(Saga etal.,1997).Previously, wehave shown thatMesp2 resulting non-segmented somitesshow caudalized properties been shown that rostro-caudal identityofsomites(Takahashi etal.,2000).Ithas Mesp2 demarcatesthenext segmental boundary anddefines the anterior PSMinthedeterminationfront(Yasuhiko etal.,2006). periodically activated bycyclic NotchsignalingandTbx6atthe somitogenesis (Sagaetal.,1997),anditsexpression is 2004). defined bytheso-calleddeterminationfront(DubrulleandPourquie, this istranslatedintospatialpatternsintheanteriorPSM,which 2005; Ridaetal.,2004).Thetemporalinformationthatresultsfrom (Besshoetal.,2001;Huppert2005;Morimoto generated byNotchsignaloscillationslinked tothesegmentation 2000; Monsoro-BurqandLeDouarin,2000).Periodicityis systems (BoryckiandEmerson,2000;Brand-SaberiChrist, which areallsomitederivatives, but alsoofthevascular andnervous the segmental architectureofnotonlythevertebra, ribsandmuscles, 2001). Thesomitesprovide thebasicaxialstructuresthat underlie hours (Iulianellaetal.,2003;Pourquie,SagaandTakeda, in bothaspatiallyandtemporallycoordinatedmannerevery two the anteriorpartofunsegmented presomiticmesoderm (PSM), Somites aregeneratedbysequentialsegregation ofcellmassesfrom INTRODUCTION show arostralizedphenotype,incontrastto the repressionofgenesundercontrolMesp2bygeneratinga Accepted 14February 2007 ‡ † 2 1 Somitogenesis,Notchsignaling,Presomitic mesoderm,Segmentation KEY WORDS: the Mesp2 patterning withinasomite.ApossibleMesp2targetgene, The Mesp2transcriptionfactorplaysessentialrolesinsegmentalborderformationandtheestablishmentofrostro-caudal Mitsuru Morimoto somite required toestablishtherostro-caudalpatterningwithina The negativeregulationofMesp2bymouseRipply2is (2007)doi:10.1242/dev.000836 Development 134,1561-1569 University SchoolofMedicine,StLouis, MO 63110,USA *Present address: DepartmentofMolecularBiologyandPharmacology, Washington Setagayaku, Tokyo 158-8501,Japan. Toxicology Division,NationalInstituteofHealth Sciences,1-18-1Kamiyoga, e-ciAizaki Ken-ichi eaiefebc opi seta o h eidcgnrto ftersr-adlpolaritywithinasomite. negative-feedback loopisessentialfortheperiodicgenerationofrostro-caudal Mesp2, leadingtoarostralizedphenotypeviathesuppressionofNotchsignaling.OurstudydemonstratesthatRipply2-Mesp2 revealed thatRipply2isanegativeregulatorofMesp2andtheloss Author forcorrespondence (e-mail:[email protected]) These authorscontributedequallytothis work SOKENDAI, Yata 1111,Mishima411-8540,Japan. Division ofMammalianDevelopment,NationalInstituteGeneticsand The Mesp2transcriptionfactor playsimportantrolesduring Ripply2 -null mouse. enhancer, indicatingthat Mesp2 3 , JunKanno Ripply2 1, -null embryosfail tosegment andthatthe * ,† , NobuoSasaki encodes aputativetranscriptionalco-repressorcontainingWRPWmotif.We findthatMesp2bindsto 3 and Yumiko Saga 3 Cellular andMolecular Ripply2 1,† Mesp2 , MasayukiOginuma is adirecttargetofMesp2.We thenexaminedwhetherRipply2isresponsiblefor -null mice.Geneexpressionstudiestogetherwithgeneticanalysesfurther 1,2,‡ Ripply2 , wasidentifiedbymicroarrayasbeingdownregulatedinthe reverse transcriptase(Invitrogen) and100pmolT7-(dT) synthesized byincubating5 according tothemanufacturer’s instructions.First-strandcDNAs were homozygous embryosatE10.5using theRNeasyMiniKit(Qiagen) and PSMofwild-type, Total RNA was purified fromcellscorrespondingtotheS–1S2somites GeneChip analysis MATERIALS ANDMETHODS withinasomite. caudal patterning regulation isrequiredfortheperiodicgenerationofrostro- the levels ofNotchsignalingin theanteriorPSM.Thisnegative by Mesp2,but alsofunctionsnegatively toward Mesp2toregulate Ripply2 proper transitionfromthePSMtosomites.We generateda that weidentified inthe affected bythe manufacturer’s instructions. Ourdetailedmethodsforthelabeling ofthe using aGeneChipSampleCleanup Module(Affymetrix), according tothe 3 Mesp2 Mesp2, wehave comparedthegeneexpression patternsof mechanisms underlyingtheregulation ofsomitogenesis by our currentstudy, whichaimedtoelucidatethemolecular which thecaudalgenesaresuppressediscurrentlyunknown. In Notch signaling(Takahashi etal.,2000).However, themannerin Uncx4.1 also known tobeastrongsuppressorofgenessuchas of al., 2005).Inaddition,Mesp2actsasthetranscriptionalactivator function asanegative regulator ofNotchsignaling(Morimotoet suppresses Notchactivity viatheactivation of GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT) knockdown analysisrevealed that zebrafish Ripply2 putative transcriptionalrepressor. Thisgeneturnedouttobe