kidney development truncations couldresultfromSALL4inhibition. caused mislocalizationofSall4intheheterochromatin;thus,somesymptomsTownes-Brocks syndromecausedbySALL1 anorectal andheartanomalies,exencephalykidneyagenesis.Sall4Sall1formedheterodimers,atruncated Sall1 of Tokyo, Tokyo 153-8904,Japan. 8654, Japan. neural differentiation andmaintainthepluripotency ofEScellsin subsequent upregulation ofIdhelix-loop-helixproteinsthatblock phosphorylation andnuclearlocalizationofSmad1, the LIF-dependent pathway forself-renewal. BMP4induces localization ofSTAT3, atranscriptionfactor essentialforthe 1999; Ying etal.,2003).LIFinducesphosphorylationandnuclear are thetwo majorextrinsic signals(Niwa etal.,1998;Matsuda inhibitory factor (LIF)andbonemorphogeneticprotein4(BMP4) differentiation bybothextrinsic andintrinsicfactors. Leukemia ES cellpluripotency requiresconstantsuppression oftheir there aresomeethicalissuestoovercome. Maintenanceofmouse human EScellsarepossiblecandidatesforcelltherapies,although vitro, EScellsdifferentiate intoavariety ofcelllineages;thus, chimeric micewhenintroducedintoablastocyst. When culturedin are establishedfromtheICMandhave the potential togenerate trophoblast celllayersoftheplacenta.Embryonicstem(ES)cells trophectoderm cellshave restrictedpotential andgive risetothe theepiblastandeventually toallthefetaltissues,while to the trophectoderm.CellsofICMarepluripotentandgive rise development oftwo distinctlineages–theinnercellmass(ICM)and Early differentiation ofthemammalianembryoleadsto INTRODUCTION KEY WORDS: Okihiro syndromearecausedby cells proliferatedpoorlywithnoaberrantdifferentiation. Furthermore,wedemonstratedthatanorectalandheartanomaliesin peri-implantation. Growthoftheinnercellmassfromknockoutblastocystswasreduced,and known asOkihirosyndrome.Inthisstudy, weshowthatatargetednullmutationinthemouse Mutations inSALL4,thehumanhomologofDrosophila Masayo Sakaki-Yumoto and cooperateswith syndrome, isessentialforembryonicstemcellproliferation, The murinehomologof Development 133,3005-3013(2006)doi:10.1242/dev.02457 DEVELOPMENT ANDDISEASE Accepted 24May 2006 † 6 1 8639, Japan. Regulation, TheInstituteofMedicalScience,UniversityTokyo, Tokyo 108- The InstituteofMedicalScience,University ofTokyo, Tokyo 108-8639,Japan. Ryuichi Nishinakamura *These authorscontributedequallytothis work Kumamoto University, Kumamoto860-0811,Japan. iouTakasato Minoru Author forcorrespondence (e-mail:[email protected]) PRESTO, JST, Saitama332-0012,Japan. Division ofIntegrativeCellBiology, InstituteofMolecularEmbryologyandGenetics, 4 3 Research CenterforAdvancedScienceandTechnology, TheUniversity Graduate SchoolofSciences,TheUniversityTokyo, Tokyo 113- Sall4, spalt, Embryonicstemcells,Okihiro syndrome, Townes-Brocks syndrome, Organogenesis,Mouse 3 , Tatsuhiko Kodama 5 Laboratory ofGeneExpression andRegulation, 1,2,6, 1, *, ChiyokoKobayashi * Sall4 ,† haploinsufficiency andthatSall4/Sall1 2 Division ofStemCell 4 Sall1 , Hiroyuki Aburatani SALL4, acausativegeneinOkihiro 1, in anorectal,heart,brainand *, AkiraSato homeotic genespalt result ofmutationsin be explained bytheformation oftruncatedSALL1proteinsasa not apparent(Nishinakamuraet al.,2001).Thisdiscrepancy could dysgenesis, but otherphenotypesobserved inthehumandiseaseare motifs oftheC2H2type.sal encodes aproteincharacterizedbymultipledoublezinc-finger cells (Takahashi etal.,2003;Nishimoto2005). in EScells,andthey playcrucialrolesintheproliferation ofstem Nichols etal.,1998).Eras pluripotency inEScells(Chambersetal.,2003;Mitsui etal.,2003; ES cellsandhave beenshown tobeessentialformaintaining Nanog the presenceofLIFsignal.Intrinsicfactors include 2003). Ithas been reportedthatSall1also functionsasa exencephaly, aswelllimbandanaldeformities (Kieferetal., exhibited moresevere defects,includingrenalagenesis, showed thatmutant miceproducingatruncatedSall1protein al., 1998).Micedeficientin anus and,lesscommonly, kidney andheartanomalies(Kohlhase et characterized bydysplasticears,apreaxialpolydactyly, imperforate with Townes-Brocks syndrome,anautosomaldominantdisease Mutations inSALL1 sensory organ development (deCelisetal.,1999). 