role in ERK/MAPK cascadeinmammals,asingle Vaillancourt, 1994). to regulate cellgrowth anddifferentiation (Johnsonand et al.,1995;Wu etal.,1995).Inmammals,thispathway isproposed laevis placentogenesis. Intheembryo, with MAPKs;and(2)apartoftheMAP2Kspecificsequence(MSS; shown inX-MAP2K1tobeinvolved intheinteractionofMAP2K1 (1) theNterminus(33%identical/66%similar),whichhasbeen Two regions ofMAP2K1show reducedhomologywithMAP2K2: MAP2K2 (Brottetal.,1993;Crews etal.,1992;Russell1995). determination in al., 1992).TheERK/MAPKpathway isimplicatedincellfate MAPK1 andMAPK3uponagonistbindingtoreceptors(Crews et In thiscascade,MAP2Ksaredualspecificitykinasesthatactivate MEK1 andMEK2),itisknown astheERK/MAPKpathway. and theERKkinases(MAP2K1MAP2K2alsoknown as ERK1 andERK2(renamedMAPK3MAPK1,respectively) factor signaling,involves theextracellular signal-regulated kinases classical pathway, which appearstobethemajoroneingrowth Mahadevan, 1995;Seger andKrebs,1995;Zanke etal.,1996).The p38 kinaseandtheJunNH2-terminal(Cano which includetheextracellular signal-regulated kinase(ERK),the Multiple MAPKpathways have beendescribedinvertebrates, cellular organelles andthenucleus(Seger andKrebs,1995). various targets scatteredinthecytoplasm, thecytoskeleton, the consist ofproteinkinasecascadeslinkingextracellular stimuli with Mitogen-activated proteinkinase(MAPK)signalingpathways INTRODUCTION KEY WORDS:MAP2K1,Mapkinasecascade,Conditionaldeletion,Placenta,Labyrinthinemorphogenesis,Syncytiotrophoblast, Mouse Map2k1 Bissonauth,SophieRoy,Vickram MathieuGravel,StéphanieGuillemetteandJeanCharron* ectoderm duringplacentogenesis Requirement for Development 133,3429-3440(2006)doi:10.1242/dev.02526 Accepted 28June 2006 *Author forcorrespondence (e-mail:[email protected]) Universitaire deQuébec,L’Hôtel-Dieu deQuébec,QCG1R2J6,Canada. Centre derecherche encancérologie del’UniversitéLaval,Centre Hospitalier to the Sequence analysisrevealed thatthemurine MAP2K1 ismorerelated Map2k1 experiments. Inaddition, cell lineageisspecifiedatthecorrecttimeduringplacentogenesis.Theplacentalphenotypecanberescuedintetraploid activation isperturbed.Abnormallocalizationofthesyncytiotrophoblastsalsoobservedin maternal sinuses,suggestinganimportantrolefortheERK/MAPKcascadeinthesecells.In of MAP2K1andMAP2K2iswidespreadinthelabyrinthineregion,MAPK1MAPK3activationrestrictedtocellslining this phenotypeisassociatedwithadecreasedlabyrinthcellproliferationandanaugmentedapoptosis.Althoughtheactiva Although two different MAP2Kproteinsarepresentinthe (Hsu andPerrimon,1994;Kornfeld etal.,1995; Umbhauer Xenopus laevis anradtselegans Caenorhabditis –/– –/– embryos dieatmid-gestationfromabnormaldevelopmentandhypovascularizationoftheplacenta.Wenowshowthat mice. Altogether,thesedataenlightentheessentialroleof anradtselegans Caenorhabditis MAP2K1 (X-MEK)thantothemouse Map2k1 , Drosophila -specific deletionintheembryoleadstonormaldevelopmentandbirthofviable Map2k1 Map2k1 , Drosophila Map2k and gene functionappearsdispensable. Xenopus laevis gene fulfillsthis and Xenopus ( Mek1 . observations indicatethat absence of providing geneticevidence that region. As thelabyrinthdevelops, itbecomeshighly foldedand the chorionwithallantois,which willgive risetothelabyrinthine In mice,placentaformationinitiates aroundE8.5withthefusionof Cross, 2000;Rossantand 2001;SimmonsandCross,2005). differentiate toachieve specializedfunctions(Coanetal.,2005; trophoblast cellsthatarisefrom theextra-embryonic tissueand network andstromacoming fromtheembryonicmesoderm,andof maternal exchanges duringgestation.Itiscomposedofavascular specific functionsinmammals. capillaries intheheadregion. However, the normally, asevidenced bythepresenceofintersomiticvessels and normal, andvasculogenesis andangiogenesisseemtotake place Map2k2 presents amarked reductionofvascularization inthelabyrinth. al., 1999).Bycontrast,the compensatory effects by region oftheplacenta. of gestationbecauseanabnormaldevelopment ofthelabyrinthine recessive lethalphenotype,themutantembryosdyingat10.5days al., 1999).Thenullmutationofthe Map2k2 characterization ofmutantmouselinesinwhichthe transduction duringmousedevelopment hasbeenrevealed bythe mammals. they have mostprobablydiverged toachieve uniquefunctionsin differences observed betweenMAP2K1andMAP2K2suggestthat Nantel etal.,1998;Papin etal.,1996).Theprotein sequence members (Catlingetal.,1995;Dang1998;Eblen2002; 2002). Itisalsoinvolved intheinteractionwithRaffamily phosphorylation siteimportantforitsfunction(ColesandShaw, al.,1997).TheMSSdomainofMAP2K1containsaPAK et from different species(Fukudaetal.,1997;Papin etal.,1996;Xu 21% identical/36%similar),whichissharedbyMAP2K1proteins presence ofnormal ) inextra-embryonic Map2k1 The placentaisahighlyvascularized organ, whichallows fetal- The differential roleofMAP2K1andMAP2K2insignal –/– gene hasbeendisrupted(Bélangeretal.,2003;Giroux in extra-embryonicectodermduring mice showed noobvious phenotype, suggesting Map2k1 Map2k2 Map2k1 (Giroux etal.,1999).Altogether, these Map2k1 Map2k1 Map2k1 Map2k1 Map2k1 –/– Map2k2 expression levels intheplacenta, –/– embryos appearmorphologically (Bélanger etal.,2003;Giroux –/– placenta, ERK/MAPKcascade –/– and RESEARCH ARTICLE placenta, eventhoughthis phenotype isobserved inthe Map2k1 Map2k2 is unabletomake upforthe Map2k1 genes accomplish gene resultsina –/– Map2k1 placenta 3429 tion or

