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11-2006

The and , when Isolated before Circulation or Chorio-Allantoic Fusion, have Hematopoietic Potential

Brandon M. Zeigler Dartmouth College

Daisuke Sugiyama Dartmouth College

Michael Chen Dartmouth College

Yalin Guo Dartmouth College

K. M. Downs University of Wisconsin-Madison

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Dartmouth Digital Commons Citation Zeigler, Brandon M.; Sugiyama, Daisuke; Chen, Michael; Guo, Yalin; Downs, K. M.; and Speck, N. A., "The Allantois and Chorion, when Isolated before Circulation or Chorio-Allantoic Fusion, have Hematopoietic Potential" (2006). Open Dartmouth: Published works by Dartmouth faculty. 734. https://digitalcommons.dartmouth.edu/facoa/734

This Article is brought to you for free and open access by the Faculty Work at Dartmouth Digital Commons. It has been accepted for inclusion in Open Dartmouth: Published works by Dartmouth faculty by an authorized administrator of Dartmouth Digital Commons. For more information, please contact [email protected]. Authors Brandon M. Zeigler, Daisuke Sugiyama, Michael Chen, Yalin Guo, K. M. Downs, and N. A. Speck

This article is available at Dartmouth Digital Commons: https://digitalcommons.dartmouth.edu/facoa/734 ac .Speck A. Nancy study, wehave investigated thehematopoietic potentialofthetwo hematopoietic cellscirculatethroughouttheconceptus.Thus,in this of hematopoieticcellemergence iscomplicatedbythefact that hematopoietic cells.Determiningwhetherany tissueistrulyasite colonization (like thefetalliver), or whether itisalsoasourceof Melchers, 1979;OttersbachandDzierzak,2005)issimplyasite of recently appreciated(Alvarez-Silva et al.,2003;Gekaset2005; whose contribution tomammalianfetalhematopoiesis was more (Houssaint, 1981).However, itisnotknown whethertheplacenta, colonization andisnotanintrinsicsourceofhematopoieticcells has longbeenacceptedthatthefetalliver isasiteofhematopoietic inthemid-gestationmouseconceptus(~10.0-11.0dpc).It hematopoietic cellnicheshave beenidentified withintheliver and 1997). et al.,1994;Palis etal.,1999;Weissman etal.,1978;Yoder etal., Medvinsky andDzierzak,1996;MooreMetcalf,1970; Müller (Cumanoetal.,1996;deBruijn2000; its precursor, thepara-aorticsplanchnopleura(PAS); andthe and definitive bloodcells;theaorta/gonad/mesonephrosregion and approaches include:theyolksac,whichgives risetobothprimitive Dzierzak, 2005;Specketal.,2002).Sitesdefinedbythese irradiated, orotherwisecompromisedadultmice(forreviews, see hematopoietic stemcells,bytransplantationintonewborn, defined usinghematopoieticprogenitorassaysor, inthecaseof sites ofhematopoieticcellemergence inmammalshave been streak stage[~7.0dayspostcoitum(dpc)](Palis etal., 1999).The sites intheconceptus,beginning inthemouseatmid-primitive- Hematopoietic cellsdevelop frommesodermlocatedinmultiple INTRODUCTION KEY WORDS:Placenta,Allantois,Chorion,Hematopoiesis,Mouse these tissues.Theseresultssuggestthattheplacentaisnotonlyanichefor, butalsoasourceof,hematopoieticcells. of the hematopoieticpotentialofallantoisandchoriondoesnotrequiretheirunion,indicatingthatitisanintrinsicproperty circulation, havethepotentialtogiverisemyeloidanddefinitiveerythroidcellsfollowingexplantculture.We that furthershow of the conceptusandthencolonizeplacenta.Here,weshowthatallantoischorion,isolatedpriortoestablishment in known whethertheplacentaisasiteofhematopoieticcellemergence,orcellsoriginatefromothersites plate. Themurineplacentacontainshighlevelsofhematopoieticstemcells,andisthereforeacellniche.However, not itis The chorio-allantoicplacentaformsthroughthefusionofallantois(progenitortissueumbilicalcord),withcho Brandon M.Zeigler or chorio-allantoicfusion,havehematopoieticpotential The allantoisandchorion,whenisolatedbeforecirculation Development 133,4183-4192(2006)doi:10.1242/dev.02596 Accepted 25August 2006 † 2 1 [email protected]) Medicine, 35Shinano-machi,Shinjuku-ku, Tokyo 1608582,Japan *Present address: DepartmentofCellDifferentiation, KeioUniversity Schoolof School ofMedicineandPublicHealth,Madison, WI53706,USA. Authors forcorrespondence (e-mail:[email protected]; Department ofAnatomy, 1300UniversityAvenue, UniversityofWisconsin-Madison Department ofBiochemistry, DartmouthMedicalSchool,Hanover, NH03755,USA. In additiontositesofhematopoieticcellemergence, two 1,† 1 , DaisukeSugiyama 1, *, MichaelChen major circulatory systemsunitenearthebase oftheallantois, region, andspreadstoward theposteriorstreak.By4-6s,three takes placeintheyolksacbloodislandsandheart primitive streak (Downs etal.,1998).Ataboutthesame time, to-proximal polarity, spreadingtoward the posteriorendofthe vascularizes denovo (~7.5dpc).Vascularization occurswithdistal- of theplacenta. Together theallantois andchoriongive risetothelabyrinthregion receptor allantois (Gurtneretal.,1995;Kwee1995)anditscounter molecule 1(VCAM1)ontheoutermesothelialsurface ofthe requires boththecell-surface moleculevascular celladhesion the maturityofallantois(Downs andGardner, 1995),and somite pairs(s),~8.5dpc].Chorio-allantoicunionismediated by make contactwiththechorionicmesoderm,andfuses it[6-8 Beddington, 1987).Ultimately, theallantoiselongatesfar enoughto 1993; Downs andBertler, 2000;Ellington,1985;Tam and primitive streakanddistalcavitation (Brown andPapaioannou, by acombinationofmitosis,continuedcellularadditionfromthe streak (~7.25dpc).Thebud enlarges withintheexocoelomic cavity and emerges firstasabud emanatingfromtheposteriorregion ofthe allantois appearsslightlylaterthanthechorionduringgastrulation, 1999; Lawson etal.,1991;Tam andBeddington,1987).The primitive streak(Beddington,1982;Coppetal.,1986;Kinder that istransformedintomesodermviapassagethroughtheposterior embryonic (Duval, 1891)derived fromproximalepiblast , whichmediatefetal/maternalexchange. component ofthechoriondifferentiates intosyncytio- and et al.,1999;Lawson etal.,1991)(Fig.1A).Theectodermal mesoderm derived fromproximalepiblast(Gardner, 1983;Kinder ectoderm derived fromtrophectodermandextra-embryonic bilayered tissueofdualorigin,composedextra-embryonic newly formedexocoelomic cavity (SnellandStevens, 1966).Itisa displacement ofextra-embryonic ectodermontothe roof ofthe . chorion, priortoestablishmentofvascular continuitywithinthe individual componentsofthemurineplacenta,allantoisand 1 , Yalin Guo Shortly aftertheallantoicbud isformed,allantoicmesoderm The murineallantoisisthoughttobecomposedwhollyofextra- The chorionarisesduringgastrulation(~7.0-7.25dpc)by ␣ 4 integrin, onchorionicmesoderm(Yang etal.,1995). 1 , Karen M.Downs RESEARCH ARTICLE 2,† and rionic 4183

