DOI: 10.1038/nc1870 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6 lk3 lk3 MELRVRTGNEKFWTLILLGV----SWIYP-S-AHANVLDTMLQRSSGKVTAGRKETNGGS.D.YR--SCLRWLCVL.I.L----LGLVE-E-NL...S..L.N...EE-.R..--...RQL------L.I.VV.T..----CLLLTLCSGAGQNP.HV..GTGV.LDS--RR--PGD.KST--------F.I.VF..LVAAVLLS.-LAEGGQNP.Y..HGTGV.PGSDPRRAQA.D DSTIAPEDAARFLSCHCSGHCPDDAKNNTCETNGQCFAINEEDENGDVILSSGCMKYEGS G..V...Y...E..T...H...II...H.EAM.T....APAL.---Q...W.Y.YH...E.ST...R.D.Y..TMV..-.G.AAVVT...LGLV.. ATSVI.---E.M.L...YH...E.ST...R.D.Y..TMA..E.G.SPV.T...LSLV.. EFQCRDTGNARLRRILECCTDQDYCNQDLHPTLPPLKTPD-VDSSVHYTALLISAMLSFI...SKQ..A...R...R..SY.VGDI..I...VSVCSF H...K.SQF.QT..TI...Q-F.F...K.E...RDSEP-P--DP.WL.F...VTVCFC H...K.SPN.QT..TI...S-T.F..R..Q...PIPGKPPFWNA.LL.F...VTVCCF ILGVIMFYCYFRYRRQGTQTRYSMGLEQDESFIPAGESLRDLIEQSQSSGSGSGLPLLVQ..TF.IIF...K.HELAP...L..HP... A.ICVTVI..Y..KW.TERQ..HRD...A...K...N...T... T.VA.TIV..Y.FKL.TGRRH.QRD.GPI.A...K...S..-T... RTIAKQIQMVKQIGKGRYGEVWMGRWRGERVAVKVFFTTEEASWFRETEIYQTVLMRHEN...K...D....R...T.RM...L...K...R......H...R...L...N...R... ILGFIAADIKGTGSWTQLYLITDYHESGSLYDYLKSTTLDTRALLRLSYSAVSGLCHLHT...N...C...S...K.A..S......N...AS...N...F...Q...AF..AC......TF...F...N...C...Y...Q...A...AC... EIFGTQGKPAIAHRDLKSKNILVKKNSTCCIADLGLAVKFISDTNEVDIPPNTRVGTKRY...R.GA...L...F..Y...I...G...N...L.LS..M..R....Y...I...G...YN...V.LS..M..R.Y MPPEVLDESLNRCHFQSYIMADMYSFGLILWEISRRCVSGGIMEEYQLPYHDLVPSDPSY.A...T...N...T...V...H..N....A...T..KN...A...I..Y..VI..MA...T...V...HV..YEM....A...T..KNQ..A...I..Y...V..MA...T...V...W.M...E... EDMREVVCIKRQRPSFANRWSSDECLRQMGKLMSECWAHNPASRLTALRVKKTLAKMSES...I...P...T......L...V.GL..TVS...N...A.L...I...V.....V.SM..VVS...N...V.L...S...V.. lk6 lk6 lk3 lk3 QDIKL...V...I...I humn ACVRL1 (Alk1) humn ACVR1 (Alk2) humn BMPR1A (Alk3) zerfish Alk6 humn BMPR1B (Alk6) humn ACVR1B (Alk4) humn TGFBR1 (Alk5) humn ACVR1C (Alk7) 43 42 66 81 49 49 47 Figure S1 Alk6 is n lk6 co-orthologue. : Sequence comprison to Alk3, Alk3, nd Alk6. Dots indicte identicl residues. Alignment performed with PROBCONS 1. : Multiple sequence lignment of Alk6 with humn TGF- type I receptors performed with CLUSTALW2 (ref. 2). Scores re mesure of similrity to zalk6. www.nture.com/nturecelliology 1 2009 Mcmilln Pulishers Limited. All rights reserved.