DEVELOPMENT and phospho-p38immunodetection. Serial sectionsof8 solution in0.1Mphosphatebuffer atpH7andthenembeddedinOCT. fixed overnight in8%paraformaldehyde, equilibratedina30%sucrose analyses describedbelow. Frozensectionswerepreparedfromspecimens according tostandardprocedures,orsubmittedimmunohistological deparaffinized, rehydratedandeither stainedwithHematoxylinandEosin care committee. Canadian CouncilonAnimalCareandapproved bytheinstitutionalanimal 1999). Allexperiments wereperformedaccordingtotheguidelinesof the 129/SvEvbackgroundhasbeenpreviously described(Girouxetal., structures allows normaldevelopment of process. Rescueofthe thereby suggestingarolefortheERK/MAPKcascadeinthis syncytiotrophoblasts areunabletoinvade theplacentallabyrinth thesyncytiotrophoblasts. However, the of mutation doesnotaffect thedeterminationanddifferentiation Despite theabsenceofMAPK1andMAPK3activation, the mainly observed inthecelllayersliningmaternal sinuses. MAPK1 andMAPK3.Inwild-typeplacenta,MAPKactivation is requirement of diminished in Moreover, theactivation oftheERK/MAPKcascadeisdramatically trophoblasts. combined toanaugmentedapoptosisoflabyrinth Map2k1 previously generatedtofullycharacterizetheplacental phenotype. development, wehave usedthe development. signaling viatheERK/MAPKpathway isessentialforplacental labyrinthine region (reviewed by RossantandCross,2001).Hence, been reportedtopresentdefectsinthedevelopment oftheplacenta cascade (GRB2,GAB,SOS1,RAF1,MAP2K1andMAPK1)have (MET, FGFR2andPDFGR)componentsoftheERK/MAPK signaling cascade.Growth factors (HGF),growth factor receptors of mouselinescarryingmutationsinmoleculesinvolved inthis placental development hasbeenhighlightedbythecharacterization the fetalbloodvessels. Contribution oftheERK/MAPKpathway to (syncytiotrophoblasts) thatseparatethematernalbloodspacefrom layer ofmononucleartrophoblastsandtwo layersofsyncytium and gasexchanges. Alarge partofthelabyrinthiscomposedone branched, generatinganimportantsurface arearequiredfornutrient 3430 1/50 dilution.The placentalvascular network was revealed with phospho-MAPK1/MAPK3 (Cell Signaling Technology) wereusedata against: phospho-p38MAPK, phospho-MAP2K1/MAP2K2and labeling (GirouxandCharron, 1998). Rabbitpolyclonalantibodies Apoptotic cellsweredetectedby terminaltransferase(TdT)DNA end H3, amitoticmarker (pH3;UpstateBiotechnology)(Aubinetal.,2002). Proliferation ratewas assessedbyimmunodetectionofphospho-histone Proliferation, apoptosisandimmunohistochemicalanalyses overnight in4%paraformaldehydeat4°C.Serial sectionsof4 inclusion orcryosection.For paraffin inclusion,thespecimenswerefixed vaginal plugasE0.5.Specimenswerecollectedandprocessedforparaffin Embryonic agewas estimatedbyconsideringthemorningofday Tissue collection The establishmentandthegenotypingof Mouse strainandgenotyping MATERIALS ANDMETHODS region. embryonic ectodermtosupportthedevelopment ofthelabyrinthine indicate thatMAP2K1actscellautonomouslyintheextra- production oflive-born animals.Altogether, theseobservations To dissectthephysiologicalroleofMAP2K1inplacental RESEARCH ARTICLE -deficient placentaexhibited areducedproliferation Map2k1 Map2k1 ␮ m weremountedonpolylysine-treatedslidesforCD31 function totransducesignalstheMAPKs, Map2k1 mutant placentaextracts indicatingthe mutation intheextra-embryonic Map2k1 Map2k1 Map2k1 mutant mouseline mutant mouselinein Map2k1 –/– embryos and -deficient ␮ Map2k1 m were those usedforimmunohistochemistry. andMAP2K1/MAP2K2werethesameas antibodies forMAPK1/MAPK3 were performedasdescribed(Bélangeretal.,2003).Thephospho-specific andthe and MAPK3 phosphorylated andnon-phosphorylatedformsofMAPK1 fromphospho-MAP2K2 to resolve phospho-MAP2K1 anti-MAP2K2 andpolyclonalanti-MAPK1antibodies.Mobilityshiftassays SDS-PAGE andprobedwithrabbitpolyclonalanti-MAP2K1, kb cDNA fragmentforthe the embryosweredissectedatE11.5orE13.5. the blastocyst stage,weretransferredintoE2.5pseudopregnant females,and described (Nagyetal.,1993).Theresultingaggregates, whichhadreached 2003). Total proteinlysates(20 Protein extracts werepreparedaspreviously described(Bélangeretal., Western blotanalysis SAS Systemwas used(Littelletal.,1998). and whileareaistherandomeffect. TheprocedurePROC MIXEDfromthe genotypes atallstagesstudiedwhengenotypeisconsideredthefixed effect the linearmixed modelwereperformedtoassessthedifference between was evaluated foraminimumoffive randomareas.Repeatedmeasuresfor apoptosis studies,ratioofpositively stainedcellsonthetotalcellnumber processed usingAdobePhotoshopCSprogram.For proliferationand camera (QICAM)andtheOpenlabsoftware (Improvision). Thephotoswere photographed onaDMRBmicroscope(Leica)usingQImagingCCD were presentedinthefigures.Bright-fieldanddark-fieldilluminations with Hematoxylin. Laboratories) was usedfordetectionandthesectionswerecounterstained hour atroomtemperature.TheVectastain HRPABCReagent(Vector binding was blocked byincubationwith10%normalgoatserumfor1 microwave for2minutesin10mMsodiumcitratebuffer. Non-specific International). Antigenretrieval was performedunderpressureina anti-CD31 antibodyusedata1/50dilution(PECAM;Pharmingen ( bredwith129/SvEvmicetotransmitthe targeted were to generatechimerasasdescribed(Bélangeretal.,2003).The null allele.Correctlytargeted EScloneswereinjectedintoMF1blastocysts third exon shouldresultinanout-of-frametranscript,creating a loxP Bam disrupt the thymidine kinasecassetteforselectionagainstrandomintegration. To 2 to4ofthe from a129/Svmousestrain-derived genomiclibrary. Itencompassesexons The targeting vector was madebyusinga12.4kbgenomic fragmentisolated Generation oftheMap2k1conditionalallele and aggregated withdiploid Tetraploid embryosofwild-typeB6CBAF1 werepreparedby electrofusion Generation oftetraploid-aggregation chimeras and were usedastemplatesforsynthesizing[ described (GirouxandCharron,1998).Thefollowing murinefragments Radioactive in situ hybridizationontissuesectionswas previously phosphatase assays In situhybridization, Map2k1 phosphatase activity was assayedbyincubatingrehydratedE10.5 embedded inparaffin wax andsectioned(7 2.4 kband4.2kb, respectively. we usedthe fragment, whereasthe genomic probe(Fig.7).The were genotypedbySouthernanalysis ofa partial excision of Mannheim). fragment. Map2k1 Six specimenswereanalyzedpergenotype.Themostrepresentative fields Map2k1 HI sitesflankingthethirdexon of sites was insertedinthethirdintron(Fig.7).Deletionof +/+ flox-neo ␤ and Map2k1 EIIa-Cre -Galactosidase stainingwas performed onwhole –/– Map2k1 ). To generatethe Map2k1 placentas (Girouxetal.,1999).Stainedspecimenswere loxP sequences, two deleter mouselinethatcanproduce mosaicmicewith gene, whichwas fusedtotheherpessimplex virus- Map2k1 –/– ␤ flanked sequences(Laksoetal.,1996).Specimens -galactosidase stainingandalkaline placenta sectionswithBMsubstrate(Boehringer Vegf Map2k1 Map2k1 floxed Map2k1 ␮ 120 isoformanda1.5kbcDNA g) wereresolved onadenaturing10% and loxP +/– endogenous allelegeneratesa2.0kb Map2k1 embryos or Map2k1 35 sequences wereinsertedintothe conditional allele( S]UTP-labeled riboprobes:a1.5 Stu ␮ m) forphotography. Alkaline I digestionusinga .A ⌬ Development 133(17) alleles producebandsat neo Map2k1 cassette flanked by Map2k1 Map2k1 –/– Map2k1 ES cellsas Map2k1 Map2k1 Map2k1 Gcm1 floxed allele +/– ), -