Ripply2 Ј ]. Aftersecond-strandsynthesis,the double-strandedcDNAs werepurified Epha4 2 , MakotoKiso +/– -knockout mouseandnow show that -knockout mouse.Unexpectedly, , whichwas recentlyreportedasamousehomologof htcne adlpoete pntesmtcclsvia that confercaudalpropertiesuponthesomiticcells and ripply1 in theanteriorPSM(Nakajimaetal.,2006).Mesp2is Ripply2 Mesp2 Mesp2 (Kawamura etal.,2005).Morpholino-mediated gene resultsintheprolongedexpressionof –/– 1 knockout. Amongthedownregulated , KatsuhideIgarashi embryos, andfoundthatseveral genesare Mesp2-GFP ␮ Mesp2 g oftotalRNA with200USuperScript II -null embryo,wefocusedona RESEARCH ARTICLE ripply1 knock-in heterozygousand Ripply2 Ripply2 is requiredforthe Lfng 3 , -null embryos , whichmight 24 is activated primer [5 Dll1 1561 and 24 Ј - -

DEVELOPMENT measured usingaDualLuciferaseAssayKit(Promega). adjusted withpcDNA3.1. After36hours,luciferaseactivities were normalize fortransfectionefficiency, andtheamountoftotalplasmidwas kinase promoter(1ng)was co-transfected asaninternalstandardto containing the (Invitrogen), following themanufacturer’s instructions.Thevector using afluorescent microscope(OlympusBX61). 488-conjugated goatanti-rabbitIgG (MolecularProbes;1:400)andobserved 4°C, incubatedwithrabbitanti-GFP (MBL;1:1000),followed byAlexa- venus, embryoswerefixed with4%paraformaldehydeinPBSovernight at described (Morimotoetal.,2005). For whole-mountdetectionofMesp2- immunohistochemical detectionof proteinswereperformedaspreviously cDNA clonecontainingintron 1.Sectioninsituhybridizationand Kitabayashi etal.,2002).For the previously (Takahashi etal.,2000;Takahashi etal.,2003;Nomura- (Takahashi etal.,2000).Theprobesusedinthisstudyhave beendescribed mount samplesandbyskeletal staininghave beendescribedpreviously Methods forgeneexpression analysisbyinsituhybridization ofwhole- Gene expression andhistochemicalanalysis 1997). by breedingwithaCAG-Cre transgenicmouse(SakaiandMiyazaki, confirmed byPCR.Thefloxed neomycincassette was laterremoved was withICRfemales.Germlinetransmissionofthetargeted allele bred pseudopregnant femalerecipients.Theresultingchimeric micewere clones wereaggregated withICR8-cellsandthentransferredto recombination was confirmed bySouthernblotting, andtargeted cell resistant cellcloneswerefurtherselectedbyPCR.Correcthomologous was thenelectroporatedintoTT2ES cells(Yagi etal.,1993).G418- codon andproduceanullallele.Theresultinglinearizedvector (25 which would introduceaterminationcodon justafterthesecondinitiation targeting vector withafloxed two putative in-frametranslationalinitiationcodons.We generateda The mouse Ripply2 complexes wereanalyzedon6%native polyacrylamidegels. Binding reactionswerecarriedoutfor30minutesonice,andprotein-DNA -DNA complexes weredetectedusingaDIGGelShiftKit(Roche). Double-stranded DNA oligonucleotideprobeswereend-labeledwithDIGand System (Invitrogen) andthencollectedviaanuclearextraction method. A 3xFLAG-Mesp2 proteinwas producedusingtheFreeStyle293Expression Electrophoretic mobilityshiftassay(EMSA) anterior-PSM enhancer( For luciferasereporteranalysisunderthecontrolof1.5kb assay Luciferase the 171bphighlyconserved region was isolatedby contained inamousegenomiclibrary. A1.5kbDNA fragmentcontaining genomic sequenceof (http://pipmaker.bx.psu.edu/pipmaker/). We cloneda5 species DNA sequencecomparisonusingthePipMaker website Highly conserved Identification ofthe (http://www.nihs.go.jp/tox/TtgSubmitted.htm). data arealsonow available onlineattheNationalInstituteofHealthSciences analysis have beendescribedpreviously (Kannoetal.,2006).Allofthese 430 2.0Array(Affymetrix), andthesubsequentwashing, staininganddata double-stranded cDNAs andhybridizationtoaGeneChipMouseGenome 1562 Foster mothersweresacrificed atE10.5andstainedfor injection andtheinjectedeggs werethenimplantedintoICRfemalemice. Fertilized eggs fromB6C3F1femalemicewerecollectedforpronuclear digestion andthensubclonedintothehsp-nlacZreporterconstruct. (0.25 Mesp2 (0,30,100ng)and3xFLAG-E47 (0,50ng)intoNIH3T3cells constructs wereco-transfectedwiththeexpression vectors 3xFLAG- identified byPCRusingDNA preparedfromtheyolksac. ( ␤ -gal) activity withX-Gal.Thegenotypesoftheembryoswerethen ϫ 10 RESEARCH ARTICLE gene targetingstrategy 5 cells perwellin24-multiwellplates)usingLipofectaminePlus Ripply2 Renilla Ripply2 gene consistsoffourexons, thefirst ofwhichharbors Ripply2 luciferase geneunderthecontrolofthymidine Ripply2 upstream regions wereidentified usingacross- Eco neo RI- somite enhancer from abacterialartificial clone(RP23) Ripply2 cassette toremove aportionofexon 1, Bam HI) fragment(20ng),reporter RNA probe,weusedafull-length Eco ␤ RI and -galactosidase Ј upstream Ripply2 Bam ␮ HI g) Ripply2 E10.5 embryos.