lk3 lk3 lk6 lk6 lk3 lk3 lk6 lk6-120 TGAGCCGTAT TTTTGATTGC TCCATCTCTA TGTTAGTGCT TGCTGACATA TTAGGAGCTT AATCAGACAT TCTTCATACT CTTACCGTGT CCATTCATCT TCACAACTGA TTGGATACTT CAGTAATTCA AAAGTGGTGC AGTCGGTGTG AAGCTGTGAT CGAGGCTGCT GTGGGGTGTG CAGTTTCTCA CATTCTGTAG AGGACACACA AAACACGGAC TGAGTGACAC ACAGGCTGCC GTCAGTGTGG AGTAGCTGTG GAGCAGTGTG TGTGTGTGTG - CATCCCTGAT CTCATCCTTA TCCCAAAGAT TACGACAAGA CAATTTGACA ATGCGTCAGC TTTTGTTCAT AGTACATCAC AACCAAAACA GCCATACAGT TCAACAACTC GACCTTCATG ATGAAAAGCA CCTTCACGAT CACATAGAGC TGAGGGATTC TCTGGCTCAC TGGAGTTCTG CAGACGCACG ATGGATCTCT ATAGGAGCTG AGAGTGTTTC CATGCCCCGG TGGTGCGGGC GGCCGGCTCC AGTCTCAGGG ATGGAGCTCC GGGTACGGAC lk3 MO lk3 MO1 lk3 MO3 lk6 MO lk6 MO1 lk6 MO2 receptor epitope Alk3-FLAG Alk8-FLAG Alk3-HA Alk8-HA...QDIKIRFKAMDYKDDDDKDYKDDDDKNSRPLEPLEL...GKTDCRFKAMDYKDDDDKDYKDDDDKNSRPLEPLEL...QDIKIYPYDVPDYA...GKTDCYPYDVPDYA linker pro domin epitope linker lignd FLAG-Bmp2 HA-Bmp2 FLAG-Bmp7 HA-Bmp7...REKRQA GGG DYKDDDDK GGG ARRGQK......REKRQA GGG YPYDVPDYA GGG ARRGQK......NAKPGA GGG DYKDDDDK GGGAA HSQVAL......NAKPGA GGG YPYDVPDYA GGGAA HSQVAL... Figure S2 Morpholinos nd epitope-tgged constructs. : Morpholinos ginst lk3/6 genes trget dissimilr, independent sequences. Binding sites re shown reltive to ATG strt of trnsltion (nucleotides +1 to +3, rrow). : Protein sequences of epitope-tgged receptors nd lignds. 2 www.nture.com/nturecelliology 2009 Mcmilln Pulishers Limited. All rights reserved.
WT C1/C2 C3 % 80% 60% 40% C4 C5 20% MO lk3 (ng) 8 6 6 6 6 6 6 4 4 4 4 lk3 (4ng) lk6 (5 ng) lk6 (ng) 4 2 2 2 2 n 76 140 72 79 111 74 227 411 178 106 39 WT MOs: 3+3 e otx2 c f dlx3;gsc 12/ 12 11/11 d g foxd3 8/8 d ` g ` n lk3 lk3 MO: lk6 lk6 P-Smd5 h 14/17 12/13 i j 11/13 j ` Actin 3+3+6 k 9/14 12/14 l m 8/19 11/19 3+3+6 13/13 12/12 11/14 Figure S3 lk3/ nd lk6/ ct redundntly during DV ptterning. : 4-somite stge emryos, lterl views, dorsl right, nterior up, of WT, mildly (C1/C2), modertely (C3), strongly (C4), or severely (C5) dorslized phenotypes. Br grph displys phenotypes cused y MOs ginst lk3, lk3, lk6, nd lk6. -m: whole mount in situ hyridiztion of WT (-d) or MO treted (e-m, MO mounts s in Figure 1-l) emryos t midgstrultion (% epioly, niml pole views with dorsl right) for dorsl mrker otx2 (nterior neurl:, e, h, k) or ventrlly expressed dlx3 (with gsc expression in midline mesoderm s positive control: c, f, i, l), or t three somite stge (oriented nterior up; d, g, j, m: lterl views; d, g : dorsl views) for foxd3 expression in crnil neurl crest (CNC, specified y intermedite BMP signling levels), showing expnded specifiction in the posterior (rrowheds in g, j) or rodly in the nterior (j ) upon prtil reduction of signling. CNC re lost upon elimintion of signling (m). Scle rs represent 0.2 mm. n: P-Smd5 levels t midgstrultion (% epioly) re reduced y lk3/6 MOs (6 ng lk3 MO, 4 ng lk3 MO, 5 ng lk6 MO, 2 ng lk6 MO). www.nture.com/nturecelliology 3 2009 Mcmilln Pulishers Limited. All rights reserved.
