Whole mount in situ hybridization, sectioning, and immunostaining In situ hybridization for tie-1 and lncrna tie-1as and sectioning of ISH stained embryos were performed according to previously published protocol. 15 CDH5, ZO-1 and anti-egfp staining was performed as previously 13 with modifications described below. 24 hpf Tg(flk: EGFP) embryos were fixed in 4% paraformaldehyde for 2 h, washed 4 5 min in PBST (PBS+0.1% Tween20) and once in PBSTX (PBST+0.1% Triton X100). After blocking in PBSTX+10% BSA+1% NGS for 2 h, embryos were incubated with primary antibodies (in PBSTX+1% BSA+0.1% NGS) overnight at 4 C. Embryos were then washed 6 1 h in PBSTX+1% BSA+0.1% NGS and then incubated with the secondary antibody (in PBSTX+1% BSA+0.1% NGS) overnight at 4 C. A final wash was performed several times in PBST. All steps were performed at RT except for antibody incubations. ZO-1 confocal stack size is 0.1221 µm for 100 200 optical sections per stack. CDH5 (1:200) confocal stack size is 0.2442 µm for 50 100 section. All images were acquired with a Leica SP1 confocal microscope (63x water immersion objective), with the slice thickness of stacks between 0.3 and 0.4 µm. All measurements were done with Imaris software using the "measurement points" option in Imaris surpass rendering. Total length of junctions and length of junctions with phenotype were measured for a total of 40-41 ISV per genotype. 5-6 ISVs per fish were measured in 6-7 different embryos of each condition. Ribonuclease protection assay (RPA) Wild type (TuAb) zebrafish embryos were grown in egg water to 24 hpf and were dechorinated before RNA isolation. Embryos were dissociated into single cells in 0.25% trypsin-1 mm EDTA. The cell suspension was centrifuged for 5 min at 1000 rpm, rinsed once with 1X PBS and resuspended in ice cold buffer RLN [50 mm Tris-HCl ph 8.0, 140 mm NaCl, 1.5 mm MgCl 2, 0.5% NP-40] for 5 min. Cells were centrifuged for 2 min at 300 rpm at 4 C and the cleared supernatant contains the Cytoplasmic RNA. The nuclear pellet was washed once with 1X PBS and re-suspended in buffer RLN and kept at 4 C for 5 10 min. Cytoplasmic and nuclear RNA were isolated using the Trizol reagent (Invitrogen). RNA integrity and genomic DNA contamination in RNA was visualized by ethidium bromide agarose gel staining. Two fractions of RNA were created. One RNA fraction was digested with 2 µl each of RNase A (100 µg/µl) and DNase I (2 U/µl) while other fraction was digested with 2 µl of DNase I alone by incubating the RNA samples at 37 C for 2 h. The digested