( Mesp2 visible for shown intheleft-handbottompanel.Two bandswere sometimes observed foreachgene,andthesebandsare mergedintheimage represent and donot artifacts intheperipheryare beneath. Thegreen signals (magenta), andmergedimagesoftheseexpressionare patterns shown Two representative examplesare shown for Mesp2 an arrowhead. ( Ripply2 ( Fig. 1.Analysisofthe are expressed inthecaudalhalfofsomite (Bettenhausenetal., 2006). Moreover, expression ofthe the presumptive somite(Morimotoetal.,2005;Nakajima such as Mesp2 isknown to functionasatranscriptionalactivator ofgenes, Ripply2 RESULTS A Comparison ofthemRNAexpression patterns ) (right-hand bottompanel).Allsampleswere prepared from and expression islostintheE9.5 during mousedevelopment.Positiveexpression isindicatedby Epha4 Mesp2 Ripply2 is apossibledirect targetofMesp2 Ripply2 B and C ) Comparisonofthespatialexpression patterns expression. Insomecases,onlyasinglebandcouldbe , theposteriorbandofwhichmergeswiththat ) Whole-mountinsituhybridizationshowingthat as revealed bysectiondoubleinsituhybridization. Lfng , whichareexpressed in therostralhalfof Ripply2 expression pattern. Mesp2 Dll1 -null embryo. and Mesp2 Uncx4.1 Development 134(8) (green) and Mesp2 genes, which and Ripply2

DEVELOPMENT DNA fragmentcontainingtheconserved E-box(RegionB,light-blueshading)from the mutant probe (showninB). ( are indicatedbytheasterisk. E47. ThisbindingofMesp2thusappears tobedifferent from itsbindingtothe means±s.d.from fourseparateexperiments. * upon transactivation.Thedatarepresent the harboring eitherthe1.5kb h ne hw ihmgiiaino h oii ein ( The insetshowshighmagnification ofthesomiticregion. localization signal).E, ofthe stretch (showninA) Ripply2 Ripply2 ( red) isevidentacross thesespecies. Aconservedregion (framedin software. around the genomic sequences eaierglto fMs2b Ripply2 Negative regulation ofMesp2by Fig. 2.Mesp2candirectly bindtotheenhancerelementof eeadtecrepnigcntutue ntetasei nlss . bDAfamn containingthishighlyconserved171 b transgenicanalyses.A1.5kbDNAfragment the gene andthecorresponding constructusedin ( genes, withinwhichahighlyconservedE-boxislocated.HCE-box, highlyconservedE-box. Eco RI; B, Ripply2 Ripply2 G Bam ) ThebindingspecificityofMesp2 wasconfirmedbysuccessfulcompetitionwithcoldprobe, but notwithanE-box Ripply2 upstream region wasligatedtoacassettecomposed ofthe HI; N, enhancer (left)orsixrepeats oftheconserved171bpfragment(right).TheadditionE47had negativeeffects eei os tpln)wt thoseinhuman,dogandchickusingMultiPipMaker sequencealignment gene inmouse(topline)with Nco .( I. D ) The Ripply2 E ) Luciferasereporter assay for enhancer drives Ripply2 B gene andactivateitstranscription. euneaineto h 7 prgo osre amongthe Sequencealignmentofthe171bpregion conserved ) Epha4 lacZ reporter geneexpression insomiticmesodermcellsatE11.0. enhancer, whichisdependantupon E47.Non-specificbands Ripply2 Mesp2 P <0.01, ** upstream region can bindMesp2intheabsenceof hsp activation, withorwithoutE47,using constructs promoter andnlacZ( C ) Thegenomicorganizationofthemouse P <0.04. ( F ) EMSAanalysesrevealing that a RESEARCH ARTICLE ( A oprsno the Comparisonof ) lacZ harboring anuclear 1563 p

DEVELOPMENT Mesp2, weperformedGeneChipanalysisusingRNAs prepared unknown. To identifynovel genesthatoperatedownstream of al., 2000).However, themolecularmechanismsunderlyingthisare indicating thatMesp2isrequiredfortheirsuppression(Takahashi et 1995; Leitgesetal.,2000),isincreasedinthe 1564 RESEARCH ARTICLE Mesp2 -null mouse, rudimental tails. those ofsegmentation-defectivemutants,featuringashorttrunkwith morphologyatE17.5issimilarto dies soonafterbirthandtheexternal Bam and establishthe allele andcrossed withaCAG-Cre mousetoremove the was thengeneratedfrom recombinant EScellscontainingthetargeted arrowheads onthelinerepresent loxPsites).Agermlinechimericmouse identical expression patterntothatof (corresponding toRIKENcDNA C030002E08) thatshowed an inthesupplementarymaterial),weselectedacDNA clone Table S1 showed areductioninexpression inthe partially deletedandreplaced withafloxed following homologousrecombination. Thefirstexonof bottom twolinesshowthepredicted structure ofthe second linerepresents thestructure ofthetargetingvector, andthe top