Papaioannou, 1993;Downs andBertler, 2000).Allantoic core mesoderm fromthestreak,anddistalcavitation (Brown and ‘allantoic core’(Downs andGardner, 1995). Stevens, 1966);andinnercells,referred tocollectively asthe epithelial cells,the‘mesothelium’(Downs etal.,2004;Snelland are identifiableintheallantoicbud: anouterlayer ofsquamous region toformtheallantois.Two morphologicallydistinctcelltypes mesoderm, someofwhichisdisplacedintotheextraembryonic epiblast ingressesintotheposteriorstreakandemerges as streak, orfutureanteroposterior(AP)axis,appears,proximal three primarygermlayers(Lawson etal.,1991).Oncetheprimitive placenta (Gluecksohn-Schoenheimer, 1944). chorion andvascularize ittoformafunctionalchorio-allantoic failure ofthedeveloping umbilicus,theallantois,tounitewith 1935; Zavadskaia andKobozieff, 1930),presumablybecauseofa that anintactcoreisessentialforvascularization.Last,abnormalitieswereobservedinthe followed bycelldeathwithinthecore.Fetalliverkinase(Flk1)-positiveangioblastsweresignificantlydecreasedin allantoic dysmorphogenesisshortlyafterthebudformed.Diminutionincellnumberandproliferationwas Yolk sac,Heart KEY WORDS:Allantois,Anteroposterior axis,Apoptosis,Brachyury, Primitivestreak,Proliferation, Patterning, Vasculogenesis, have investigatedtheroleof core oftheallantois,whereitsfunctionisnotknown.Here,usingmolecular, geneticandclassicaltechniquesofembryology, development hasremainedobscure.Tproteinwasrecentlyidentifiedinseveralnewsitesduringmousegastrulation,includingth chorion. Ultimately, mutantembryosdieduringmid-gestation.Inthe60yearssincethisdiscovery, theroleof Mouse conceptuseshomozygousformutationsinbrachyury( Kimberly E.InmanandKaren M.Downs* the murineallantois Brachyury isrequiredforelongationandvasculogenesisin Development 133,2947-2959(2006)doi:10.1242/dev.02454 Accepted 23May 2005 *Author forcorrespondence (e-mail:[email protected]) Public Health,1300UniversityAvenue, Madison,WI53706,USA. Department ofAnatomy, –MadisonSchoolofMedicineand UniversityofWisconsin Zavadskaia, 1927). affecting taillengthinheterozygousadults(Dobrovolskaia- this family, Papaioannou, 2001;Showell etal.,2004).Thefoundingmemberof unexplained, rolesduringdevelopment (Naicheetal.,2005; genetic alterationssuggestthatthey playimportant,although largely exhibit dynamicexpression patternsduringembryogenesis,and shares acommonDNA-binding domain,the T-box. T-box genes The evolutionarily conserved T-box family oftranscriptionfactors INTRODUCTION microsurgical perturbationofthewild-typeallantoiccorephenocopied (Pecam1), whosespatiotemporalrelationshiptoFlk1suggestedaroleinpatterningtheumbilicalvasculature.Remarkably, and didnotcoalesceintoendothelialtubules,possiblyasaresultoftheabsenceplateletcelladhesionmolecu suggest aglobalrolefor elongation andvascularpatterning.Inaddition,morphologicaldefectsinotherextraembryonicembryonic organs reported sitesofTlocalization.Ourfindingsrevealthatisrequiredtomaintaintheallantoiccore,whichessentialfor The allantoicbud thenenlarges bycellproliferation,additionof The allantoisisderived fromepiblast,the progenitor tissueofall T , was identifiedinmiceasasemi-dominantmutation T / T mutants diedduringmid-gestation(Chesley, T in vascularizationoftheconceptus. T in allantoicdevelopment.Conceptuseshomozygousforthe T ) exhibitashort,misshapenallantoisthatfailstofusewiththe expression studiesplaced notochord, nascent mesodermandgutendoderm (Wilkinson etal., mutants, namelytheprimitive streakanditsderivatives, thenode, (Downs etal.,2004). Vcam1 chorio-adhesive celldomaintothedistalallantoicregion streak; nonetheless,information fromthestreakconsigns is intrinsictotheallantoisandnotdependentuponprimitive angioblasts/endothelium, mesotheliumandchorio-adhesive cells, allantoicmesoderm intoitsthreeknown celltypes, of al., 1995).Recentstudieshave suggestedthatdifferentiation molecule 1(Vcam1)(Downs, 2002;Gurtneretal.,1995;Kwee and identifiedbygraduallocalizationofvascular celladhesion located inthedistalallantoicregion (Downs andGardner, 1995) allantoic unionismediatedbychorio-adhesive mesothelialcells, maturity oftheallantois(Downs andGardner, 1995).Chorio- 6- to8-s,inaprocessthatisdependentuponthedevelopmental allantois fromtheyolksac(Downs etal.,1998). amalgamation takes place,primitive erythroidcellsenterthe accompaniedbyprimitive erythropoiesis;oncevascular not et al.,1998;Downs etal.,2004).Allantoicvasculogenesis is allantois andmerges withthoseoftheyolksacandembryo(Downs pairs (-s),thenascentFlk1vascular plexus reachesthebaseof regulated distal-to-proximalsequencesuchthat,by4- to 6-somite coalescence intoendothelialtubules thenfollows aspatiotemporally et al.,1998).Continuedformationofangioblastsandsubsequent distal region oftheallantois,farthest away fromtheembryo(Downs (Vegf) (Millaueretal.,1993;Yamaguchi etal.,1993),initiallyinthe tyrosine kinasereceptorforvascular endothelialgrowth factor angioblasts containingFlk1(Kdr–MouseGenomeInformatics),a vasculogenesis, asevidenced bytheappearanceofscattered mesoderm differentiates denovo intotheumbilicalvasculature by Subsequent tomolecularcloning of The elongatedallantoiscontactsthechorionandfuseswithitby T C / T C vascularization defect,providingfurthersupport T T C Curtailed /T T C heart andyolksac,recently within mostsitesdefective in ( T C ) mutation( RESEARCH ARTICLE T (Herrmann etal.,1990), T in allantoic T T C C / / T T C C ) exhibited allantoises Mouse, allantoic le 1 we 2947 T e / T

DEVELOPMENT Thus, toelucidatetheroleof ( brachyury 2 (Herrmann etal.,1990)thateliminatesatleastoneothergene, selected forstudy, itencompassesa200kilobase(kb)deletion the nodeandnotochord. disappeared, but itpersistedinthestreakanditsanteriorderivatives, chorion (Downs, 2002;Downs andGardner, 1995),allantoicThad By 6-to8-s,whenallnormalallantoiseshave unitedwiththe uninterrupted continuumwithembryonicTintheprimitive streak. dpc), Tidentifiedanallantoiccoredomainthatformed coincides withallantoicelongationtoward the chorion(~7.5-8.5 neural plate/lateallantoicbud (LB)and6-sstages,aperiodthat was notpresentintheearlyallantoicbud (EB).Then,betweenthe ectoderm, welocalizedTtoanovel domain,theallantoiccore.T extraembryonic andembryonicvisceralendodermchorionic protein intheheartandavariety ofnon-mesodermal sites,including allantois (InmanandDowns, 2006).Inaddition to identifyingT mount, immunohistochemistry, andpayingparticularattentiontothe within themurinegastrula,employing sectional,ratherthanwhole allantois. MATERIALS ANDMETHODS conceptus. also towidespreadTfunctioninvascularization ofthemurine a majorroleforTinformationoftheumbilicalvasculature, but novel Tsites(InmanandDowns, 2006), ourdatapointnotonlyto in theheartandyolksac,bothofwhichwererecentlyidentifiedas and defective vasculogenesis. Asabnormalitieswerealsoobserved major consequencesofwhichwereforeshorteningtheallantois proliferation was reducedintheallantoisandcorecellsdied, 1994). Ourfindingsrevealed that,intheabsenceofT, cell C-terminally truncatedproteinproduct(HerrmannandKispert, STOCK- Madison. Animals (PublicLaw 99-158)asenforcedbytheUniversity ofWisconsin- Health Service(PHS)Policy onHumaneCareandUse ofLaboratory unless otherwisenoted.AllanimalsweretreatedinaccordancewithPublic (Downs etal.,2004),wesetouttodiscover theroleof Wilson etal.,1993).Inlightofarecentmodelallantoicontogeny observed within development, confoundedtheinterpretationofabnormalities understanding ofthefundamentalparametersallantoic (Herrmann, 1991;Wilkinson etal.,1990),togetherwithinadequate However, discrepanciesin embryonic body(Rashbassetal.,1991;Wilson etal.,1993). suggested that 1990). Resultsofchimericstudiesin 2948 eei akrudscesu aig*o itr fltesprpi)( ihaa tei)( ihaa tei)Mendelianinherita (%withanalatresia) (%withanalatresia) † oflitters perpair) offspringThe totallive-born recovered from oflitters B6.C3(Cg)- C3.Cg- successfulmatings* Genetic background Table 1.Fertilityandincidenceofanalatresia in For allexperiments, B6.C3(Cg)- 8 of radiation-induced (Searle,1966)19basepair(bp)deletioninexon *At least24breeding pairsofeachbackground were paired continuously for4months.Failure toproduce alitterwithin thisw 8.33%, Although theoriginal We recentlyre-investigated thespatiotemporallocalizationofT T T that resultsinacodonframe-shiftpostulatedtoproduce ‡ T RESEARCH ARTICLE C 75.0% and C T /J C T Down and STOCK- C T2 T ) (Rennebecketal.,1998;Rennebeck1995). T § 28.5% ofthesealso displayedhindlimbdefectsandagrossly shortenedbody. acted inacell-autonomousmannerwithinthe n / у T T 3 specimensforallstagesandgenotypespresented, C chimeric allantoises(Rashbassetal.,1991; /J ecnaeo Ttlnme (vrg ubr +/+Offspring (averagenumber Total number Percentage of Down T , 921181(.)56()557(6.29 81(19.8 73(33.0 586(0) 117(0) 165(0) 8.1(3.9) 6.6(2.7) 6.2(2.2) 141 30 38 69.2 45.8 48.9 ␹ 2 mutation hasmostfrequentlybeen =35.56, 6.55and0.74,respectively. T T in theallantois,weselected expression withintheallantois T C /+ ϫ +/+ matingswasusedtodetermineexpected valuesfor T / T } +/+ conceptuses T C /+ mice associatedwithgeneticbackground Average littersize T in the T C , a Signal Amplification(TSA)Kit(Perkin-Elmer, Shelton,CT)accordingto dilutions, respectively. For Vcam1,signalwas amplifiedusingtheTyramide 315g; andBmp4,sc-6896)wereusedat1:100,1:90,1:501:90working Biotechnologies, SantaCruz,CA;T, sc-17743;Vcam1,sc-1504;Flk1,sc- only inSTOCK- were generated(Table 1).Expected 1:1Mendelianratioswereexhibited 248-bp assigned theaverage stageof+/+and staged bydevelopment ofanteriorstructures,afterwhichthey were between theLB-and6-sstages. females oftheinbredhybridstrainB6CBAF1/J; conceptuseswerecollected CD1 backgroundwas generouslyprovided byDrT. N.Satoandmatedto months old)werepairedwith 13:00). Animals weremaintainedundera12-hourlight/darkcycle (lightsout genotyping Animal husbandry, intercross mating,dissection,stagingand 2006). Stockconcentrations(200 previously described(Downs, 2002;Downs etal.,2004;InmanandDowns, Histology andsectionalimmunohistochemistrywerecarriedout as Histology andsectionalimmunohistochemistry J oipoensigadbedn efrac.Threelinesof NJ) toimprove nestingandbreedingperformance. months old,andwereprovided withplasticigloos(BioServ, Frenchtown, days, indicatingthat recovered fromthreelittersoftimedintercrossmatings,andnoneonlater dark cycle. Pregnant femaleswerekilledbyCO 15 hourslater, andthetimeofconceptionwas taken asthemidpointof start ofthedarkcycle. Femaleswereexamined foracopulationplug4and 6-s, 8.25-8.5dpc;6-to8-s,8.5dpc).Until4- numbers ofsomitepairs(1-to2-s,8.0-8.25dpc;2-4-s,8.254- headfold (EHF, LHF)stages,~7.75-8.0 dpc.Thereafter, stagingwas by and lateallantoicbud (EB,LB)stages,~7.25-7.5dpc;andearlylate were: early, mid-andlatestreak(ES,MS,LS)stages,~6.75-7.0dpc;early Briefly, stagesandtheirequivalent approximatedayspost-coitum(dpc) (Downs, 2006;Downs andDavies, 1993;Downs andGardner, 1995). selection, dissectionandembryostagingwereaspreviously described Bioexpress, Kaysville,UT).By~9.5dpc,onlyone (hereafter referredtoasFlk1 To obtain other two mouselines(Table 1),andwerethususedthroughoutthestudy. increased fertilityanddecreasedincidenceofanalatresiarelative tothe al., 1993),withprimers(5 protocol usedwas amodificationofthat describedbyStottetal.(Stott Biosciences, SanDiego, CA).Thepolymerasechainreaction(PCR) 5.2), andprecipitationusingsodiumacetatePelletPaint (EMD NaOH (20minutes,95°C),followed byneutralizationwith40mMTris (pH Louis, MO)beforeprocessing(below). DNA was extracted in25mM conceptuses wererinsedinphosphate-buffered saline(PBS,Sigma,St small pieceofyolksacoranteriorembryonictissuewas taken, afterwhich whole-mount expression studies(Fig.6A),amaleKdr Laboratories) was used(Downs, 2006).For theFlk1/Pecamallantois matings betweentheinbredhybridstrainB6CBAF1/J (Jackson bisected allantoicexplants (Fig.5B,partsg-m),theF2generationof 2). For apoptosispilotexperiments andproductionof‘wild-type’whole ACATCGGAGAACCAGAAGACGA-3 T T C C allele. Reactionswererunona4%GQASieve agarosegel(ISC /+ breedingcoupleswereestablishedwhenanimals3- T C / T ␹ C 2 analyses oneachgeneticbackground. ForC3H.Cg- conceptuses, STOCK- T C T /J C Down / T C Ј mutants diedbetween~9.5and10.5dpc(Table -TGCAAAGCCCTGTGATGCAA-3 (Table 1);furthermore,offspring exhibited lacZ T C T /+ males(3-to10-monthsold)priorthe ) (Shalabyetal.,1995)re-derived onthe indow wasclassifiedasinfertile. C +Ofpig analysesof /+ Offspring ␮ T g/ml) ofantibodies(SantaCruz C Ј ) thatidentifieda267-bp T /+ littermates.For genotyping,a § ‡ † C 0.26 ) ) ) /J Down Development 133(15) T T 2 C C T asphyxiation. Estrus /+ females(3-to6- / T C C / T P conceptuses were -value for р C р 1 conceptus was ϫ 0.001 tm1Jrt T 10 C , –13 Ј T T and 5 ␹ + C mouse 2 and a mice nce Ј -

DEVELOPMENT counterstaining withhematoxylin. (DAB) inchromogensolution(Dako, Carpinteria,CA)and was followed by variances wereassumed,andthesignificancelevel ( and Fig.5A,partd)was assessedbytwo-way Student’s *One (from ~10.5-13.5dpc),resorbing conceptuseswere genotyped.Deathoftheconceptuswasdefinedasabsenceheartbeat. Flk1 Whole-mount immunostainingforPecam1 the manufacturer’s instructions.Detectionwas with3,3 Requirement forbrachyuryinvascularization Metavue andsuperimposed withbrightfieldusingPhotoShop 7.0software. Corporation, San José,CA).Fluorescentimageswere pseudocoloredin cube (excitation, 535 nm; emission,590ChromaTechnology camera attachedtoaNikon Diaphotinverted microscopewithaG2Afilter and thenafterpeelingitaway toexpose theallantois usingaSpotRT-Slider were taken oftheposteriorendconceptus,firstwithyolksacintact, returned toculturefor6hours.At12 hoursafterculture,fluorescentimages in HEPES-buffered dissectionmedium(Downs, 2006),photographed,and 2006). After6hours,conceptuseswereremoved fromtheincubator, placed and conceptuseswereplacedintowholeembryoculture(WEC)(Downs, field andfluorescenceimagesweretaken immediatelyfollowing injection in 0.3Msucroseintothemidlineofposteriorprimitive streak.Bright- tetramethylindocarbocyanine perchlorate,MolecularProbes, Eugene,OR) 0.05mg/mlCellTracker CM-DiI(1,1 of Cell movements weretracedbymicroinjecting asmallvolume (~0.5 DiI labeling,wholeembryoandexplantcultures sections (4- Cell countsandmitoticindex werefromsagitallyorientedhistological Cell counts,mitoticindex(MI)andmorphologicalmeasurements significance of carried outaspreviously described(Downs etal.,2004).Statistical Measurements ofallantoiclengthforascertainingtheVcam1domainwere tissue whereclearboundariesbetweenepiblastandmesodermwereabsent. respectively. Within thisdomain,theprimitive streakwas taken asdense and thenodedefinedposterioranteriorlimitsofprimitive streak, diagrammed (Downs andHarmann, 1997).Boundarieswiththeallantois boundary betweentheallantoisandprimitive streakwas previously Institutes ofHealth,Bethesda,MD)was usedtovisualizethesections.The proximal domainswerenotdistinguished,andImageJsoftware (National previously reported(Downs andBertler, 2000),except thatdistal, mid-and / 4(50 1(25 2 2.)3 2.)7(18 2.)6(16 4 (20.0) 6(31.6) 3(27.3) 7(31.8) 31(23.8) 123(25.7) 31(22.5) 14(25.0) † All genotypeswere confirmedbyPCR.Forallstagestheexpectedpercentage of+/+, T +/+ Genotype Table 2.Recoveryofconceptusesfrom intercross matingsof eopin 89 50 5(.)5(.)005 2.)8 5*(26.3) 0 0 5(3.8) 25(5.2) 7(5.0) 5(8.9) Resorptions T ubro itr 75 03322 2 3 3 20 54 17 6 Number oflitters viewed andphotographedusing acompoundmicroscope. (which