RNA samples were used for cdna synthesis using oligo dt primers and superscript III (Invitrogen). Primer pairs (P1 and P2) were used to detect the overlapping region of anti-sense RNA while a second set of primers (P3 and P4) were designed specific to non-overlapping single stranded region of non-coding RNA. The cdna was PCR amplified using Phusion polymerase (Finnzymes) and probed for specific bands corresponding to tie-1 non-coding RNA. Collagen gel tube formation assay HUVEC were re-suspended (final concentration 1 10 6 ) in a mixture of 1 ml containing rat tail type I collagen (1.5 mg/ml), 1/10 volume 10 M199, and 1M Hepes and neutralized with NaOH. Droplets (0.2 ml each) of the cell/collagen mixture were placed in non-tissue culture dishes and allowed to polymerize for 15 min at 37 C. M199 medium containing either vehicle or recombinant human VEGF (100ng/mL) plus 1% FBS was added to each well and HUVEC s allowed to form tube-like structures for 1 2 d. Tube-like structures were photographed by using Leica DMIL imaging system with QCapture Program (QImaging, Inc, Surrey, BC, Canada), and
total-network length (defined as an elongation of cell into tube-like structures typically seen in 3D cultures) was quantified in five fields for each triplicate per experiment by using the measurement tools provided with NIH ImageJ. Western blotting Endothelial lysates were generated 72 h post transfection and western blots performed with antibodies for tie-1 (Polyclonal, Abcam), hsp90 (Assay Designs, Inc.) and actin (Sigma) as described previously. 37 Bioinformatics analysis RNA secondary structures and energies were predicted using RNAfold and RNAcofold from the Vienna RNA package. 20,21
Table S1. Primer sequences used in this study Name Usage Sequence DU6447265 O1 tie-1 5 RACE AAGATGTCAATGCAAAGTCCCCACAG DU6447265 I1 tie-1 5 RACE GCCCTGAGATATCAATCCTTCCTGAATCTG DU6447263 O1 tie-1 3 RACE GGATTGATATCTCAGGGCATGT DU6447263 I1 tie-1 3 RACE GTTGACCTTGGATTACTCA DU6447265 O2 tie-1as 5 RACE TGAAAGGACACTTCAGATTCAGGAAGGA DU6447265 I2 tie-1as 5 RACE CTCAGGGCATGTTGACCTTGGATTACTCA DU6447263 O2 tie-1as 3 RACE TAATACTGGCGCCTTTGTCC DU6447263 I2 tie-1as 3 RACE CCAGTAAAAAGATGTCAATG ztie1as5 end PCR/probe TAATACGACTCACTATAGGGGAAAATTTTCAACTGAAGAAAACATC ztie1as3 end PCR/probe ATTAACCCTCACTAAAGGGATTTTTTGGCATTGGTGTGGAC P1 RPA GGCTTATGTCAACATGGCTCTC P2 RPA CTTGGGCTATGACCCCTTCT P3 RPA, qpcr TGCATTATCCTTACTGAAAGTCATC P4 RPA, qpcr CTGAAGAAAACATCTTACCAACTGA ztie1f qpcr CATGGAGATCGCTGTCGTAA ztie1r qpcr TGCATTTGCCTTTGTTCTTG ztie2f qpcr