lineshowsthegenomicorganizationof external morphologyoftheresultingexternal knockoutmouse. Thetargetingstrategyusedforthe Fig. 3. the anteriorPSMasapairofbandsby8.0dpc,similarto somitogenesis (Fig.1A).However, itsexpression becameevident in expression appearedtobeabsentorvery weakpriorto to berestrictedthenascentmesodermatE7.0,but (Kawamura etal.,2005).Theinitialexpression of mouse 11.5 dpcembryos.ThiscDNA was subsequentlyrevealed tobethe from bothwild-typeand HI; H, Ripply2 Hin dIII; N, Ripply2 eerecentlyreportedbyKawamura etal. gene sections oftailregions (C,D). Hematoxylin andEosinstainingofparasagittal morphology(A,B)andbythe by external that the E17.5 stainedwithAlizarinRed-AlcianBluereveal segmental border. ( display irregularly sizedmyotomes,andanunclear segmentation defects. Fig. 4.The Ripply2 phenotype ofthe ribs (F; neural arches andlackscomponentsoftheproximal Nco -knockout mouseline.Ssp, I; K, Mesp2 n –/– =4), whichissimilartotheaberrant Ripply2 embryos ( Kpn Ripply2 -null embryos.Amongthegenesthat I; X, –/– Psen1 fetus harborsfewerpediclesof Xho E-G n -knockout mouseexhibits Mesp2 =3 atE10.5)were compared I. ( ) Skeletalpreparations at -null fetus(G; neo Mesp2 ( B A-D ) The Ripply2 Ripply2 by insituscreeningof cassette (the Development 134(8) Ripply2 ) Ripply2 nl mro (see -null embryos Ripply2 Ssp Mesp2 Ripply2 gene andthe Ripply2 I; E, –/– gene, the neo n +/– embryos =2). -null mouse Eco was found and cassette ( locus A Ripply2 ) The RI; B, was Mesp2

DEVELOPMENT and pattern forgenesexpressed intheanteriorPSM,including expression inseveral segmented somites(Fig.2D),which is atypical out ofninePCR-positive embryos,wedetectedspecific transient transgenicanalysesusing a bp sequence(Fig.2B)possessed enhanceractivity, weperformed –6747,Fig.2B). To (–6917to investigate whetherthe1.5kbregion containingthisconserved 171 conserved region a identified (Fig.2A).Fromtheseanalyses,we sequence alignmentsoftware human, dogandchickgenomicsequencesusingMultiPipMaker regulatory sequencesinthe indicated that (Fig. 1C),aspredictedfromourGeneChipanalysis.Thesedata thus addition, precedes thatof Mesp2 two completely merged, whereas theotherisofasingle patterns areshown inFig.1B.Oneshows singlebandsthatare double insituhybridizationofembryonictailsections.Two typical expression domainsof during thesomite-formingperiod(Fig.1Aanddatanotshown). The (Fig. 1A).Theexpression of Ripply2 Negative regulation ofMesp2by To examine thispossibility, wesearchedforpossiblecis- Epha4 Ripply2 band. Thisobservation indicatesthat Ripply2 (Haraguchi etal., 2001;Nakajimaetal.,2006). We next bands inwhichthecaudalbandismerged withadistinct Ripply2 Ripply2 expression was lostinthe might beatarget ofMesp2. Mesp2 , but that Ripply2 Ripply2 and Ripply2 Ripply2 ␤ then continueduntil12.5dpc, -gal reporter(Fig.2C).Infive gene bycomparingmouse, were next comparedby persists forlonger Mesp2 Mesp2 Mesp2 -null embryo expression band and Mesp2 ␤ -gal . In subsequent transactivation. Ripply2 the ideathatMesp2bindsto this E-boxintheenhancerof weakened bytheaddition of E47.Theseresultsareconsistentwith luciferase reporterassay. Furthermore,thebindingofMesp2 was not befunctionalbindingasnoassociated activity was detectableby (Fig. 2F,G). E47was alsofoundtobindthisE-box, but thismight didnot confirmed thatthisbindstoMesp2,whereasamutated form containing ahighlyconserved E-boxCATCTG sequence,and to Mesp2withorwithoutE47.We identified aDNA fragment sequence forE-boxes, orforanN-boxwhichiscapableofbinding or N-boxmotifs,wescreenedthe171bp family, whichisknown tobindeitherE-box heterodimeric partner. SinceMesp2belongstothebHLH-type the reporteractivity onlyinthepresenceofE47,apossible al., 2006),inwhichMesp2was observed tobindandtransactivate findings ofourprevious studyofthe Genome Informatics)(Fig.2E).Thisresultwas different fromthe but notinconjunctionwithE47(alsoknown asTcfe2a–Mouse sequence. Bothconstructswereactivated bytheadditionofMesp2, fragment andtheotherharboringsixrepeatsof171bpconsensus constructs weregenerated–onecontainingthe1.5kbgenomic enhancer activity was dependantuponMesp2. Two reporter employed aluciferasereporterassaysystemtoascertainwhetherthe gene, andthatthisenhancer does notrequireE47for Ripply2 The oscillatoryexpression of atE10.5. ItoIII) (indicatedby different cyclicphases the expression bands(brackets)are slightlyexpandedin posterior PSMwasunaffected, buttherostral-most expression of ( be reduced, whereas rostral genessuchas ( usinganti-GFPantibodies. by