sometimeshadtheeffect ofdistortingrelative allantoiclengths),and PBS, equilibratedin70%glycerol,gentlysquashedbeneathacover slip temperature afterwhichthey werere-fixed inparaformaldehyde,rinsed Biotechnologies). Conceptusesweredeveloped for5minutesatroom biotinylated goatanti-ratIgGdiluted1:500(sc-2041,SantaCruz a 1:100 (537355,BDBiosciences,SanJose,CA).Secondaryantibodywas Primary antibodywas monoclonalratanti-mouse Pecam1(Mec13.3)diluted Laboratories, Burlingame,CA)was applied priortotheDAB colorreaction. contained goatserum(Chemicon),andready-to-useABCreagent(Vector (Schlaeger etal.,1995)withthefollowing modifications.Blockingsolutions and specimenswereprocessedstainedaccordingtopublishedmethods (Downs etal.,2004).Yolk sac,amnion andchorionweredissectedaway, rinsed inPBS,andthenX-galstainedfor2hours,aspreviously reported Two C C +2 4.)6 4.)24(90 6(31 3.)7(36 4.)8(40.0) 8(42.1) 7(63.6) 8(36.4) 56(43.1) 234(49.0) 68(49.3) 24(42.9) /T /+ C lacZ T T C C /+ conceptuseswere deadandresorbing; allotherdead, resorbing conceptuseswere /+ conceptuswasdeadandresorbing; allotherdead,resorbing conceptuseswere conceptuses werefixed in4%paraformaldehyde(2hours,4°C), ␮ m thickness)andwerecalculatedintheentireallantoisas T C / + and T 3(32 2(32 6(01 8(92 3.)1(.)00 0 1(9.1) 7(31.8) 38(29.2) 96(20.1) 32(23.2) 13 (23.2) 67 p ~. p ~. p ~. p ~. p ~. p ~05dc ~13.5dpc ~10.5dpc ~9.5dpc ~9.0dpc ~8.5dpc ~8.0dpc ~7.5dpc ~6.75 dpc C / n T C (%) compared with+/+(Fig.3B-D,Fig.4D,E, n (%) Ј P dioctadecyl-3,3,3 ) was taken as Ј -diaminobenzidine n (%) t -test; equal р 0.05. Ј ,3 ␮ T Ј l) C - n /+ T (%) C /+ and mice AAC-3 as describedabove. culture, yolksacsphereswerepreparedforsectionalimmunohistochemistry, yolk sacswereplacedintoWEC(Downs, 2006)for24hours.Attheendof one below thechorionandone above theamnion(Fig.7B,parta).Liberated exocoelomic cavity, afterwhichglassscalpelswereusedtomake two cuts, (1:100 dilution). wells ofa24-wellplate.ExplantswerestainedwithantibodiesagainstFlk1 which allbisectedpieceswerereleasedandpipettedindividually intothe allantois whileitwas stillattachedtothe posteriorendoftheembryo,after (Beddington, 1987).To ensureorientation,initialcutsweremadeintothe bisected transversely orlongitudinallyusinghandcraftedglassscalpels described (Downs, 2006).For halved allantoises,wholeallantoiseswere similar toprevious fate mappingthisregion (Smithetal., 1994). allantois ( T cells werefoundintheposteriorprimitive streakalone( labeled Not allspecimensforgenotypescontainedlabeledallantoiccells; spanning primers.Primerswere: (Access RT-PCR kit,Promega, Madison, WI)usinggene-specific,intron- RNA (100ng)served astemplateinallreverse-transcription PCRreactions RNA isolationusingtheRNeasy MicroKit(Qiagen,Valencia, CA).Total pooled bystageandgenotype(3-5 conceptuses/stage/genotype)during for genotyping,afterwhichconceptuses wereindividually snap-frozen, and Anterior tissuebeneaththeheartwas cutbymeansofglassscalpelsandused RNA isolationandRT-PCR STOCK- allantoic core(datanotshown). Amongstthethreegenotypesof within theneurectoderm(LBtoLHFstages),withafew positive cellsinthe Briefly, apoptoticcellsweredetectedinB6CBA/J F2conceptusesprimarily F2 conceptusesconfirmedthespecificityofLysoTracker Reduptake. (Ghatnekar etal.,2004;Zucker etal.,1999),pilotexperiments inB6CBA/J LysoTracker was detectedwithaG2Acube.Asreportedinotherstudies AMA filtercube(excitation, 360nm;emission,469 Chroma)and dewaxing, Hoechst-stainednucleiwereimagedusingaDAPI/Hoechst/ above onsamplesateachoftheEB,LB,EHF, LHFand1-sstages.After cell deathintheprimitive streak,6 sometimes hadtheeffect of distortingrelative allantoiclengths.To assess Allantoises wereisolatedandgentlysquashedbeneathacoverslip, which fixation, conceptuseswererinsedinPBSandequilibrated70%glycerol. PBS, andfixed in4%paraformaldehydeat4°Cfor1hour. Following labeling, conceptuseswererinsedthreetimesindissectionmedium,then mg/ml Hoechst(bis-benzimide,SigmaH33258)for30minutes.After ␮ Conceptuses (EB-to6-s)weretransferredWECmediumcontaining5 LysoTracker Redstaining death in the primitive streak(datanotshown). Thus,subsequentevaluation ofcell CCTGACCGAGCG-3 T C M LysoTracker RedDND-99(MolecularProbes,Eugene,OR)and0.01 C Age / For yolksacexplants, theallantoiswas firstaspiratedfromthe Allantoises wereexplanted, culturedandprocessedaspreviously / + T T C C T / . and C T / C T Ј . , 5 C T T n conceptuses was25%,50%andrespectively. Where possible T C n Ј C / C (%) -CCCCTTCATACATCGGAGAA-3 =1, 7and4for+/+, /J T / T C Down C mutants focusedontheallantois. , respectively), orinboththeposteriorstreakandproximal strain, nodifferences incelldeathwereobserved within Ј n , 5 (%) Ј -TACTTGCGCTCAGGAGGAGC-3 T T ␮ C or / m transverse sectionswerepreparedas + n T and (%) C RESEARCH ARTICLE , 5 T Ј -TGCTGCAGTCCCATGAT- C / T Ј ; C ␤ , respectively), thelatter -actin, 5 † n (40.0) (%) n =1, 3and4for+/+, Ј -ATGAAGAT- Ј . Cycling 2949

DEVELOPMENT T reduced cellproliferation Allantoic dysmorphogenesisisassociatedwith endoderm, itisnotmaintainedinthehomozygousmutants. initially correctlylocalizedbut, withtheexception ofectoplacental using primersthatflanked the ectoplacental endoderm,wherehighlevels ofT not intheallantoisatthisstage.With theexception ofthe endoderm (datanotshown), but, inaccordwithourprevious findings, (Fig. 1B,partsa,c),includingtheprimitive streakandectoplacental product are produced in mesothelial cells andsomeunderlyingcorecells, few ofwhich (Inman andDowns, 2006)in+/+and protein was observed inallrecentlydescribedsitesattheEBstage gene products,itcandetecttheunalteredNterminusofT A mutant RESULTS agarose gel. 68°C for2minutes.Five percentofthereactionwas runon a4%GQASieve conditions were35cycles of94°Cfor30seconds,60°C1minuteand 2950 that, ratherthanelongatingtoward thechorion, of +/+littermates(datanotshown). BytheEHFstage,itwas evident T the appropriatesiteatcorrectdevelopmental time(~7.25dpc), appearance ofthebud and1-s(Fig.2).Althoughthey emerged from Allantoic dysmorphogenesiswas examined betweentheonsetof conceptuses duringthisperiodofdevelopment. LHF- to5-sin T the cytoplasm ofthistissue(datanotshown), T from was carriedoutontotalRNA from+/+, rncitwsdtce n++samples, transcript was detectedin+/+ of Tand,althoughnotcapabledistinguishingbetweenand The antibodyusedrecognizesapeptidesequenceattheNterminus sites inthe mutant contained T(Fig.1B,partsb,e-g). Thereafter, thehomozygous allantoic core(Fig.2,compare A-CwithD-F),whichnormally Flattening oftheallantoicprojection seemedtobeduelossofthe histological sectionimpossible (Fig.2,compareA-CwithD-F). display oftheentirelength mutantallantoisesinany given allantoises tendedtospreadtoward theamnion(Fig.2A,D),making differences werenotedforany featurebetween misshapen allantoisattheEHFstage(datanotshown). Nogross not shown), theearliestdiscerniblegrossdefectwas ashort, (~9.0 dpc)andmid-/hindgutneuraltubeclosure(~9.5dpc;data dpc), andfailure ofchorio-allantoicunion(~8.5dpc),axialrotation 2). Althoughgrossdefectsincludedanabsenceofsomites(~8.25 T No differences werenotedinproteinlocalizationbetween+/+and point weendedthisstudy(e.g.Fig.1B,partsb,d;datanotshown). detected in that the 1994; MacMurrayandShin,1988;Stottetal.,1993)have suggested Results ofDNA analysisandgeneticstudies(HerrmannKispert, protein areproducedin status of C C C C The remainderofourstudyfocusedon Immunohistochemistry was thencarriedoutonsectionedmaterial. /+ embryosatthesestages.We concludethatthe / / / T T T C C C T samples atallstagesexamined (LHF-to8-sin+/+and conceptuses wereexamined priortoembryonicdeath(Table allantoic buds wereimmediatelylesspronouncedthan those RESEARCH ARTICLE T C T T C C transcripts (seeMaterialsandmethods).Asingle / C T T T allele producesamutantgeneproduct.To clarifythe C expression inhomozygotesusedthisstudy, RT-PCR C C / T T /+ samples,andasingle allantois appearedtoconsistpredominantly ofouter C T C C transcript andtransientprotein conceptus betweentheEHF-to4-sstages,atwhich / T C ; Fig.1A). T C / T C T conceptuses, andthattheproteinis C T deletion andthusdistinguished C / T T C C T / T mutants T C C C T T transcript was detectedin /+ and -related allantoicdefects. conceptuses, respectively and C T protein persistedin C T C C was absentinall T transcripts were / T C T T C transcript and C C conceptuses +ad+/+ /+ and / C T . Tand C mutant T C /+; T T C C ( used asaloadingcontrol foreachcorresponding TRT-PCR reaction. M indicatesmarker, a100bpgeneruler. Minus reverse-transcriptase control (–RT) wasnegativeforallreactions. was 258 highlight theT-defined core domain.Thetotallengthofthisallantois immunohistochemical sections(6 posterior primitivestreak (DownsandHarmann,1997).