AGCACACTCTCCTCACAGCA ztie2r qpcr TTCGCCACAAAGTTCTCTCC zactinf qpcr AATCCCAAAGCCAACAGAGA zactinr qpcr CACACCATCACCAGAGTCCA htie1ex22f1 human tie-1as TGACCAGTCTGACCCTTACAGCCTCTG 5 RACE htie1ex22f2 human tie-1as CTCTGACTTAAGCTGCCTCAAGGAAT 5 RACE htie1ex22r1 human tie-1as GGGGCAGCTGAACTAGAAAG 3 RACE htie1ex22r2 human tie-1as GATAGCTATGAGACACCTACA 3 RACE hcd31f qpcr CCAGTTTGAGGTCATAAAAGGA hcd31r qpcr AGGCAAAGTTCCACTGATCGA htie1f qpcr CCTGAGCTACCCAGTGCTAGA htie1r qpcr TTTCAGAGGCATACTCTTTCAGC htie1asf qpcr CAGCAGACACAGAAAAAGCATC htie1asr qpcr TGGCTAGGACCTCCAGTATGAT mtie1ex23f1 mouse tie-1as 5 RACE GAACATGTCGCTGTTTGAGAACT mtie1ex23f2 mouse tie-1as 5 RACE AAATTGCTTCCACCTGTGACTT mtie1ex23r1 mouse tie-1as 3 RACE GGGGCTAAACAGGGTGTTAGTA mtie1ex23r2 mouse tie-1as 3 RACE TAGGTGGAGGATCAGAAG ztie1mof PCR/MO Efficacy CAAGACCCCATCATATTCACATC ztie1mor PCR/MO Efficacy GTTGCTACATCAGCAGCAAACT Long non-coding RNA sequences for Tie-1 (zebrafish, mouse, and human) zebrafish tie-1as lncrna (shorter) GAAAATTTTCAACTGAAGAAAACATCTTACCAACTGAAGAAAACAACTCATGCACA GTACATCTTGGATGGTCTGAAAATGAGTAAACTAACAGCAAATCTTAATTTTATGAG CAGAACAGATGACTTTCAGTAAGGATAATGCATCGTTTTCTCAAGTGCTACATTTAA TAATACTGGCGCCTTTGTCCATGGTATGAACTGCATTTGATTTATTTACATTTCAAAA ACCTCCCAGTAAAAAGATGTCAATGCAAAGTCCCCACAGACCCGATGCGCTACAAT
GGTAGCTTATATCGTAGGTTCCATTTCCTCTTGAGTAATCCAAGGTCAACATGCCCTG AGATATCAATCCTTCCTGAATCTGAAGTGTCCTTTCACAGTCTGTTCTCTATAGCCAT GTGGCCTTGGTGGAGTGTCTCTTTGAGCGATGATGGAACCTGGCCACAGGGAAGTG ACAGTAACGCAGAGCCCTACTGCGATGAGCCTCCCTTGGGCTATGACCCCTTCTTGG GTCTGGGGGGGCTGGTAGTCAGGCCTCTTCAGCGGTAGCATCTATTCCAGCATAAGT GAAGTTCTCAAAGAGAGCCATGTTGACATAAGCCTAGAATACAAAAAAGAAGTAGT GGTTTGGAGGGAACAGGCAGGGGGAACTGAATAAATAACCTTAATTGGAGCTCAAA TGCAATATGTTTGTGCTACATGAGGAATATTGAAAAACTATCAAATATACGGAAATC TATTCGATTTCATGTCTATTTTTTTTTCCAAAACAGCTTTTAGGGTCCACACCAATGC CAAAAAAAAAAA Mouse tie-1as lncrna TTTTTTTTTTTGGTGATGTTGGTCCATCTTTTTCTTCATTTGCTTTTTTTCTTTCCTTTTT AATATGTTTTTGCTTCTGGTTTTCAGATTCTTCTTCTTCATCTGAACTACTTTCTGGTT TCTTGGTTTTATGCTTAGGAACTTTCTTTGGTGGCTGCAAGTTAATTCCAGCATCTGC TGTCCAATCAGAATAATCACTGGAGTAGTCACTGGAGCTGCCATCACTGTGCCATGC TCGCTCCTCTTCCTCAGAAGTCCCACCACTGACAGCAAGTACTTCACCTTCATCTGA AGAACTAGTTCCGTTTTCTAGCTCCTCTAAGGGCCTGGGTGTTTCTTCCAGTGCAGAT CTTGTACGGTAATTGTGCTGATTCGCTTGCTGCTTTTTAGAATCCCCAAGATCCAGGA AATGCTCATGAGCATGATTCTTTGAGACAGTAAGTATTTTATTCTCTTTTGCAACAGT TAAGTGTTTCCTTTTCTCTTCTGA has-1 TGGCTAGGACCTCCAGTATGATGTTGAATAAAAGTGAGGAGGGCACGTATCCTTGTC TTATTCCTGATGTTAGGGAGAAAGCATTCAGTCTTTTACTATTAAATATGATGCTAAC TGTATTTTTTTTGGTAAACATCTTTTATCAGATTAACAAAGATTCCATTTGCAGTTTG TTAAAGCTTTTTTTTTTTAAATCACAAAGGGTGTTATATTTTGTCAGATGCTTTTTCTG