fluorescence, detected backgrounds. Theconfocalimageswere visualized E10.5 for analyzed culturing for20minutes.Bothwere then immediately andtheotherfixedafterexplant separated intotwohalves,withonebeingfixed Ripply2 Ripply2 were employedtocharacterizesomitogenesisinthe null embryos. Ripply2 by Mesp2-venus,wascompared betweenthe embryos (J,L).Mesp2protein expression, visualized Fig. 5.Altered geneexpression inthe the expressionof patterns such as expression bandappearsrostrally inthe levels (M,N),atE10.5.Anadditional andintron (K,L)probes, andprotein by exon(I,J) Ripply2 maintainedforlongerinthe anterior PSMis E O , , F P Lfng ) and Ripply2 ) Comparisonofthe Ripply2 +/– +/– –/– –/– –/– Uncx4.1 RA Theexpression of mRNA. Epha4 embryos atE11.5.( embryo. Theexpression ofcaudalgenes embryos. embryos (O).( (M, –/– +/– Lfng embryos (P),ascompared withthe n =2) and Whole-mount insituhybridizations ( (Q) and ( G A , nteatro S.ThePSMof in theanteriorPSM. RESEARCH ARTICLE , H B Epha4 ) show an expanded pattern in ) showanexpandedpattern ) and Ripply2 Q Ripply2 Lfng Ripply2 , Mesp2 R Dll1 enhancer (Nakajimaet ) Theprolonged Lfng I-N expression at patterns –/– ( –/– C ) Comparisonsof , (asterisks) inthe RA detected mRNA, (N, D Lfng (R) embryoswas gene consensus ) wasfoundto Mesp2 n =3) genetic in the Ripply2 Ripply2 Tbx18 1565 –/– -

DEVELOPMENT patterns werepatterns compared betweenwild-type(A,C,E)and protein (C, revealed by skeletal staining,differed fromthatof Ripply2 homozygotes. Asexpected fromtheexpression patterns,the performed timedintercrossmatingstoanalyzethephenotypesof the Since theheterozygousmicewerefoundtobenormal, we knockout miceusingEScell-mediatedgenetargeting (Fig.3A). somitogenesis. To elucidate thispossibility, wegenerated whether Ripply2mightfunctioninthisMesp2pathway during involved intheformationofsomiteboundary, wespeculated Because Mesp2confersrostralpropertiestothesomitesand is phenotype The and activities intheanteriorPSM)antibodiesusingsectionsofwild-type(G) (green) andanti-activeNotch1(magenta;thewhitelinesindicate (B,D,F) embryosatE11.0.( Ripply2 Fig. 6.Notchsignalingisreduced intheanteriorPSM 1566 However, thevertebral phenotype ofthe null embryo,withashorttrunkand tail(Fig.3B)(Sagaetal.,1997). of the17.5-dpcfetuswas found tobesimilarthatofthe These homozygousmicealsodiedsoonafterbirth.Themorphology and theembryosdisplayednoclearsegmental borders(Fig.4A-D). expression isupregulated butNotchactivityisreduced. mesoderm (Saga etal.,1997). arches owing to the caudalizedcharacteristicsofsomitic embryos asitfeaturesextensive fusionofthepediclesinneural Ripply2 Ripply2 RESEARCH ARTICLE –/– –/– embryo. n embryos failed toproceedthroughnormalsomitogenesis –/– =2; D, (H,I) E11.0embryos.Inthe -knockout mouseexhibitsarostralized n =2) and ( A-F ) G-I Notch1 Hes5 ) Doubleimmunostainingwithanti-Mesp2 mRNA (A, mRNA (E, Ripply2 n n =2; F, =2; B, Ripply2 –/– background, Mesp2 n n =4) expression =2), Notch1 –/– Ripply2 embryos, as Mesp2 Ripply2 –/– Mesp2 -null - - was intactinthe theexpression intheposteriorPSM although or intheanteriorPSM, addition, no greatly reducedinthe Ripply2 is prolongedorthatthe type and bothwild- intheanteriorPSM, examined. Duringsomitogenesis observed that cyclic expression stage(Fig.5Ianddatanotshown). However, we expression bandofvariable widthornoband,dependingonthe expansive in (Fig. 5A,C).Theexpression ofthesegenesisincreasedandmore somites was restrictedtothecaudalcompartmentsofsomites expression ofthecaudalmarkers of markers werefoundtobepresentinthe examined). Inaddition,wedidnotobserve any frequently observed inthemore-rostralregion (fouroutofsix one ortwo bands(Fig.5J).Inotherwords, anadditionalbandwas embryo (Fig.5G),andthisexpression inthe segmental borderinthe detectable intheareajust caudal tothenext presumptive singleMesp2-venus bandwas 2005). Inatypicalcase, is viableandshows normalsomitogenesis(Morimotoetal., Mesp2 proteinsinvivo becausethehomozygousknock-inmouse previously, Mesp2-venus canbeusedtovisualizefunctional consistent withtheseskeletal defects.In studies usingbothrostralandcaudalmolecularmarkers are signaling (Koizumi etal.,2001).Thefindings ofourgeneexpression presenilin 1( arches (Fig.4E,F),andthephenotyperesembledthatof band inthe necnetdbnscudb icre (Fig. 5N).Thesedata interconnected bandscouldbediscerned without the rostralcompartmentofS0andS1somitesin pattern was evident (Fig.5F).Inaddition, expanded throughouttheentiresomiteregion andnoclearsegmental in