(e-g)Transverse indicates thepreviously definedboundarybetweentheallantoisand conceptuses. Asingle and T T Fig. 1. Bertler, 2000;Kinderetal., 1999;Tam andBeddington, 1987),with the latter’s pre-chorionicfusionstages(~7.25-8.5dpc)(Downs and C withF). distal allantoicregion by 1-s(Downs etal.,1998)(Fig.2,compare showed signsofendothelialization, whichistypicallyseeninthe was notdetectedinthe reactions for+/+(LHFto8-s), persistent mRNAtranscriptandtransientprotein. with theexceptionofectoplacentalendoderm(notshown),T LHF, T(b)waslocalizedtotheallantoiccore andprimitivestreak, and, samples, andasingle 48 (39.5% ofthetotalfixedallantoiclength).(f)Amid-region sectionat strong g, 100 extraembryonic visceralendoderm.Scalebars:ind,100 endoderm; ps,primitivestreak; x,exocoelomiccavity;xve, allantois; am,amnion;bi,yolksacbloodisland;eve,embryonicvisceral B C Immunohistochemical detectionofTand ) / As theprimitive streakcontributes cellstotheallantoisthroughout T ␮ C m. (g)Themostproximal sectionat6 C (c,d) conceptusesinsagittallyorientedhistologicalsections.T(a) T T ␮ (c) were localizedtotheprimitivestreak (ps)attheEBstage.At C m fore-g. and afaint ␮ /T m afterfixation.(e)ThedistalmostT(+)sectionat102 C homozygous mutantsproduce asinglebut T C T T C band were detectedatallstagesin transcript wasdetectedin+/+samples,a T transcript wasdetectedin C / T C T conceptus. Theslantedblacklineinb C /+ ␮ (LHF to8-s)and m) through a2-sallantoisto ␤ -actin RT-PCR product was ␮ m. ac,amnioticcavity;al, C Development 133(15) (brown) in+/+(a,b)and T T C C /T / T ( C C A ␮ (LHF, 5-s) samples. ) RT-PCR m fora-d;in T C /+ ␮ C m (d)

DEVELOPMENT numbers in b; datanotshown). differed significantlyfrom+/+atany stage examined (Fig.3B,part stained sectionsofallantoises(al)in+/+(A-C)and Abbreviations asinFig.1.ScalebarF:100 C, arrow), afeature thatwasnotreadily apparent in allantoises exhibitedsignsofendothelializationindistalregions (inset, for furtherexplanation). Thus, hours ofculture(Fig.3A;datanotshown; seeMaterialsandmethods were observed intheproximalallantoissomespecimens after12 lineage tracerDiI(Fig.3A).Inallgenotypes,labeleddescendants was notclearlyestablishedormaintainedin LHF stage, striking reductionincellnumberwas observed (Fig.3C).Bythe EHF transition(Fig.3B,parta)suchthat,bythestage,a to +/+attheEBstage,itwas significantlydecreasedbytheLB- number norMIintheprimitive streakof Requirement forbrachyuryinvascularization T streak intotheallantois.EHF-stageposteriorcellsof+/+, allantoic stuntinginvolved afailure ofcelldisplacementfromthe 2000; Downs etal.,2004),weinvestigated thepossibilitythat the largest numberaddedatheadfoldstages(Downs andBertler, of allantoicelongation. the primitive streakandenteringtheallantoisthroughoutperiod T conceptuses. Mesothelialcells(arrowheads) were observedin+/+and endothelialization in Fig. 2.Allantoicdysmorphogenesisandabsenceof the proliferation defectwas allantois-limited. nascent streakmesodermprior to enteringtheallantois,orwhether conclude whetherdefective allantoicproliferationoriginatedwithin calculate theMIinnascentstreak mesodermand,thus,couldnot previous study(Downs andBertler, 2000),wecould notaccurately in sagitallysectionedmaterial to make directcomparisonswitha those of+/+and (Fig. 3D). observation, theMIof levels werenever achieved (Fig.3D).Inaccordancewiththis Although numbersof correlated withtheonsetofreducedcellnumbers(Fig.3C,D). C C / Thus, decreasedcellproliferationresultedinreduced Although themitoticindex (MI)of / T + C allantoises. Thedensecore of+/+allantoises(blackoutline,A-C) and T T T C C T /+ allantoisesalsoexhibited areductioninlengththat C / C T / / T C T C C T conceptuses werelabeledwiththefluorescent allantoises. However, astheMIswerecalculated allantoises weresignificantlyshorterthan+/+ C / T T C C T T / (Fig. 3B,parta).Bycontrast,neithercell T C C C /+ allantoiseswas intermediatebetween /+ cellsincreasedthereafter, wild-type allantoises. T C / T C T cells werecapableofexiting C ( A-F / T T T C ␮ C C /+ or / m forA-F. Hematoxylin andeosin- ) T allantoises was similar C . By1-s,+/+ T C T T / T C C C / /T T (D-F) C C . conceptuses T C / T C expression patternsin 2006) inextraembryonic ectodermand itsderivative, chorionic (Rivera-Perez andMagnuson,2005)protein(InmanDowns, others, have recently demonstratedthepresenceof far enough,they might fusewiththechorion.However, we,and a minimalinboth was the the allantoiccore. present inallcellsremaining by sectionalimmunohistochemistryinthecurrentstudy, Bmp4was blood vessels (Downs etal.,2004;Lawson etal.,1999).Asjudged layers untilabout4-s,afterwhichexpression extends toallantoic expressed intheallantoicmesotheliumandunderlying several cell required forformationoftheallantois(Winnier etal.,1995),isfirst previously demonstratedthatbonemorphogeneticprotein4(Bmp4), formation ormaintenanceoftheouterlayermesothelium.We had toward thechorion.However, lossofthecoredidnotappear toaffect Loss ofthecorecoincideswithfailure oftheallantoistoelongate Collectively, ourdatasuggestthatTmaintainstheallantoiccore. or chorio-adhesivecells T doesnotaffect theformationofmesothelium allantoises(Fig.4A,partsh-j). indistinguishable from+/+ detected in allantoises at5-s(Fig.4C,parta;datanotshown), Vcam1was not Given that The allantoiccore diesintheabsenceofT T rare inmesotheliumandimmediatelysubjacentcelllayersofthe cells wereobserved (Fig.4A,partsh,j).Atallstages,apoptosiswas much reducedinsizecomparedwith+/+andonlyafew apoptotic elongated far enoughtocontact thechorion. contained Vcam1(Fig.4C,parts c,d;Fig.4D,E),althoughthey never These resultsinitiallysuggestedthat (Gurtner etal.,1995;Kwee1995)(Fig.4C,partb,inset). despite correctlocalizationwithinthemyocardiumofheart primitive streak(Downs etal.,2004).Incontrastto+/+and Vcam1 domainismaintainedatadistanceof 1995; Kweeetal.,1995).We hadpreviously demonstratedthatthe union withthechorion(Downs andGardner, 1995;Gurtneretal., distal chorio-adhesive mesothelialsubpopulationthatmediates iia o++and similar to+/+ (~8.5-9.25 dpc), was notlongenoughtoexhibit Vcam1(Fig.4D).By8-to16-s T the coreat1-s,althoughitwas morevariable andlessrobust thanin T the entirelengthofcore,includingdistal-mostpart the allantoiccore(Fig.4A,partsa,b).By3-s,theseextended along were foundwithinthe confirmed bysectionalanalysis(datanotshown). At 1-s,dyingcells 4A), andtheapparentsitesofexpression weresubsequently assessed usingLysoTracker Redinwhole-mountpreparations(Fig. shown). Patterns ofcelldeathinmutantallantoiseswerethen caspase-3 inpilotexperiments (seeMaterialsandmethods;datanot marker ofapoptosiswas verified bycomparisonwithactivated specificity ofuptake oftheacidotropicdyeLysoTracker Redasa whether thecoredomainwas maintainedinthemutants.The However, aftermeasuringit,wefoundthatthe5-s Vcam1 C C C To provide additional supportforthepresenceofmesotheliumin Thus, theseresultssuggestthat, could / / / T T T T C C C C / allantois (Fig.4A,partsf,g).By6-s,mutantallantoiseswere allantois. In T (Fig. 4A,comparepartsc-ewithb).By6-s,celldeath , actingupstreamofitinanallantois-specificmanner. C mutants, wenext examined Vcam1,whichidentifiesthe T T was expressed withintheallantoiccore,weinvestigated C / T C T C allantoises atequivalent stages(Fig. 4C, partb), T T / T C C C /+ allantoises,celldeathwas alsoconfinedto /+ (Fig.4B,partsa-d);comparisonofBmp4 T T mutant allantoiseshadexceeded 220 C C / / T T T C C C / allantois, initiatinginthemid-region of with +/+furtherhighlightedthelossof + and T RESEARCH ARTICLE C T T / T C was inthesamepathway as C / T T allantoises atintensities C C / T allantoises, andwas C у allantoises elongate 220 T C ␮ / T m fromthe C T allantois ␮ mRNA m and 2951 T C /+

