, and transcribed using T7 polymerase. ETV2 TACgtaTTTTGCGAA was in the pCS2 plasmid, and transcribed using Sp6 polymerase. FOXC2 was in the pCR2.1 plasmid and transcribed using T7. mouse dll4in3ΔTBX R:TTCGCAAAAtacGTAAAATACAG- RBPJ was in the pcDNA3.1 plasmid and transcribed using T7. GAAGAGGCCCGTCAG To label the probe, double-stranded oligonucleotides were la- mouse Dll4in3ΔRBPJ-a F:CcccGGgtCGCGGGGAGCACGG- 32 fi ′ beled with P-dCTP, using klenow to ll in overhanging 5 ends, CGGTGAG and purified on a nondenaturing polyacrylamide-Tris/Borate/ EDTA (TBE) gel (2). Twenty-microliter binding reactions con- mouse Dll4in3ΔRBPJ-a R:acCCgggGCCAAAAATAACCC- sisted of 3–5 μL of protein or lysate control and 2 μLof10× binding GCAG buffer (40 mM KCl, 15 mM Hepes, pH 7.9, 1 mM EDTA, 0.5 mM mouse Dll4in3ΔRBPJ-b F:GCTGCAGAAGGCCGAGGCTG- DTT, 5% glycerol). For ETS factors, RBPJ, MEF2C, and TBX2, CCAG 0.5 μg of poly-dI-dC was used. For FOXC2, 0.25 μg of poly- Δ deoxyinosinic-deoxycytidylic acid (dI-dC) was used, and for SOX7 mouse Dll4in3 RBPJ-b R:TTCTGCAGCGCCGTGCTCCC- and SOX18, 0.3 μg of poly-deoxyguanylic-deoxycytidylic acid (dG- CGCG dC) was used. For competitor lanes, a 50-fold excess of compet- mouse Dll4in3ΔMef2C F:TGCGTTTCCTGCGGGTcccgggTG- itor DNA was added in a volume of 1 μL. Binding reactions were GCGTGGGAACG incubated at room temperature for 10 min before the addition of mouse Dll4in3ΔMef2C R:CGTTCCCACGCCAcccgggACCC- radiolabeled probed, after which they were incubated for an GCAGGAAACGCA additional 20 min. Gels were electrophoresed on a 6% non- denaturing polyacrylamide gel (except the ETS competition gel, mouse Dll4in3ΔSOX-a F:GCCTGGACTCAGAGCcCgggT- which was run on a 10% nondenaturing polyacrylamide gel). GCGTTTCCTG mouse Dll4in3ΔSOX-a R:CGCAGGAAACGCAcccGgGCT- Immunohistochemistry. Primary antibodies were β-galactosidase (β-gal) (MP Biomedicals), Neuropilin1 (Nrp1) (Abcam), Ephrin- CTGAGTCCAG B2 (Efnb2) (R&D systems), Endomucin (Emcn) (Santa Cruz), mouse Dll4in3ΔSOX-b F:AAAGGAAAAATAAAAACCAT- and Caspase-3 (Sigma). TACCTACG