DEVELOPMENT indicating thatit isanintrinsicpropertyofboth tissues. hematopoietic potentialisnotdependent onchorio-allantoicfusion, following explant culture.Furthermore,weshow that this of thecirculation,express isolated fromearlyheadfold-stage conceptusesbeforeestablishment their hematopoieticpotential. followed bymethycellulosecolony forming assays,todetermine in thepresenceofhematopoieticcytokines, orasexplants onplastic Second, weculturedallantoisesandchorionsonOP9stromal cells et al.,2002),but hasnotbeenreportedinthepre-fusion allantois. fusion (Northetal.,1999)andinthechorionpriorto(Lacaud (Ottersbach andDzierzak,2005),atthesiteofchorio-allantoic galactosidase unlabeled hoststoformchimericallantoises,notriplylabeled(␤- thymidine, andthengraftedintotheexocoelomic cavity of were labeledintrinsicallywithlacZandextrinsically withtritiated et al.,1998).Incontrast,whenheadfold-stagemurineallantoises and thushasthepotentialtoformdefinitive (adult)blood(Caprioli marrow following engraftmentintothecoelomofhostembryos, extensively tohematopoieticandendothelialcellsintheadultbone colleagues alsoshowed thattheavian allantoiscouldcontribute (Caprioli etal.,1998;Caprioli2001).Dieterlen-Lièvreand tissue andcontainsblood-island-like clustersofhematopoieticcells apparently prevascularized allantoisappearstobeahematopoietic or notthey originatedfromtheallantois.Inavian ,the in earlysomitepairconceptuses,itwas notentirelyclearwhether (Downs etal.,1998). erythroid cellscanbeseenfreelycirculatingwithintheallantois 1998; InmanandDowns, 2006).By8s(~8.5-8.75 dpc),primitive forming avascular continuumthroughouttheconceptus (Downs, 4184 al., 1996).Runx1 North etal.,2002;1999;Okuda1996;Wang et Uitz etal.,2000;Kalev-Zhylinska etal.,2002;Lacaud hematopoietic cellemergence intheembryo(Caietal.,2000;Ciau- stem cellformation,andisalsoanearlymarker for sitesof cultures. Runx1isatranscriptionfactor requiredforhematopoietic expression ofRunx1inboththeintactconceptusand inexplant cardiovascular systemsintheconceptus.First,wefollowed the the vascular link-upbetweentheumbilical,yolksacand and thechorionpriortotheirfusionand,moreimportantly, priorto experiments toexamine thehematopoieticpotentialofallantois 2005; OttersbachandDzierzak,2005),weundertookaseriesof placenta several dayslater(Alvarez-Silva etal.,2003;Gekas experiments suggestingthathematopoieticcellsemerge fromthe hematopoieticcells werenotexamined. Inlightofrecent of out duringonlyashortperiod(24hours),andearlymarkers al., 1998). cells, theoriginofwhichhasnever beenaccountedfor(Downs et exhibited aconstantbut relatively smallpopulationoferythroid and 4sstages(~7.5-8.25dpc),orculturedinisolationfor24hours potential: 5-10%ofallantoisesexplanted betweenthe neural plate suggested thatthemurineallantoismighthave erythropoietic of erythroidcells(Downs etal.,1998).However, somedata suggesting that,inthemouseconceptus,allantoisisnotasource yolk sacwereentirelydevoid ofbenzidine-positive cells, chorion anddidnotexhibit vascular continuitywiththefetusand/or that werefree-floatingintheexocoelom orfusedonly withthe culture(Downs etal.,1998).Furthermore,thoseexplants were detectedinthehostallantoisesfollowing 24 hoursofwhole Here weshow that boththeallantoisandchorion,having been Although erythroidcellscouldbedetectedinthemurineallantois The aforementionedstudiesofthemurineallantoiswerecarried RESEARCH ARTICLE + /tritium expression hasbeendocumentedintheplacenta + /benzidine Runx1 and exhibit hematopoietic potential + ) donor-derived erythroidcells genotype ofexplants from Institutions’ AnimalCareandUseCommittees. Downs etal.,2004).Allmouseprocedureswereapproved byour (Downs, 2006;Downs andDavies, 1993;Downs andHarmann,1997; staging andmorphologicalscoringwereperformedaspreviously described examined them4hourslaterforthepresenceofavaginal plug.Dissections, mated them5minutesbeforethebeginning ofthedarkcycle andthen B6CBAF1/J mice(JacksonLaboratory).We identifiedfemalemiceinestrus, mice werebredtofemaleC57BL/6J,C57BL/6J endoderm. separate theanteriorneuro-ectodermandmesodermfromhead minutes andthendissectedusingaglassneedlemouthaspiratorto saved. Thetissuesweredigestedintrypsin/pancreatinat4°Cfor10 was thenmadethroughtheepiblast; anteriorportionoftheepiblastwas embryonic fromcomponentsoftheconceptus.Asagittalcut was madebelow theamnionusingaglassscalpeltoseparateextra- ConA 488tolabeltheanteriorheadendoderm(Fig.2C).Atransverse cut isolated embryonicectodermbyincubatingheadfold-stageconceptusesin 488-positive tissue fromthechorion. endoderm bydippingtheconceptusintoConA488andremoving theConA Germany). Oncethistechniquewas established,welabeledonlythe ConA 488byfluorescencestereomicroscopy (Leica,Ernst-Leitz-Strasse, endoderm was confirmedbytheabsence ofConA594andthepresence scalpels andamouthaspirator. Separationofthechorionfromvisceral (Downs, 2006)for10minutes at4°Candseparatedwiththeaidofglass microcapillary pipette.Tissues weredigestedusingpancreatin/trypsin Concanavalin A(ConA488)intotheexocoelomic cavity usingamouth-held exocoelomic cavity was labeledbyinjectingAlexaFluor488-conjugated Probes/Invitrogen, Carlsbad,CA)(Fig.2B).Themesodermliningthe (ConA 594)[5mg/mlinphosphate-buffered saline(PBS)](Molecular conceptus for1minuteinAlexaFluor594-conjugated Concanavalin A embryonic andvisceralendodermwas labeledbysubmerging theintact with glassscalpels. in somecasessubdivided theallantoisesintodistal,midandproximalthirds We isolatedallantoisesbymouth aspiration(Downs, 2006)(Fig.2A),and Embryo dissection Runx1 lights on:0:00).Runx1 All miceweremaintainedinlight-reversed conditions(lightsoff: 12:00/ Mouse husbandry MATERIALS ANDMETHODS (2-ME, 1 cytokines fromR&DSystems,Minneapolis, MN)and2-mercaptoethanol ng/ml), granulocyte monocyte stimulatingfactor (GM-CSF, 5ng/ml)(all ligand (Flt3-L,10ng/ml),granulocyte colony stimulatingfactor (G-CSF, 5 50 ng/ml),interleukin3(IL3,5 interleukin6(IL6,5ng/ml),Flt-3 containing 20%fetalbovine serumsupplementedwithstemcellfactor (SCF, wells of12-wellplatesandgrown in1ml explants wereaddedtoa freshlypreparedlayerofOP9stromalcellsinsingle Individual allantois,chorion,PAS, visceralendodermandneuroectoderm OP9 explantcultures stained for6hours␤ paraformaldehyde for2hoursonice;they werethenwashed withPBSand stage, withaminor1-2scomponent.We fixed theexplants in4% suspensions for48hours(Downs, 2006).Mostmaterialwas atheadfold Well CellCultureCluster, Corning,NY)orinrollerdrum Allantoises andchorionswereculturedasexplants directly onplastic(24 Explant cultures forRunx1andLy6A expression Runx1 primers andamplificationconditions(Northetal.,1999). by PCRfromcorrespondingepiblastDNA usingpreviously described ME), B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J (JacksonLaboratory, BarHarbor, 1999) (formaldesignationsoftheRunx1 We isolatedPAS asdescribedbyGodinetal.(Godinal.,1993).We To isolatethechorion,Reichert’s membrane was removed andthe Tg(Ly6a/GFP) tm1Spe rd/+ ϫ females (Wang etal.,1996)with 10 and –5 M) (Sigma)at37°C in5%CO Runx1 (Ottersbach andDzierzak,2005),or tm2Spe -deficient explants weregeneratedbymating -galactosidase activity (Milesetal.,1997).The Runx1 , respectively). For markingexperiments, lz/+ ϫ Runx1 Runx1 ␣ rd rd/+ 2 -MEM (Gibco/Invitrogen) (Nishikawa etal.,1998)to Development 133(21) and matings was determined ϫ lz/+ 129S1/SVImJ F1,or Runx1 males (Northetal., Runx1 lz alleles are lz/+ male