DEVELOPMENT Our previous characterizationof labyrinthine region oftheplacenta Map2k1 RESULTS Map2k1 2004; Yu etal.,2004),wehypothesizedthatthedisruptionof et al.,2003;Pages etal.,1993;von Giseetal.,2001;Wu etal., ERK/MAPK cascadeincellularproliferationandsurvival (Huynh (Giroux etal.,1999).Given thewell-documentedroleof 1 daybeforethedeathof placenta was reducedinsize,aphenotypealreadyobvious atE9.5 1999). However, thelabyrinthineregion ofthe spongiotrophoblasts andlabyrinthinetrophoblasts(Girouxetal., lineages,asshown bythepresenceofgiantcells, cell not perturbthedifferentiation oftheplacental trophoblast development oftheplacenta.Theabsence revealed theessentialroleplayedbyMAP2K1innormal role inextra-embryonicectoderm is required fortheexpansionof Map2k1 Map2k1 mutant embryos(Fig.1A) Map2k1 mutant conceptuses Map2k1 function did mutant reduced growth ofthe assays wereperformedtodetermineifapoptosiscontributes tothe not statisticallysignificant( Map2k1 Map2k1 Cross, 2001).Bycontrast,theproliferationratewas halved in in extensive labyrinthinedevelopment (Fig.1B,D)(Hemberger and proliferation rateinthelabyrinthineregion was elevated resulting to E10.5placentas(Fig.1B).Inwild-typeplacentas,the using phospho-histoneH3antibodytodetectmitoticcellsinE8.5 proliferation and/orsurvival. We firstperformedimmunostaining Map2k1 decreased proliferationobserved inE8.5 E8.5 andE9.5 (Fig. 1C,E).Conversely, apoptosiswas increasedbyfivefold in apoptotic cellsweredetectedinE8.5toE10.5wild-typespecimens –/– –/– might affect labyrinthineexpansion bylimitingcell placenta atE9.5andE10.5( embryos weredyingandinresorption(Fig.1E). Map2k1 –/– Map2k1 specimens andbytenfoldatE10.5when P> 0.09; Fig.1D).Inparallel,TUNEL –/– labyrinthine region. Very few RESEARCH ARTICLE ( of E10.5wild-type Map2k1 E9.5 andE10.5wild-type paraffin sectionsfrom E8.5, detected byTUNELassayson and ( phospho-histone H3antibody by immunostainingwith Map2k1 observed forE9.5andE10.5 proliferating cellratio(D)was significant reduction ofthe represented. Astatistically ( and increased apoptosis. from reduced proliferation Map2k1 labyrinthine region in underdevelopment ofthe Fig. 1.The ( placental sections. in A;50 labyrinth. Scalebars:100 trophoblast giantcells;l, allantois; d,deciduum;g, stages analyzed(E).al, Map2k1 significantly increased in apoptotic cellratiowas ( trophoblasts inproliferation The percentage oflabyrinthine chorioallantoic region atE8.5). in thelabyrinthineregion (or cells (B)andapoptotic(C) indicate examplesofmitotic Map2k1 A B D Proliferation wasdetected ) Hematoxylin-Eosin staining ) ) andapoptosis( Map2k1 P< C ) apoptosiswas –/– –/– –/– ␮ 0.01; Fig.1D).The –/– +/+ m inB,C. placenta atall placentas. The placentas. Arrows ) and placenta results –/– labyrinth was Map2k1 E ) is 3431 –/– ␮ m

DEVELOPMENT E10.5 pMAPK3 levels (Fig.2A).Thisdecreasewas moredramaticin extracts, themutationcausedadiminutionofpMAPK1 and MAPK3 phosphorylation(pMAPK1/pMAPK3),whileinplacenta extracts, lackofMAP2K1didnotseemtoaffect MAPK1and MAPK1, respectively (Fig.2A).InE9.5 the gelwerecontrolledwithantibodiesagainstMAP2K1and absence oftheMAP2K1proteinandlevel ofproteinsloadedon read-out oftheirstateactivation (Gopalbhaietal.,2003).The MAP2Ks atspecificresiduesoftheactivation loopisusuallyagood MAP2K1/MAP2K2 antibodies.ThephosphorylationofMAPKsand using phospho-specificMAPK1/MAPK3and extracts fromE9.5andE10.5wild-type western blotanalyseswithembryonicandplacentalwholeprotein placenta was duetoperturbedERK/MAPKsignaling,weperformed Loss ofERK/MAPKactivationin placenta. reduced expansion ofthelabyrinthineregion in Therefore, perturbedcellproliferationandapoptosisunderlaythe 3432 embryos weredyingandintheprocessofresorption. between E9.5andE10.5embryoscouldbethat explanation forthedifference inpMAPK1andpMAPK3levels cellular proliferationandsurvival intheplacenta.Alikely placentas, itcouldnotactivate theERK/MAPKpathway andpromote To definewhethertheproliferationdefectobserved in embryo andstronglyinthelabyrinthineregion (Girouxetal.,1999). Both Map2k1 reduction inpMAPK1andpMAPK3levels was detectedin phosphorylation (pMAP2K2;Fig.2B).Similarly, animportant although MAP2K2was phosphorylatedandactivated in those ofwild-typespecimens(Fig.2B).Theseresultssuggestedthat pMAPK3 andpMAP2K1proteinswereunchangedcomparedwith Map2k1 Map2k1 RESEARCH ARTICLE –/– embryos. In and –/– Map2k2 placentas, despitethepresenceofMAP2K2 Map2k2 genes areubiquitouslyexpressed inthe –/– specimens, thelevels ofpMAPK1, Map2k1 Map2k1 Map2k1 –/– –/– –/– placenta Map2k1 embryonic Map2k1 Map2k1 specimens Map2k1 –/– –/– –/– –/– maternal sinusesoftheplacentallabyrinth.In pathway was stronglyactivated inthecellpopulationlining Map2k1 (Fig. 3A;seeFig.S1inthesupplementarymaterial).Bycontrast, cells inE10.5 at E9.5,pMAPK1/pMAPK3stainingwas restrictedtotheallantoic endothelium oftheembryonicbloodvessels (Fig.3B).As observed the maternalsinuses,whilenosignalwas observed inthe pMAPK1/pMAPK3 stainingwas mainlydetectedinthecellslining despite thewidespreadMAP2K1andMAP2K2activation, allantois andatthechorioallantoicinterface (Fig.3B).Interestingly, specimens, thehigheststainingwas restrictedtothecellsof Map2k1 we performedimmunostainingonE9.5andE10.5wild-type, the ERK/MAPKcascadeisactivated duringplacentaldevelopment, In ordertoidentifyinwhichcelltypeofthechorioallantoicregion sinuses the cellsliningmaternal Robust activationoftheERK/MAPKpathwayin chorioallantoic interface, phosphorylationofMAPK1and placentas, even thoughMAP2K2activation was observed atthe embryonic bloodvessels inwild-typeand allantoic cellsand,inaddition,theendotheliumlining the labyrinthineregion, thecellsliningmaternalsinuses, allantois. pMAPK1/pMAPK3 stainingwas observed only