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 2of15 Δ mouse Dll4in3 SOX-b R:CTGGTGAAATATCCTTTTCCT- Lowercase indicates nonendogenous sequence added for la- CTGTG beling purposes, or mutated to ablate binding sites (as described mouse Dll4in3ΔFOXC F:cGggTTTTACACCTTTTGCGA- in Generation of Mutant Dll4 Enhancer Constructs). ATTC Primers for EMSA. The following are the primers for EMSA: mouse Dll4in3ΔFOXC R:AAccCgGGAAGAGGCCCGTC Δ mouse Dll4in3 ETS-a WT EMSA F:ctagAATTGCGTTTCC- mouse Dll4in3 FOX,ETS-b F:TGACGGGCCTCTaaCTcaAT- TGCGGGTT TTTACACCTTTTGCG Δ mouse Dll4in3 ETS-a WT EMSA R:ctagAACCCGCAGGA- mouse Dll4in3 FOX,ETS-bR:CGCAAAAGGTGTAAAATtg- AACGCAATT AGttAGAGGCCCGTCA Δ Δ mouse Dll4in3 ETS-a EMSA F:ctagAATTGCGTcTg- mouse Dll4in3 ETS-a F:cTgCaGCGGGTTATTTTTGG- CaGCGGGTT CGTG Δ Δ mouse Dll4in3 ETS-a EMSA R:ctagAACCCGCtGcAgA- mouse Dll4in3 ETS-a R:CtGcAgACGCAATTGTGCTCTG- CGCAATT AGTC Δ mouse Dll4in3 ETS-b/TBX WT EMSA F:ctagCGGGCC- mouse Dll4in3 ETS-g/h F:GAGctAAAGctTATTTCACCAG- TCTTCCTGTATTTTACACC TTTTGCGAA CACAACAATTC Δ mouse Dll4in3 ETS-b/TBX WT EMSA R:ctagTTCGCAA- mouse Dll4in3 ETS-g/h R:TAagCTTTagCTCTGTGTCAG- AAGGTGTAAAATACAGGA AGAGGCCCG AACAACATC mouse Dll4in3 ETS-b Δ EMSA F:ctagCGGGCCTCTaaCTca- mouse Dll4-12 F:TCCTAAGTCCTCCCTGTTCTG ATTTTACACCT TTTGCGAA mouse Dll4-12 R:CTTAATGTTAGGTGAGCTCTC mouse Dll4in3 ETS-b Δ EMSA R:ctagTTCGCAAAAGGTG- mouse Dll4-16 F:CCAGCACTAAGTCAAAGTTGCCCAG TAAAATtgAGttAG AGGCCCG mouse Dll4-16 R:CCAGGGAGGTCACAGCATTTATGTT- mouse Dll4in3 TBX Δ EMSA F:ctagCGGGCCTCTTCCTG- TTG TATTTTACgtaTTTT GCGAA mouse Dll4+14 XhoI F:ACCTGGAAGCTTATGCCAGAG- mouse Dll4in3 TBX Δ EMSA R:ctagTTCGCAAAAtacGTA- ACACA AAATACAGGAAGAG GCCCG mouse Dll4+14 XhoI R:GAAGGACTCGAGGTAGATG- mouse Dll4in3 ETS-c WT EMSA F:ctagGTTTGCGAAT- GACTT TCCGCTGGTTT mouse Dll4-12ΔSOX-a/RBPJ F:CGGCCTGGTTCCCGCAGTC mouse Dll4in3 ETS-c WT EMSA R:ctagAAACCAGCGGA- ATTCGCAAAC mouse Dll4-12ΔSOX-a/RBPJ R:ATTTAGGAGCCGGAACCT- CAGG mouse Dll4in3 ETS-d/e WT EMSA F:ctagGCTCCTTTGGA- AAGGGAATAATGGC mouse Dll4-12ΔSOX-b F:CccgGgGTATGGTCGCCCCATG Δ mouse Dll4in3 ETS-d/e WT EMSA R:ctagGCCATTATTCC- mouse Dll4-12 SOX-b R:cCcggGGGGATTCCGTCCATTCTG CTTTCCAAAGGAGC Δ mouse Dll4-12 SOX-d F:ccCGGGGCCCCCCAGAAGG mouse Dll4n i3 ETS-f WT EMSA F:ctagTGGCTTTGGGA- mouse Dll4-12ΔSOX-d R:CCCGggTGTCTCCCCGGCCC TGTTGTTCT human Dll4-12 ChIP F:GGTGACGACATTGTTGTTCTTA- mouse Dll4in3 ETS-f WT EMSA R:ctagAGAACAACATCC- TACTACAG CAAAGCCA human Dll4-12 ChIP R:CCAGCTGAGCTCACTACCATTG mouse Dll4in3 ETS-g/h WT EMSA F:ctagGACACAGAG- GAAAAGGATATTTCACC human Dll4in3 ChIP F1:GCTCGCTGCCTGGACTCAG mouse Dll4in3 ETS-g/h WT EMSA R:ctagGGTGAAATA- human Dll4in3 ChIP R1:TGTGTCAGAACAACATCCCA- TCCTTTTCCTCTGTGTC AAGC mouse Dll4in3 ETS-g/h Δ EMSA F:ctagGACACAGAGcAAA- human Dll4in3 ChIP F2:ACGCTCGGATTCCGCTCGCTG AGGccATTTCACC human Dll4in3 ChIP R2:TGTGTCAGAACAACATCCCA- mouse Dll4in3 ETS-g/h Δ EMSA R:ctagGGTGAAATggCC- AAGC TTTTgCTCTGTGTC mouse Dll4mP SpeI F:AGTAGACTAGTCTGGAAAGGAA- mouse Dll4in3 Mef2C WT EMSA F:ctagCCTGCGGGTTA- AGGGAGATC TTTTTGGCGTGGGA mouse Dll4mP BamHI R:ATAATGGATCCCCCTTGGG- mouse Dll4in3 Mef2C WT EMSA R:ctagTCCCACGCCAA- GTGTCCTCTC AAATAACCCGCAGG mouse Dll4in3 NotI R:ATATGCGGCCGCAGAGTTTCCT- mouse Dll4in3 Mef2C Δ EMSA F:ctagCCTGCGGGTcgccc- GGCGAAGT TTGGCGTGGGA LacZ genotyping F:GTCGTTTTACAACGTCGTGACT mouse Dll4in3 Mef2C Δ EMSA R:ctagTCCCACGCCAAgg- LacZ genotyping R:GATGGGCGCATCGTAACCGTGC gcgACCCGCAGG