TGTCTGCTGAAATGATCATGTAGTTTTTGTTTTTTAGTCTATTGATATAGTGTATGGA TTTTCAGATGTTAAACCAACCTTGCATTCTTGGGATAAATTCCACTTTATTATGATGT ATAATCTTTTTTTTATGTTTCTGGATTCTTCCTACTAGTGTTTTGTTGAGAATTTTTGC ATCCATATTCATAAGAGATATTGGTCTGTAGTTTTCTTTTCTTGTGCAATCTTTGTCTG TTCTGGGTATCAGGGTAATACTGGCCTCACAGAATAAATTGGGAAGCCTTCTCTCGT TTTTTATTGTTTGGAAAAGTTTGTATAGAGTTTTTGTATTTATTCTTTAAATGTTTGGT AAAATTCACCATTGAAGCATTTGGGCCTGGGCTTTTTCTTTGTGGGTGTTTTTTTTTTT TAATTATTATTACTAATTCAATCTCTTTTTTTGTTTAAGTCTATTTAGATTGGTGAATT CCTTCTGGAGTCAGTTTCAGTAGTTTGTGTATTTTTAGGAATTTATTCATCTCATCTA AGGTTTCTAATTTATAGCCATGCTCCTCACCTTTACTGTTTTTCCTATATAATCTGTTC TCCACAGGGTAGCCAGAGTGACTTTAAAAAAATGCAAATCTCATTATGACTCACCCC TGCTTATAGCTCTTCTGTGGCTCCTCTGCTCTTAGGATAAAGATTCATATCCTATAAG GCTCAGCATAGCCTGGCATCCGTCACCTCTCTGGGTTCATTACCCTGTTTTCCCTTTG CTCCAGCCACAGAGAACTATTTGCAGCTCCTGAACATGCCACATATCACAGGTGTTG TTTCCTGTGCCAGGGTCTCTTCCTTTGGTGAAACCCTGCTTCCTCTTCATGGTCCAGA TTAAAACATCACTTTCTCTGGGAAACTGTTCTGACTCCAGTCCTGTTTAAGCCTTCCC CAGCTTCCCCCGCTCTCTTAGAGCATACACCGCTGTATTTTGTTGCTTGTTAACATAG CTGTCACTTCACAGAGGCATCCTTGTTTACATATTGGTCTCCATCAAAGACAATGGTT TAATCTCCACAGCCTGGACCTGGCAAAATCCCTGCAGTGAATGTGGATCTGAAATGA
AGTTTTATTCTAGCAGTTCCCATCTCCCTCATCCTACGTAGCCTCCCATTTAAATTCA TCCTCCTCCAACCATCCAAGTATTTTCAGAATACAGATCCTAATCATTACCTCCCTGC AGCTTCCTGCATTTGTATATAATGATATCAGCTGACACTGTGTACTTACTATGGCCCA GGCCCAGTGCTGTGAAAGATGGGCCTGTGTGGTCTCATTTAACACAAACAACGAGG TAGCTGTTACTAACTCCATTAATTAGATGAGAAAACTGAAGCTCAGAGACTTGACCA AGGCCACACAGATGATAAGTGACATAGCAGGGACTTGAACACAGGTTGGTGTGATC CTAGAACCTGTGCTCTTCATCACAGTGCCACACCATGTTATGCCTTCTTTGATGTCTT CTGATTCACTTGGCTGGCTGAAGTTGGGAGTGGCCTCTGCCCTCCTCCTTTTGGGCTC ATACATTGGGCAGTGAAGTGGCTACCAAGCCAGGGAAGGCTGCCTTCCTGGCTTCCA GCATGCGGCCTAGCTGTAGCGCAATCTGGGCAAAGGGGGGTCGCTCATAGGGACGG TCCCGCCAGCACTGACGCATCAGCTCGTACACTTCATCGTCACAGTTTCGAGGCTGC TCCATGCGGTAGCCCTGGGGCAGCTTTTCATAGAGCTCGGCACAGGTCATGCCACAG TAGGGTGTACCTCCAAGGCTCACTATCTCCCAAAGAAGGACTCCAAAGGACCAGCT GAAATAAGAAGGATGCACTTAGTTACCTTCTAGGCCCTGGTCCCTAGCAGTTTAGCT has-2 AGGGGAAAACTGGCCTTCTAATCAGGACAGAGGTAGGGGCTGGTGGTGTTTGTGTA AAATGAGTCTTCCAGAGCACCTCCTATCCTGGACAGGCAACCTCAACAGAGGGCTG AGGCCTCAGACAGTGTTTCTTTTCCTAGAAAGCCCAGTTTCTGTCTCTTTTTTTTTGA GACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGTGCGATCTTGGCTCACT GCAAGCTCCGCCTCCCAGGTTCAGGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTG GGACTACAGGTGCCCGCCACCACTTCCAGCTAATTTTTTGCATTTTTAGTAGAGACG GGGTTTCACCATGTTCGCCAGGATGGTCTCAAGCTCCTGACCTCGTGATCCACCCGC CTCGGCCTCCCAAAGTGCTGGGATTACAGGAGTGAGCCACCACGCCCGGCCAGTTTC TGTCTCTTGGGTTTAACTACGCCCCTCACCCCAAGATTGACCAGCAGTAAAAGGTAG ACATTGAATGGAGCTTATTAGAACATTTATTTTTTTCTGAATGACAGGCCGGGAGTG