incellsofthe phosphorylated inlabyrinthinetrophoblastsandtheallantois, 3B; seeFig.S1inthesupplementarymaterial).In cells oftheallantoisinbothwild-typeand trophoblasts, incellsliningthematernalsinuses(Fig.3A)and pMAP2K1/pMAP2K2 stainingwas detectedinlabyrinthine antibodies. AtE9.5,thepresenceofpMAPK1/pMAPK3and MAPK1/MAPK3 andphospho-specificMAP2K1/MAP2K2 Altogether, theseresultsindicatedthattheERK/MAPK One daylater, pMAP2K1/pMAP2K2positive signalwas seenin –/– –/– and specimens, even thoughtheMAP2K2proteinwas Map2k1 Map2k2 –/– –/– placentas. E10.5 ( total protein extractsfrom E9.5( were blotanalysisof evaluatedbywestern MAPK1/MAPK3 andMAP2K1/MAP2K2 Expression andphosphorylationlevelsof Map2k1 ERK/MAPKactivationin Fig. 2. E10.5 and MAPK3wassignificantlyreduced in and placentas.PhosphorylationofMAPK1 placentas. observed inthecorresponding E9.5 Map2k1 and remained unchangedinE9.5 placenta sectionswithphospho-specific Map2k1 B –/– ) wild-typeand –/– embryos. Areduction wasalso placentas andembryos. –/– Map2k2 embryos andplacentas, Development 133(17) Map2k2 Map2k1 –/– placentas (Fig. –/– Map2k1 Map2k1 specimens A –/– ) and embryos –/– –/–

DEVELOPMENT the activation statusofthep38/MAPK cascadein 2000; Rousseauetal.,1997).Therefore,wedecidedtoinvestigate in angiogenesisandactivated inresponse toVEGF(Mudgettetal., Thep38/MAPKsignalingpathway was shown tobeimplicated 3). cascade maynotplayadirectroleinlabyrinthineangiogenesis(Fig. the cellsliningmaternalsinuses,indicatingthatERK/MAPK wild-type placentaswhencomparedwiththeactivation observed in embryonic vascular endothelialcellsofthelabyrinthineregion in the ERK/MAPKpathway was notstronglyactivated inthe al., 1999).However, thedatapresentedinFig.3demonstratedthat suggesting thatplacentaangiogenesiscouldbedefective (Girouxet Map2k1 are excluded fromthechorioninE9.5 We have previously shown thatthefetalvascular endothelialcells placentas Normal VEGFangiogenicsignalingin ERK/MAPK cascade. the specificrequirementforMAP2K1inactivation ofthe MAPK3 proteinswas greatlyreduced,reinforcingthenotionof activation ofthep38/MAPK cascadebyinsituhybridization and labyrinthine angiogenesis,we thenstudied the deciduum.To evaluate theVEGFsignalingcascadein cells correspondingtomaternal bloodvessels werealsodetectedin confined tothechorioallantoic plate(Fig.4A,B).CD31-positive embryonic bloodvessels originatingfromtheallantoiswere the wholelabyrinthregion, whilein antibody. Inwild-typespecimens,thefetalbloodvessels invaded immunostaining onE10.5placentasectionswithananti-CD31 placentas. To studythevascular endothelialnetwork, wefirstperformed role inextra-embryonicectoderm Map2k1 Map2k1 Vegf –/– Map2k1 Map2k1 expression and specimens the –/– placentas, mutant –/– Gal stainingandlocalize vector usedtogeneratethe advantage ofthe activation coincidewiththedomain of actionMAP2K1,wetook al., 2005). to hypoxicstress(Blaschke etal.,2002;Conrad2000; Fan et p38/MAPK cascadewas detected(Fig.4F),indicatinga response endothelial cellswereconfinedandwhereactivation ofthe chorioallantoic plateandintheallantois,wherevascular Map2k1 Gal stainingwas observed inthelabyrinthregion, whereasin the maternalbloodcells(Fig.4G,H). Thus,in chorioallantoic plate,forminga barrierbetweentheembryonicand and Map2k1 supporting aroleforp38/MAPKinangiogenesis(Fig.4C).In endothelial cellsofthelabyrinthineregion andinthe allantois, p38/MAPK cascadewas specificallyactivated inthefetalvascular immunolocalization, respectively. Inwild-typespecimens,the dramatic increaseof exchanges betweentheembryoand themothercausedby thelatter mostprobablyresultingfrompoor blood stress, capacitytogenerateanangiogenic responsetothehypoxic a endothelial cellsblocked atthechorioallantoicplate,indicating the p38/MAPKcascadewas activated intheembryonicvascular of wild-typespecimens(Fig.4E).In probably correspondingtovascular endothelialcellsinthelabyrinth functional (Fig.4D). interface, suggestingthatthep38/MAPKcascadewas still To assessiftheincreased Map2k1 –/– –/– specimens, p38remainedactivated atthechorioallantoic specimens, X-Galstainedcellswereconfinedtothe –/– placentas. In lacZ Vegf cassette oftheROSA Vegf wild-type, MAP2K2 stainingofE9.5( MAPK1/MAPK3 andanti-phospho-MAP2K1/ Map2k1 interface (red arrows) andintheallantoisof activation ofMAP2K2atthechorioallantoic vessels (blackarrowheads). Despitethehigh endothelial cellsliningtheembryonicblood was alsoobservedinthevascular E10.5, phospho-MAP2K1/MAP2K2signal only intheallantois(red arrowhead). (B)At placentas, MAPK1/MAPK3were activated Map2k1 labyrinthine trophoblasts ofwild-type, the allantois(red arrowheads), andinsome sinuses(blackarrows),maternal incellsof were phosphorylatedincellsliningthe placentas. (A)AtE9.5,MAP2K1/MAP2K2 Map2k1 MAP2K1/MAP2K2 inwild-typeand MAPK1/MAPK3 andphospho- Fig. 3.Localizationofphospho- Map2k2 labyrinthine trophoblasts inwild-typeand sinuses (blackarrows) andinsome was primarilyfoundaround maternal However, phospho-MAPK1/MAPK3signal 50 sinus.Scalebar: labyrinth; ma,maternal allantois; ev, embryonicbloodvessel;l, only intheallantois(red arrowhead). al, phospho-MAPK1/MAPK3 were detected expression was detectedindiscreteislets Map2k1 Map2k1 ␮ Map2k1 Vegf m. expression was observed atthe –/– –/– –/– –/– expression andthep38/MAPK expression inE10.5 Map2k1 placentas (red arrowhead), specimens. In and RESEARCH ARTICLE placentas. +/– mutant alleletovisualizeX- placentas, awidespreadX- Map2k2 Map2k1 –/– and ␤ Map2k1 Anti-phospho- -geo promotertrap –/– Map2k1 Map2k2 A placentas. –/– ) andE10.5( –/– placentas, a Map2k1 –/– placentas, –/– 3433 B +/– )