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 3of15 mouse Dll4in3 RBPJ-a WT EMSA F:ctagTTATTTTTGGCG- mouse Dll4-12 SOX-a Δ EMSA R:ctagGAACCAGGCCGG- TGGGAACGCGGGG AGCAC CccgGgCCTGGGGACC mouse Dll4in3 RBPJ-a WT EMSA R:ctagGTGCTCCCCG- mouse Dll4-12 SOX-b Δ EMSA F:ctagACGGAATCCCC- CGTTCCCACGCCAAAAA TAA ccgGgGTATGGTCGC mouse Dll4in3 RBPJ-b WT EMSA F:ctagGAGCACGGCG- mouse Dll4-12 SOX-b Δ EMSA R:ctagGCGACCATACcC- GTGAGAAAGGCCGAGGC cggGGGGATTCCGT mouse Dll4in3 RBPJ-b WT EMSA R:ctagGCCTCGGCCT- mouse Dll4-12 SOX-c WT EMSA F:ctagCGGACATGCCA- TTCTCACCGCCGTGCTC ACAAACAGCTCATTGA mouse Dll4in3 RBPJ-b Δ EMSA F:ctagGAGCACGGCGc- mouse Dll4-12 SOX-c WT EMSA R:ctagTCAATGAGCTG- TGcagAAGGCCGAGGC TTTGTTGGCATGTCCG mouse Dll4in3 RBPJ-b Δ EMSA R:ctagGCCTCGGCCTTctg- mouse Dll4-12 SOX-d WT EMSA F:ctagCCGGGGAGAC- CAgCGCCGTGCTC AACAATGCCCCCCAGAA mouse Dll4-12 RBPJ WT EMSA F:ctagCTCCTAAATG- mouse Dll4-12 SOX-d WT EMSA R:ctagTTCTGGGGGG- GTGGGAATAAGGGTCCCCA CATTGTTGTCTCCCCGG mouse Dll4-12 RBPJ WT EMSA R:ctagTGGGGACCCTTA- mouse Dll4-12 SOX-c Δ EMSA F:ctagCGGACATGCCccgg- TTCCCACCATTTAGGAG gACAGCTCATTGA mouse Dll4-12 SOX-a WT EMSA F:ctagGGTCCCCAGGA- mouse Dll4-12 SOX-c Δ EMSA R:ctagTCAATGAGCTGT- CAAAGCCGGCCTGGTTC cccggGGCATGTCCG mouse Dll4-12 SOX-a WT EMSA R:ctagGAACCAGGCCG- mouse Dll4-12 SOX-d Δ EMSA F:ctagCCGGGGAGACcc- GCTTTGTCCTGGGGACC gggTGCCCCCCAGAA mouse Dll4-12 SOX-b WT EMSA F:ctagACGGAATCCC- mouse Dll4-12 SOX-d Δ EMSA R:ctagTTCTGGGGGGCAccc- CATTGTGTATGGTCGC ggGTCTCCCCGG mouse Dll4-12 SOX-b WT EMSA R:ctagGCGACCATACA- CAATGGGGATTCCGT Morpholinos. Morpholinos are as follows: mouse Dll4-12 RBPJ Δ EMSA F:ctagCTCCTAAATGccc- sox7-MO1:ACGCACTTATCAGAGCCGCCATGTG (11) GGgtTAAGGGTCCCCA -MO1:TATTCATTCCAGCAAGACCAACACG (11) mouse Dll4-12 RBPJ Δ EMSA R:ctagTGGGGACCCTTA- MO:CAAACTTCCCTGTCACAACAGGCGC (12) acCCgggCATTTAGGAG scrambled MO:CCTCTTACCTCAGTTACAATTTATA (12) mouse Dll4-12 SOX-a Δ EMSA F:ctagGGTCCCCAGGc- CcggGCCGGCCTGGTTC dll4 MO:GTTCGAGCTTACCGGCCACCCAAAG (12)

