example, shown toplayaroleinthedevelopment ofbothcelllineages:for the hemangioblast.Anumberoftranscriptionfactors have been originate fromacommonprogenitorcell,whichhasbeentermed hematopoietic andendothelialcells,ithasbeenspeculatedthat they 2002). Given theclosephysicalproximity ofthevery earliest budding fromtheendotheliumofdorsalaorta(de Bruijnetal., definitive hematopoiesis–hematopoieticstemcellscanbedetected -gonads-mesonephros region –theinitialembryonic siteof angioblasts surroundingprimitive erythrocytes. Similarly, inthe the formationofmaturevascular network. vasculature occursviapruningofunneededbranches,resultingin (Risau,1997).Furthermaturationofthisnew endothelial cellsandvessels aregeneratedlaterviaaprocess called the vitellinearteryandvein, andtheendocardialtubes. New network, whichconsistsofpaireddorsalaortae,thecardinalveins, , resultsintheformationofinitialvascular blood islandsoftheyolksac.Thisprocess,known as vasculature develops fromangioblaststhatareassociatedwiththe functional priortolymphangiogenesis.Themurineblood are organized inparallel,thebloodvasculature develops andis and lymphaticsystems.Althoughthebloodsystems In vertebrates, thevascular network iscomposedofseparateblood INTRODUCTION Scl hematopoietic progenitorsinzebrafish (Stainieret al., 1995)and Accepted 26February 2007 ‡ † 3 1 KEY WORDS: regulatory hierarchygoverning been ascribedthepropertyofregulatingexpressionnumerousendothelium-specificgenes.However, thetranscriptional GATA-2, atranscriptionfactorthathasbeenshowntoplayimportantrolesinmultipleorgansystemsduringembryogenesis, Melin Khandekar regulation A (2007)doi:10.1242/dev.001297 Development 134,1703-1712 USA *These authorscontributedequallytothis work Genetics, UniversityofMichiganMedical School,AnnArbor, MI48109-2200, USA. Ritsuko Shimizu cells, andthepan--specificexpressionof bind thecrucialEboxinenhancervitro.Thus,GATA-2 isexpressedearlyinlymphatic,cardiacandbloodvascularendot in transgenicmice.DevelopmentoftheendotheliumisknowntorequireSCL(TAL1), andanSCL-E12(SCL-Tcfe2a) heterodimercan within theenhancerdiminisheditsactivity, theablationofitssingleEboxextinguishedendothelialenhancer-directed expres enhancer isrestrictedtotheendocardial,lymphaticandvascularendothelium.WhereasdisruptionofthreeETS-bindingmotifs transgenic analyses,weidentifieda endothelial expressionduringembryogenesisbyfollowingGFPin Author forcorrespondence (e-mail:[email protected]) Present address: Aveo Pharmaceuticals, 75 SidneyStreet, Cambridge, MA02139, TARA Centre, UniversityofTsukuba, 1-1-1Tennoudai, Tsukuba 305-8577, Japan. Department ofCellandDevelopmentalBiology In theyolksac,bloodislandsconsistofathinlayer (also known as Gata2 cloche Gata2 is requiredfortheformationofendothelialand , Endothelium,Cardiovascular, Lymphatic, Enhancer, ETS,SCL,Mouse intronic enhancerconfersitspan-endothelia-specific 1,3 Tal1 1, , MasayukiYamamoto *, WilliamBrandt – MouseGenomeInformatics),which Gata2 Gata2 activation inendothelialcellshasnotbeenfullyexplored.Here,wedocumentGATA-2 2 Department ofHuman 1, endothelium enhancerinthefourthintronandfoundthat *, Yinghui Zhou 3 , Kim-ChewLim Gata2 is controlledbyadiscreteintronicenhancer. 1,† , SusanDagenais lymphatic vessels afterE14.5(Kaipainen etal.,1995). during earlyembryogenesis, but becomeslargely restrictedto which bindsVEGFC,isdetected inbloodandlymphaticvessels Lmo2 et al.,1994).Analysisofchimericmicebearingcontributions from embryonic day(E)9.75secondarytohematopoieticfailure (Warren erythropoiesis intheembryo; member oftheLIMdomainfamily, isrequiredforprimitive Scl consequences of Transgenic expression ofSCLisabletorescuethephenotypic and morphogenesisofendothelialcells(Lazraketal.,2004). vasculogenesis (Patterson etal.,2005),aswellinthemigration (Visvader etal.,1998),andithassincebeenshown toplayarolein requirement forSCLintheremodelingofyolksacvasculature of thehematopoieticdefectin et al.,1995;Shivdasani etal.,1995).Subsequenttransgenicrescue shown toberequiredforhematopoieticdevelopment inmice(Robb encodes abasichelix-loop-helixtranscriptionfactor, was initially 3 ( Expression ofthemurinevascular endothelialgrowth factor LYVE1 , isexpressed several hours laterinasubsetof lymphatic regulatory gene competent tobecomelymphaticendothelialcells(LECs). The the anteriorcardinalvein isthefirst signthatthesecellsare at E9-9.5inendothelialcellslining – MouseGenomeInformatics) endothelial hyaluronanreceptorgene( 1904; Wigle andOliver, 1999).Expressionofthelymphatic vasculature arisesfromthebloodvasculature (Sabin,1902;Sabin, for lymphaticdevelopment hasproposed thatthelymphatic 1996). angiogenesis andhemorrhagingthroughouttheCNS(Wang etal., eliminates definitive hematopoiesis andresultsindefective Similarly, targeted disruptionofthetranscriptionfactor remodeling ofbloodvessels requires 1 and JamesDouglasEngel The most-widelyacceptedandexperimentally supportedmodel functions downstream of –/– + Vegfr3 embryonic stem(ES)cellsrevealed thatangiogenic cells intheanteriorcardinal vein (Oliver, 2004). Gata2 , alsoknown as -GFP knock-inembryos.Usingfounder cloche 2 , ThomasW. Glover mutation inthezebrafish, suggestingthat cloche Flt4 Lmo2 Prox1 1,‡ – MouseGenomeInformatics), Scl RESEARCH ARTICLE Gata2 (Liao etal.,1998).LMO2,a Lmo2 , encodingahomeobox ablation resultsindeathat -null embryosrevealed a Lyve1 regulation bythis 2 (Yamada etal.,2000). ; alsoknown as , NorioSuzuki Runx1 helial Xlkd1 sion 1703 3 ,