DEVELOPMENT injection ofDiI(red) intothe primitivestreak of+/+(a-c)and hour windowduringwhichallantoic lengthandcellnumberwere reduced in Angioblasts are reduced in possibility thatawaits furtherinvestigation. to 1-s.( with respect to+/+are reported beloweachdatapoint.( allantoic coreprevented differentiation ofallantoicmesoderm umbilical vasculature, wenext investigated whetherlossofthe Because amajorfunctionoftheallantoisistoestablish between theallantoisandprimitive streak; theyellowlineinc,foutlinesextentofallantois. Scalebarind:500 yolk sac;(c,f)liveimagesofthedorsal allantoicsurfacesaftertheyolksacwaspeeledaway. Thegreen lineina,b,d,erepr homozygous Thus, chorio-allantoicfusionmaynonethelessbeprecludedin period, suggestingthatTmayhave aroleinchorio-allantoicunion. ectoderm (InmanandDowns, 2006),duringthe allantoic fusion 2952 Fig. 3.Migrationofmesoderminto s.e.m.; length are reduced. allantois; ch,chorion.( P D RESEARCH ARTICLE -values forthemutant genotypeswithrespect to+/+are reported aboveeachbar. ) Bargraphofallantoiclengths(in T C / T C ( B A mutants becauseofchorionicdefects,a ) Mitoticindex(MI)calculatedfor the allantois(a)andprimitivestreak (b)for+/+, ) Whole-mountimagesoftheallantois(al)andprimitivestreak (asterisk)atthetimeof(a,d)and12hoursafter(b,c,e,f) T T C C / T /T C C ␮ mutant allantoises allantoises from theprimitivestreak isnormal,but proliferation, cellnumberandallantoic m) in+/+, T C C /+ and ) Bargraphofallantoicnucleicounts in+/+, T C /T C T df H conceptuses.(a,b,d,e)Live imagesoftheposteriorend(up)through the (d-f) EHF C / T C genotypes fortheintervalEHFto 1-s. InCandD,error barsrepresent the By contrast,althoughFlk1was correctlylocalizedto angioblasts didnotdiffer significantlyfrom+/+(Fig. 5A,partd). allantoises atthisstage(Fig.3C),thepercentageofFlk1-positive part d). Flk1-positive angioblastswas significantlyreduced(Fig.5A, of cells(Fig.5A,partc;datanotshown), thepercentage core allantoiccoreof+/+and the al.,1998),Flk1was detectedinscatteredcellsthroughout et bloodvessels. Inaccordancewithprevious results(Downs into (Fig. 5A,partsa,b).Despitedecreasedcellnumbersin T C / T C . Error barsrepresent s.e.m.; T C /+ T C /+ and and T C P T -values foreachmutantgenotype /+ allantoisesattheEHFstage C / T T C C / T genotypes fortheintervalLB ␮ C m fora-e,250 genotypes overthe~6- esents theboundary Development 133(15) ␮ m forf.al, T T C C / T /+ C

DEVELOPMENT allantoises, butwasdetectedinmesothelialandseveralunderlying celllayers.Bycontrast,allofthecellsin bar. Abbreviations asinFig.1. beneath thefieldofview);‘ys’indicatesremnants oftheyolksacafterdissectiontoexposeallantois.Scalebarinj:10 were labeledwithHoechst.Whitelineineachpaneloutlinestheallantois;green barmarksthebaseofallantois(whichin Requirement forbrachyuryinvascularization death in+/+, Fig. 4.LossofTresults inlossoftheallantoiccore butmaintenanceoftheallantoicmesotheliumandchorio-adhesivecells. proximal (d)regions ofasingleallantoiswere Bmp4positive.Scalebarind:100 maintenance ofthe~220 appropriately localizedVcam1, similartothatshowninC,d.( allantoises unitedwiththechorion (ch)andcontainedVcam1inthedistalmesothelialcore cells(arrowheads). (d) boundary betweentheposteriorstreak andtheallantoisina,bd.Scalebard:100 fused withthechorion,butVcam1 wasobservedinthedistal-mostmesotheliumandsomecore cells(arrowheads). Blacklinesdis Vcam1 wasnotdetectedin ( of Vcam1in+/+(a,c)and of allantoiclengthinVcam1-stained sectionsreveals that B ImmunohistochemicalanalysisinsectionsshowingthatBmp4(brown) wasexcludedfrom thedenseproximal region of+/+(a)and ) T C /+ and T C / T T ␮ C C m Vcam1-negativeregion in T / whole-mount allantoisesassessedbyLysoTracker Red(pinksignal)at1-s(a-e),3-s(f,g)and6-s(h-j).Nuclei(blue) T C C / T (b,d) allantoises.(a)Vcam1localizedtodistalmesothelialandcore cells(arrowheads) of+/+allantoisesat5-s,but C (b) allantoises,althoughitwasdetected inthedeveloping T T C C / T / T C E C ) BargraphofVcam1-negativeregions inthesamehistologicalsectionsshowing specimens. Error barsrepresent s.e.m.inDandE; allantoises attainlengths>220 ␮ m forc,d;200 ␮ m fora,b,d;200 T C / ␮ T m (red horizontalline),allof whichcontained C ␮ heart (ht,insetinb).(c)At16-s(~9.5 dpc),+/+ m fora,b.( ␮ m forcandinsetinb.( C T ) Immunohistochemicaldetection C P RESEARCH ARTICLE / -values are reported aboveeach T C allantois indistal(c)and T C / T 0 C ␮ allantoises were not m. panelhliesjust tinguish the D T ) Bargraph

C ( /+ (b) A ) Cell 2953

DEVELOPMENT All explantswere immunostainedforFlk1(brown) and counterstainedinhematoxylin.(a-c)24-hourallantoicexplantcultures fr methods), transversely(T1/2)orlongitudinallybisectedwild-typeallantoises alongthepresumptive dorsoventralaxis(DV1/2), allantoic explantsare indicatedatthelowerleftofpanelsa-f.Otherindications(g-m)are WT(wild-typeF2,s distal core of+/+(a), detection andquantificationofFlk1-positiveangioblasts(brown) insectionsoftheallantois.Flk1-positiveangioblastswere p 2954 allantoises. Error barsrepresent s.e.m.; The Fig. 5. in anisolatedcentralcore of anEHF-stageallantois.Scalebarinm:500 90° tocut‘1’abovej.(j,m)24-hour explantsoflongitudinallybisectedEHF(j)and1-s(m)axialA1/2allantoises. Theinset from transversely-bisected EHF (h)and1-s(l)allantoises.(i)24-hourlongitudinallybisected dorsoventralDV1/2explants primitive streak region. (g,k)Undividedwholeallantoisexplantfrom EHF(g)or1-s (k)wild-typeconceptus.(h,l)24-hourpro DV1/2, topanelsk-m.‘1’and‘2’, andthered lines,indicatethesequenceofcutsmadeby glassscalpels.Thetissuebene allantoises (g)andtheorientation ofeachplanebisection(h-j);corresponds tothepanel directly below(g-j)and,w matings ofEHFconceptuses;(d-f) 48-hourallantoicexplantsfrom EHFconceptuses.Schematic drawingsaboveg-jdemonstrateund 1. m,mesoderm; mt,mesothelialcells. RESEARCH ARTICLE T -maintained allantoiccore isrequired forexpansionandcoalescenceofFlk1-positiveangioblasts. T C /+ (b)and T C / T C P (c) allantoises(arrowheads). (d)Bargraphshowingreduced percentage ofFlk1-positivecellsin -values withrespect to+/+are reported aboveeachbar. Scalebarinc:100 ␮ m (allpanelsinB,exceptforinset inj:1,250 ␮ m fora-c.( ␮ m). Abbreviations asinFig. ( injshowsvascularization ee Materialsand A ith theexceptionof ) Immunohistochemical Development 133(15) ; cut‘1’wasmadeat ximal T1/2explants rominent intheEHF oraxial(A1/2)plane. ath ‘2’istheposterior om B ) Genotypesof T ivided C /+ T C intercross / T C