GGAGTAGCTGAACAGGGCATGTTAGTGGTGTGAGTGCTGGCTTAAGCGAAGGAGCT GGAGTGGGGGTTTGTGTTGGGGGAGCAGTGGGATGGGAAACACACCTGTGGGGCAG CTGAACTAGAAAGAAGGATGCCCAGGATAGCTATGAGACACCTACAAAGCATGGGA ACCCAGTTCCCCTAAGCCCACCCCATCCCCAGATCCCTTTTTTTCTCCCTTAAGTTAA AAAAATTCCTTGAGGCAGCTTAAGTCAGAGGCTGTAAGGGTCAGACTGGTCACAGG TTAGACAGCAGAGTTTGCTCCGGCCAGCAGAGCCACGTTCTGGCTGGATGGCAGCTC AGGCCTCCTCAGCTGTGGCATCAATGCCCGCGTAAGTGAAGTTCTCAAACAGCGAC ATGTTCACATAGGCCTGGGGGCAGAGCAAGCTTTAGTGGGGGCCAGGAATGGGGGT GCATCTGGCCTCAAGCTCCTGGACTTCCATGTGGTGGGGGCATTTGAACTGACACTG ATTTGTTCTGGAGACCTAAGATGGCCCCATGGCCAAGAGTGGCCTTTACCAGCCCAG GGCAGTGAAGGCATCAGGCCACAGTTGGGGCCTCAGGTGAGAGGCTCTCAATGCCA TTGTTAGAGGAGGGTTTTTACATGCACTCTTTCAATGAATT has-3 TGGCTAGGACCTCCAGTATGATGTTGAATAAAAGTGAGGAGGGCACGTATCCTTGTC TTATTCCTGATGTTAGGGAGAAAGCATTCAGTCTTTTACTATTAAATATGATGCTAAC TGTATTTTTTTTGGTAAACATCTTTTATCAGATTAACAAAGATTCCATTTGCAGTTTG TTAAAGCTTTTTTTTTTTAAATCACAAAGGGTGTTATATTTTGTCAGATGCTTTTTCTG TGTCTGCTGAAATGATCATGTAGTTTTTGTTTTTTAGTCTATTGATATAGTGTATGGA TTTTCAGATGTTAAACCAACCTTGCATTCTTGGGATAAATTCCACTTTATTATGATGT
ATAATCTTTTTTTTATGTTTCTGGATTCTTCCTACTAGTGTTTTGTTGAGAATTTTTGC ATCCATATTCATAAGAGATATTGGTCTGTAGTTTTCTTTTCTTGTGCAATCTTTGTCTG TTCTGGGTATCAGGGTAATACTGGCCTCACAGAATAAATTGGGAAGCCTTCTCTCGT TTTTTATTGTTTGGAAAAGTTTGTATAGAGTTTTTGTATTTATTCTTTAAATGTTTGGT AAAATTCACCATTGAAGCATTTGGGCCTGGGCTTTTTCTTTGTGGGTGTTTTTTTTTTT TAATTATTATTACTAATTCAATCTCTTTTTTTGTTTAAGTCTATTTAGATTGGTGAATT CCTTCTGGAGTCAGTTTCAGTAGTTTGTGTATTTTTAGGAATTTATTCATCTCATCTA AGGTTTCTAATTTATAGCCATGCTCCTCACCTTTACTGTTTTTCCTATATAATCTGTTC TCCACAGGGTAGCCAGAGTGACTTTAAAAAAATGCAAATCTCATTATGACTCACCCC TGCTTATAGCTCTTCTGTGGCTCCTCTGCTCTTAGGATAAAGATTCATATCCTATAAG GCTCAGCATAGCCTGGCATCCGTCACCTCTCTGGGTTCATTACCCTGTTTTCCCTTTG CTCCAGCCACAGAGAACTATTTGCAGCTCCTGAACATGCCACATATCACAGGTGTTG TTTCCTGTGCCAGGGTCTCTTCCTTTGGTGAAACCCTGCTTCCTCTTCATGGTCCAGA TTAAAACATCACTTTCTCTGGGAAACTGTTCTGACTCCAGTCCTGTTTAAGCCTTCCC CAGCTTCCCCCGCTCTCTTAGAGCATACACCGCTGTATTTTGTTGCTTGTTAACATAG CTGTCACTTCACAGAGGCATCCTTGTTTACATATTGGTCTCCATCAAAGACAATGGTT TAATCTCCACAGCCTGGACCTGGCAAAATCCCTGCAGTGAATGTGGATCTGAAATGA AGTTTTATTCTAGCAGTTCCCATCTCCCTCATCCTACGTAGCCTCCCATTTAAATTCA TCCTCCTCCAACCATCCAAGTATTTTCAGAATACAGATCCTAATCATTACCTCCCTGC AGCTTCCTGCATTTGTATATAATGATATCAGCTGACACTGTGTACTTACTATGGCCCA GGCCCAGTGCTGTGAAAGATGGGCCTGTGTGGTCTCATTTAACACAAACAACGAGG TAGCTGTTACTAACTCCATTAATTAGATGAGAAAACTGAAGCTCAGAGACTTGACCA AGGCCACACAGATGATAAGTGACATAGCAGGGACTTGAACACAGGTTGGTGTGATC CTAGAACCTGTGCTCTTCATCACAGTGCCACACCATGTTATGCCTTCTTTGATGTCTT CTGATTCACTTGGCTGGCTGAAGTTGGGAGTGGCCTCTGCCCTCCTCCTTTTGGGCTC