1. Kawakami K (2005) Transposon tools and methods in zebrafish. Dev Dyn 234(2): 8. Bussmann J, Bakkers J, Schulte-Merker S (2007) Early endocardial morphogenesis 244–254. requires Scl/Tal1. PLoS Genet 3(8):e140. 2. De Val S, et al. (2004) Mef2c is activated directly by Ets transcription factors through an 9. Gering M, Patient R (2005) Hedgehog signaling is required for adult blood stem cell evolutionarily conserved endothelial cell-specificenhancer.Dev Biol 275(2):424–434. formation in zebrafish embryos. Dev Cell 8(3):389–400. 3. Leslie JD, et al. (2007) Endothelial signalling by the Notch ligand Delta-like 4 restricts 10. Sacilotto N, Espert A, Castillo J, Franco L, López-Rodas G (2011) Epigenetic angiogenesis. Development 134(5):839–844. transcriptional regulation of the growth arrest-specific 1 (Gas1) in hepatic cell 4. Thompson MA, et al. (1998) The cloche and spadetail differentially affect proliferation at mononucleosomal resolution. PLoS ONE 6(8):e23318. hematopoiesis and vasculogenesis. Dev Biol 197(2):248–269. 11. Cermenati S, et al. (2008) Sox18 and Sox7 play redundant roles in vascular 5. Rowlinson JM, Gering M (2010) Hey2 acts upstream of Notch in hematopoietic stem development. Blood 111(5):2657–2666. cell specification in zebrafish embryos. Blood 116(12):2046–2056. 12. Siekmann AF, Lawson ND (2007) Notch signalling limits angiogenic cell behaviour in 6. Patterson LJ, Gering M, Patient R (2005) Scl is required for dorsal aorta as well as developing zebrafish arteries. Nature 445(7129):781–784. blood formation in zebrafish embryos. Blood 105(9):3502–3511. 7. Zhong TP, Rosenberg M, Mohideen MA, Weinstein B, Fishman MC (2000) gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science 287(5459): 1820–1824.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 4of15 Fig. S1. (A) Schematic representation of the Dll4 locus from University of California, Santa Cruz (UCSC) ENCODE Browser (1), HUVEC-specific H3K27Ac in light blue, K562 leukemia cell line in pink. On the bottom, the potential vascular-specific enhancers are depicted with their respective positions (in kb) with respect to the TSS (except for the intron 3, indicated as in3). (B) Schematic representation of the mouse Dll4 locus (Upper line, exons are black boxes) and Dll4in3end transgene (Lower line). Prom, endogenous promoter. (C–G) A representative X-Gal–stained E15 Dll4in3end transgenic embryo. X-Gal staining is detected in arterial (a) but not venous (v) cells in whole-mount embryo (C and D), yolk sac (E), and transverse section (F). Expression of the venous marker Endomucin (Emcn) did not overlap that of the reporter gene (G). (H–O) Further examples of transgenic mice expressing the mouse Dll4in3hsp transgene at E8 (H), E19 (I), and tissues from a P6 pup (J–O), all showing X-Gal staining in arteries (a) but not veins (v). (P) Time course of the tg(Dll4in3:GFP) zebrafish line between 1–4 dpf.