the 2005). To identifytheunderlyingcauseofrostralizedphenotype Notch signalingpathway intheanteriorPSM(Morimotoetal., somites isgeneratedbytheinteractionbetweenMesp2and As wehave previously reported,therostro-caudalpolarityof expression Ripply2 isanegativeregulator of displays arostralizedphenotype. (Fig.5H).We thusconcludethatthe wild type increased andtheexpression domainexpanded ascomparedwiththe somites (Fig.5E).In 2001), was expressed intherostralcompartment of thesegmented rostral propertiesofthesomites(Bussenetal.,2004;Kraus Ripply2 signal obtainedwas low, wefrequentlydetectedtwo bandsinthe Althoughthe usinganintronprobe. performed insituhybridization H). However, inthe influenced inthe cannot yetbeexcluded. mRNA stability exist intheir differences although thepossibilitythat The enx xmndhow theexpression oftheMesp2protein is We next examined Mesp2 Tbx18 Ripply2 Ripply2 –/– –/– Mesp2 Ripply2 , whichisknown tobeinvolved inthemaintenanceof embryos (Fig.5L;inallthreeexamined), but onlyone per ob prolongedintheabsenceofRipply2, appears tobe background. To distinguish thesepossibilities,we Ripply2 Dll1 Mesp2 Mesp2 -null embryo,the Psen1 expression, suggestingeitherthat –/– Ripply2 +/– stripe couldbeobserved withinthesomiticregion mouse fetusshowed fewer pediclesofneural embryos generallyshowed eitherasingle +/– nl mro Tkhsie l,20) but was etal.,2000), -null embryos(Takahashi Ripply2 )-null mouse(Fig.4G),whichlacksNotch is expressed inthe Ripply2 mro Fg K.Hne thetranscription Hence, embryos (Fig.5K). Ripply2 –/– Ripply2 embryo (Fig.5D).Bycontrast,therostral Mesp2 –/– Ripply2 –/– akrud Aswehave shown background. Mesp2 –/– Uncx4.1 -null embryos,(Fig.5B,D).In transcripts arestabilizedina embryos, thisexpression was embryo, two broader and +/– gene expression profile was Ripply2 Ripply2 akrud(Fig.5M). background and Ripply2 Epha4 Ripply2 Mesp2 Development 134(8) Dll1 –/– Ripply2 Ripply2 –/– Mesp2 embryo (Fig.5E- was expressed in embryo aseither in thesegmented +/– –/– embryos, the embryo was -null mouse –/– expression Ripply2 embryos Mesp2 +/–

DEVELOPMENT prolonged Mesp2and/orLfngexpression inthe even inthepresumptive caudalcompartmentofthesomites,by These resultssuggestthatNotchsignalingmightbesuppressed, half-PSM(Fig.5Q,R). a with explant cultureexperiments minute persistentexpression of 5P). This PSM of further understandthemolecular events operatingintheanterior elevated activity (Huppertetal.,2005;Morimoto 2005).To PSM andstabilizesasaclear stripeintheanteriorPSMwith In somite-stageembryos,Notch activity oscillatesintheposterior patterning suppression necessaryforrostro-caudal Mesp2, butnotLfng,isresponsible fortheNotch embryo. Ripply2 disappearing fromtherostralendofexpression domaininthe embryos. Thewidthofthis in theanteriorPSMissimilartothatof a cyclic wave-like patternintheposteriorPSM,but itsexpression suppression ofNotchsignaling.Moreover, Mesp2 intheanteriorPSMandissubsequentlyinvolved inthe of Mesp2. these factors formanegative-feedback looptorestrictthelevels suggest that Ripply2 Negative regulation ofMesp2by for muchlongerascomparedwiththe the anterior-most We previously reportedthat +/– Ripply2 embryo (Fig.5O).However, inthe Mesp2 –/– Lfng embryos, wefirst examined the expression of is negatively regulated byRipply2,andthat band was foundtobewiderandpersist Lfng Lfng Lfng band becomesthinnerbefore expression isactivated by a loeietfo 20- was alsoevident from Ripply2 Lfng Mesp2 Ripply2 +/– expression shows embryos (Fig. in –/– Ripply2 Ripply2 embryos, +/– –/– becomes thefuturecaudalcompartment.In compartments ofthesomites,wherebyNotchactive site activities leadstothegenerationoffuturerostralandcaudal boundary (Fig.6G).Inaddition,thecontrastbetweenNotch a sharpboundarywithMesp2thatdeterminesthenext segmental wild-type embryos,theNotchactivity intheanteriorPSMexhibited Intriguingly, the vertebral morphology ofthe the expression ofthecaudal molecularmarker specimens, andthenexamined thesomitepropertiesbyanalyzing double-knockout embryo fromwhichwepreparedskeletal embryo. To testthispossibility, wegenerateda expanded intheanteriorPSM embryos. Interestingly, theseexpression patternswerefoundtobe Notch1 result oftheprolongedactivation of speculated thatthesuppression ofNotchsignalingmightbethe (Fig. 6H,I). expression bandingwas foundtoupregulated, asshown previously appeared toberepressedinthesenullembryos,whereastheMesp2 6H,I). However, theelevation ofNotchactivity intheanteriorPSM the