DEVELOPMENT determine whetherthesyncytiotrophoblast precursorsof blocked atthechorioallantoicjunction(Fig.5D,F).Inorderto strongly suggestedthatthe a similarpatterntothe alkaline phosphataseactivity was detectedinpatchesandfollowed E9.5 andE10.5wild-type probe usedasamarker forsyncytiotrophoblast precursorsonE8.5, were duetothemisspecificationofsyncytiotrophoblasts. reported, Map2k1 hypovascularization. Thedefective angiogenesisobserved in 3434 Map2k1 performed alkalinephosphataseassaysonE10.5wild-typeand placentas wereabletodifferentiate intosyncytiotrophoblasts, we Map2k1 Normal determinationanddifferentiation of problem. was normallyactivated in from extra-embryonic tissues. Bycontrast,thep38/MAPKpathway from theembryo.Thesedatasuggested thatthe (Fig. 5C,E),whilein type placentas, sinuses inwild-typeplacentas(Fig.5G,I).In observed astrongalkalinephosphataseactivity aroundmaternal activity (Matsubaraetal.,1993;Wu etal.,2003).Asexpected, we the maternalsinusesexpress endogenousalkalinephosphatase greatly reducedinthe Our resultssuggestthattheactivation oftheERK/MAPKcascadeis Tetraploid rescue of morphogenesis bythesyncytiotrophoblasts. appears tobenecessaryforthechorioallantoicbranching likely toberequiredforsyncytiotrophoblast differentiation but labyrinth. Thus,theERK/MAPKsignalingviaMAP2K1is not chorion, allowing theformationofvascular network ofthe phosphatase activity. However, they wereunabletoinvade the were alsoabletodifferentiate, asdemonstratedbythealkaline phenotype hasan extra-embryonic origin thataffects thelabyrinthine as wellthevascularization defectobserved in (Fig. 3),weinvestigated whetherthe lackofpMAPK1andpMAPK3, MAPK3 arestronglyactivated inthecellsliningmaternalsinuses (Coan etal.,2005;SimmonsandCross,2005).AsMAPK1 sinuses toisolatethefetalbloodcirculationfrommaternalone Syncytiotrophoblasts andmononucleartrophoblastscoatthematernal vessels arisingfromtheallantois(RossantandCross,2001). allow thevascularization ofthelabyrinthbyembryonicblood allantois andinvolved thesyncytiotrophoblast cellline.Thiswillthen of thelabyrinthproceedsinresponsetoinstructive signalsfromthe before thechorioallantoicfusion,suggestingthatmorphogenesis syncytiotrophobasts, atthesiteofallantoicmesodermevagination chorionic platestartstoexpress (Rossant andCross,2001).Asubgroupoftrophoblastsinthe epithelium contributes actively tothechorioallantoicbranching labyrinth. Thereisincreasingevidence toindicatethat thetrophoblast morphogenic andvascularization processeswill generatethe fuses withthechorionaroundE8.5.Atthisstage,complex The vascularization ofthelabyrinthisinitiatedwhen theallantois determined attherightmomentin detected in chorions (Steccaetal.,2002),andasimilarexpression profilewas We firstperformedinsituhybridizationexperiments witha RESEARCH ARTICLE –/– –/– Gcm1 –/– placentas maythereforebesecondarytoamorphogenic Map2k1 specimens asdifferentiated syncytiotrophoblasts lining syncytiotrophoblasts Gcm1 was expressed incellclustersE8.5wild-type –/– Map2k1 -positive cellsstartedtoinvade thelabyrinth Map2k1 chorions (Fig.5A,B).InE9.5andE10.5wild- Map2k1 Gcm1 Map2k1 –/– Gcm1 –/– Map2k1 placentas, signal (Fig.5H,J).Theseresults –/– syncytiotrophoblasts that originate Gcm1 vascular endothelial cellsderived -positive cellswerecorrectly -deficient embryos –/– Map2k1 , anearlymarker ofthe Gcm1 placentas. Aspreviously Map2k1 Map2k1 Map2k1 –/– -positive cellswere placentas. They –/– –/– –/– specimens, Map2k1 placentas, placental Gcm1 –/– specimens andatthechorioallantoicinterfaceof p38 MAPKintheembryonicbloodvesselsoflabyrinthwild-type arrows). In Gal stainingof Eosin-stained sectionsofspecimensEandFare presented inFig.1A.X- For placentalstructure identification,nearadjacentHematoxylinand Scale bars:50 chorionic plate;d,deciduum;g,tropohoblast giantcells;l,labyrinth. staining isrestricted tothechorioallantoicplate.al,allantois;ch, The labyrinthineregion ispresented. In define the (black arrowheads). wild-type ( at thechorioallantoicplate(B).Detectionofp38MAPKactivationin Fig. 4.Abnormalvascularizationof region of arrows). High placentas wasanalyzedbyinsituhybridization(red signalandblue aggregated withembryonicstem(ES) cells(Nagyetal.,1993).ES contribute tothetrophectoderm anditsplacentalderivatives once playing anessentialrole,weperformed tetraploidrescueexperiments. which structure,embryonicor extra-embryonic, MAP2K1was consequence ofabnormalchorioallantoic branching.To definein morphogenesis. Thevascularization defectobserved mightbea labyrinth. Map2k1 Tetraploid embryosdevelop poorlybut they have thepotentialto –/– Map2k1 C Map2k1 ( Map2k1 The embryonicvascularnetworkofE10.5wild-type( B ) and ) placentaswasrevealed byanti-CD31staining(black Vegf ␮ Map2k1 m inA-D,G,H;100 Map2k1 –/– expression levelswere detectedintheallantoic –/– expression profile innormalandmutantplacentas. Vegf placentas, indicatingaresponse tohypoxicstress. placentas, thefetalbloodvesselswere restricted +/– expression inwild-type( –/– ( G ( ) and D ) placentasrevealed ahighactivationof Map2k1 ␮ m inE,F. Map2k1 Mapk2k1 –/– ( H Development 133(17) –/– ) placentaswasusedto –/– Map2k1 E ) and placental specimens, X-Gal Map2k1 –/– placentas A –/– ) and ( F )

DEVELOPMENT h rescuewas incomplete. TheE13.5chimerawas slightly the and thehistologyofchimera obtainedatE13.5suggestedthat obtained atE11.5werewelldeveloped, whereasthegrossmorphology idtp lcna(i.6-) The wild-type placenta(Fig.6U-W). intermingled withthematernalsinuses similarlytothoseobserved in embryos, fetalbloodvessels migratedintothelabyrinthand Map2k1 lineages. Basedonthesecharacteristics,aggregation of mass derivatives but nottotrophectodermorprimitive endoderm with tetraploidcellsinchimeras.They canparticipatetoallinnercell cells have essentiallythereciprocaldevelopmental potentialcompared allowed thesurvival of Anti-CD31 stainingshowed thatin revealed thatthelabyrinthdeveloped normally(Fig.6H,J,M,O,R,T). morphology andhistologicalstainingofthechimericplacentas (four andonechimericembryos,respectively; Fig.6C,E).Gross combination of of thetissueresponsibleforplacentalabnormality. The cells towild-typetetraploidembryosshouldleadtheidentification role inextra-embryonicectoderm Map2k1 –/– Map2k1 ES cellsandtetraploidwild-typeembryos –/– embryos uptoE11.5andE13.5 Map2k1 Map2k1 –/– –/– tetraploid chimeras tetraploid-rescued Map2k1 –/– ES Conditional deletionof trophoblasts forthenormaldevelopment ofthelabyrinthineregion. MAP2K1 isrequiredinextra-embryonic-derived structureslike the dispensable forthenormaldevelopment oftheembryo,andthat rescued Normal activationofMAPK1and MAPK3in embryo, itcanprobablybereplaced byitshomologue, and