DEVELOPMENT 57°C, 30seconds;72°C,45seconds. (Keller etal.,1993).PCRwas performedfor29cycles: 94°C,30seconds; ACT AGA ACA CCTGC-3 Hprt CTC TTGGG-3 erythropoietin (EPO)and1ϫ10 well plateinthepresenceof50ng/mlSCF, 10ng/mlFlt3-L,4U/ml equivalents ofallantoisesorchorionsonOP9cellsinasinglewell24- counted andselected7dayslater. Methocult (StemCellTechnologies, Vancouver, Canada).Colonieswere pipette. Cellswerecollectedbycentrifugationandculturedin3.3mlof3434 Basel, Switzerland)for25minutesandmechanicallydisassociatedusinga washed inPBS,thenincubatedpre-heated (37°C)DispaseII(Roche, medium was replacedafter24hours and at48hourstheexplants were 10 dayslater. MethoCult (StemCellTechnologies). Colonieswerescoredandharvested single-cell suspensionsasdescribedabove andplatedin3.3mlof3434 proliferation oferythroidprogenitors.Explantsweredissociatedinto Rockville, MD)withFloJosoftware (Tree StarInc.,Ashland,OR). cells wereanalyzedonaFACSCalibur flow cytometer (BectonDickinson, cells wereexcluded withTO-PRO-3 (MolecularProbes/Invitrogen), and and re-suspendedin200␮ FITC (RB6-85C)(BDPharmingenoreBioscience,SanDiego, CA),washed CD45-PerCP (30-F11),Mac1-PE(M11/70),c-kit-FITC(2B8),orGr-1- (BD Pharmingen,SanDiego, CA)for20minutesat4°Cthenincubatedwith (Fiering etal.,1995),␤ containing 500␮ equivalents ofallantoisesorchorionsinasinglewell24-wellplate Two-day explant cultureswereperformedbyplatingsixconceptus Progenitor assays a concentrationof2ϫ followed bypassagethrougha40- minutes in500␮ Single-cell suspensionswerepreparedbyincubatingtheexplants for10 Flow cytometry culture. cell sorting(FACS) analyseswereperformedafter14daysofexplant uptake orCD45expression after7daysofculture.Fluorescenceactivated were analyzedforDiI-Ac-LDL(BiomedicalTechnologies, Stoughton,MA) of themediumandcytokines werereplacedevery otherday. Explantcultures encourage theproliferationanddifferentiation ofmyeloidlineagecells.Half The allantoisandchorionhavehematopoieticpotential headfold stages,17at1-16sandtwo at9.5dpc. the figurelegends. We examined five conceptusesatbud stages,eightat galactosidase was performedfor6hoursat37°C,unlessotherwisenotedin (Vector Labs,Burlingame,CA)orleftunstained.Stainingfor and 5␮ wereassayedfor␤ Histological analysis CTG CCATAA 3 kit (Qiagen,Valencia, CA).⑀ mouse bonemarrow, and~9.0dpcmouseyolksacusinganRNA extraction or colony-forming unitgranulocyte-macrophage (CFU-GM)colonies,adult RNA was extracted fromindividual burst-forming uniterythroid (BFU-E) 3 hoursatroom temperatureandstainedbyMay-Grunwald Giemsa. selected colonies werecentrifugedontoslides(14g (Roper Scientific,Tucson, AZ). stereomicroscopy andphotographedusingamonochromedigitalcamera 90 minutesatroomtemperature.Explants wereobserved byfluorescence with Pacific Blue-conjugated streptavidin (MolecularProbes/Invitrogen) for CD45-biotinylated antibody (BDPharmingen)overnight at4°Candthen 4 hoursat37°C,fixed in4%paraformaldehyde,andthenincubated firstwith ␤ -Globin expression analysis Five-day explant cultureswereperformedbyplatingsixconceptus Explants fromOP9cultureswereincubatedin1 Single-cell suspensionsofexplants (1 primers (5 m paraffin sectionswereeithercounterstained withNuclearFast Red Ј GCT GGTGAAAAG GAC CTCT-3 Ј l ofCellDissociationBuffer (Gibco/Invitrogen) at37°C L of50%ratserum/DMEM(Downs etal.,2001).The and 5Ј-CAC AAACCC CAG AAACAG ACA-3 Ј) and Ј and 5Ј 10 6 -major globinprimers(5 cells permlweretreatedwithFcblockingserum l FACS buffer (BioSure,GrassValley, CA).Dead AGC GGACAC ACA GGATTGCTG3 -Globin (⑀ Ј ) wereusedtoanalyzeglobinexpression –5 -galactosidase activity (Milesetal.,1997), M 2-MEtopreferentiallyencouragethe ␮ m strainer. Explantcellsuspensionsat y) primers(5 ϫ 10 4 Ј -CTG ACA GAT GCT cells) orindividually ␮ Ј , 8minutes),dried for TGT CCTCTG l/ml DiI-Ac-LDLfor Ј and 5Ј -CAC AGG -CAC ␤ Ј ) - Runx1 fusion hasoccurreditisnolonger possibletodeterminewhetherthe expression was much lessintense.Inaddition,oncechorio-allantoic in thechorionpriortochorio-allantoicfusion,allantoicmesothelial Although ourresultsofhistologyclearlyrevealed Runx1expression chorion, independentoftheirunion Runx1 isexpressed in boththeallantoisand (Fig. 1B). embryonic endodermor‘ectoplacental’(Duval, 1891)] visceral endodermoverlying thechorionicectoderm [‘distal’extra- described (Northetal.,1999)(notshown), andinextra-embryonic and mesodermalcomponentsofthevisceralyolksacaspreviously circulation. cells, incellsunderlyingtheendotheliumandwithin is expressed inthelabyrinthregion oftheplacentainendothelial (Ottersbach andDzierzak,2005)showed thatby~11.0dpc,Runx1 analyzed theexplants forRunx1expression. tissues priortotheirfusion,cultured theminvitroasexplants and both theallantoisandchorion express Runx1,weisolatedboth from thechorion,orboth tissues.Therefore,toconfirmthat studies withthesameRunx1 1K,L) aspreviously described(Northetal.,1999).Inprevious in endothelialcellsandintra-aortichematopoieticclusters(Fig. forms fromtheallantois,expressed Runx1throughoutits lengthboth conceptus (Fig.1I,J).By~10.5dpc,theumbilicalartery, which expression inallantoicmesotheliumwas clearlyseeninthe9.5dpc 1G,H) but notinthemid-region oftheallantois (Fig.1G).Runx1 endothelial cellsliningtheproximalallantoicvasculature (Fig. both attheplaneofchorio-allantoicfusion(Fig.1G)andin 1E,F). Atlatertimes(~16s,~9.25dpc),Runx1expression was seen also inasmallclusterofcellswithinthebaseallantois(Fig. where theallantoisintersectschorion(Fig.1E,bluearrows) and cells atthechorio-allantoicinterface (Fig.1C-E),atexternal sites fusion (12s,~9.0dpc),Runx1expression was seenwithininternal mesothelium insomespecimens(notshown). Afterchorio-allantoic within thedistalportionofallantois,includingouterlayer wherethesetwo tissuesjoin,aswellfaint bluestain found convincing bluestainwithintheproximalallantoisand ectoderm was negative atallstagesexamined. Intheallantois,we mesodermal cellsbythelateheadfoldstage(Fig.1B).Chorionic at theearlybud stage(notshown), andwas intenseinmostchorionic seen aspunctatebluestaininginsomemesodermalcellsbeginning same locations(Fig.1B).Runx1expression inthechorionwas first visualized fromalacZ‘knock-in’allele(Northetal.,1999),inthese mesoderm (Lacaudetal.,2002).We observed Runx1expression, as islands (Lacaudetal.,2002;North1999),andinchorionic 1999), thevascular andhematopoieticcomponents ofyolksacblood Runx1 isexpressed inyolksacvisceralendoderm(Northetal., We andotherspreviously reportedthatatstagespriortofusion, to andshortlyafterchorio-allantoicfusion(~7.5-9.25dpc)(Fig.1). We examined theexpression ofRunx1intheintactconceptusprior chorion Runx1 isexpressed intheallantoisand RESULTS stage material. Sterling Heights,MI). photographed usingadigitalcolorcamera(DiagnosticInstruments, Cytospin preparationswereobserved bycompoundmicroscopy and All explant experiments wereperformedatleastthreetimesonheadfold- Faint, punctatebluestainingwas observed inbothendodermal + cells atthefusionjunctionoriginated fromtheallantois, lz allele, OttersbachandDzierzak RESEARCH ARTICLE 4185