DEVELOPMENT expression continuedpostnatally. We alsoobserved GFP expression vessels formedduringvasculogenesis andangiogenesis,thatits was expressed incellsliningarterialandvenous We foundthatGFP vasculature of we systematicallyexamined GFPexpression inthedeveloping leaving theroleforGATA-2 in endothelialfunctionundefined. time oftheirearlyembryonicdemise(~E10)(Tsaietal.,1994), embryos failed toreveal any obvious defectsinthevasculature atthe al., 2000).Surprisingly, however, theanalysisof Vegfr2 abolished itsactivity intransgenicreporterassays,indicatingthat 2000) and site inthe (also known as al., 1997), (Dorfman etal.,1992;Yamashita etal.,2001), specific genespreproendothelin(immatureform ofEDN1) sites have beenimplicatedintheregulation oftheendothelium- GATA-binding sites(Dorfmanetal.,1992).For example, GATA crucial forendothelialdevelopment andfunctionareregulated via expectation was earlyevidence thatmany genesthatappearedtobe function would resultinvascular defects.Adding furthertothis endothelial celllines,itwas originallybelieved thatlossofGATA-2 in vascular isstronglyexpressed development in andthatGATA-2 conclusions fromtheinitialloss-of-functionexperiments. 1993; Kitajimaetal.,2002;TsaiandOrkin,1997),underscoringthe proliferation ofvery earlyhematopoieticprogenitors(Briegel etal., of examination of due toablockinprimitive hematopoiesis(Tsaietal.,1994).Further endothelial cells. a widevariety oftissues,includinghematopoietic,neuronaland library (Yamamoto etal.,1990),andwas shown tobeexpressed in Molkentin etal.,2000;Morrisey etal.,1998). developmental events (Molkentin, 2000;Molkentin etal.,1997; involved incardiac,genitourinary andmultipleendodermal Similarly, GATA-4, GATA-5 andGATA-6 have beenshown tobe activities (Pandolfi etal.,1995;Pevny etal.,1995;Tsai1994). progenitor andT-lymphoid, respectively –amongmany other of different hematopoieticlineages–erythroid, hematopoietic GATA-1, GATA-2 andGATA-3 areallimportantinthedevelopment which have historicallybeensubdivided intotwo subfamilies. in development. TherearesixGATA family membersinvertebrates, zinc-finger transcriptionfactors thatplaydemonstrablycrucialroles Prox1 of humanhereditary-lymphedemacausative genesindicatethat development, but gene-ablationstudiesinmiceandtheidentification Little isknown aboutthemolecularevents leadingtolymphatic (Oliver, 2004;Oliver andAlitalo,2005;Sabin,1902;1904). and maturationintheterminalstagesoflymphaticdevelopment Oliver, 1999).Finally, thelymphaticplexus undergoes remodeling sprout andspreadintothesurroundingtissuesorgans (Wigle and eventually fusetoformprimitive lymphsacsfromwhichnew LECs anterior cardinalvein inapolarizednon-randommanner, and 1704 2004; Petrova etal.,2004). Irrthum etal.,2003;Karkkainen2000; (Wigle etal.,2002;Wigle andOliver, 1999;Fang etal.,2000; To begin toinvestigate theroleofGATA-2 inendothelialfunction, Given thatmany genesinvolved inhematopoiesisalsoparticipate GATA-2 was originallyclonedfromachicken reticulocyte cDNA Beginning atE10.5,LECsbud andmigrateaway fromthe GATA factors belongtoanevolutionarily conserved family ofC Gata2 , expression isdependentonGATA activity invivo (Kappelet Vegfc RESEARCH ARTICLE –/– Icam2 Vegfr2 ES cellsshowed thatGATA-2 playsapivotal roleinthe , Vegfr2 Vegfr3 Gata2 Nos3 Gata2 (Cowan etal.,1998).MutationofaGATA-binding Gata2 (Kappel etal.,2000;Minami2004), , -GFP knock-inembryosduringembryogenesis. – MouseGenomeInformatics)(Germanetal., endothelium-specific enhancercompletely Foxc2 gain-of-function andinvitrodifferentiation -null mutantembryosdieatmid-gestation and Sox18 are requisitestotheprocess Pecam1 (Gumina et Gata2 eNOS -null 4 (5 SCL bindingingelshiftassays(Kappel etal.,2000).PrimersEcSCLmut(f) 5 mutagenesis was performedtointroducemutations(from5 verified bysequencing. 167 bpamplicon(defined bythearrows inFig.4A,5A),whichwas to generatea3 previously (Zhouetal.,1998). photographed aswhole-mountorcryosectionedspecimensdescribed as previously described(Zhou etal.,1998).Transgenic embryoswere d16Z transgeniclinewereharvested forX-galstainingandPCRgenotyping 2004). Attheindicatedtimes,embryosfromfostermothersora microinjection intofertilizedova aspreviously described(Khandekaretal., described (Khandekaretal.,2004). indicated inthefigure legends. Digitalimageswererecordedaspreviously using theappropriatefluorochrome-conjugatedsecondaryantibodies,as Covance) andrabbit anti-LYVE1 (1:400;Upstate)antibodieswas detected goat anti-VEGFR3(1:20;R&DSystems),rabbitanti-PROX1 (1:800; GFP (1:1000;MolecularProbes),ratanti-PECAM(1:200;Pharmingen), as previously described(Khandekar etal.,2004).Reactivity torabbitanti- indicated inthetext andfigure legends, andprocessedforimmunostaining in mice, whichhadGFPinserted(inframe)atthetranslationinitiationcodon Wild-type CD1micewerematedwith Transgenic mice endothelium-specific enhancerin postnatal lymphaticvessels. We thenfunctionallyidentified an in budding LECsduringearlylymphaticdevelopment, aswellin natural positioninthe thymidine kinase(TK)genepromoterinTK fragments examined herewerecloned3 restriction enzymes(seelegend toFig.3)(Zhouetal.,2000).Other For microinjection,plasmidGR22- Expression-plasmid construction activators ofendothelium-specific in vivo Altogether, thesedataimplicateETSfamily membersandSCLas bind withhighaffinity tothiscrucialenhancerEboxinvitro. known asTCFE2A–MouseGenomeInformatics) heterodimers of thevasculature, andwedemonstratethatSCL-E12 (E12isalso experiments showed thatSCLactivation isrequiredforelaboration quantitatively reduced,but didnotabolish,enhanceractivity. Prior motif. Bycontrast,disruptionofthreeETS-bindingsites specific enhanceriscruciallydependentonanEbox(CANNTG) specific mutagenesis,thatthepotency oftheminimalendothelium- and lymphaticendothelialcells.Additionally, wefound,usingsite- the expression ofacis-linked reportertransgenein cardiovascular (Stratagene) andthenexcised with Sal MATERIALS ANDMETHODS TK polymerase beforeself-religation.To constructTKAA site (5 Fig. 5),primersEndocons(f)andEndocons(r)containinganengineered endothelium-specific (VE)enhancerintoTK sequentially with polymerase beforebeingclonedintoTK Apa ACTGAGTCGAGGTGGCTCTG-3 was clonedintoTK To deletetheinternal TKAN polymerase beforeself-religation.Theresultant0.8kbp the 1.2kbp Ј Ј -CAcccG-3 Gata2 To mutatetheEboxinVEenhancer, oligonucleotide-basedPCR For foundertransgenicanalyses,expression constructswerepurified for -CGGACAcccGCAGCCG-3 I fragmentfromplasmidGR22was first subclonedintopBluescriptII ␤ I fragmentwas excised fromplasmidGR22andtreatedwithT4DNA . To generateTKSX Ј ␤ -ggtctagaCCATGGAGTCACCTATACTGTG-3 was generatedfromTKAA exon 2(Suzukietal.,2006).Embryoswereharvested atthetimes Sfi Ј Ј ; mutationsarelowercase) thathadbeenshown toeliminate I- fragment usingGR22 plasmidastemplateinaPCRreaction. Xba Xba I ␤ Alw Gata2 Gata2 I andwithKlenow polymerase.For microinjection, to generateTKSX ␤ NI- , TKBX 4was treatedwith Apa locus. To generateTKBX Ј ) andEndocons(r)(shown above) wereused I fragment,aplasmidsubclonecontaining Xba ␤ ␤ was treatedwith Ј , respectively) wereusedtogeneratea Gata2 by I beforere-cloninginto lacZ Gata2 Ј Nco to theherpessimplex virus(HSV) ⌬ ␤ was digestedwithdifferent I restriction-enzymedigestion. AA intron 4thatcouldregulate ␤ -GFP knock-inheterozygous ␤ (thereby generatingTKVE , whichhadbeentreated Gata2 (Clontech) tomimictheir ␤ . To clonethevascular Development 134(9) Spe ␤ Alw transcription. ␤ , a2.9kbp Sfi Ј I- , a460bp and 5 Ј Sfi I- NI- -CATCTG-3 Xba I andT4DNA Xba Apa Gata2 Ј I fragment -ggtctaga - I-digested I andT4 Bam Alw Gata2 YAC Xba NI- HI- Ј ␤ to I ,

DEVELOPMENT transfected or withEF-1 2000) from293T humanembryonickidney cellsthatwereeithermock Nuclear extracts wereprepared asdescribedpreviously (Tanimoto etal., Electrophoretic mobility shiftassay CGcg-3 to cloningintothe verify incorporationofthemutationandwerethendigested with Endocons(r) asprimers.Thegel-purified PCRproductsweresequencedto purified andpooledastemplatesinaPCRreactionusingEndocons(f) were usedtogeneratea5 Similarly, EcSCLmut(r)(5 A al., 2003)intoallthreeETS-bindingsitesinthe TTGCCGGcgCGGAAACA-3 cgCATCTGCAG-3 and thenclonedintothe either thefirst two orallthreeETS-bindingmotifsweredigestedwith incorporation ofthedesiredmutations.Fragmentscontainingmutations in as describedabove, andtheenhancerwas thensequencedtoverify primers Ecmets1(f)(5 3 not shown) orTKVE (5 Ј Ј ), Ecmets3(f)(5 Gata2 A similarstrategy was usedtointroduceamutation(5 -ATAGGAAACcgCGGCAGGAG-3 Ј ) thathadbeenshown toeliminateETS-factor binding(O’Reillyet endothelial enhancer Xba Ј Ј -TGTTTCCGcgCCGGCAA-3 ), Ecmets2(r)(5 ␤ I siteofTK mEts1,2,3 (Fig.5),respectively. Ј Xba -CTCCTGCCGcgGTTTCCTAT-3 Ј Ј -CGGCTGCgggTGTCCG-3 I siteofTK fragment. Theresultantampliconsweregel- Ј ) wereusedtomutagenizetheVEenhancer, ␤ to generateTKVE ␣ Ј -CTGCAGATGcgCGGA TAGGAA- rmtrdrce C cDNA alone promoter-directed SCL ␤ Ј ), Ecmets2(f)(5 to generateTKVE Gata2 Ј ) andEcmets3(r)(5 ␤ mScl (Fig.5). Ј Ј VE enhancer. The ] andEndocons(f) Ј -TTCCTATC CG- -CGGA-3 ␤ Ј ), Ecmets1(r) mEts1,2 (data Xba Ј I prior to 5 Xba Ј Ј - - I in thelegend toFig.6.After1hourofpre-incubation,2 lowercase)] orantibodies(Rodriguezetal.,2005)wereaddedasindicated (Molecular Dynamics). electrophoresis, thegelsweredriedandrecordedusingaPhosphorImager electrophoresis onneutral6%TBE/polyacrylamidegels.After incubated foranadditional30minutes.Allsampleswerefractionated by radiolabeled wild-typeoligonucleotideprobewas addedtoeachsampleand we first investigated whetherornot endothelial celllines(Dorfman etal.,1992;Umetani2001), hadbeenreportedinseveral Because theexpression ofGATA-2 factor GATA-2 Early, pan-endothelialexpression oftranscription RESULTS or nuclearextract (35 or withaCMVpromoter-directed E12expression plasmid.Eithernoextract embryos. expression in Fig. 1.EndothelialGATA-2 (5 at 4°C.Unlabeledoligonucleotides[20-or200-foldmolarexcess; wildtype TCCGGACAcccG HEPES (pH7.9),1mMMgCl artery; cv, cardinal vein;da,dorsalaorta. ventricle; mv, mesencephalicvesicle;uv, umbilicalvein;ua, R were co-labeledwithDAPI.h,;tb,tailbud;as,aorticsac;ven, vessels thatstainedonlyforGFP(arrows). ThenucleiinpanelsL,Oand of staininginlymphaticvessels(arrowheads) isdistinctfrom blood (P)andGFP(Q)expression asdescribedabove.Coincidence for VEGFR3 P1postnatalintestinesandmesenterywere sectionedand stained (P-R) nuclear staining(arrowhead), thusconfirmingtheirLECidentity. respectively. NoticethatVEGFR3-positivecellsdisplayedanti-PROX1 antibodies usingCY2-orCY3-conjugatedsecondaryantibodies, adjacent sectionwasco-stainedwithanti-PROX1andanti-VEGFR3 weakly, incomparisontoLECs,withanti-VEGFR3antibody. (O)An dorsal aortaandcardinal vein,bothofthesebloodvesselsstainedonly GFP immunofluorescence wasdetectedstrongly inendotheliaofthe expressed bothVEGFR3andGFP(arrowheads). Noticethat,although respectively. Clustered cellsinthevicinityofanterior cardinal vein VEGFR3 (M)usingCY2-orCY3-conjugatedsecondaryantibodies, Transverse embryoniccryosectionswere stainedforGFP(N)or (M,N) endothelial cells(I,L).Boxedareas inG-Iare magnifiedinJ-L. PECAM andanti-GFPstainingdemonstratesthat conjugated secondaryantibodies,respectively. Coincidenceofanti- (G,J) orPECAM(H,K)antigensusingCY3-AlexaFluor488- vessels andtheaortaintailregion ofanembryoco-stainedforGFP angiogenesis (D).(E)Aphase-contrastimageofF. (G-L)Theintersomitic begin toinvadetheneuraltube,atypicalexampleofsprouting the oftheheartventricle(C),aswellinvesselsthat thin-walled umbilicalveinandthick-walledartery(E,F),in region ofanembryo,intheendothelialiningaorticsac(C), was detectedintheintersomiticvessels(B,arrowheads) inthetail Alexa Fluor488-conjugatedsecondaryantibody. GFPimmunoreactivity Transverse embryoniccryosectionswere stainedforGFPusing (B-D,F) its head(notshown)facingtotheleft,towards thetailbud. vasculogenesis (arrowhead), ofawhole-mountembryoorientatedwith visualized intheheartanddorsalaorta,avesselformedby (B-D,F-R) orlight(E)microscopy. (A)RobustGFPfluorescence is was monitored bydirect fluorescence (A),indirect immunofluorescence E10.5 (B-D,G-L),E11.5(M-O),E18.5(E,F)andpostnatalday1(P1;P-R) 2006). Inwhole-mount E9.5embryos,robust GFPfluorescencewas vasculature of inthe endothelial cellsinvivo byanalyzingGFPstaining Ј -TCCGGACATCTG ( A-R ) GFPexpression in Gata2 CAGCCGGT-3 ␮ CAGCCGGT-3 -GFP knock-inheterozygotes(Suzuki etal., g) was addedtobindingbuffer containing20mM 2 , 0.5mMDTTand37.5ng/ Ј ; Eboxunderlined,mutatednucleotides Gata2 Ј RESEARCH ARTICLE ; Eboxunderlined)ormutant(5 -GFP embryosatE9.5(A), Gata2 Gata2 was expressed inall Gata2 -GFP knock-in ␮ l (2 is expressed in ␮ ϫ l poly(dI-dC) 10 5 cpm) of 1705 Ј -