Blenis,2001; Brunetetal.,1995;Zhang andLiu,2002). required forcellproliferationandsurvival in mammals(Ballif be The activation oftheERK/MAPKcascadehasbeenshown to phosphatase activity(J;arrowheads) andthe There isacorrelation betweenthepunctuatedlocalizationofalkaline produced highlevelsofalkalinephosphataseactivity(arrowheads). type specimens(I),cellssurroundingbloodsinuses thematernal E10.5 (E,F),wild-type(A,C,E)and Gcm1 chorioallantoic trophoblasts insyncytiotrophoblasts wasassessedby 7A; seeMaterialsandmethods). of theMAP2K1catalyticdomain,wereflanked by mutation. Thethirdexon sequences,encodingtheATP-binding site development, wegeneratedmicecarryingaconditional Map2k1 To circumvent theplacentalphenotypeanddeterminewhether embryo in thetetraploidrescueexperiments, wecannotexclude aclonaleffect. With thisbreeding,normalMendelianratio oflive-born primordial roleof positive cellswere detectedasearlyE8.5inwild-typeand bars: 100 conversely tothoseseenin normal, nofetalbloodcellwas detectedinthebloodvessels Moreover, even thoughthevascularization ofthelabyrinthwas underdeveloped andpresentedsignsofhemorrhage(Fig.6E). Map2k1 adult micewereperformed.NoMAP2K1proteinwas detected in protein extracts fromdifferent organs of mice weredevoid ofMAP2K1protein,westernblotanalyses animals was obtained(Table 1;Fig.7B).To verify thatthe chorioallantoic interfacein at E9.5andE10.5, placentas, withnomajordifference betweenthegenotypes.However, in Fig. 5.Determinationanddifferentiation ofsyncytiotrophoblasts activity inE10.5wild-type(G,I)and sinuses(C,E;arrows).to linethematernal ( whereas incontrol placentas, aggregation experiments rescuedpartially the These observations raisedthepossibilitythattetraploid- allele (Fig.7C).Both phenotype. Alternatively, asone Map2k1 E10.5, exhibiting thesamephenotypeasoneobserved inthe and whenthey wereintercrossed,they generated littersthatdiedat Sox2Cre inembryonicderivatives (Hayashietal.,2002).To doso, sequences Sox2Cre chorioallantoic interfaceof Map2k1 Altogether, thesedataindicatedthat in situhybridization(red signal)inE8.5(A,B),E9.5(C,D)and –/– ⌬ ; ␮ Map2k1 / gene functionisrequiredfornormalmouseembryo transgenic micetospecificallydelete ⌬ embryos (Fig.8A).Altogether, theseresults confirmedthe m inA-H;50 –/– Map2k1 organs, demonstratingthatthe placentas. +/ Gcm1 Map2k1 ⌬ males werebredwith ⌬ Map2k1 / ⌬ ␮ labeling remained restricted tothe ( m inI,J. embryos A-F Map2k1 Map2k1 Map2k1 in placentadevelopment, whereas,inthe Gcm1 ) Thefunctionaldifferentiation of ⌬ / Map2k1 ⌬ Map2k1 Map2k1 –/– Map2k1 Map2k1 female andmalemicewerefertile –/– -positive cellsinvadedthelabyrinth +/– specimens (D,F;arrowheads), placentas (F;arrowhead). Scale RESEARCH ARTICLE tetraploid chimeras(Fig.6S,T). –/– G-J –/– Map2k1 –/– floxed (B,D,F) placentas. Gcm1 gene inthe Map2k1 ) Alkalinephosphatase ES cellclonewas tested (H,J) placentas.Inwild- Map2k1 mice werebredwith Map2k1 expression inthe Map2k1 +/+ Map2k1 ⌬ and loxP +/floxed allele isanull Map2k2 Map2k1 Map2k1 Map2k1 Map2k1 might be –/– sites (Fig. females. Gcm1 Map2k1 exon 3 mutant 3435 . - ⌬ ⌬ ⌬ –/– / / / ⌬ ⌬ ⌬

DEVELOPMENT and Genotype ofthetetraploidembryos andyolksacsrevealed thattheembryoswere embryos showninC,E. genotype ofthetetraploid performed toconfirmthe (lower panel)analyseswere blot (upper panel)andwestern tE10.5was duetothefact thatthe at inability ofMAP2K2toactivate theERK/MAPKpathway activated MAP2K2(Fig.2B).Thesedatasuggestedthatthe were included ascontrol. NoMAP2K1 protein wasdetectedin MAPK1 andMAPK3inE10.5 issuedirectly, weanalyzedthephosphorylationstatusof this dying fromunderdevelopment oftheplacenta.To address and diploid tetraploid wild-typeembryos chimeras were prepared from ( presented forcomparison. Map2k1 wild-type (A,F,K,P,U) and cells. Forcomparison,E10.5 embryo and from wild-typetetraploid (C,H,M,R,W) E11.5chimeras magnification imagesofK-O. images ofF-J.( ( and Eosinstaining. were analyzedbyHematoxylin Corresponding placentas( E13.5 (D,E)foranalysis. recovered atE11.5(C)or foster mothers,chimeraswere cells (C,E).Aftertransferinto embryos (D)or and fetuses. Map2k1 Fig. 6.Tetraploid rescue of E9.5 In 3436 ,deciduum;g,tropohoblast giantcells;l,labyrinth. y, yolksac;e,embryo.Scalebars: 1mminA-J;100 d, from theembryonicand extra-embryonictissuescontainedboth immunostaining. ( ( some developmentaldefects. (arrows inS;T)suggested blood cellsinthelabyrinth and theabsenceofembryonic morphology oftheembryo(E) (D,S). However, thegross compared totetraploidcontrol region oftheplacenta(T)when vascularization ofthelabyrinth E13.5 (E)alsoshowednormal tetraploid chimeraobtainedat Map2k1 tetraploid rescue ofthe (M,R,W) indicatedthe the histologyofplacenta aggregated embryos(C)and Map2k1 normal appearanceofE11.5 specimens are shown.The were detectedin whereas 1daylater, nopMAPK1-andpMAPK3-specificbands C-E K-O U-W ) Tetraploid-aggregation Map2k1 Higher magnification ) Anti-CD31 ) RESEARCH ARTICLE –/– –/– –/– Map2k1 -deficient placentas phenotype. The tetraploid- (B,G,L,Q,V) Map2k1 Map2k1 –/– ( A P-T Map2k1 embryos are , B –/– X ) Higher ) Wild-type ) PCR +/– Map2k1 –/– embryos, thecascadewas normallyactivated, –/– ES F-J ES ) –/– embryos, despitethepresenceof Map2k1 Map2k1 ⌬ / ⌬ embryos andtheir –/– embryos were Map2k1 Map2k1 –/– ES cells(ES26-4) orin breeding of MAPK3. Thus,inabsenceofanormalplacenta,E10.5 activation. embryos weredyingandhencecouldnotachieve ERK/MAPK and pMAPK3in females. Western blotanalysesrevealed nodecreaseinpMAPK1 corresponding rescued placental extracts (Fig.8B).Therefore,inE10.5conditionally alleles. Map2k1 Map2k1 Map2k1 –/– Sox2Cre , whiletheyolksac,whichreceived acontribution –/– ⌬ Map2k1 Map2k1 / ⌬ ES cells(ES26-4)and Map2k1 embryos, MAP2K2canactivate MAPK1and ; Map2k1 floxed/ ⌬ ␮ –/– / m inK-O;50 ⌬ tetraploid chimera presented inC. embryos, andinthecorresponding ⌬ +/ placentas obtainedfromthe ⌬ males with Map2k1 ␮ m inP-W. Development 133(17) +/– embryo DNAs Map2k1 Map2k1 +/floxed –/–