DEVELOPMENT directions fortheextra-embryoniccompartment.( posterior (p)boundariesoftheprimitivestreak (ps)are indicated.Compassshowsanterior(ant),posterior(pos),proximal (pro) anddistal(dis) The chorionconsistsofchorionicmesoderm(chmes)andectoderm (chect),thelatterofwhichisderivedfrom trophectoderm. anterior(a)and The (mes) andectodermlayer(ect).Theconceptusissurrounded (visend). byembryonic(emb),yolksac(ys)andectoplacental(ep)visceralendoderm (bi)andallantois(al).Shownincolorlabeledaremesoderm layer bothembryonicandextra-embryonicendodermlayer(end), embryo(emb),yolksac showing locationsoftheectoplacentalcavity(epc),chorion(ch),exocoelomic cavity(ecc),amnion(am),amniotic(ac), in thedistalregion (arrowhead) andwithintheallantoicbase(box)ina16s hematopoietic clustersina10.5dpc Runx1 endoderm. ␤ chorionic (ch)mesoderm(blackarrow) andbloodislands(bi).Thebluearrow indicatesarea visceral ofpunctatebluestaining inectoplacental showing Runx1 length oftheumbilical artery(ua)ofa10.5dpc 4186 Fig. 1.Runx1expression before andafterchorio-allantoic fusion. in putativeendothelialcells(arrowheads) liningabloodvesselwithintheallantoicbase.( (boxed region). ␤ chorio-allantoic fusion(blackarrow), atthejunctionsbetweenallantoisandchorion(blue arrows) withinthebase andinaclusterofcells lz/+ RESEARCH ARTICLE conceptus. (D -Galactosidase stainingwasperformedfor18hours.( + cells inallantoicmesothelium(arrow). ( -Galactosidase stainingwasperformed for18hours.( ) DetailofRunx1expression from boxedregion inC.( Runx1 lz/+ conceptus. (L Runx1 B ) SagittalsectionofanEHF-stage lz/+ J ) Detailofboxedregion from I.( conceptus. ) WholemountshowingRunx1expression inthechorionicdisk(cd)andalong C ) Runx1expression (arrowheads) atthechorio-allantoic fusionjunctionina12s F ( ) DetailofRunx1expression from boxedregion inE.( A ) Schematicdiagramofanearlyheadfold(EHF)stagemouseembryo, Runx1 E ) Runx1expression ina12 s lz/+ Runx1 conceptus. (H I K ) Sectionthrough placenta ofa9.5dpc ) Runx1expression inendothelium andumbilicalartery lz/+ conceptus showingRunx1expression (blue)inthe ) DetailofRunx1expression (boxedregion inG) Runx1 lz/+ conceptus attheplaneof Development 133(21) G ) Runx1expression Runx1 lz/+ embryo