DEVELOPMENT appeared tobeweakerintheumbilicalvein(uv)thanartery(ua). remodeled vasculature oftheyolksac(C),andinembryoproper (D,E),aswellintheumbilicalvessels(E),where thes (B) specimens.Strong d16 GFP initially sprout(Fig.1M,arrowheads). To confirm furtherthat the scattered cellslyingnearthecardinalvein, fromwhereLECs However, VEGFR3immunoreactivity was mostpronouncedin lying neartheanteriorcardinalvein (Fig.1N,arrowheads). the cardinalvein anddorsalaorta,aswellinclusteredcells (Fig. 1N)antibodies.GFPfluorescencewas detectedstrongly in embryo werestainedwithanti-VEGFR3(Fig.1M)oranti-GFP vasculature, serialtransverse cryosections ofanE11.5 endothelial cells. verifying that immunofluorescence werecompletelycoincident(Fig.1I,L), antibody (Fig.1H,K),theanti-GFPandanti-PECAM PECAM antigenusingAlexa Fluor488-conjugated secondary When thesamesectionswereco-stainedfordetectionof vascular structuresinthetailbud region stainedstrongly(Fig.1G,J). an anti-GFPantibodyandaCY3-conjugatedsecondaryantibody, molecule. WhenE10.5embryoniccryosectionswerestainedwith against GFPandPECAM,anendothelialcell-specific cell-adhesion specific, weperformedco-immunostainingwithantibodiesdirected resulting fromthegreatersurface areaofthevein. expression level, orsimplyduetoa difference intheopticaldensity differential GFPstainingmightbeduetoagenuinedifference in could bereadilyvisualizedinbothbloodvessels (Fig. 1F).The difference inthefluorescenceintensity, GFPimmunoreactivity andartery(Fig.1E).Althoughtherewas anapparent which aclearmorphologicaldistinctioncanbemadebetween and arteries,weanalyzedtheumbilicalcordsofE18.5embryos,in during primaryvasculogenesis andangiogenesis. showed thatGATA-2 isabundantly expressed invessels formed (Fig. 1D),asiteofintensesproutingangiogenesis.Thesedata ventricular endocardium(Fig.1C),andthedeveloping neuraltube intersomitic vessels (Fig.1B,arrowheads), theaorticsacand of E10.5embryos,GFP-immunopositive cellswereseenliningthe well asinthedeveloping heart(Fig.1A).Intransverse cryosections detected inthedorsalaorta,avessel formedbyvasculogenesis, as Fig. 2.YAC d16Zcontains 1706 blood andlymphatic vasculatures oftheintestineand arrowhead). Similarly, GFP-expressing cells wereidentified inthe which was locatednear theanteriorcardinalvein (Fig.1O, immunofluorescence co-localized inthesamecellpopulation, (Fig. 1O).Nuclearanti-PROX1 andcytoplasmic anti-VEGFR3 co-stained fortheLEC-specific markers VEGFR3andPROX1 To investigate whether To verify that the GFPexpression was indeedendothelium- To determinewhether lacZ + /VEGFR3 RESEARCH ARTICLE -tagged yeastartificialchromosome (Zhouetal.,1998)were stainedfor Gata2 + cells wereindeedLECs,asecond sectionwas lacZ is expressed quitespecifically invascular Gata2 expression wasobservedinthedevelopinghearttube(arrowhead, A),intheaortaandendocardium (ec;B),inthe Gata2 Gata2 was expressed differentially inveins endothelium regulatory sequences. is expressed inthelymphatic Gata2 +/GFP ( A-E While investigating enhancer Localization ofa within theboundariesofthese YACs. regulatory element(s)directing (data notshown). Thisledtothetentative conclusionthatthe displayed anidenticalvascular derivative ofd16Z (Zhouetal.,1998;Zhou2000)– Furthermore, YAC d18Z(–40to+73kbp)–asmaller, 5 umbilical vein andarteryof differential GFPintensityofexpression observed earlierinthe respectively). Thisdifferential stainingwas reminiscentofthe fainter thanintheumbilicalartery(Fig.2E,uv andua, E), althoughthestaininginumbilicalvein appearedtobe vascular systemoftheyolksacandinembryoproper(Fig.2C- 2B). ByE12.5, (Fig. 2A)and,byE10.5,intheaortaandendocardium et al.,1998),X-galstainingwas very prominentinthehearttube embryos bearingthesame(d16)YAC, but taggedwith gal) staininginthedeveloping vasculature ofE8.5transgenic previously, wegeneratedtheplasmidGR22- 0kbpofthe 20 Zhou etal.,1998).Whenwere-examined the homozygous is theunderlyingcauseofearlyembryoniclethalityin Gata2 to +73kbp(withrespectthetranslationinitiationsite)of (YAC) ,containingsequences from–198 We previously reportedthata271kbp expression inthevasculature A transgenicYAC recapitulates endogenous systems, embryonic vascular system.Inthecardiovascular andlymphatic angiogenesis, aswellinthearterialandvenous branches ofthe blood vessels thatareformedduringvasculogenesis and cells. Inthebloodvasculature, itisindiscriminatelyexpressed in expressed earlyinlymphatic,vascular andendocardialendothelial PROX1 andLYVE1 immunostaining (datanotshown). cells (Fig.1R,arrowhead). Similarresultswereobtainedwith to LECsafterE14.5,co-localizedwithasubsetofGFP-positive day 1(P1)pup.Expressionof mesentery (Fig.1Q),andtheskin(datanotshown) ofapostnatal We concludethatGATA-2 isapan-endothelial marker thatis ) EmbryosatE8.5(A),E10.5(B)andE12.5(C-E)bearingthe ␤ -galactosidase activityaswhole-mount(A,C-E)orcryosectioned locus was capableofrescuingthehematopoeticfailure that Gata2 Gata2 Gata2 expression persistspostnatally. lacZ Gata2 expression was pronouncedthroughoutthe genomic sequence (from–9kbptoslightly mutant embryos(Khandekaretal.,2004; Gata2 activity inthedeveloping nervous system Gata2 Vegfr3 endothelium-specific lacZ Gata2 -GFP knock-inheterozygotes. pattern intransgenicembryos (Fig. 1P),whichisrestricted endothelial expression lay Gata2 taining superficially Development 134(9) lac ␤ -galactosidase ( Z, whichcontains yeast artificial lacZ Ј -deletion Gata2 Gata2 (Zhou ␤ -