1. Rosenbloom KR, et al. (2013) ENCODE data in the UCSC Genome Browser: year 5 update. Nucleic Acids Res 41(Database issue):D56–D63.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 5of15 Fig. S2. (A and B) RBPJ, NICD, SOX7, and SOX18 bind the Dll4in3 enhancer. (A) Representative ChIP against SOX7 and SOX18 (right) and Notch intracellular domain (NICD) and RBPJ (left), from HUVECs. RNA Polymerase II (Pol II) and IgG were used as positive and negative controls, respectively. (B) Radiolabeled oligonucleotide probes encompassing Dll4in3 RBPJ-a (lanes 1–4), SOX-a (lanes 5–11), and SOX-b motifs (lanes 12–18) were bound to recombinant RBPJ, SOX7, and SOX18 proteins. All proteins efficiently bound to labeled probes (lanes 2, 6, 9, 13, and 16), were competed by excess unlabeled self-probe (wt, lanes 3, 7, 9, 14, and 17), but not by mutant self-probe (mu, lanes 4, 8, 10, 15, and 18). (C) Radiolabeled oligonucleotide probe encompassing the Dll4in3 TBX2 site was bound by recombinant TBX2 protein (lane 2) and was competed by an excess of unlabeled probe (wt) but not mutant (mu) self-probe (lanes 3 and 4). Ra- diolabeled oligonucleotide probe encompassing the Dll4in3 site was bound by recombinant MEF2C protein (lane 6) and was competed by an excess of unlabeled probe (wt) but not mutant (mu) self-probe (lanes 7 and 8). An unlabeled oligonucleotide of Dll4in3 containing the RBPJ mutation found in Dll4in3ΔRBPJ was still able to efficiently compete for binding of MEF2C (lane 9), suggesting that the RBPJ-a mutation did not affect MEF2C binding. The binding of recombinant RBPJ (RB) to a control RBPJ site (lane 11) was not efficiently competed by excess unlabeled Dll4in3 RBPJ-b probe (lane 12), nor was radiolabeled Dll4in3 RBPJ-b site probe able to bind recombinant RBPJ (lane 14). The binding of recombinant FOXC2 (FC2) to a control binding site (CTRL FOX) (lanes 15–17) was not competed by excess unlabeled Dll4in3 FOXC probe (Dll4, lane 18), nor was radiolabeled Dll4in3 FOXC site probe able to bind recombinant FOXC2 (lanes 19 and 20). Radiolabeled oligonucleotide probes encompassing a known ETS binding motif (MEF2C ETS) were bound by recombinant ETS1-DNA binding domain (ETSDBD) (lanes 21–23) and efficiently competed by excess unlabeled Dll4in3 ETS-a, ETS-b, and ETS-g/h. The ETS motifs within the Dll4in3 RBPJ- a site, and in ETS-c, ETS-d/e, and ETS-f did not effectively compete for binding (lanes 24–30). (D) Radiolabeled oligonucleotide probes encompassing the control ETS (lanes 1–6), Dll4in3 ETS-a (lanes 7–12), RPBJ-a (lanes 13–18), ETS-b (lanes 19–24), ETS-d/e (lanes 25–30), ETS-f (lanes 31–36), and ETS-g/h (lanes 37–42) were incubated with recombinant ETSDBD, ETV2, ELF1, ERG, and ELF2. Probes ETS-a, ETS-b, and ETS-g/h bound in a manner similar to control ETS whereas little binding was detected for the RBPJ-a site (which contains an ETS motif), ETS-d/e, and ETS-f. In all cases, white vertical lines indicate where regions of EMSA gelhavebeen deleted from image for clarity.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 6of15 Fig. S3. (A–J) Representative 2–2.5 dpf Tol2-mediated mosaic transient transgenic zebrafish generated using WT or mutant versions of the Dll4in3:GFP transgene. (A) mouse WT Dll4in3:GFP, (B) zebrafish WT dll4in3:GFP, (C) ΔETS-a/b/g/h Dll4in3:GFP, (D) ΔMEF2C Dll4in3:GFP, (E) ΔRBPJ-a Dll4in3:GFP, (F) ΔSOX- a Dll4in3:GFP, (G) ΔSOX-b Dll4in3:GFP, (H) ΔSOX-a/b Dll4in3:GFP, (I) ΔRBPJ-a/SOX-a Dll4in3:GFP, (J) ΔRBPJ-a/SOX-a/b Dll4in3:GFP.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 7of15 Fig. S4. Whole-mount image of every transient transgenic mouse embryo generated with either WT or mutant Dll4in3 and Dll4-12, in which any X-Gal staining could be detected (and counted as an expressor in Fig.3C). The numbers simply indicate the designated identifier for each embryo during analysis, and correspond exactly to those described in Table S1.