DEVELOPMENT 2001). Platedallantoicexplants gave risetoRunx1 streak (Downs andGardner, 1995;Downs etal.,2004;Downs etal., normal behavior even whenremoved fromcontactwiththeprimitive appear tohave aninternaltimingmechanism revealed bytheir explants weretheequivalent of~9.75dpc(~20s),asallantoises Fig. 3.Runx1expression intheallantoisandchoriondoesnotrequire chorio-allantoic fusion. ectoderm withpancreatin/trypsin digestfollowedbyexcisionwithaglassscalpel. the amnion;2,asagittalcuttoisolateanteriorportionofembryo;3,embryonicendodermandmesodermwere removed from the (control forTable 1).TheendodermwaslabeledwithConA594(green). Three cutswere cutproximal madewithglassscalpels:1,atransverse to isolatedinthiswayhadnovisibletracesofgreen fluorescent ve-derivedcells(Fig.4A,B).( hand panels.We thentrimmedvisceralendoderm(ve,green) awayfrom thechorion(ch,red) ofexplantculture, withaglassscalpel.After24hours cavity. bi,bloodisland;epc,ectoplacentalcavity. Two incisions(dottedlines)were isshownintheright madetoisolatethechorionicplate,which submerging theconceptusinConA594(green) andinjectingConA488(red) thatlinesthe intotheexocoelomiccavity(ecc)tolabelmesoderm Several celllayerswere trimmedfrom thebasewithaglassscalpel.am,amnion;ys,yolksac.( (48 hours)from aRunx1 the surfaceofsphere. ( fast red stainedhistological sectionsofwholemountpreparations ofallantoicspheres showingRunx1expression predominantlyin mesotheliumon derived cells.AllantoisesfromRunx1 to ensurethattheallantoiseswerenotcontaminatedwithyolk-sac- several celllayersfromthebaseofallantoiswithaglassscalpel pipette (Fig.2A).Following aspiration,wecarefullytrimmed allantoises fromearlyheadfoldto1-2sconceptuseswithaglass allantois canberemoved fromtheseinsertionsites.We aspirated When aspiratedviaamouth-heldmicrocapillarypipette,theentire allantois withtheamnionandyolksac(Downs andHarmann,1997). posterior primitive streakwas taken asthesiteofinsertion The allantoisandchorionhavehematopoieticpotential assayed for␤ cultured for48hoursonplastic,afterwhichtimetheexplants were Fig. 2.Isolationoftheallantoisandchorion. conceptus (3.2ϫ OP9 cells. speiul end theboundaryofallantoiswith As previously defined, -galactosidase activity. We estimatedthat48-hour ). (C ) Allantoissphere cultures (48hours)from lz/+ D conceptus showingRunx1expression (blue)(3.2 ) Tg( Ly6a/GFP ) (Sca-1)expression from thefreshly isolatedallantoisandchorionfollowing 24hoursofculture on lz/+ conceptuses werethen ( A ) Schematicdiagramillustratingisolationoftheallantois(al)withaglassmicrocapillary pipette. + cells (Fig.3A). Runx1 lz/+ ( lz/+) andwild-type(+/+)conceptuses (left). Righthandpanelsare nuclear ϫ stage allantoisesintodistal,midandproximalthirdscultured allantois gave riseto Runx1 with expression inmesothelium.To discover whichregion ofthe cell layersaroundthesurface ofthespheres(Fig.3C),consistent spheres revealed thatRunx1was expressed intheouteronetotwo under thesecultureconditions(Fig.3C).Sectioningthroughthe expression was observed inthespheresthatformed dpc). Runx1 tubes andculturedtheminvitrofor48hours(equivalent to~9.75 of theculturewell,wesuspendedexplanted allantoises inroller was inducednonspecificallythroughcontactwiththeplasticsurface mesothelial componentoftheallantois. Runx1 confirming thatatleastsomeoftheRunx1 to originatewithintheallantoicmesothelium(Downs etal.,2004), These motilecellswerepreviously shown bylimitedfate mapping as mesenchymal-like cellsthathadcrawled off theexplant (Fig.3A). ). (B To ruleoutthepossibilitythatRunx1expression intheexplants ) Chorion(ch)explantculture onplasticfrom a + cells includedthatremainedwithintheexplant, aswell B ) Chorion(ch)isolation.Endodermwaslabeledby C ) Isolationofanteriorembryonicectoderm ( A ) Allantois(al)explantculture onplastic + cells, wealsosubdivided headfold- RESEARCH ARTICLE + cells camefromthe Runx1 lz/+ 4187