Notch1signalsoscillatednormallyinposteriorPSM(Fig. the expression of but theNotchactivity appearedtobelostasjudgedfromthefact that Mesp2 antibodiesinbothwild-typeand double immunostaininganalysisusinganti-active Notch1andanti- activity anditsrelationshiptoMesp2expression, weconducted was absent(Fig.6E,F).To furtherconfirm thisreducedNotch1 Since theexpression of mRNA (Fig.6A,B)andNotch1protein6C,D) inthese Hes5 , aNotchtarget gene(Ohtsukaetal.,1999), shown. representative imagesofE9.5embryosare skeletal defectsinthe E17.5 fetusesorE9.5embryos.The C, B, null ( that ofthe fetus (E)showsasimilarmorphologyto independently examinedatE10.5(A, Uncx4.1 contrast, the absent inthiscompound-nullfetus(C).By the neuralarches were almostcompletely or ( additional lossof found tobefurtherenhancedbythe knockouts of Fig. 7.Geneticanalysesusingdouble- compared amongwild-type( Uncx4.1 Lfng B ), n n Mesp2 =2; C, Ripply2 =2; D, D is underthecontrolofMesp2,we ) and expression was pattern expression were patterns RESEARCH ARTICLE Ripply2 . n n Mesp2 / The skeletalmorphologiesand Lfng =1) andE9.5(A, =4; E, Ripply2 Ripply2 Ripply2 Ripply2 Lfng double-null ( Lfng –/– single-null fetus(D).The n / =2). Only Mesp2 / Mesp2 embryo (Fig.6B,D), , andthepediclesof Ripply2 Ripply2 –/– in the and either Uncx4.1 embryos. Inthe double-null ( Ripply2 Ripply2 double-null A n –/– C =4; B, ), ), –/– Ripply2 fetus were Lfng Mesp2 (Fig. 7). embryo, Lfng -null n / / 1567 n Lfng Lfng =2; =2; - E –/– )

DEVELOPMENT (blue line)regulation surrounding Mesp2.Thetranscriptionof wild typeand isestablishedordisruptedintheanteriorPSMof caudal polarity system forMesp2viaRipply2.( dependent pathway. We herein propose anewnegativeregulatory expression. Mesp2proteins are alsorapidlydegraded viaaproteasome- suppresses Notchsignalingbyactivating enhanced bybothNotchsignalingandTbx6.Atthesametime,Mesp2 retained becauseofthelackMesp2.In compartment ofS0,both phenotypes. As expected, thevertebral morphologyofthesefetuses Mesp2 mainly duetothefunction ofMesp2,wealsogenerated diminished inthedouble-knockout embryos. (compare Fig.5Bwith7C), andwas foundtobecompletely Uncx4.1 the somites. incomplete. Thisresults intheexpansionofrostral properties within compartments, althoughthesuppression onNotchsignalingis expression persistsforalongerperiodinboththerostral andcaudal rostralized ascomparedwitheitherthe double-knockout mousewas notrecovered, andwas more somitogenesis. Fig. 8.GeneticcascadesintheanteriorPSMregulating 1568 Ripply2 signaling through thesuppression of localized intherostral compartmentofS–1andsuppresses Notch 4F withFig.7C)or wild type A B To determinewhetherthesuppressionofNotchsignaling is / RESEARCH ARTICLE Ripply2 -/- was alsonotrecovered bytheadditionallossofLfng neirposterior anterior Ripply2 Ripply2 S+1 double-null miceandanalyzedthe resultingskeletal ( A ) Schematicofthepositive(red line)andnegative –/– Tbx6 Lfng proteasome mediatedpathway mutants. IntheanteriorPSM,Mesp2is Ripply2 rostral rostral Dll1 Mesp2 Mesp2 -null fetus(Fig.7B).Theexpression of expression andNotchsignalingare Mesp2 B 0S-1 S0 ) Schematicillustratinghowtherostro- signaling

Notch caudal Dll1 signaling Notch caudal Dll1 Dll1 Lfng . Bycontrast,inthecaudal Mesp2 Mesp2 signaling Notch signaling Notch Dll1 Dll1 Ripply2 Notch signaling and suppressing Ripply2 Ripply2 L-fng –/– –/– embryo, Mesp2 (compare Fig. Dll1 Mesp2 Dll1 is mutants. the somitesbecomingcompletely rostralizedinthesedouble that suppressesNotchsignaling intheanteriorPSM,andresults of thelackLfng.Thisinturn enhancesthefunctionofMesp2 across amuchwiderareaalong theanterior-posterior axisbecause Mesp2 isprolongedowing tothelackofRipply2,andisdistributed distribution defect.In thisdouble-nullmouse,theexpression of Ripply2 background (ourunpublisheddata).Thephenotypeof the Mesp2-venus patternshows adiffuse distribution inthis absence ofLfng,suchabiasedgradientisnotgenerated,and the higher inthepresumptive rostralcompartment. However, inthe Mesp2-venus expression patternshows acleargradient,being the Mesp2-venus knock-inmouse.Inthewild-typeembryo, of LfngduringMesp2distribution, baseduponourobservations of signaling activity. We speculatethatthisiscausedbythefunction loss ofLfng,and,infact, thisresultsinafurtherreduction inNotch null embryoasthissuppressionwas notrescuedbytheadditional to becrucialforthesuppressionofNotchsignalingin boundaries viatheactivation ofLfng.However, Lfngappearsnot Mesp2 suppressesNotchsignalingtoestablishsegmental potent negative