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 8of15 Fig. S5. (A and B) The Dll4-12hsp drives arterial-specific expression. (A) Whole-mount E15 X-Gal–stained transgenic embryo. The values on the bottom right represent the number of expressing embryos per number of transgenic embryos. (B) Representative immunofluorescence on a paraffin-embedded section from an expressing Dll4-12 transgenic mouse. Expression of the venous marker Endomucin (Emcn) did not overlap with reporter gene. (Scale bar: 25 μm.) (C) Multispecies alignment using ClustalW of the conserved region of the Dll4-12 enhancer. Colored sequences depict confirmed consensus binding motifs, and gray sequences depict motifs identified in silico that did not bind in EMSA analysis. (D and E) RBPJ, NICD, SOX7, and SOX18 bind the Dll4-12 enhancer. (D) Radiolabeled oligonucleotide probes encompassing the Dll4-12 RBPJ (lanes 1–4), SOX-b (lanes 5–11), and SOX-d sites (lanes 12–18) were used in EMSA with recombinant RBPJ, SOX7, and SOX18 proteins. All proteins efficiently bound labeled probes (lanes 2, 6, 9, 13, and 16), were competed by excess unlabeled self- probe (wt, lanes 3, 7, 9, 14, and 17), but not by mutant self-probe (mu, lanes 4, 8, 10, 15, and 18). (E) Representative ChIP against NICD and RBPJ (Upper)and SOX7 and SOX18 (Lower) from HUVECs. RNA Polymerase II (Pol II) and IgG were used as positive and negative controls, respectively. White vertical lines between input and αSOX7, and αSOX7 and αSOX18, indicate where data lanes have been deleted from the gel image to remove duplicated experiments using alternative antibodies.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 9of15 Fig. S6. (A) Analysis of GFP expression in 3-dpf tg(Dll4in3:GFP) zebrafish embryos after scrambled, rbpj, double sox7;sox18, and triple sox7;sox18;rbpj MO injections. Triple sox7;sox18;rbpj MO results in total ablation of tg(Dll4in3:GFP) expression. We used 0.25 pmol for sox7 and sox18 and 0.30 pmol of rbpj MO. (B–E) Analysis of GFP and endogenous dll4 expression in tg(Dll4in3:GFP) (34 hpf) and WT zebrafish (26 hpf) after chemical inhibition of Notch signaling with 100 μM N-[N-3,5-Difluorophenacetyl]-L-alanyl-S-phenylglycine Methyl Ester (DAPM) alone (B and C) and in combination with sox7;sox18 MO (D and E). sox7; sox18 MO injection in combination with DAPM results in total ablation of both GFP and endogenous dll4 expression. (F–I) Chemical inhibition of VEGF sig- naling in tg(Dll4in3:GFP) zebrafish embryos with 1 μM SU5416 alone or in combination with rbpj or double sox7;sox18 MO abolishes the GFP expression. Values on the bottom right indicate number of embryos with the predominant and displayed phenotype/ total number of embryos analyzed. In all cases, 0.125 pmol of sox7 and sox18 and 0.15 pmol of rbpj MO were used.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 10 of 15 Fig. S7. (A) Whole-mount in situ hybridization against fli1a in 26 hpf WT zebrafish embryos after injection of control (Left) and triple sox7;sox81;rbpj MO (Right). Values on the bottom right indicate number of embryos with the predominant and displayed phenotype per total number of embryos. (B) Analysis of GFP expression in 26 hpf tg(fli1a:GFP) zebrafish embryos after rbpj and sox7;sox18 MO injection. Values on the bottom right indicate number of embryos with the predominant and displayed phenotype per total number of embryos. (C and D) Whole-mount double immunofluorescence against activated caspase- 3 (red) and GFP on tg(fli1a:GFP) zebrafish embryos after control (Left) and triple sox7;sox18;rbpj MO (Right) injection at 21 hpf (C) and 26 hpf (D). Nuclei were counterstained with DAPI. No significant apoptosis was detected. Values on the bottom right indicate number of embryos with the predominant and Legend continued on following page