DEVELOPMENT characteristically migratory, we suspectthattheplatedRunx1 PAS Chorion Allantois Allantois Embryonic ectoderm chorions isolatedfromRunx1-deficientconceptusesfailed toyield hematopoietic territory(Table 1).Incontrast,allantoisesand were equivalent tothoseseenwiththePAS, anestablished to abundant roundnonadherentcells(Fig.4C,E)atefficiencies that Both theallantoisandchorionfromwild-typeconceptusesgave rise differentiation ofmyeloidlineagecells(IL3,IL6,G-CSF, GM-CSF). presence ofhematopoieticcytokines thatsupportthe growth and explants onOP9stromalcells(SuzukiandNakano,2001) inthe systems intheconceptus.We firstculturedallantoicandchorionic circulatory continuumisestablishedbetweenthemajorvascular chorions isolatedfromearlyheadfoldto1-2sconceptuses,beforea We next assessed thehematopoieticpotentialofallantoisesand potential The allantoisandchorionhavehematopoietic two tissues. chorion isindependentoffusionandthereforeintrinsictothese Furthermore,Runx1expression intheallantoisand mesoderm. expressed inboththeallantoisandchorionic is Runx1, oneoftheearliestmarkers ofdefinitive hematopoiesis, expressed Ly6a/GFP (Fig.3D). 24 hoursofexplant culture,boththeallantoisand chorion the freshlyisolatedchorionbut notintheallantois(Fig.3D).After Consistent withtheirfindings,weobserved Ly6a/GFP expression in ectoplacental conebeginning inpre-streak-stageconceptuses. Ly6a/GFP isexpressed intheextra-embryonic ectodermand and Dzierzak(OttersbachDzierzak,2005)reportedthat allantoises andchorionsbeforeafterexplant culture.Ottersbach transgene (deBruijnetal.,2002;OttersbachandDzierzak,2005)in hematopoietic marker, Sca1,asvisualizedwithaLy6a/GFP population was derived fromchorionicmesoderm. plastic. Allthreepiecesoftheallantoisgave risetoRunx1 these piecesseparatelyas24-or48-hourexplants ontissueculture 4188 also Fig.2C). ¶ § ‡ † for thepresence ofabundantnonadherent cellsasseeninFig.4C,E. *Cultures includedSCF, Flt3-L,IL3,IL6,G-CSF, GM-CSF and2-ME,were scored Allantois Tissue hematopoietic cells(HC) Table 1.Efficiency atwhich OP9explantcultures yielded component gave risetotheRunx1 mesoderm fromectoderm,wedonotknow which chorionic most hadcrawled off theexplant. Aswedidnotseparatechorionic plastic (Fig.3B).Runx1 chorions andculturedthemasexplants for48hoursontissueculture throughout thelengthofallantois. shown), indicatingthatRunx1expression mayultimatelyinitiate given thatchorionicmesodermisRunx1 PAS, para-aorticsplanchnopleura. Late headfoldstage. Early headfoldstage. Isolation oftheembryonicectodermisdescribed intheMaterialsandmethods(see Thus, onthebasisoftheseobservations, weconcludethat In additiontoRunx1,wealsoanalyzedtheexpression ofanother To assesswhetherthechorionalsoexpressed Runx1,weisolated § RESEARCH ARTICLE ¶ + H o12s27 EHF to1-2s H o12s10 22 EHF to1-2s EHF to1-2s cells persistedinchorionicexplants, and tg fepat yieldingHC* ofexplants Stage EHF LHF 1-2 s ‡ † + cells intheexplant culture,but + Number % , andthatmesodermis 22 17 8 + cells (not 100 90 10 96 90 77 + major globinbut not or definitive erythroidcells.Thecoloniesexpressed PCR todeterminewhethertheBFU-Ecoloniescontainedprimitive We analyzed␤ conditions astheexpression ofRunx1intheexplants (Fig.3A,B). serum (Fig.5C);they appearedatthesametimeandunder thesame found inexplants following 2daysofcultureonplasticin50%rat explant cultures(Fig.5C).Hematopoieticprogenitors werealso (CFU-GM) anderythroid(BFU-E)progenitorswerepresentinthe monocyte, megakaryocyte (CFU-GEMM),granulocyte-macrophage erythroid progenitors.We foundthatgranulocyte, erythrocyte, and IL3inordertopreferentiallyencouragetheproliferationof SCF, Flt-3ligandandEPO,whileomittingG-CSF, GM-CSF, IL6 assays following 5daysofOP9explant cultureinthepresenceof accordingly revealed myeloidlineagecellsinthecultures(Fig.5B). markers Gr-1 andMac-1(Fig.5A),cytospin preparations G-CSF andGM-CSFexpressed eitherorboththemyeloid cell from allantoisandchorionexplants culturedinSCF, IL3,IL6,Flt3-L, mother (Fig.4G,I).SomeofthenonadherentGFP indicating thatthey werederived fromtheconceptusandnot Runx1 isrequiredfortheemergence ofCD45 macrophages basedontheirabilitytouptake DiI-Ac-LDL(Fig.4H,J). expressed thepan-leukocyte marker CD45,andmany appearedtobe Runx1-deficient conceptuses.Many oftheCD45 while cellsexpressing thesemarkers wereabsent inculturesfrom allantois andchorionexplants fromwild-typeconceptuses(Fig.4K), antigens weredetectedinnonadherentcellscollectedfromboththe et al.,1996;Wang etal.,1996).Cellsexpressing CD45,c-kit,orboth (Lacaud etal.,2002;Li2006;Mukouyama etal.,2000;Okuda sac macrophages,but itisnotrequiredforprimitive erythropoiesis chorions fromconceptusesderived fromcrossingatransgenic conceptus andnotamaternalsource,weisolatedallantoises (Okuda etal.,1996;Wang etal.,1996). requirement forRunx1inestablishingdefinitive hematopoiesis round nonadherentcells(Fig.4D,F),consistentwiththestrict tissues was revealed after culturingthemasexplants onOP9 the chorionhave hematopoieticpotential.Thepotentialofboth and/or chorionthemselves was notknown. and -gonad-mesonephros(AGM) region, orfromtheallantois other hematopoieticsiteswithin theconceptus,suchasyolksac Dzierzak, 2005).However, whetherthese cellswerederived from hematopoietic stemcells(Gekasetal.,2005;Ottersbach and dpc, theplacentaisalsoasourceoflong-termrepopulating potential colony-forming cells(Alvarez-Silva etal.,2003).By11.0 including CFU-GM,CFU-GEMM,BFU-Eandhigh-proliferation- placenta isanenrichedsourceofhematopoieticprogenitors, mother takes place.Recentstudieshave suggestedthatthemurine form thechorioniclabyrinth,whereexchange betweenthefetusand initially physicallywellseparatedintheconceptus,andthenfuse to progenitor tissuesoftheplacenta,allantoisandchorion, are of two organs: theumbilical cordandthechorionicdisk.Two The chorio-allantoicplacentaofeutherianmammalsisacomposite DISCUSSION definitive erythroidcells. Wong etal.,1983),indicatingthattheBFU-Ecoloniesconsistedof primitive, yolk-sac-derived erythroidcells(Brothertonetal.,1979; allantois andchorionculturesthenonadherentcellswereGFP actin/GFP malemousewithawild-typefemalemouse.Inboth As asecondapproachweperformedhematopoieticprogenitor To demonstratethatthenonadherentcellswerederived fromthe In thisstudy, wehave demonstratedthatboth theallantoisand -globin geneexpression inindividual coloniesbyRT- ⑀ y, thelatterofwhichisaspecificmarker of Development 133(21) + cells, includingyolk + cells generated + cells also ␤ ␤ + - - ,