DEVELOPMENT were mesothelial,thecellpopulationthatdidnotdieinintact than areduction incellnumber, perturbationoftheTcore domain vascularization (Fig. 5B,partj,inset).Thus,weconcludethat,rather j). Preservation ofthe centralaxialallantoiccoreresultedinnormal timing. VEGF vascular plexus inthemutantwas notsimplyanissueofdelayed at 24hours(Fig.5B,partf),suggestingthatfailure toforma vasculature. Bycontrast,themajorityofcellsin et al.,2004)ontopofwhichwas anexpanded Flk1-positive consisted ofanadherentlayermesothelial-derived cells(Downs Within the interconnected seriesofendothelialcelltubules (Fig.5B,partd). explants displayedexpansion oftheFlk1-positive plexus intoan present, assemblyintoavascular plexus was notobserved. allantoises (Fig.5B,partc).Althoughsomeangioblastswere T Allantoic angioblastsdonotendothelializein Requirement forbrachyuryinvascularization between +/+and immunohistochemistry, noobvious differences weredetected for24hours.AsjudgedbymorphologyandFlk1 isolation allantoises wereexplanted attheEHFstageandculturedin T Disruption oftheallantoiccore phenocopiesthe shown). therefore hadnodiscernibleeffect onthemutants(datanot 2001), didnotrescueangioblastsorendothelializationand little difference in interconnected plexus (Fig.5B,parte).Bycontrast,therewas present, but they werediscontinuousandnotassembledintoan population ofFlk1-positive angioblastsin (Gurdon etal.,1993),weinvestigated whetherareducedstarting localized thresholdlevels offactors requiredfordifferentiation As a‘communityeffect’ isrequiredforcellpopulationstoachieve To examine theendothelializationof vasculogenesis similar tothatseenin striking contrast,allA1/2 explants exhibited afailure of roughly halftheamountofmesothelium aswholeallantoises.In vasculature adheres (Downs etal.,2004),allantoichalves contained crawls off theallantoisandformsacell baseontowhichthe type (Fig.5B,partsg-i).Notunexpectedly, asthemesothelium convincing vascular plexi, althoughtheseweresmallerthanwild numbers ofcellsandFlk1-positive angioblasts. embryonic axis,wereassumedtocontainroughlyequivalent longitudinal explants, whatever theirpolarityrelative tothe allantoic domains(Downs etal.,1998).Thus,attheoutset, previously beennotedinthepresumptive dorsoventral orleft-right coordinates, noobvious differences in Flk1 localizationhad carried outtoidentifydistinctdorsoventral or left-right allantoic experiments ofallantoisesintransverse section have notbeen (Downs etal.,1998).However, althoughcarefulsystematic more Flk1-positive angioblasts,thebasecontainedmorecells that, althoughthedistalhalfofEHF-stageallantoiscontained half was thenplatedinisolation.Previous results haddemonstrated [presumptive dorsoventral halves (DV1/2); Fig.5B,parti].Each withrespecttothisaxis (A1/2); Fig.5B,partj],orat90° either indirectregister withtheembryonicaxis[axial halves bisected transversely (T1/2;Fig.5B,parth)orlongitudinally, cell number, wild-typeEHF-stageallantoisesweremicrosurgically reason forthefailed vasculogenesis. To decreaseoverall allantoic C C Explants werethenculturedfor48hours,afterwhich+/+ All EHF-stageT1/2andDV1/2 bisected explants formed / / T T C C vascularization defect mutants T C /+ explants, elongatedFlk1-positive tubules were 165 , addedtotheculturemedium(Downs etal., T T C C / / T + C explants at48hourscomparedwiththose explants (Fig.5B,partsa,b).Explants T T C C / / T T C C T explants (Fig.5B,part C allantoic angioblasts, / T C explants was the T C / T C explant T C / T C T in Absence ofPecam1andvascularpatterning angioblast survival andendothelialization. a roleintheformationofmesotheliumbut profoundlyaffects on plexus formation.We concludethattheaxialcoredoesnotplay had begun, disruptionoftheaxialcoredidnothave astrikingeffect endothelialization parts k-m).Thus,onceangioblastclusteringand then inlittermatesof the embryo(Fig.6B,partaand inset).In surface, aswellonthesurface ofthedeveloping dorsalaortaeof the centralallantoicvessel, whereitwas alsopresentonthecell midline nordorsalaortaewereobserved in delay invasculogenesis. NeitherthePecam1-positive allantoic stages examined (2-and5-s),although anoccasionalPecam1- Given that Pecam1 stainingwas absent inthedorsalaortae(Fig.6B,partsb,c). Pecam1-positive vessel was presentbut ofvariable length,and domain intheallantoisbetween~7.25and8.5dpc. contrast withFlk1,Pecam1definesaspatiallypatternedvascular with theembryonicvasculature until6-s(Fig.6A,partd).Thus,in angioblasts didnotcoalesceintoacentralvessel andamalgamate individual cells(e.g.Fig.6A,partc).Intriguingly, Flk1-positive Pecam1 was mostclearlyobserved onthecellsurface, outlining embryonic dorsalaorta(Fig.6A,partc).Between2-and 6-s, formed arobust centralvessel thathadamalgamatedwiththe around theproximalallantoiccore(Fig.6A,partb),but, by6-s,it central trackofproximalPecam1seemedtosplitandtriangulate increased branchingwithincreasingage(Fig.6A,partc).At2-s,the (Fig. 6A,partsa,b),formingacentralvessel thatshowed signsof inset). FromtheLBcluster, thePecam1domainextended distally previously, werescatteredthroughoutthecore(Fig.6A,parta, cells attheLBstage,whereasFlk1angioblasts,asdescribed vascular plexus similartothosefoundin+/+and EHF-stage andT1/2explants, they didformanendothelialized 2004). AlthougholderA1/2explants werenotasrobust asundivided evidence ofendothelialization(Downs etal.,1998;Downs etal., allantoises atslightlylaterstageswhentheseboremorphological specific totheheadfoldstage,wethenlongitudinallybisected embryo. of bilateralsymmetrythatisinregister withtheAPaxisof appears toaffect allantoicendothelialization,but onlyalongtheaxis reporter wild-type allantoiseswhoseFlk1angioblastsweremarked witha angioblasts wereseverely diminished,weexamined Pecam1firstin the allantoiccorediedin relationship betweenPecam1andFlk1isnotknown, but, given that 2003; Newman etal.,1992;Wong etal.,2000).Thespatial intercellular junctionsinnon-migratingcells(Gratzingeretal., migrating endothelialcells,but localizestoendothelialcell and Newman, 2003).Pecam1isdispersedacrossthecellsurface in endothelial cellsurvival andmigration(Newman, 1999;Newman tyrosine-based inhibitorymotif(ITIM),andplayingrolesin (Ig) superfamily, containing acytoplasmic immunoreceptor Papaioannou, 2003).Pecam1isamemberoftheimmunoglobulin as earlyheadfoldstages(Ilicetal.,2003;Naicheand observed anecdotallywithinacentralmidlinevessel oftheallantois during pre-fusionstages(seeDowns etal.,2004), Pecam1 hasbeen In contrasttoFlk1,whichisscatteredthroughouttheallantoiccore C In +/+littermatesof Pecam1 was initiallyobserved inaclusteredgroupofallantoic To determinewhetherperturbationtotheaxialallantoiccorewas / T C mutant allantoises lacZ T C construct (Flk1 / + pups areviable,theseobservations mayreflecta T C T / + C T / T intercrosses. C C / T lacZ mutants, Pecam1was detectedalong C mutants, andthatnumbersofFlk1 ; seeMaterialsandmethods), RESEARCH ARTICLE T C / T T C C / + conceptuses atthe allantoises, the T C / + (Fig. 5B, 2955