ATACATTGGGCAGTGAAGTGGCTACCAAGCCAGGGAAGGCTGCCTTCCTGGCTTCCA GCATGCGGCCTAGCTGTAGCGCAATCTGGGCAAAGGGGGGTCGCTCATAGGGACGG TCCCGCCAGCACTGACGCATCAGCTCGTACACTTCATCGTCACAGTTTCGAGGCTGC TCCATGCGGTAGCCCTGGGGCAGCTTTTCATAGAGCTCGGCACAGGTCATGCCACAG TAGGGTGTACCTCCAAGGCTCACTATCTCCCAAAGAAGGACTCCAAAGGACCAGAC ATCACTCTTGGTGGTATAGACACTGTAGTTCAGGGACTCAATGGCCATCCAGCGCAC AGGGAGACGCCCCATCGTCTTCTTCACATAAACCTCCTCTCCCCGAGAAAGGCCGAA GTCTGCAATCTTGGAGGCTAGGTTCTCTCCGACCAGCACATTCCGGGCAGCCAGGTC CCTGTGGATGAACTGCTTCTCACTCAGGTACTGCAT
Figure S1. Cloning of zebrafish, mouse and human orthologs of tie-1 antisense long noncoding RNA (tie-1as lncrna) (A) A new vascular EST (DU644726, black rectangle) was initially mapped to 73 bp down stream of tie-1 locus on zebrafish chromosome 6. The primers derived from the EST were utilized to perform RACEs to get the full sequence. (B) Zebrafish tie-1as lncrna was confirmed by RT-PCR. The primers correspond to the introns of tie-1. A miniature version of the tie-1 genomic locus is depicted in reference to the location of tie-1as lncrna. The brown and blue color boxes indicate previously published and novel sequences respectively, which is also indicated in a larger view in panel C. (C) Zebrafish tie-1as lncrna encompasses a 804 bp sequence, which contains a 148 bp unique 5 sequence, followed by a 456 bp region that is complementary to tie-1 s last exon, and a 200 bp 3 region complementary to tie-1s last intronexon junction. The 5 rectangle region in brown color indicates previously published sequence (accession # XM_001334637.2). The rectangle region in blue color indicated new sequences. (D) Two tie-1as s were identified in zebrafish. (E) A part of tie-1as lncrna was identified in mouse E9.5 embryos. (F) Three human tie-1as lncrna (has-1, -2, -3) were identified in HUVECs and HPAECs. The total RNAs were extracted using Trizol followed by DNase I treatment. The RNAs were recovered by RNeasy kit (Qiagen). RACEs were performed by kit (Ambion) or SMART (Clontech). Figure S2. Prediction and confirmation of RNA duplex of tie-1 and tie-1as (A) RNA secondary structures of the tie-1 mrna and its duplex with tie-1as lncrna. These structures were predicted using the Vienna RNA package. (B) The distribution of the energy ratios of tie-1as lncrna and zebrafish mrna duplex to the sum of the singular fold energies. The marker indicates the energy ratio for tie-1as lncrna and tie-1 mrna. (C) The energy distribution of tie-1 mrna and permuted tie-1as lncrna duplexes. The marker indicates the energy of the tie-1 mrna and tie-1as lncrna duplex.