DEVELOPMENT Note thepresence ofabundant,nonadherent round cells.( (ve) after48hoursonOP9cells.( of culture onOP9stromal cells.ConA488positivecells thatcrawledoff theexplantare boxed.( of theintactconceptus. hematopoietic progenitorcellswithinboththeallantoisandchorion resultsstronglysuggestthatRunx1expression identifies our erythroid cellswhereasRunx1-deficientexplants didnot.Thus, formed myeloidlineagecellsandprogenitorsfordefinitive CFU-C assaysfollowing a2dayexplant cultureonplastic.Explants stromal cellsinthepresenceofhematopoieticcytokines, orby The allantoisandchorionhavehematopoieticpotential Fig. 4.Thepre-fusion allantoisandchorionhavehematopoieticpotential. seven Runx1 analysis intheseplots.Cellseach gatewere analyzedintheplotsbelow. Theexperiment wasperformedthreetotal often+/+and times,witha CSF and2-MEstainedforCD45 andc-kit.Theexplantswere cultured separately, and three allantoisesorchorionswere forFACS pooled yellow andGFP round cellsare present. (E ( conceptus. (G explants from +/+andRunx1 deficient( transgenic conceptus,labeledwith DiI-Ac-LDLandanti-CD45asinpanelH.( H AllantoisOP9culture from ) rd/rd ) AllantoisOP9culture from a + allantoises andchorions. CD45 + cells are cyan.( ) ChorionOP9explantculture, sameconditionsasinpanelC.( ␤ -actin/GFP transgenicconceptus,stained withDiI-Ac-LDL(red) andanti-CD45(blue).GFP C ) Allantoiscultured for7daysonOP9cellsinthepresence ofSCF, IL3,IL6,Flt3-L,G-CSF, GM-CSFand2-ME. I Runx1 ) ChorionOP9culture from a␤ ␤ -actin/GFP transgenicconceptus,showing thatnonadherent cellsoriginatefrom theconceptus. rd/rd ) conceptusescultured onOP9 cellsfor14daysinthepresence ofSCF, IL3,IL6,Flt3-L,G-CSF, GM- D ) AllantoisOP9culture from aRunx1deficient( -actin/GFP transgenicconceptus.( extensively totheendotheliumofdorsalaortaand middle andproximalportionsoftheallantoisdidcontribute However, ectopicallytransplantedallantoiccellsfromthedistal, conceptuses (Downs etal.,1998;Downs andHarmann,1997). exocoelomic cavity orintotheallantoisofunlabeledhost produced very few, ifany, erythroidcellswhengraftedintothe K ) Representative FACS analysis ofthree pooledallantoisandchorion Downs andcolleaguesoriginallyshowed thatthemurineallantois ( A ) ExplantofConA488(red) labeledchorionfollowing48hours F ) ChorionOP9culture from aRunx1deficient( B ) ExplantofConA594labeledvisceralendoderm J ) ChorionOP9culture from a␤ Runx1 lz/rd RESEARCH ARTICLE ) conceptus.Nononadherent + DiI-Ac-LDL + cells are Runx1 -actin/GFP 4189 lz/rd )

DEVELOPMENT n pooled chorionexplantsfollowingculture for14daysonOP9stromal cellsinthepresence ofSCF, IL3,IL6,Flt3-L,G-CSF, and2-ME. GM-CSF used asanegativecontrol. chorion methylcellulosecultures. Yolk sacwasisolatedfrom ~9.0dpcconceptusesasa positive control for 4190 Fig. 5.Myeloidanddefinitiveerythroid potentialofallantoisandchorionexplants. experiments. (D serum onplastic,orafter5daysof explantculture onOP9cellsinthepresence ofSCF, Flt3-L and EPOwere averagedfrom thr bar=10 ␮ Grunwald Giemsa-stainedcytospin preparations ofindividualcoloniesfrom theallantois andchorioncultures.m, myeloidcell; e,erythryocyte; cultured togetherasexplants,disaggregated andplated inmethycellulose.Thetopthree panelsare representative May- colonies.(Bottom) myelocytes. (C OP9 cellsundertheconditionsdescribedinA.a-h,allantoisexplants; i-p,chorionexplantcultures; macrophages; a-c,i-n,monocytes;d-e, f-h,o-p, experiments RESEARCH ARTICLE m. Colonynumberspersixconceptus equivalents(±s.e.m.inparentheses) following2daysof explantculture inthepresence 50% rat of =4, n ) Methylcellulosecolonyformingassaysfrom theallantoisandchorion.Sixconceptusequivalentsofallantoiseschorionswere allantois ) RT-PCR forglobingeneexpression ( =32, n chorion =25. (B ) May-GrunwaldGiemsastainednonadherent cellsisolatedfrom allantoisandchorionexplantscultured on ⑀ y and␤ major). BFU-EorCFU-GMrepresents individualcoloniesfrom eithertheallantois or ( A ) FACS analysisoftenpooledallantoisandnine ⑀ y expression, andbonemarrow was Development 133(21) ee independent