DEVELOPMENT in Flk Pecam1 (brown) andFlk1(X-gal,blue) of pyknotic cellswereobserved in myocardium (Fig.7A,partsa,b).By16-s(~9.5dpc),large numbers +/+,onlylow levels ofFlk1were detectedinthemutant to reduced myo-andendocardium(Fig.7A,partsa,b).Incontrast appeared dysmorphic,exhibiting adecreasedpericardialcavity, and myo- andendocardium(InmanDowns, 2006),theheart mass (datanotshown). By6-s,whenlevels ofTweremaximalin breeding datarevealed that represent thethreemajorvascular systemsintheconceptus.Our The allantois/umbilical,yolksacandcardiovascular systems the conceptus T playsaglobalrole invascularizationthroughout loss ofvascular patterningintheallantois. differentiation ofFlk1orPecam1angioblasts,itsabsenceresultsin Thus, weconcludethat,althoughTisnotrequiredforthe involved structuralabnormalitiesofdeveloping heartlayers. cell surface), (a)(insetshowslocalizationonthe +/+ until 6-s(d).( centralvessel localized toapatterned (da; c).Flk1-positivecellswere not developing embryonicdorsalaortae ultimately convergeduponthe distally andproximally (a-c),and defined acentralvesselthatextended panels inA).BytheEHFstage,Pecam1 allantois (arrowheads inthisandall positive cellswere dispersedinthe the LBstage(inset),whereas detected inasmallclusterasearly methods). (a)Pecam1(arrows) was A singlePecam1-positivecellwasdetectedinthe T examined thesemajorvascular organs forevidence ofaberrancies in blood islandsoftheyolksac(InmanandDowns, 2006),we within theextraembryonic visceralendodermoverlying nascent protein was recentlydescribedwithinthedeveloping heartand and thepresenceofarobust circulation(Downs etal.,1998).AsT precisely withactivity ofthechorio-vitellineplacenta(Copp,1995) dependence onthechorio-allantoicplacenta,andcoincidingmore 10.5 dpc(Table 2),slightlyearlierthanpresumptive embryonic positive cellwas foundinthedistal in vascular scaffolding thatismissing Fig. 6.Pecam1identifiesanascent 2956 when Twas recentlyreportedinextraembryonic visceralendoderm observed insomehistological sectionsasearlytheEHFstage 100 in a-c)wasvariable Pecam1-positive centralvessel(arrows s conceptuses.Formationofthe Similar toprevious resultsin T histological sectionsof T was firstobserved intheheart(InmanandDowns, 2006), (datanotshown). Asearlyas4-s,when indistinguishable from+/+ T C C C /+ or In addition,aberrant Until 2-to3-s,thedeveloping / T / T T C ␮ C C /T lacZ m forA,partsa,b,Ba-d;250 mutant conceptuses. C hearts wereobserved during thetimeofheartloopingand T RESEARCH ARTICLE conceptuses (seeMaterialsand allantoises. C / T C T conceptuses (arrowheads markthesitewhere continuitywiththeembryonicdorsalaortaewasexpected).ScalebarinB,partd: B C ) Pecam1detectionin /+ (b,c),and ( T A C T ) Whole-mount /+ allantoises. T C C / / T T T C C lacZ T C / T yolk sacbloodislandmorphology was C T hearts revealed areducedcardiogenic / C / T T - (d) 2- C mice (Kingetal.,1998),defects in embryos diedbetween~9.5and T T T C C C / T / / T T C ␮ C C allantois (Fig.6B,partd). m forA,partsc,d;25 hearts (datanotshown). heart fieldwas grossly T C / T C allantois (arrow ind).Pecam1wasnotdetectedtheposteriorembryonicregion of ␮ m forinsetinA,parta;50 phenotype intermediatebetween+/+and evidence ofpyknosis (Fig.7B,partsd,e). in acompactclusterwithindiscontinuousFlk1vessels and exhibited (Fig. 7B,partsd,e).Presumptive hematopoieticprecursorsremained abnormally highlipophiliccontentwithincytoplasmic vacuoles mutant yolksacswas discontinuous,andappearedtocontainan Fig. 5A,partc). however, abnormalitiesweremostpronouncedby1-s(e.g.Fig.2F, (InmanandDowns, 2006); overlying bloodislandmesoderm that landmarkstudy, presumptive +/+, not fusewiththechorion(Gluecksohn-Schoenheimer, 1944).In mutant embryosexhibited ashortandmisshapenallantoisthatdid over sixtyyearsagothathomozygous brachyury( made The startingpointforthecurrentstudywas theobservation DISCUSSION dysmorphic (Fig.7B,partg). appeared relatively normal(e.g.Fig.7B,part f),whereasotherswere blood islandsandassociatedextraembryonic visceralendoderm cells (Fig.7B,partsb,c).Bycontrast,thevisceralendodermof positive vascular channelscontainedanabundance ofhematopoietic yolk sacsexhibited continuityofthevisceralendoderm, andFlk1- rest oftheconceptus(Fig.7B,parta)andculturedfor24hours.+/+ sacs of+/+, positive endothelium. endoderm layersbut werenotpackagedinsideacontinuousFlk1- found betweentheextraembryonic mesodermand visceral approximate locationofdeveloping bloodislands,wherecellswere allantois inthe embryo. elucidation oftheroleTin theallantois,androleof genotype witheachclassof allantoicdefectprecludedan resultant development. Itwas concluded,however, thatthevariability inthe coelom toelucidatetherole oftheallantoisinembryonic were explanted andcultured withinthechickextraembryonic To examine development ofmutantyolksacsinisolation, T allantoic phenotypesandthe inabilitytocorrelate T C /+ and T C ␮ / T m forinsetinB,parta. C T C conceptuses hadathickened region inthe / T C conceptuses wereseparatedfromthe T T T /+ and C C /+ yolksacsexhibited a Development 133(15) / T C ; forexample, some T / T conceptuses T T C / / T T) C

DEVELOPMENT ␮ former exocoelomiccavityoftheintactconceptus.Scalebarind:100 endoderm offoregut; se,surfaceectoderm;mt,mesothelium;‘x’, phenotype (seetext).Abbreviations asinFig.1.ht,heart;en, different regions ofthesame cluster andcontainedpyknoticcells(red arrowheads). (f,g)Two not uniformlythickin endothelium (arrows) wasdiscontinuousandtheendodermlayer Requirement forbrachyuryinvascularization T et al.,1990),wewereabletoreadily genotypeconceptusesbearing were present between in c).Presumptive hematopoieticprecursor cells(black arrowheads) cells (arrowheads) were observedinthe Fig. 7. vasculature in+/+(c), through a+/+yolksacsphere. (c-g)Highermagnification ofyolksac isolate theyolksac(seeMaterialsandmethods).(b)Histologicalsection spheres. 1and2indicatethelevelofcutsmadeinconceptusto stage conceptuses(a)andcultured for24hourstoproduce yolksac Scale barinb:100 ( myocardial (my)andendocardial (ec)layersin hearts showareduction inthepericardial cavity(pc)andboth advances inallantoicdevelopment. Theseincluded knowledge of the combine thisinformationwith recenttechnicalandintellectual A Curtailed m forc-g;400 Equivalent sections(insetsinaandb)of+/+(a) ) Thanks tothe‘tour-de-force’ molecularcloningof T C , a /T C T conceptuses exhibitdefectsinheartandyolksac. -limited allele(Searle,1966;Stott etal.,1993),andto ␮ m forb. ␮ m fora,b.( T T T C C C / / T T / T C C C (d,e) and yolk sacs(compare dandewithwildtype yolk saclayers,butremained inatight T C /+ sphere reveal anintermediate B ) Yolk sacswere isolatedfrom EHF- T C /+ (f,g)spheres. Flk1-positive T C / T C mutant endocardium. T C / T C . AfewFlk1-positive T C T / T C (Herrmann (b) 6-s promoter region of T protein(Schmidtetal.,1997).Ourresultsreveal thattheunaltered ES cells,the factor (Fgf) family ( feedback loopinvolving Yamaguchi etal.,1999)indicatedanindirect autoregulatory 1995) andmouse(Clementset al.,1996;Galceranet2001; (Casey etal.,1998;Isaacs etal.,1994;Schulte-Merker andSmith, yolk sacaswell.Despitebeingshorterthanwildtype,all mitotic index, celldeathandvasculogenesis, thelatteraffecting the parameters examined, includingallantoicelongation,cell number, T environment. Thisfindingissimilartoreports ofanother demonstrated that with respecttothewild-typeproteinproduct. that mayinterferewith protein (HerrmannandKispert,1994;MacMurrayShin,1988) transport (Brunsetal.,1985).Itwas previously shown that,in sinuses ofDuval (Duval, 1891),thought tobeinvolved incalcium ectoplacental (distalextraembryonic) endoderm,whichwilllinethe throughout theperiodexamined here.Bycontrast,theT maintained intheheterozygousandhomozygousgenotypes The vascularization ofthemouseconceptus. thesefindings,weconcludethat of islands inthemouse(Belaoussoff etal.,1998).Thus,onthebasis the chick(MiuraandWilt, 1970;Wilt, 1965)andyolksacblood endoderm isrequiredfortheformationofyolksacbloodvessels in within yolksacendoderm(InmanandDowns, 2006).Yolk sac be requiredforcorrectyolksacvascularization, as Twas identified that Tisrequiredautonomouslywithintheheart. cardiogenic mass(InmanandDowns, 2006).This findingsuggests dysmorphology beginning at4-swhenTwas first detectableinthe of Taffected development of theheart,firstsignsofcardiac in themouseconceptus.Althoughnotyetextensively studied,loss allantois, ourresultslinkTfunctiontowidespreadvascularization appropriately vascularize. failure oftheallantoistoelongatetoward thechorionand We show herethat,intheabsenceofT, thecoredied,resultingina of robust expression of the LBand4-sstages,aperiodthatcoincideswithourrecentfinding (see Downs etal.,2004). we madeuseofanemerging developmental context fortheallantois explant