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 11 of 15 displayed phenotype per total number of embryos. (E) Analysis of GFP expression in 2.5-dpf tg(kdrl:GFP);tg(:dsRed) zebrafish embryos after control and sox7;sox18;rbpj MO injection. Pooled blood cells could be detected in the trunk and tail of the sox7;sox18;rbpj MO-injected fish. Values on the bottom right indicate number of embryos with the predominant and displayed phenotype per total number of embryos. In all cases, 0.125 pmol of sox7 and sox18 and 0.15 pmol of rbpj MO were used.

Fig. S8. (A–D) Whole-mount in situ hybridization of arterial markers on control, rbpj, double sox7;sox18, and triple sox7;sox18;rbpj MO at 26 hpf. Triple sox7; sox18;rbpj knockdown results in the loss of all of the arterial markers analyzed. Black arrowhead shows dorsal aorta. White arrowhead indicates where dorsal aorta should be. Black arrow indicates posterior cardinal vein, and white bracket indicates single axial vessel/expanded posterior cardinal vein. Black bracket indicates notochord. Probes used are depicted in the bottom left of each picture. Values on the bottom right indicate number of embryos with the pre- dominant and displayed phenotype/total number of embryos analyzed. In all cases, 0.125 pmol of sox7 and sox18 and 0.15 pmol of rbpj MO were used. (E–K) Whole-mount in situ hybridization of arterial (A–F) and venous (G) markers on control and dll4 MO-injected WT zebrafish embryos at 26 hpf. Probes are depicted in the bottom left. Values on the bottom right indicate number of embryos with the predominant and displayed phenotype per total number of embryos. Six nanograms of dll4 MO were used.

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 12 of 15 Table S1. Summary of the reporter gene expression pattern detected in all transient transgenic mice generated by WT or mutant Dll4in3 and Dll4-12 transgenes in which X-Gal staining could be detected (recorded as expressors in Fig. 2C) Staining intensity Staining detected Staining in arteries: * least, Ectopic expression Method of Construct Stage ID in arteries detected in veins ***** most (tissue) analysis