DEVELOPMENT seta roleof essential associated withitshypovascularization andrevealing the lethal phenotypecausedbytheunderdevelopment ofthelabyrinth The disruptionofthemurine DISCUSSION Map2k1 et al.,2003).We have previously shown thatboth can compensateforthelackof Map2k2 etal.,1999).Bycontrast,theabsenceofphenotypein (Giroux MAPK3), revealed that,althoughMAP2K1andMAP2K2canbe MAP2K2, aswelltheirdownstream effectors (MAPK1and 1999). Immunostainingoftheactive formsofMAP2K1and Map2k2 mutants suggeststhat genes arewidelyexpressed intheplacenta(Girouxetal., role inextra-embryonicectoderm Map2k1 Map2k1 Map2k2 during embryonicdevelopment Map2k2 gene leadstoanembryonic is dispensable,as gene function(Bélanger Map2k1 Map2k1 and erepressedbythephosphorylationofSer be activity ofactivated MAP2K1andMAP2K2proteinscan require ERK/MAPKcascadeinthelabyrinthmayspecifically the of chorioallantoicinterface, suggestingthattheactivation the pMAPK1 andpMAPK3wererestrictedtotheallantoiccellsat tSer at 2003),suggestingthatthephosphorylationofMAP2K2 al., trophoblasts ofwild-typeplacentas.In maternal sinuses,theallantoiccellsandsomelabyrinthine pMAPK1 andpMAPK3wereobserved inthecellslining limited tospecificlineages.For example, detectablelevels of activated inseveral celltypesoftheplacenta,theiractionis 212 Map2k1 may accountforthelackofMAPK1/MAPK3 . Ithasrecentlybeenshown thatthecatalytic type (+/+)and different organsof8-week-oldwild- analyses ofproteins extractedfrom with a pancreas; Th,thymus. kidney; Li,liver;Lu,lung;Pa, MAP2K2. Br, brain;He,heart;Ki, against MAP2K1,MAPK1and probed withantibodiesdirected littermates. Triplicate blotswere ( Map2k1 Fig. 7.Generationofthe third third andfourth loxP neo represented bythewhiteboxes.A translated exons2to4are conditional allele. for thegenerationof of thepanels.( alleles isindicatedontherightside panel). Thepositionofthedifferent panel) anda Map2k1 blotted andhybridizedwitha Tail DNAwasdigestedwith breeding a DNA from alitterobtainedafter added atthe5 selection cassette(graybox)was virus-thymidine kinase(HSV-TK) respectively. Theherpessimplex and 6.9kbof targeting vectorcontains5.5kb the secondandthird exons.The ( 3 genomic sequencesonthe5 A B Ј ) Southern blotanalysisoftail ) Southern ) sides ofthe RESEARCH ARTICLE Map2k1 selection cassetteflankedby sites wasinsertedbetweenthe loxP Sox2Cre genomic probe (A;upper conditional allele. site wasinsertedbetween Map2k1 Map2k1 gene targetingstrategy Cre Map2k1 Ј Map2k1 neo C +/– 212 end ofthevector. ) Westernblot Map2k1 probe (lower ; Map2k1 Map2k1 insertion, +/floxed (Gopalbhai et –/– ⌬ homologous / ⌬ placentas, exons. A female +/ ( ⌬ ⌬ Stu / Ј ⌬ male. and 3437 ) I,

DEVELOPMENT arrowhead) characteristicofthe phospho-MAPK1/MAPK3 andphospho-MAP2K1/MAP2K2. were probed withantibodies againstMAPK1,MAP2K1,MAP2K2, Map2k1 Comparative Hematoxylin-EosinstainingofE10.5wild-typeand ubro us6844666 6 15% 12.5% 15% 6 25% 15% 12.5% 4 10% 12.5% 4 10% 12.5% 20% 12.5% detection levels. 8 MAP2K1 functioninthelabyrinthtrophoblastsareunder that thelevels ofactivated MAPK1andMAPK3sufficient for 12.5% phosphorylation inthelabyrinth.Alternatively, itisalsopossible 15% 6 Male % expected % obtained Number ofpups Table 1.Rescueoftheplacentalphenotypein 3438 Map2k1 Fig. 8.NormalactivationoftheERK/MAPKcascadeinrescued Gross morphologyof cascade activationin sinusesinthelabyrinthineregion,maternal respectively. ( arrows andarrowheads indicatethefetalbloodvesselsand and fetalbloodvesselsinterminglinginmutantspecimens.Theblack produced from mating between development oftheirextra-embryonic structures. E10.5embryos Map2k1 Map2k1 RESEARCH ARTICLE +/floxed ⌬ ⌬ / ⌬ / ⌬ placenta sections showed the absence of maternal sinuses placenta sectionsshowedtheabsenceofmaternal +/ embryos. ⌬ ;Tg females were usedforprotein extracts.Westernblots +/ Sox2Cre Sox2Cre Map2k1 ( were crossed withfemale Map2k1 A ) Placentalphenotypeof Tg +/ ; + Map2k1 ⌬ +/+ / ⌬ Map2k1 Sox2Cre ; embryos revealed hemorrhages(white ⌬ / ⌬ Map2k1 –/– ; Map2k1 embryos havinganormal Tg null phenotype. +/ Map2k1 + +/ ⌬ ; +/ Map2k1 ⌬ +/floxed Sox2Cre males with Map2k1 B . ) ERK/MAPK ⌬ / ⌬ Tg embryos. +/ transgene-mediated recombination floxed/ + Genotype of live born pups Genotype ofliveborn ⌬ ; Map2k1 However, in and othertrophoblastsfromthelabyrinth)werepresentin trophoblast giantcells,spongiotrophoblasts,syncytiotrophoblasts trophoblastic celllineagesderived fromtheTScells(including MAPK3 arenormallyphosphorylatedinE9.5 Tanaka etal.,1998).However, thelackofphenotypein presence ofFGFsignalingandTScellniches(Corsonetal.,2003; observed inregions oftheextra-embryonic ectodermknown forthe agreement withthis,astrongactivation ofMAPK1andMAPK3was stem (TS)cellestablishmentandmaintenance(Rossant,2001).In MAPK cascadewas shown toberequiredfor invitrotrophoblast diverse mammaliancellularprocesses. reflects thecomplexity ofthecontrolsrequiredforregulation of presence ofvarious isoformsatdifferent levels ofthepathway MAPK1 andMAP2K1throughtheERK/MAPKcascade.The specific signalstransitsviadistinctproteinkinaseisoformssuchas which placentadevelopment requiresthatthetransductionof Saba-El-Leil etal.,2003).Together, theseresultssupportamodelin developmental anomaly(Hatanoetal.,2003;Pages etal.,1999; mutants, whereasthe partial development ofthe MAP2K1 maycontribute invivo totheseprocesses,allowing the establishment andmaintenance,otherfactors independentof even iftheERK/MAPKcascadeisrequiredinvitroforTS placentas (Fig.5)(Girouxetal.,1999).Theseresultssuggestthat embryos but notfrom we wereabletogenerateTScellsfromwild-typeand proliferation andmaintenanceisfurthersupportedbythefact that Theroleof establishment andgrowth. mutants stronglyindicates in embryo. underdevelopment ofthelabyrinthandultimately tothedeathof the chorionandlabyrinthineregion wereimpaired,leadingtothe Map2k1 perturbed in unpublished; datanotshown). Even thoughtheplacentagrowth is completely abolishedinboth reduced. AtE10.5,activation oftheERK/MAPK cascadeisalmost whereas intheirplacenta,activation of theERK/MAPKcascadeis ERK/MAPK activation inE10.5 embryo doesnotrequireMAP2K1 protein.Theabsenceof 8B), indicatingthatMAPK1andMAPK3phosphorylationin the MAPK3 iscomparablewiththatseeninwild-typespecimens(Fig. defects inmouse mutantsforseveral