DEVELOPMENT Bam cardiovascular endothelium-specific expression. Whena2.3kbp whether they couldfunctionasclassicalenhancerstodrive Gata2 tentatively localizedtowithina1.8kbp Hence, the complete lossofendothelialX-galstaining(0/7embryos;Fig. 3C). Most instructively, GR22- respectively; Fig.3A,Banddatanotshown) (Zhouetal.,2000). the majorityof fragments reproducedthesameendothelium-restrictedactivity in postnatal brain (Fig.3J)ofTKSX artery andvein (Fig. 3H,I), andthevascular network ofthe 3E,F), thebloodvessels oftheyolksac(Fig.3G),umbilical evident intheendothelial cellsliningtheaortaandheart(Fig. activity (Fig.3Dand datanotshown). Discrete transgenic founders,eachretained endothelium-specific enhancer endothelium-specific enhancer. Both transgenic analysis,overlapping fragmentsforthepresenceofan with different restrictionenzymesinordertotest,byfounder (Zhou etal.,2000).PlasmidGR22- reporter geneinsertedinframeattheinitiationcodonexon 2 beyond exon 6,withrespecttothetranslationalstartsite)a A Gata2 Next, wetestedvarious fragments intheTK HI- fourth intron. Xba endothelial enhancer Gata2 I (BX)ora1.2 lacZ endothelium-specific enhanceractivity couldbe transgene-positive embryos(9/10and5/5, aZKpn lacZ Sfi I- Xba I (SX)fragmentwas tested in I- lacZ Sfi Xho I transgenicsdisplaceda ␤ was separatelydigested Sfi I- transgenic animals. I- Sal Sal ␤ lacZ and I I interval inthe vector toassess staining was Kpn I- Kpn lacZ I the GR22- lacZ 1G; blackboxes)are represented. OverlappingfragmentsoftheGR22- non-codingfirstexons(1Sand (e2)-e6; blackboxes]andtwoalternative constructs. The representative transgenicembryosgeneratedusingtheGR22- 1.2 kbpSXfragmentfromonlythe3 the the boundariesofaminimumenhancerelementrequiredtoachieve lymphatic vascular systems. endogenous developing vasculature, recapitulatingthefullextent of Furthermore, asubsetof ( Fig. 3.Localizationofanendothelialenhancerin in transgenicembryosgeneratedusingTKBX Kpn Sfi Sal embryos. ( with endothelialstainingamongthetotalnumberoftransgene-positive indicated. Thenumbersonthefarrightrefer tothenumberofembryos transgenic analyses.Thesizesofthe termini (inTKAA enhancer anddirected the 1.2kbpSXfragmentwas abletofunctionasa classical enhancer isalsoactive inthelymphaticendotheliallineage.Thus, (Fig. 3K,L,arrowheads), indicatingthattheendothelium-specific transgenic embryostainedwiththeLEC-specific marker LYVE1 anterior cardinalvein (Fig.3K,L,cv)ofanE10.5 TKSX (J) gestationagesfrom aTKSX (D). ( remained active inLECs(data notshown). demonstrated thattheminimal endothelium-specific enhancer immunostaining ofE10.5 TKAA endothelium-specific enhancer asdefined by theAArestrictionfragmentissufficient for activity (0/15;Fig. 4A,D).Thus,theminimalendothelium-specific from theSXenhancerfragment eliminatedallendothelialenhancer contrast, deletionoftheinternal specific expression inE10.5transgenicembryos(Fig.4B,C).By postnatal brain(J).Interestingly, clustersofround endocardium (F);andinthevascularnetworkofyolksac(G) (I);inthe lining thedorsalaorta(E),umbilicalartery(H)andvein (G,J) specimens.X-galaccumulationwasdetectedintheendothelia ␤ endothelia. the endothelialenhancerisactiveinLECsaswellcardiovascular cardinal vein(cv)stainedforbothproteins (arrowheads), indicatingthat marker LYVE1. Noticethatsomecellsinandaround theanterior simultaneously for Discussion). (K,L)Ananteriortransverseembryonicsectionstained which issuggestiveofearlyhematopoieticcellformation(see could beseenbuddingintothelumenofdorsalaorta(arrow, E), A -galactosidase activityascryosectioned(E,F,H,I,K,L) orwhole-mount ) Schematicrepresentations of I fragment(C).( We testednext aseriesofsmallerconstructs(Fig.4A)toestablish I (datanotshown)or Gata2 I, K; plasmid were excisedusingdifferent restriction enzymes( E-L ) EmbryosofE10.5(E-F;K-L),E12.5(G),E18.5(H,I)orpostnatal Sal lacZ B endothelium-specific expression pattern.Deletionofthe I, Sa; , C Gata2 ) ExtensivevascularX-galstainingwasobservedin plasmid (Zhouetal.,2000)andTK lacZ Sfi D ␤ I, S; ␤ ) Asimilar reporter gene(graybox),the -galactosidase activityandfortheLEC-specific expression intheendocardial,bloodand didnotalterthecardiovascular endothelium- ) Xba Kpn Gata2 lacZ I, X; I- Kpn ␤ ␤ lacZ -gal Xho stable transgeniclinewere stainedfor Gata2 expression throughouttheentire I fragment(B),butnotwiththe Alw enhancer activity. Anti-LYVE1 expression wasreproduced pattern I, Xh)andwere assayedinfounder + Gata2 RESEARCH ARTICLE NI- cells inandsurroundingthe Ј YAC d16Z(Zhouetal.,1998), end (inTKSR Apa -enhancer testfragmentsare ␤ ␤ I (AA)460bpfragment (not shown)andTKSX transgenic embryos ␤ lacZ Gata2 expression -positive cells Gata2 ␤ coding [exon2 rfromboth ) or intron 4. Bam aZXho lacZ Kpn 1707 HI, B; ␤ I- ␤ I-

DEVELOPMENT (see Fig.5).( positions oftheprimerpairsusedtoamplify167bpVEenhancer number oftransgene-positiveembryos.Thearrows represent the indicate thenumberofembryoswithcardiovascular fragment lengths(inbp)are indicated.Thenumbersontheright TKSX showedwidespread endothelial ( reporter gene.Thepositionsofrelevant restriction enzymesites A; 3 convergent arrows shown inFig.4Aand Fig.5A).Interestingly, the within acentral167bpcoreregion (asdelineatedbythetwo restriction fragmentshowed anunusualclusteringofbindingsites might bindtothisenhancer(Fig.5A).Acloserexamination ofthis database, identified anumberofcandidateregulatory moleculesthat consensus transcriptionfactor-binding motifsfrom theTRANSFAC element usingMatinspector2.2(Quandtetal.,1995),whichuses the implies anassociatedfunctionalsignificance. Analysis ofthe460bp extreme degree ofevolutionary sequenceconservation strongly 58 bpregion thatharbored96%identity(datanotshown). This AA fragmentdisplayed92%sequenceidentity, aswellanearby the humansequencedemonstratedthata355bpregion withinthe the mouse sequences thatsurroundthem(Lootsetal.,2000).Comparisonof Regulatory elementsarethoughttodiverge moreslowly than Gata2 Identification ofkeyregulatory motifswithinthe ␤ endothelial X-galaccumulation.Inthelatterembryos,onlyectopic Gata2 the TKSR enhancer. Fig. 4.Finelocalizationofthe 1708 none oftheseembryos (0/17)exhibited endocardium-specific that didnotdiffer from theTKAA endothelial expression in arangeofvascular tissue(14/17;Fig.5B) transgenic foundersshowed thatTKVE test itsabilitytorecapitulateendothelial expression. Analysisof was identified previously asdefining the3 bearing thehighesthuman-mouse identitywas clonedintoTK endothelium-specific activity (Fig.4A,B). Ј -gal activitywasobserved. The mouse167bpendothelium-specific fragment(calledVE) terminus ofthisregion correspondedcloselytothe Apa Gata2 ␤ ⌬ , TKAA intron 4were individuallycis-linkedtoaTKpromoter- AA I, Ap; RESEARCH ARTICLE minimal endothelium-specificenhancer ␤ endothelium-specific enhancerelement.Sub-fragmentsof ( Gata2 A (D) andTKAN B-E Nco ␤ ) Schematicillustrationsoftransgenicconstructs(TKSX , TKSX ) E10.5embryosbearingTKSR I, N; 460 bpendothelium-specific sequencetothatof Rsr ⌬ AA II, R, ␤ ␤ (E) transgenicembryoswere devoidof and TKAN Sfi I, S; Gata2 Xba ␤ ␤ construct. Strikingly, however, ) usedtofunctionallylocalize I, X)andtherestriction endothelium-specific ␤ -gal staining,whereas the Ј ␤ ␤ functional boundaryof (B) orTKAA was abletoconfer ␤ -gal staining/total Rsr ␤ lacZ II sitethat (C) Alw ␤ ␤ -gal ␤ NI, , to the endocardiumwhileadjoining3 radiolabeled probe usedforEMSAstudies(seeFig.6).( two convergentarrows. Theitalicizedsequencescorrespond tothe TKVE The 167bpminimalvascularendothelium-specific(VE)enhancerin ( arrow), endothelial recapitulates widespread vascular(14/17),butnotendocardial (0/17, bp candidate regulatory effectors withintheevolutionarilyconserved460 endothelium enhanceractivity. Fig. 5.IdentificationofacrucialEboxfor will requirefurther investigation. via whichthey contribute toendocardialendothelialdevelopment, any ofthesefactors, anddeterminationoftheunderlyingmechanism 2005). Whetherornottheseserve asbonafide bindingmotifsfor endocardial differentiation (NemerandNemer, 2002;Zhou et al., regulate endocardialenhancers andtobeinvolved inregulating factors, someofwhichhave beenshown previously todirectly 5), SMAD,AP2(alsoknown asTcfap2a) andMEF2Ctranscription consensus bindingsitesfortheNFAT, Nkx2.5 (alsoknown asNkx2- 5A). Examinationoftheformersequencesrevealed thepresenceof direct transgeneexpression inbloodendothelia(Fig.4AandFig. Alw endothelium-specific activity islargely containedwithinthe290bp conjunction withdatapresentedearlier(Fig.4),that the staining (Fig.5B,arrow). We surmisefromthesedata,in of the TKSR the ventricularchamberofTKVE Notice theconspicuousabsenceofX-galstaininginendocardium of ( vascular endothelium-specificX-galaccumulation(0/24). the singleSCL-bindingsite(A)inTKVE displayed vascularendothelium-specific in TKVE C E-G Simultaneousmutationofallthree ETS1-bindingconsensussites(A) ) Alw NI- Transverse sectionsthrough theheartsofE10.5embryosbearing ) ␤ ␤ Alw NI- (E; Fig.4B),TKAA (B) wasgeneratedusingthePCRprimerpairsindicatedby ␤ Rsr mEts1,2,3 resulted infarfewer(3/25)transgenicembryosthat Apa NI- II fragmentof endothelium-specificenhancersequenceare highlighted. I Rsr II fragmentarerequiredfor lacZ expression inE10.5transgenicembryos. ␤ (F; Fig.4C)orTKVE Gata2 intron 4andthatthe5 ( A ␤ Consensusbindingmotifsfor ) ␤ embryo. Slcompletelyabrogated mScl lacZ Ј 167 bpcanautonomously expression. ( Gata2 ␤ (G; Fig.5B)transgenes. Development 134(9) Gata vascular B 2 expression in ) TKVE D Ј ) Disruptionof -most 155bp ␤

DEVELOPMENT directing endothelium-specific enhanceraloneisincapableofindependently staining (Fig.4A,E).We concludethatthe5 endocardial X-galstainingdespiteexhibiting variably ectopicX-gal that, of11recovered transgenicembryos,nonedisplayed blood endothelium. extension, intheregulation of a profoundeffect on theactivity oftheVEenhancerand,by These datademonstratethatthe single embryos (9/24)exhibited ectopicstaininginthespinalcordorhead. of transgeneintegration site,becausesomeofthetransgenic Fig. 5D).Thislackofstainingwas presumablynotduetotheeffect transgenic embryos,nonedisplayedendothelial the TKVE eliminate SCLbinding(Kappeletal.,2000)was incorporated into (5 167 bpVEenhancerisimportantforitsoverall activity. Amutation shown andFig.5C). endothelium-specific, staining(1/12and3/25,respectively; datanot the overall numberofembryosdisplaying weak,albeit 2003). Mutationofeithertwo orallthreeETS-bindingsites reduced previously shown toeliminateETS-factor binding(O’Reillyetal., amutationthatwas individually mutatedfromCGGAtoCGcg, directly addressthishypothesis,thethreeETS-bindingsiteswere multiple Etsfamily membersmightmodulateitsactivity. To within the167bp Kappel etal.,2000).Theexistence ofthreeETS-binding sites endothelium-specific enhanceractivity (Gottgensetal.,2002; al., 1997),andhave alsobeenreported toplayprominentrolesin (Ayadi etal.,2001;Wang et crucial rolesinvascular development A Alw for endocardium-specific reportergeneactivation, wetestedthe antibody. but notmutant,competitorsaswellbytheadditionofananti-SCL were present intheextractandwasspecificallydisruptedbywild-type, lower-mobility complexwasobservedonlywhenbothSCLandE12 (lane 11)were addedtoseparatebindingreactions. Formationofa box, anti-SCLantibody(1or3 excess) containingwild-type(wt;lanes5,6)ormutant(mut;7,8)E demonstrate bindingspecificity, unlabeledcompetitors(20-or200-fold incubated withradiolabeledEboxoligonucleotideprobe. To (lane 3)orwithSCLplusE12expression plasmids(lanes4-11),were cells, whichwere mocktransfected(lane2),withSCLalone enhancer Ebox. Fig. 6.AnSCL-E12complexbindsspecificallytothe Ј Gata2 Next wedeterminedwhetherthesingleEboxmotifpresentin The Etsfamily oftranscriptionfactors have beenshown toplay To ascertainwhetherthe5 -CATCTG-3 NI- Nco endothelial enhancer Gata2 ␤ I fragmentinthecontext ofTK plasmid. Amongthe24recovered TKVE Ј endocardium expression. No extract(lane1)ornuclearextractsfrom 293T to 5 Gata2 Ј -CAcccG-3 VE enhancersuggestedthatoneor ␮ Ј -most 155bpfragmentwas sufficient l; lanes9,10)orcontrol mouseIgG Gata2 Ј ) thatwas previously shown to Gata2 expression throughout the ␤ Ј (Fig. 4A),andfound intron 4Eboxexerts -most 155bpofthe ␤ -gal staining(e.g. Gata2 VE- ␤ mScl regulatory effectors of thus implicatingEtsfamily member(s)andSCLascandidate and Ebox-bindingsitescontribute tothepotency oftheVEenhancer, serve asausefuladditionalmarker inthelymphaticfield. BothETS- to theLEClineage, expression. Given thepaucityofmolecular markers