80% lk8/+ X lk8/+ 80% lk8/+ X lk8/+ c 80% WT X WT C5 60% 60% 60% C4 40% 40% 40% C3 20% 20% 20% C2 lk3 MO (ng) +/+ lk8 +/+ lk8 nd nd lk8/+ lk8 lk8/+ lk8 n 97 72 24 66 19 Genotype: All 6 6 8 8 lk3 RNA (pg) 0 10 20 n 121 216 65 lk3 MO + + + + lk3 MO + + lk6 MO + + lk6 MO + + lk8 RNA n 57 59 95 53 C1 WT Figure S4 Alk3/6 nd Alk8 ct nonredundntly nd independently. : Emryos from crosses of lk8 heterozygotes injected with 8 or 6 ng lk3 MO, scored for phenotype, then genotyped. : zlk3 mrna does not rescue lk8 zygotic mutnts. c: lk8 mrna ( pg) cnnot rescue lk3/6 knockdown (6 ng lk3 MO, 4 ng lk3 MO, 5 ng lk6 MO, 4 ng lk6 MO2). 4 www.nture.com/nturecelliology 2009 Mcmilln Pulishers Limited. All rights reserved.
5% input Alk3-HA + Alk3-FLAG + + + + IP: + + + -HA + + + + + WT 2-/- 7-/- WT WT WT 2-/- 7-/- 5% input Alk8-HA + Alk8-FLAG + + + + IP: + + + -HA + + + + + WT 2-/- 7-/- WT WT WT 2-/- 7-/- IB: -FLAG 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1% input 5% input IB: -HA Figure S5 Alk3 nd Alk8 form lignd-independent homo-oligomeric complexes. Anti-HA IP followed y nti-flag IB from lystes of crosslinked WT, Bmp7 protein null, or mp2 MO treted emryos expressing either Alk3-HA nd Alk3-FLAG () or Alk8-HA nd Alk8-FLAG (). www.nture.com/nturecelliology 5 2009 Mcmilln Pulishers Limited. All rights reserved.
Figure 1m Figure 3 Figure 3 20 15 Figure 5 Figure 5 Figure S6 Full gel scns of western lots. Brs indicte sizes of moleculr weight stndrds in kilodltons. Closed rrowheds denote unprocessed Bmp7 or Bmp2 proproteins. Under nonreducing conditions, Bmp2 proprotein is poorly detected. Bmp7 homomeric complexes sometimes pper s high moleculr weight species (open rrowheds). 6 www.nture.com/nturecelliology 2009 Mcmilln Pulishers Limited. All rights reserved.
Tle S1. Numer of emryos processed for co-ip. Figure 3 lne # Numer of emryos Figure 5 lne # Numer of emryos Figure S5 lne # Numer of emryos 2, 3 2-4 2 4-8 2 1, 4, 5 8-10 2 4-8 1, 2 2 5-9 3 4 0 2009 Mcmilln Pulishers Limited. All rights reserved.
Supplementl References 1. Do, C. B., Mhhshym, M. S., Brudno, M. & Btzoglou, S. ProCons: Proilistic consistency-sed multiple sequence lignment. Genome Res 15, 330-40 (2005). 2. Lrkin, M. A. et l. Clustl W nd Clustl X version 2.0. Bioinformtics 23, 2947-8 (2007). 2009 Mcmilln Pulishers Limited. All rights reserved.