intermediates of the development hasbeensuggestedbytheidentificationofplacental undergoing resorption. was mostprobablyduetothe fact thattheseembryosweredyingand Tg Using specificMAP2K1andMAP2K2inhibitors,theERK/ A similarplacentalphenotypehasbeenreportedforthe The characterizationoftheactivation of theERK/MAPKcascade The importanceoftheERK/MAPK cascadeinplacental Map2k1 +/+ +/floxed function, proliferationandsurvival ofthetrophoblastsin ; –/– Map2k1 embryos andplacentasindicatesthatMAPK1 Map2k1 Tg Map2k1 +/ Sox2Cre ⌬ / ⌬ +/+ Map2k1 -rescued embryos,activation ofMAPK1 and ; –/– Mapk3 Map2k1 specimens (Fig.1),thedifferent Map2k1 Map2k1 Tg –/– Map2k1 +/ Sox2Cre blastocysts (V.B., M.G.andJ.C., mutation doesnotcauseany Map2k1 –/– +/ ⌬ as akey playerinTScell ; placenta. Intheabsenceof –/– embryos andplacentas. –/– Development 133(17) Map2k1 Map2k1 Tg conceptuses (Fig.2) Map2k1 +/ Sox2Cre ⌬ / ⌬ ; –/– in TScell Map2k2 Map2k2 Map2k1 embryos, Mapk1 –/– –/– –/–

DEVELOPMENT Bélanger, L.F., Roy, S.,Tremblay, M.,Brott, B., Steff, A.M.,Mourad,W., Ballif, B.A.andBlenis,J. Aubin, J.,Déry, U.,Lemieux,M.,Chailler, P. andJeannotte,L. case of Mudgett etal.,2000;Tamura etal.,2000;Yang etal.,2000).Inthe vascularization oftheplacentabyangiogenesis(Adams etal.,2000; impaired, suggestingaroleforthep38/MAPKpathway inthe in wild-typeplacentas.In activated intheendothelialcellsliningembryonicbloodvessels autonomous. We showed thatthep38/MAPKcascadewas strongly vascularization defectassociatedwiththismutation isnon-cell tetraploid-aggregation experiments, suggestingthatthe in chorioallantoic interface. Thespecificdeletionof explain whythesyncytiotrophoblasts arestalledatthe might syncytiotrophoblast celldifferentiation and/orfunction. syncytiotrophoblasts shouldallow thedefinitionofitsrolein Blaschke, F., Stawowy, P., Goetze,S.,Hintz, O.,Grafe,M.,Kintscher, U., mutants (known as Watson andCross,2005).For example, the pathways suchasthep38/MAPKcascade(RossantandCross,2001; ERK/MAPK cascade,aswellinmutantsaffecting otherMAPK Map2k1 Adams, R. H., Porras, A., Alonso, G., Jones, M., Vintersten, K.,Panelli,S., Adams, R.H.,Porras,A.,Alonso,G.,Jones,M.,Vintersten, References D’Amore andJ.C.Cross forthe Drs. R.EriksonandH.Greulich forthemurine for ERK2antibody;andF. Harel forstatisticalanalyses.We are alsogratefulto Carter forskilledtechnicalassistancewithblastocystmicroinjection; L.Caron manuscript; DrD.Dufortforhelpfuladvicesontetraploidexperiments;M. We thankDrsL.Jeannotte,J.AubinandHuotforcriticalreading ofthe and MAP2K1inlabyrinthinemorphogenesis. during placentaformation,withp38beinginvolved inangiogenesis Map2k1 function. Asthep38/MAPKcascadeisactivated inabsenceof the abnormalmorphogenesisoflabyrinthinabsence as syncytiotrophoblasts toinvade thelabyrinthineregion mayreflect chorioallantoic interface. Thisincapacityofendothelialcellsaswell cascade was stillactivated intheendothelialcellsblocked atthe number oflabyrinthinetrophoblastsin to theundergrowth ofthelabyrinth.Indeed,lackasufficient out thepossibilitythatsyncytiotrophoblast defectmight bedue syncytiotrophoblasts (Girouxetal.,1999).However, wecannotrule fibroblasts mightreflectthelackofmigration maternal, bloodvessels in labyrinthine layer. Thedevelopment ofembryonic,but notof http://dev.biologists.org/cgi/content/full/133/17/3429/DC1 Supplementary materialforthisarticleisavailableat Supplementary material (MOP-67208) andtheCancerResearch Society(J.C.) mice. ThisworkwassupportedbytheCanadianInstitutesofHealthResearch Westphal, F. W. AltandA.P. McMahonforthe migration deficiency observed in syncytiotrophoblast celldeterminationanddifferentiation. Thecell vessels derived fromtheallantois but itisnotessentialfor labyrinth leadingtoitsvascularization bytheembryonicblood in theextra-embryonic ectodermforthemorphogenesis ofthe growth anddevelopment. Hugo, P., Erikson,R.andCharron, J. signals. mitogen-activated protein kinase(MAPK)(MEK)-MAPKcell survival signaling. Stomach regional specificationrequires Hoxa5-driven mesenchymal-epithelial development. of p38alphaMAPkinaseinplacentalbutnotembryoniccardiovascular Valladares, A.,Perez, L.,Klein,R.andNebreda, A. R. Altogether, ourresultsindicatethatthe Cell GrowthDiffer. Mapk14 function, thissuggeststhatbothpathways actindependently role inextra-embryonicectoderm Development Mol. Cell mutants, placentadevelopment canberescued Mekk3 6 129 (2001). Molecularmechanismsmediating mammalian , 109-116. Mol. Cell.Biol. 12 , 4075-4087. , 397-408. and Map2k1 Vegf Map3k3 p38 and (2003). Mek2isdispensableformouse ␣ , respectively) presentareduced 23 Gcm1 –/– Map2k1 –/– , 4778-4787. specimens, thep38/MAPK Map2k1 EIIaCre labyrinth isdramatically RNA probes; andtoDrsH. Map2k1 –/– Map3k3 Map2k1 and gene; toDrsP. mouse embryonic (2000). Essentialrole Sox2Cre gene isrequired Map2k1 (2002). and –/– transgenic placenta Mapk14 Map2k1 in the Cross, J.C. Crews, C.M.,Alessandrini,A.andErikson,R.L. Hemberger, M.andCross, J.C. Hayashi, S.,Lewis,P., Pevny, L.andMcMahon,A.P. Huynh, H.T., Nguyen,T. T., Chow, P. K.,Tan, P. H.,Soo,K.C.andTran, E. Hsu, J.C.andPerrimon,N. Johnson, G.L.andVaillancourt, R. Dang, A.,Frost, J.A.andCobb,M.H. Corson, L.B.,Yamanaka, Y., Lai,K.M.andRossant,J. Conrad, P. W., D.E.andBeitner-Johnson, Millhorn, D. Brunet, A.,Brondello, J.M.,L’Allemain, G.,Lenormand,P., McKenzie,F., Brott, B.K.,Alessandrini,A.,Largaespada,D.Copeland,N.G.,Jenkins, Catling, A.D.,Schaeffer, H.J.,Reuter, C.W., Reddy, G.R.andWeber, M.J. Cano, E.andMahadevan,L.C. Giroux, S.,Tremblay, M.,Bernard, D.,Cardin-Girard, J.F., Aubry, S., Hatano, N.,Mori,Y., Oh-hora,M.,Kosugi,A.,Fujikawa, T., Nakai,N.,Niwa, Gopalbhai, K.,Jansen,G.,Beauregard, G.,Whiteway, M.,Dumas,F., Wu, C. Lakso, M.,Pichel,J.G.,Gorman,R., Sauer, B.,Okamoto,Y., Lee,E.,Alt,F. K., Guan,K.L.andHorvitz,H.R. Kornfeld, Eblen, S.T., Slack,J.K.,Weber, M.J.andCatling,A.D. Coles, L.C.andShaw, P. E. Coan, P. M.,Ferguson-Smith,A.C.andBurton,G.J. Littell, R.C.,Henry, P. R.andAmmerman,C.B. Giroux, S.andCharron, J. Fukuda, M.,Gotoh,Y. andNishida,E. Fan, B.,Wang, Y. X.,Yao, T. andZhu,Y. C. 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