andtoolsunique endothelial cells,thuspreciselymimickingendogenousGATA-2 invascular, endocardialandlymphatic invivo enhancer elementthatiscapableofautonomouslydirectingreporter embryos, continues unperturbed. development afterE10,thenominaltimeofdeath (data notshown), it remainstobedeterminedwhetherlymphatic The existence ofanendothelialenhancerfor enhancer Identification of functionally defined 460bp In summary, thedatapresentedheredelimitboundariesofa DISCUSSION transcription factor SCL. enhancer canbestronglyboundbythebasichelix-loop-helix complex formation.Thus,thecrucialEboxmotifin but notcontrolIgG(Fig.6,lane11),significantly reducedEMSA showing thatadditionofananti-SCLantibody(Fig. 6,lanes9,10), 7,8). Thebindingspecificity andproteinidentitywereconfirmed by lanes 5,6),but notbymutantEboxoligonucleotide(Fig.6,lanes the additionofanexcess ofunlabeledEboxoligonucleotide(Fig.6, complex (Fig.6,lane4),whichcouldbespecifically competedby containing SCL-E12,but notSCLalone,resultedinalow-mobility radiolabeled Eboxoligonucleotideprobeswithnuclearextracts either SCL,orSCLplusE12,expression vectors. Incubationof (EMSAs) usingnuclearextracts from293Tcellstransfectedwith To doso,weperformedelectrophoreticmobilityshiftassays determine whetherSCLcouldbindtotheEboxinVEfragment. endothelial differentiation (Visvader etal.,1998),wewishedto Because SCLhadbeenshown previously tobeessentialfor motif inthe An SCL-E12heterodimer avidlybindstotheEbox PROX1 postnatal lymphaticvasculature. Althoughtheinitialbudding of at mid-gestation,andthatit continues tobeexpressed inthe , whenLECs bud fromtheanteriorcardinalveins erythroid cells(Grassetal.,2006)(seebelow). the recentobservation thatthisenhancerisactive indefinitive Gata2 identified heremayplaysomeroleintheappropriate regulation of (Gottgens etal.,2004),suggeststhatthe enhancer of to bedetermined.However, theexistence ofanendothelium-specific cells orsimplyendothelialbeingsloughedintotheaortais yet (Fig. 3E,arrow). Whetherthesecellsarebonafide hematopoietic lacZ generated fromtheaorticendothelium.Suggestively, weidentified enhancer couldalsotarget asubsetofhematopoieticcellsthatare (Choi etal.,1998),onemightspeculatethatthisendothelial hemangiogenic cellsarecloselyrelatedtotheendotheliallineage hematopoietic stemcells(Suzukietal.,2006).Given thattheearliest (Tsai etal.,1994)andisindeedcapableofspecifically marking that GATA-2 playsacrucialroleinearlyhematopoieticdevelopment intriguing questionsaboutitsfunction.Earliergeneticdataindicated We reportherethat -positive cellsthatappeartobe‘budding’ fromtheaorticwall in hematopoieticprogenitorsaswell,particularlylightof + LECs appearedtobenormalinGATA-2-deficient embryos Scl Gata2 , whichalsomarkshematopoieticprogenitors Gata2 Gata2 Gata2 Gata2 VE enhancer Gata2 is alsoexpressed attheearlieststageof vascular endothelialexpression. endothelium-specific enhancermay pan-endothelium-specific fourth intronendothelium-specific RESEARCH ARTICLE Gata2 Gata2 intron 4enhancer raises several Gata2 Gata2 1709 -null VE

DEVELOPMENT and recent generationofa the cranialandintersomiticvasculature, aswellhemorrhaging.The embryos displayedalessintricateweave, whichwas disorganized, of display cardiovascular defects(Xinetal.,2006).Notably, these possible 1998; Umetanietal.,2001),makingGATA-6 aprimecandidatefor precursors aswellinmatureendothelialcells(Hatzopoulosetal., (Hatzopoulos etal.,1998). 2001), althoughitisexpressed inendothelialprogenitors appear tobeexpressed inmatureendothelialcells(Umetanietal., Gata4 member doesseemtobesporadicallyactive (ourunpublisheddata). appear tobewidespread,but thiscloselyrelatedGATA family lacZ generate arobust vascular phenotype.Basedontheanalysisof mutant embryossimplydietooearly(~E10)(Tsaietal.,1994)to endothelium. Anotherequallyplausiblepossibilityisthat GATA-6, maycompensateforany lackofGATA-2 inthe redundancy ofotherGATA family members,includingGATA-4 and Gata2 1710 the Within aninitialfunctionallydefined restrictionfragmentdescribing endothelium-specific enhanceractivity Role ofEtsfamilytranscriptionfactorsin GATA-6 involvement, ifany, inendothelialdevelopment. et al.,2006)shouldnow permitexploration ofcellautonomous enhancers, including important intheactivation ofanumberendothelial-specific (Sumanas andLin,2006)have beenshown tobefunctionally shown toplayanimportantroleinvascular development invivo VE enhancer. TheEtsfamily oftranscriptionfactors have been binding sitesimplicatedaroleforthesefactors inthecontrolofthis expression. Theexistence ofthreeputative Etsfamily-member- core enhancerthatwas sufficient to recapitulatevascular endothelial et al.,2003).Furthermore,disruptionof as in endotheliumdevelopment. factors mayeitherplaynoroleoralsobefunctionallyredundant factors hasnotrevealed vascular deficiencies, suggestingthatthese vascular remodeling.However, targeted mutationofotherETS (Wang etal.,1997),suggestingthatTELplaysanimportantrolein one Etsfamily member, resultsindefective yolksacangiogenesis affinity bindingsites. endothelial activation despitemutationswithincanonicalhigh- able tobindothersequences withintheenhancer, enabling of ETS-bindingsitessuggests that somefamily membersmaybe endothelium (Lelievre etal.,2001).Additionally, theheterogeneity the multipleEtsfamily members thatareexpressed withinthe out thepossibilitythatmutation doesnotabolishthebindingof some membersofthefamily (O’Reillyetal.,2003),wecannot rule has beenshown inasimilarassaytoeliminateDNA bindingfor may serve toaugmentitsoverall potency. Becausethiscismutation are notessentialforthetissuespecificity oftheenhancer, but rather remain endothelium-specific, indicatingthattheETS-binding sites the weakstainingpatterndetectedintheseembryosappeared to transgenic embryosdisplayingendothelial as indicatedbythenumberof,andX-gal-stainingintensity in, disrupt ETS-bindingsitessignificantly attenuatedenhancer activity, In thebloodvasculature, thelackofany reportedphenotypein In the Tek Gata2 Gata6 knock-in mice, (Minamietal.,2003)and ) –/– , whichplaysacrucialroleinheartdevelopment, doesnot RESEARCH ARTICLE Gata2 Gata2 embryos ledtotheinitialconclusionthatfunctional endothelial enhancer, wesubsequentlyidentified a167bp heterozygotes arenormal,compound -complementing endotheliumactivity. Whereas VE enhancerdefined here,mutationspredictedto Gata3 Gata6 Scl (Gottgens etal.,2004), conditional loss-of-functionallele(Sodhi expression intheendotheliumdoesnot Gata6 Flk1 is expressed inbothendothelial (also known as Tel ␤ -gal staining.However, (also known as Tie2 (also known Kdr Gata2 (Elvert ) Gata3- Gata2 Gata4 Etv6 ), envisaged, ETSfactors activate constitute aregulatory circuitrywherein,inthesimplest scenario endothelium, wesurmisethatETS,SCLandGATA-2 together human there isnoGATA-binding siteintheendothelialenhancerof responsible foractivating cell linesappearstobeGATA-2, suggestingthatGATA-2 is The factor responsibleforbindingtothesesitesinhematopoietic has GATA sitesthatarecrucialfor Interestingly, theenhancerspecific forhematopoieticprogenitors (Barton etal.,2001;Gottgens2002;Sinclair1999). specific enhancersthatarerequiredforitsappropriateexpression transcriptional regulation of ontogeny ofthehemangioblast.Acomprehensive analysisofthe leading tothespeculationthatSCLmaybeimportantfor development (Patterson etal.,2005;Visvader etal.,1998), in bothhematopoiesis(Shivdasani etal.,1995) andvascular The transcriptionfactor SCLhasbeenshown toplaycrucialroles endothelial enhanceractivity An Ebox-bindingfactorisrequired for feedback loop. collaborate withGATA-2 toreinforce are alsoconsistentwiththepossibilitythatEtsfamily members cooperate toactivate both Gata2 The datapresentedhereareconsistentwiththepossibilitythat acting withoutdirectlybindingtoDNA (Gottgensetal.,2004). lethality of selective rescueofhematopoiesis,itseems likely thattheearly vascular defectsin remodeling, whereasGATA-2 hasnot.However, becausethe SCL hasbeenshown toplayaprominentroleinvascular similarity hasnotbeendemonstratedinthevascular system,where lethality (Shivdasani etal.,1995;Tsai1994).Thisphenotypic primitive anddefinitive hematopoiesis,resultinginmid-gestational system. Targeted mutationofbothgenesresultsindefects underscored bythesimilarityoftheirfunctionsinhematopoietic the activity oftheenhancerintrans clearly notexcluded fromthedatapresented. developmentally relatedtissues,althoughotherinterpretationsare specific interesthere,Grassetal. recently identified this same auto-regulated byGATA-2, oreven byanotherGATA factor. Of enhancer alsoraisestheissue of whetherthisenhancermightbe explore possiblefunctions ofGATA-2 inthevasculature. underway tocircumvent theembryonichematopoieticlethalityand function ofGATA-2 inthevascular system.Experimentsare enhancer of Ets-family activity (Gottgensetal.,2004). Theendothelial that development. Lendingfurthercredencetothispointistheevidence these two factors areoftenintimatelyintertwinedduring However, thesedataalsounderscorethepointthatfunctionsof factors maybedependentonthespecific tissueinquestion. that thenatureofepistaticrelationshipbetweenthesetwo and hematopoieticprogenitors(Gottgensetal.,2002),suggesting regulated byGATA factors inboththeCNS(Sinclairetal.,1999) bp endothelium, wemutatedthesingleEboxpresentwithin167 The similaritiesbetweentheregulation of To assesstherelationshipbetween The presenceofmultipleGATA-binding motifswithintheVE Gata2 Scl Scl and SCL expression intheendotheliumiscruciallydependenton and Gata2 VE enhancer. Scl Scl , althoughitremainspossiblethataGATA factor is Gata2 reciprocallyreinforcingactivators inthese contains five ETS-bindingsitesthatarerequiredfor -null miceprecludesamorepreciseanalysisofthe appear toberegulated byETSfactors inthe Gata2 Scl -null mutantswereonlyrevealed after Scl Scl in endothelialcells.Similarly, thedata has previously beenshown tobe Scl in earlyhematopoiesis.However, Scl has identified several tissue- - Scl activation assays.Given that Scl , andETSSCLthen enhancer activity invivo. expression inapositive- Scl Scl Development 134(9) and and Gata2 Gata2 Gata2 are also in the

DEVELOPMENT Choi, K.,Kennedy, M.,Kazarov, A.,Papadimitriou,J.C.andKeller, G. Ayadi, A.,Zheng,H.,Sobieszczuk,P., Buchwalter, G.,Moerman,P., Alitalo, References grants from theNIH(GM28896,J.D.E.andHL71206,T.W.G.). of MichiganCellandMolecularBiologyNIHTraineeship (W.B.); andbyresearch University MedicalScientistTraining Program Fellowship(M.K.);byaUniversity and stimulatingdiscussion.ThisworkwassupportedbyaNorthwestern technical assistance;andmembersoftheEngellaboratoryforhelpfuladvice We thankWeimin Song,MistiThompsonandXiaJiangforoutstanding further investigation. hemangioblast-responsive element)awaits resolutionfollowing hematopoietic andendothelialcells(asonemightimaginefora that canbeabonafide target forGATA-factor activation inboth activated inerythroidcellsorwhethertheelementrepresentsone endothelial-enhancer activity thatcansimplybe surreptitiously Grass etal.andtheactivity wedefined hererepresentsan cells inarigorousvivo assay. Whethertheactivity identified by element isatleastequallyasactive andasspecific forendothelial abrogated theerythroidenhanceractivity. Here,weshow thattheVE transfected erythroidcellsandthateliminationoftheGATA sites sequences overlapping theVEelementexhibited robust activity in (Grass etal.,2006).Inthatstudy, theauthorsdemonstratedthat analysis ofGATA-1 regulation ofthe element throughitsevolutionary sequenceconservation duringan A Cowan, P. J.,Tsang, D.,Pedic,C.M.,Abbott,L.R.,Shinkel,T. A.,d’Apice,A. Briegel, K.,Lim,K.C.,Plank,Beug,H.,Engel,J.D.andZenke,M. Barton, L.M.,Gottgens,B.,Gering,Gilbert,J.G.,Grafham,D.,Rogers, de Bruijn,M.F., Ma,X.,Robin,C.,Ottersbach,K.,Sanchez,M.J.and Elvert, G.,Kappel,A.,Heidenreich, R.,Englmeier, U.,Lanz,S.,Acker, T., Dorfman, D.M.,Wilson,B.,Bruns,G.A.andOrkin,S.H. Fang, J.,Dagenais,S.L.,Erickson,R.P., Arlt,M.F., Glynn,M.W., Gorski,J.L., German, Z., Chambliss, K. L., Pace, M. 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