DEVELOPMENT integrin the intra-embryonic splanchnopleuralmesoderm andcontains the allantoisproper. Theallantoisofavian embryosemanates from allantois was thoughttooriginate fromthecoreofendodermwithin Dieterlen-Lièvre, 1999).The endodermalsignalintheavian formation ofhematopoieticcells inthedorsalaorta(Pardanaud and become splanchnopleuralmesoderm thatcouldcontribute tothe somatopleural mesoderm,which hasnohematopoieticpotential,to embryo, abriefexposure toendodermcanre-specifyaxialand formation ofbloodislands(Belaoussoff etal.,1998).Intheavian endoderm provides solublesignalsthatarerequiredforthe endoderm. For example, inthemammalian ,visceral hematopoietic programinmesodermrequiresasignalfrom murine conceptusesclearlyshow thattheinductionof program intheallantoisandchorion.Studiesbothavian and the sourceofsignal(orsignals)thatinduceshematopoietic contribute tohematopoiesisintheplacenta. as Runx1 allantois thatgives risetohematopoieticcellsisnotclear;however, the hematopoieticcellssuppliedtochorion.Thesitewithin mesothelium isinvolved, withRunx1 mediates chorio-allantoicunion,itislikely thattheallantoic al., 2002),lendingsupporttothenotionthatRunx1 hematopoietic stemcellsintheembryo(Gribietal.,2006;North integrin andRunx1areexpressed onalllong-termrepopulating ␣ between theallantoisandchorionvery closelyresembles thatof expression inthechorionicmesodermandatsiteof intersection the chorionicplate.Oneinterestingobservation isthatRunx1 then entertheallantoicvasculature duringorafteritspenetrationinto derived fromallantoicmesotheliumand/orchorionicmesodermmay staining, thattheRunx1 ectoderm, wesuspect,basedonourresultsof embryo proper. from theallantoisandnotviacirculationyolksacor Therefore thehematopoieticprogenitorsmusthave arisendenovo continuity betweentheallantoisandyolksacwas achieved. subregions ofearlyheadfoldstageconceptuses,beforevascular of theallantoiswas demonstratedhereinallthreeallantoic in vitrothecurrentstudy. Importantly, thehematopoieticpotential 1997) isconsistentwiththehematopoieticpotentialdemonstrated aortic mesenchymeupontransplantation(Downs andHarmann, extensive contribution oftheallantoistodorsalaortaandperi- (Cumano etal.,1996;Cumano2001).Inretrospect,the could berevealed following explant cultureonstromalcells and 10sstages,containsmultipotenthematopoieticactivity that intra-embryonic hematopoieticterritoryand,betweentheheadfold embryo (Downs andHarmann,1997).ThePAS isanestablished mesenchyme directlyadjacenttoitwithinthePAS ofthehost The allantoisandchorionhavehematopoieticpotential these fusingsurfaces persist.Inthisway, Runx1 findings presentedhere,wesuggestthatcellsfromoneorbothof or differentiate intoothercelltypes(Downs, 2002).Basedon the fusingsurfaces oftheallantoisandchorionbreakdown, persist Once theallantoisfuseswithchorion,itisnotknown whether receptor mesothelium (Gurtneretal.,1995;Kwee1995)anditscounter fusion (Downs andGardner, 1995)viaVCAM1ontheallantoic chorionic mesodermotherthanthatthey mediatechorio-allantoic base oftheallantoisbeingasecondpotentialsource. 4 integrin, bothtemporallyandspatially(Downs, 2002).Both One oftheissuesraisedbythesestudiesconcernslocation Although wedidnotseparatethechorionicmesodermfrom Very littleisknown abouteitherallantoicmesotheliumor + + ␣ cells atthechorio-allantoicfusionjunctionultimately cells werelocatedontheoutermesothelialsurface, which 4 integrin onchorionicmesoderm(Yang etal.,1995). + mesodermal componentisthesourceof + endothelial cellswithinthe + hematopoietic cells ␤ -galactosidase + and ␣ ␣ 4 4 Belaoussoff, M.,Farrington,S.M.andBaron, M.H. B.M.Z. issupportedbyT32AI-07519. Public HealthServicegrantRO1CA58343toN.A.S.,RO1HD042706K.M.D. hematopoietic potentialoftheavianallantois.Thisworkwassupportedby We acknowledgeFrançoiseDieterlen-Lièvre forherpioneering workonthe the yolksaccDNA,andbothSteveFieringJimPalisfortheirhelpfuladvice. Fujiwara forsendingthem.We thankXiaoHuandSteveFieringforproviding Brown, J.andPapaioannou, V. E. Brotherton, T. W., Chui,D.H.,Gauldie,J.andPatterson,M. Beddington, R.S. Cumano, A.,Dieterlen-Lièvre, F. andGodin,I. Cai, Z.,deBruijn,M.F. T. R.,Ma,X.,Dortland,B.,Luteijn,T., Downing,J.R. hematopoietic emergence awaits furtherinvestigation. emerges denovo fromtheplacenta,orcomesothersitesof is ahematopoieticorgan. To whatextent hematopoieticactivity chorionic mesodermarecompletelyunclearatthistime. and ectoderm,thesignalsresponsibleforRunx1expression inthe this notion.Moreover, aslittleisknown aboutchorionicmesoderm (Downs etal.,2004),furtherexperimentation isneededtoaddress about theroleofstreakinspecificationallantoicmesoderm different timesintheprimitive streak.However, assolittleisknown and Beddington,1987),thesesignalsmaybeencounteredat 1986; Downs, 2002;Kinderetal.,1999;Lawson etal.,1991;Tam prolonged period(6.75-8.5dpc)(Beddington,1982;Coppetal., these two sitesoriginateswithintheprimitive streakover a within thebase.Itistemptingtospeculatethat,asmesodermat activate Runx1expression in the distalportionofallantoisand hypothesize thatsignalsseparatedeithertemporallyorspatiallymay the baseofallantoisemerges later(Kinderetal.,1999).We et al.,1999;Lawson etal.,1991),whereastheclusterofcellswithin emerged firstfromtheprimitive streak(Downs etal.,2004;Kinder component usingmoderntechniquesandmarkers maybewarranted. re-examination ofthemurineallantoisforanendodermal A layer (Duval, 1891;InmanandDowns, 2006;Mossman,1937). positive cellsandissurroundedbyamesoderm-derived mesothelial embryonic mesodermthatcontainswithinitacoreofBrachyury- allantois ofthehousemouseisthoughttoconsistentirelyextra- exhibits anendodermalcomponent(Mossman,1937),the al., 1998).However, whereastheallantoisofmany eutherian within itendodermderived fromtheprimitive hindgut(Caprioliet Caprioli, A.,Jaffredo, T., Gautier, R.,Dubourg,C.andDieterlen-Lièvre, F. D. Alvarez-Silva, M.,Belo-Diabangouaya,P., Salaun,J.andDieterlen-Lievre, F. References We thankElaineDzierzakforproviding the Caprioli, A.,Minko,K.,Drevon, C.,Eichmann,A.,Dieterlen-Lievre, F. and Copp, A.J.,Roberts,H.M.andPolani, P. E. Ciau-Uitz, A.,Walmsley, M.andPatient,R. the mouseembryo. induction andrespecification ofA-Pidentitybyvisceralendodermsignalingin Embryol. Exp.Morphol. Embryol. different regions oftheembryonicectodermduringgastrulationinmouse. during earlymousedevelopment. Acad. Sci.USA Hemoglobin ontogenyduringnormalmousefetaldevelopment. 5437-5444. 95-115. grafting ofposteriorprimitivestreak cellsinvitro. germ cellsintheearlypostimplantation mouseembryofollowingmicrosurgical embryonic generationofmousehematopoieticstemcells. and Dzierzak,E. the allantois. seedingbyhematopoieticandendothelialprecursors(1998). Blood-borne from 431. (2003). Mouseplacentaisamajorhematopoieticorgan. and molecularaspects. 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DEVELOPMENT Fiering, S.,Epner 4192 Cumano, A.,Ferraz,J.C.,Klaine,M.,DiSanto,P. andGodin,I. Gardner, R.L. de Bruijn,M.F. T. R.,Speck,N.A.,Peeters,M.C.E.andDzierzak, E. Keller, G.,Kennedy, M.,Papayannopoulou,T. andWiles,M.V. Kalev-Zhylinska, M.,Horsfield,J.A.,Flores, M.V. C.,Postlethwait,J.H., Inman, K.E.andDowns,M. Houssaint, E. Gurtner, G.C.,Davis,V., Li,H.,McCoy, M.J.,Sharpe, A.andCybulsky, M.I. Gribi, R.,Hook,L.,Ure, J.andMedvinsky, A. Godin, I.E.,Garcia-Porrero, J.A.,Coutinho,Dieterlen-Lièvre, F. and Gekas, C.,Dieterlen-Lievre, F., Orkin,S.H.andMikkola,K. de Bruijn,M.,Ma,X.,Robin,C.,Ottersbach,K.,Sanchez,M.-J.andDzierzak, Kwee, L.,Baldwin,H.S.,Shen,M., Stewart,C.L.,Buck,C.,A. Kinder, S.J.,Tsang, T. E.,Quinlan,G.A.,Hadjantonakis, A.K.,Nagy, A.and Downs, K.M.andBertler, C. Downs, K.M.andHarmann,C. Downs, K.M.andGardner, R.L. Downs, K.M.,Temkin, R.,Gifford, S.andMcHugh,J. Downs, K.M.,Gifford, S.,Blahnik,M.andGardner, R.L. Downs, K.M.andDavies,T. Downs, K.M. Downs, K.M. Downs, K.M. 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