regulator of 2006). Inaddition,ourcurrentstudyhasidentified Ripply2asa regulated negatively bytheproteasomepathway (Morimotoetal., activation andinhibition.Previously, wereportedthat Mesp2is (Fig. 8A).However, theseprocessesmustberegulated byboth crucial fortheperiodicinductionof cooperation betweenTbx6andcyclic activated Notch-signalingis correct rostro-caudalpolarity. Duringthisactivation step,the the periodicsuppressionofNotchsignaling,andalsotoestablish anterior PSM,thelevels ofMesp2arestrictlyregulated toachieve Cole etal.,2002;Hirata2004;Morales2002).Inthe destabilization ofmRNA (Besshoetal.,2003;Chen2005; including transcriptionalsuppression,proteindegradation and Hes7 mouse posteriorPSM(Ridaetal.,2004),inwhichtheclockgenes mechanisms basedonthenegative regulation ofseveral genesinthe caudal patterning.Theperiodicityofsomitogenesisisestablishedby component oftheregulatory network involved inestablishingrostro- feedback regulation ofMesp2byRipply2constitutesacore Our currentstudyestablishesthehypothesisthatnegative- DISCUSSION the functionofMesp2. Uncx4.1 exhibited acaudalizedphenotype(Fig.7D,E).Theexpression of was foundtobevery similartothe these decrease intheactivated formofNotch1intheanteriorPSM caudal markers. Immunohistochemicalanalysisfurtherrevealed a rostral marker genesbut alsoareductionintheexpression of additional lossofMesp2. the exclusively tonegatively regulate Mesp2,becausethephenotypeof 8B). Itisnoteworthy inthisregard thatRipply2mightfunction longer periodandleadstothesuppressionofcaudalproperties(Fig. In theabsenceofRipply2,Mesp2expression ismaintained over a is requiredforthecorrectestablishmentofrostro-caudalpatterning. that thedefectobserved inthe suppresses theexpression ofthisgeneindependentRipply2,and embryo (Fig.7E).Theseresultsclearlyshowed thatMesp2 The Ripply2 and Ripply2 Ripply2 / Lfng was alsoupregulated tosimilarlevels asinthe Lfng -knockout mouseisalmostcompletelyreversed bythe double-knockout mouseappearsalsotoreflectthis -null embryos.Previously, wehave shown that -null mutantexhibits notonlyanexpansion of are negatively regulated byseveral mechanisms, Mesp2 Ripply2 rncito,andasafactor that transcription, Mesp2 Mesp2 –/– mouse canbeattributed to (Yasuhiko etal.,2006) single-null fetus,and Development 134(8) Mesp2 Ripply2 -null -

DEVELOPMENT Bessho, Y., Sakata,R.,Komatsu,S.,Shiota,K.,Yamada, S.andKageyama,R. References http://dev.biologists.org/cgi/content/full/134/8/1561/DC1 Supplementary materialforthisarticleisavailableat Supplementary material the MinistryofEducation,Culture, Sports,ScienceandTechnology, Japan. Organized Research CombinationSystemandNationalBioResource Project of was supportedbyGrants-in-AidforScienceResearch onPriorityAreas (B),the Tamura forpermittingustousetheFreeStyle 293expression system.Thiswork Randy Johnsonforgenerously providing the technical supportandformaintainingthemiceusedinthisstudy. We thank We are particularlythankfultoYuki Takahashi andAya Satohfortheirvaluable Ripply2 Negative regulation ofMesp2by and zebrafish, theunderlyingmechanismsmightwellbedifferent. upon Mespgeneexpression appearstobesimilarbetween mouse embryos. Hence,althoughtheimpactoflossRipplyproteins Dll1 However, Tbx6expression isunchanged(datanotshown) andthe signaling isinvolved inthegeneactivation (Yasuhiko etal.,2006). PSM whereT-box factor bindingincombinationwithNotch sequence requiredforthefaithful expression of it isplausibletoassumethatsuppresses Moreover, becauseRipply2hasnoapparentDNA-binding domain, the terminationofMesp2expression atanappropriatetime. Ripply2 arecurrentlyunknown. Ripply2appears to berequiredfor Bessho, Y., Hirata,H.,Masamizu,Y. andKageyama,R. bp upstreamregion ofthe mespb deltaC regions, andthisisaccompaniedbytheupregulation of morphants alsodisplayupregulation of Inthezebrafish, of-function studieshave yetbeenreported. andnoloss- (ourunpublisheddata), etal.,1995) segmental (Dehni not are expression patterns but their to beexpressed inthePSM, (Kawamura etal.,2005)(datanotshown). revealed previously byinvitro assaysinbothzebrafish andmouse Groucho homologTle1and/orTle3viatheWRPWmotif,as en,G.,Liu Dehni, Brand-Saberi, B.andChrist, Borycki, A.G.andEmerson,C.P., Jr Bettenhausen, B.,HrabedeAngelis,M.,Simon,D.,Guenet,J.L.and Chen, J.,Kang,L.andZhang,N. Bussen, M.,Petry, M.,Schuster-Gossler, K.,Leitges,M.,Gossler, A.and Cole, S.E.,Levorse,J.M.,Tilghman,M.andVogt, T. F. segmentation. (2001). 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DEVELOPMENT