system(Downs etal.,1998;Downs etal.,2001).Moreover, method ofwholeembryoculture(Lawson etal.,1991),andan staging system(Downs andDavies, 1993),areliableandpractical origin ofallantoicmesoderm(Lawson etal.,1991),amorphological Thus, theseresultssupportanantimorphicnatureofthe allantoises ultimatelygrew far enoughtofuse withthechorion. only transientlyexpressed inthe although initiallylocalizedproperlytotheTexpression domain,was restore taillengthin where introductionofafull-length phenotypes intermediatebetweenthoseof+/+and Previous studiesindicatedthatthe (antimorphic) actionofthe some degree ofautoregulation of presented hererevealed that Wis To provide insight intothemolecularnatureofT, we Whilst themajorfocusofourstudywas ontheroleofTin We reportthatTmaintainsanovel allantoiccoredomainbetween (Herrmann, 1991;KispertandHerrmann, 1994),andsupports T C allele produces amutantgeneproduct T promoter canbeactivated intheabsence offunctional T Xenopus T C C T expresses asinglemRNA transcript that is T is activated invivo inacompletelymutant C T /+ animals(Stottetal.,1993).Thework in thislocation(InmanandDowns, 2006). T in n ebr ftefirbatgrowth and membersofthefibroblast ) andtheWntfamily (mouse).Together, T T T C C C / /+ conceptuses.Dominant-negative allele was revealed inexperiments + heterozygotes exhibited allantoic T. T T T RESEARCH ARTICLE C A seriesofstudiesin transgene failed tocompletely / C T deletion producesamutant C T conceptus, except within plays aglobalrolein T also appearedto T C / T C T Xenopus C T protein, C for all allele, allele 2957 T C T / / + T

DEVELOPMENT deinkinase(FAK; Ptk2– Mouse GenomeInformatics)exhibit adhesion 2000). Inaddition,mutantsbearingadisruption infocal al., et heterophilic interactionsbetween Pecam1andintegrins (Wong (Bohnsack andHirschi,2003). Thisprocessmayinvolve implicated inangioblastmigration andassemblyintocords (Downs etal.,1998). primitive erythroidcellsintotheallantois takes placeatabout5-s previous resultsthatdemonstratedinfiltrationofyolksac the vascular systemsoftheconceptusby6-sisconsistentwith connection was notcertainuntil6-s(Fig.5A-C).Amalgamation of to extend toward theembryonicregion at2-s,althoughitsembryonic Pecam1 cellsimmediatelyassembledintoacentralvessel thatbegan not formacentralvessel until6-s(Fig.5A,partd).Bycontrast, dispersed Flk1-positive cellscanassemble. Flk1-positive cellsdid Pecam1-positive cellsprovide avascular scaffolding uponwhichthe within theallantoisisnotyetknown, ourresultssuggestthat allantoic midlinevessel innormalallantoises,was abolished. patterning, asrevealed byanincreasinglybranchedPecam1-positive In theabsenceof patterning into angioblasts,butisrequired forvascular T isnotrequired fordifferentiation ofmesoderm developmental window thatwas overlooked inthoseprevious studies. cells hadcontributed tothecore,lattermighthave diedduringa likely contributed totheallantois.Inthosechimeraswheremutant allantois, whichweshowed was notthecase (Fig.3A), 1993): insteadoffailure tobedisplacedfromthestreakinto allantoises ofotherwisechimeric why et al.,2004).Finally, theseresultsprovide aplausibleexplanation for allantoic Vcam1isdependentuponsignalingfromthestreak(Downs activity, aswehave previously demonstratedthatpositioningof within thedistalallantoicregion furthersupportednormal streak examined. ThatVcam1was correctlyexpressed andpositioned normal forbothcellproliferationandsurvival duringthestages contrast, theprimitive streak,whichalsoexpresses T, appeared Together, thesehadtheeffect ofabrogatingallantoicelongation.By proliferation was diminishedandcellsintheallantoiccoredied. cell layersweremaintained.Inaddition,overall allantoiccell (Fig. 1B,partse-g).Intheabsenceof immunohistochemical sectionsthatTlieswithintheallantoiccore and Downs, 2006).Here,wehave confirmedbytransverse section, was continuouswiththeembryonicprimitive streak(Inman between LBand6-s(~7.25-8.25dpc),which,asobserved insagittal demonstrated thatTislocalizedtoanovel allantoiccoredomain As discussedinprevious sections,recentresultsfrom ourlaboratory required forallantoicelongation T maintainsanovelallantoiccore domain the machinerytodegrade defective proteins. very littleisknown aboutthistissue,onepossibilityisthatitlacks protein persistsonlyinectoplacentalendodermisnotclear;although required tosustainfurtherproductionofT. WhythemutantT the initiationof of these studiesproposedthatFgfandWntfamily membersaretargets 2958 cell was foundinhomozygous of failure tosurvive. Moreover, althoughtheoddPecam1-positive expand, eitherbecauseofdiminishedproliferationand/or because Localization ofPecam1tothe surface ofangioblastshasbeen Although thepreciserelationshipbetweenFlk1andPecam1 T . Furthermore,althoughneitherFgfsnorWntsarerequiredfor T / RESEARCH ARTICLE T mutant cellswerenotfoundwithinsomedefective T T , angioblastsformed,but theirpopulationdidnot expression, signalingfromthese T C / T T / C T T mutant allantoises,vascular , onlytheouterfew allantoic conceptuses (Wilson etal., T targets is T / T cells C to rescue affinity forheparin(FerraraandDavis-Smyth, 1997),was notable Belaoussoff, M.,Farrington,S.M.andBaron, M.H. Flk1/ providing uswiththe We thankDrDavidStottforassistancewithgenotyping,Tom Satofor of aT-activated pathway forvascular patterning. patterning mayidentifyintegrins andFAK asdownstream members investigation intothemolecularhierarchiesinvolved invascular patterned allantoicvasculature (Ilicetal.,2003). Thus,further differentiation ofangioblastsbut lackanendothelializedand Beddington, R.S.P. References aligned withrespecttotheembryonicAPaxis. observations suggestthattheallantoiccorepossesses polaritythatis perpendicular toithadnoaffect onvascularization. Thus,these tothisaxisor endothelialize. Longitudinalbisections90° endothelialization defect:angioblastsformed,but they didnot plane ofcontinuitywiththeembryonicmidlinephenocopiedT within theallantoiccore.Physicallydisruptingcoreits matrix (Jacobs-Cohenetal.,1983). with observations that may beabsentfromordefective inmutantallantoises,whichaccords further studyisrequired,heparinorheparansulfate proteoglygans angioblasts culturedinlow serum(Downs etal.,2001).Although Brown, J.andPapaioannou,V. E. Bohnsack, B.L.andHirschi,K. Institutes ofChildHealthandDevelopment(K.M.D.). degree (K.E.I.)andwassupportedbyRO1HD042706from theNational on themanuscript.ThisstudywascarriedoutinpartialfulfillmentofPhD Clements, D.,Taylor, H.C.,Herrmann,B. G.andStott,D. Chesley, P. Bruns, M.E.,Kleeman,Mils,S.D.E.andHerr, J.C. Downs, K.M.andGardner, R.L. Downs, K.M.andBertler, C. Downs, K.M.andHarmann,C. Copp, A.J. Casey, E.S.,O’Reilly, M.A.,Conlon,F. L.andSmith,J.C. Dobrovolskaia-Zavadskaia, N. Downs, K.M.,Gifford, S.,Blahnik, M.andGardner, R.L. Downs, K.M.andDavies,T. Downs, K.M. Downs, K.M. the mouseembryo. induction andrespecification ofA-Pidentitybyvisceralendodermsignalingin Approach implantation mouseembryos.In Exp. Zool. during earlymousedevelopment. Circ. Res. Dev. suggests separationofmesodermlineagesearlyinmousegastrulation. regulatory control oftheBrachyurygeneinaxialandnon-axialmesoderm non-palindromic response element. transcription factorBrachyuryregulates expression ofeFGF through bindingtoa mouse placentaandyolksac. Immunochemical localizationofvitaminD-dependentcalcium-bindingprotein in Trends Genet. Anat. Embryol. Anat. Embryol. regulated. ontogeny: allantoicattachmenttothechorion isselectiveanddevelopmentally 1266. morphological landmarksinthedissectingmicroscope. allantois. viable. souris nouveau-néeetsurl’existenced’uncaractère (facteur)hereditaire, non- 121 allantoic explantsandfusionwiththechorion. 131. 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DEVELOPMENT