Dll4in3 WT E9 1 Yes No **** Yes (fascia) Histology WT E9 3 Yes No **** No Histology WT E11 2 Yes No **** No Histology WT E11 4 Yes No *** Yes (drg) Dissection WT E11 6 Yes No *** Yes (drg) Dissection WT E11 9 Yes No **** No Histology WT E11 10 Yes No **** No Dissection WT E11 11 Yes No **** No Dissection WT E11 14 Yes No **** Yes (fascia) Histology Zebrafish WT E11 12 Yes No **** Yes (drg) Histology Zebrafish WT E11 13 Yes No * Yes (fascia) Histology Zebrafish WT E11 14 Yes No **** No Histology Zebrafish WT E11 17 Yes No **** No Histology ΔMEF2 E11 6 Yes No ** No Dissection ΔMEF2 E11 7 Yes No ** No Whole mount ΔMEF2 E11 9 Yes No **** Yes (fascia) Histology ΔMEF2 E11 13 Yes No * Yes (drg) Whole mount ΔMEF2 E11 15 Yes No ***** No Dissection ΔMEF2 E11 18 Yes No ***** No Histology ΔMEF2 E11 21 Yes No *** No Dissection ΔRBPJ-a E11 1–4 Yes No **** No Histology ΔRBPJ-a E11 1–5 Yes No ** No Whole mount ΔRBPJ-a E11 1–19 Yes No ** Yes Histology ΔRBPJ-a E11 1–28 Yes No ** No Whole mount ΔRBPJ-a E11 2–3 Yes No **** Yes (fascia, muscle) Histology ΔRBPJ-a E11 2–4 Yes No ***** Yes (sympathy-etic trunk, Histology fascia) ΔRBPJ-a E11 2–17 Yes No *** No Whole mount ΔRBPJ-a E11 2–27 Yes No *** No Histology ΔSOX-a E11 3 Yes No ** Yes (fascia) Histology ΔSOX-a E11 4 Yes No **** Yes (fascia) Histology ΔSOX-a E11 5 Yes No *** No Histology ΔSOX-a E11 6 Yes No ** Yes (fascia, drg, muscle) Histology ΔSOX-b E11 6 Yes No ***** No Histology ΔSOX-b E11 11 Yes No ***** No Histology ΔSOX-b E11 16 Yes No ***** No Histology ΔSOX-b E11 20 Yes No ***** Yes (fascia) Whole mount ΔSOX-b E11 21 Yes No ***** No Histology ΔFOXC E11 1 Yes No **** Yes (fascia) Histology ΔFOXC E11 2 Yes No * Yes (drg) Whole mount ΔFOXC E11 3 Yes No *** Yes (drg) Whole mount ΔRBPJ-a/b E11 1 Yes No *** No Histology ΔRBPJ-a/b E11 7 Yes No * No Whole mount ΔTBX E15 7 Yes No * No Whole mount ΔTBX E15 9 Yes No **** No Histology ΔTBX E15 10 Yes No *** No Histology ΔSOX-a/-b E11 1–3 Yes No ** Yes (drg) Histology ΔSOX-a/-b E11 2–5 Yes No ** No Histology ΔRBPJ-a/SOX-a E11 1 Yes No ** No Histology ΔRBPJ-a/SOX-a E11 3 Yes No * No Histology ΔRBPJ-a/SOX-a E11 6 Yes No ** Yes (drg, fascia) Histology ΔRBPJ-a/SOX-a E11 14 Yes No **** Yes (drg) Histology ΔRBPJ-a/SOX-a/b E9 15 No No — Yes (gut tube) Histology ΔRBPJ-a/SOX-a/b E11 2 No No — Yes (neural tube) Whole mount ΔRBPJ-a/SOX-a/b E11 4 No No — Yes (neural tube) Histology ΔRBPJ-a/SOX-a/b E11 10 No No — Yes (drg) Whole mount ΔRBPJ-a/SOX-a/b E11 11 No No — Yes (drg, muscle) Histology ΔRBPJ-a/SOX-a/b E11 15 No No — Yes (neural tube) Whole mount

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 13 of 15 Table S1. Cont. Staining intensity Staining detected Staining in arteries: * least, Ectopic expression Method of Construct Stage ID in arteries detected in veins ***** most (tissue) analysis

Dll4-12 WT E11 4 Yes No ** No Dissection WT E11 11 Yes No **** Yes (hindbrain, fascia) Dissection WT E11 15 Yes No **** No Histology ΔRBPJ/SOX-a/b E11 6 No No — Yes (drg) Histology ΔRBPJ/SOX-a/b E11 7 No No — Yes (neural tube) Histology ΔRBPJ/SOX-a/b E11 12 No No — Yes (forebrain) Whole mount

Each individual embryo was given an ID number, and assessed for X-Gal staining in arteries, veins, and away from the vasculature (ectopic). The method of analysis is also described. Histology indicates that the embryo was paraffin embedded, sectioned, and examined by light microscopy. Dissection indicates that the embryo was dissected and examined under a binocular dissection microscope, and whole mount indicates that expression pattern was assessed as a whole- mount X-Gal stained embryo. This latter analysis was conducted only when the staining pattern was near-identical to other expressing embryos that were more thoroughly investigated. drg, dorsal root ganglia. Dashes denote when no staining was detected.

Movie S1. Three-dimensional rendering of confocal Z-stacked images of a 50-hpf control MO-injected tg(fli1a:GFP) embryo.

Movie S1

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 14 of 15 Movie S2. Three-dimensional rendering of confocal Z-stacked images of a 50-hpf rbpj MO-injected tg(fli1a:GFP) embryo.

Movie S2

Movie S3. Three-dimensional rendering of confocal Z-stacked images of a 50-hpf sox7;sox18 MO-injected tg(fli1a:GFP) embryo.

Movie S3

Movie S4. Three-dimensional rendering of confocal Z-stacked images of a 50-hpf sox7;sox18;rbpj MO-injected tg(fli1a:GFP) embryo.

Movie S4

Sacilotto et al. www.pnas.org/cgi/content/short/1300805110 15 of 15