1996; Nellenetal.,1996),trachea(Kuhnlein andSchuh, 1996)and formation andcellfate decisionsinthewingdisc(deCelisetal., During thelaterstagesofdevelopment, during earlydevelopment (Jurgens, 1988;Kuhnlein etal.,1994). and isrequiredforthespecificationofheadtailregions (known as et al.,1996;Nellen1996). expression iscontrolledbythe at theanteroposteriorboundaryofwingimaginaldiscs,and its 4 The Humans andmiceeachhave fourknown Sal-relatedgenes , MakotoAsashima 2 , SayokoFujimura spalt and heterozygotes exhibitedanincreasedincidenceof Eras. SALL1-SALL4 ( ( sal) genewas firstisolatedfrom sal), causetheautosomaldominantdisorder Oct3/4 on chromosome16q12.1have beenassociated SALL1, ascomparisonwith and and 3 in humansandSall1-Sall4 acts asaregion-specific homeoticgene, 1 , NobuakiYoshida Sall4 Sall1 , Yuko Matsumoto Nanog dpp Sall4-null embryonicstem(ES) Utf1 gene leadstolethalityduring (decapentaplegic) gene(deCelis show kidney agenesisorsevere RESEARCH ARTICLE are alsoexpressed abundantly are expressed athighlevels in sal Drosophila regulates pattern 5 sal Sall1-null mice 2 and , is expressed in mice). Oct3/4 and it 3005 ,

DEVELOPMENT MATERIALS ANDMETHODS syndrome thatiscausedbytruncationsofSALL1. the underlyinggeneticmechanismsinvolved inTownes-Brocks heterodimerization ofSall4andSall1invivo, whichcouldexplain proliferation ofEScells.We furtherreveal theimportanceof unexpectedly essentialforearlyembryogenesis,and forthe this study, wereportthatthemouse redundancy inorganogenesis, wegeneratedSall4 kidney anomalies(Al-Baradieetal.,2002;Kohlhase etal.,2002). movement deficitsand,lesscommonly, anorectal,ear, heartand as Okihirosyndrome,whichischaracterizedbylimbdeformity, eye Mutations inSALL4 abnormalities intheoralstructuresarepresent(Parrish etal.,2004). postnatal dayanddeficienciesinthecranialnerves and abnormalities (Strathdeeetal.,1997). mental retardation,midfacial hypoplasia,delayedgrowth andlimb Individuals withthisdeletionexhibit hearingloss,cardiacproblems, in casesof18qdeletionsyndrome(Kohlhase etal.,1999). to (Sato etal.,2003).Althoughnodiseasesarethusfar directlylinked kidney phenotypescomparablewiththoseof no apparentphenotype,andmicelackingboth suppressor (Lietal.,2001;Li2004). however, it hasbeenreportedthatSall2functionsasatumor (MTA)1 andMTA2 (Kieferetal.,2002). associated protein46/48(RbAp46/48),metastasis-associated such ashistonedeacetylase(HDAC)1, HDAC2, retinoblastoma- interacting withcomponentsofchromatinremodelingcomplexes transcriptional repressorbylocalizingintheheterochromatinand 3006 resistance (Neo a vector thatcontainedthe EcoRI 8.2-kb A Generation of Sall4-targeting vector was constructedbyincorporatingthe5Ј To investigate therolesofSallfamily genesandtheirfunctional It isstillunclearifSALL2 SALL3, thisgeneislocatedinaregion thatiscommonlydeleted RESEARCH ARTICLE Sall4 r Sall4 ) gene(pGK-Neo)andthediphtheriatoxinAsubunit fragment andthe3 -deficient mice( cause anautosomaldominantdisorderknown ␤ -galactosidase gene( is associatedwithhumandisease; Ј BamHI-EcoRV 2.3kbfragmentinto Sall4-del) Sall3-null micedieonthefirst Sall4 Sall2-deficient miceshow gene was foundtobe Sall1 Sall1 lacZ), theneomycin -deficient mice.In and knockout mice Sall2 Aor51HI- show by usingtheSall4- homologous recombinantswereobtainedatafrequency of68.8%(11/16) homologous recombinants).Indeed,whentestedinwild-typeEScells, expressed inEScells,including when they areincorporatedintothepromoterregions ofgenesthatare generated byasimilarmethod.Thesevectors conferdrugresistanceonly fibroblasts inthepresenceofLIF(10 ES cellsweremaintainedonmitomycinC-treatedprimaryembryonic generate germlinechimerasthatwerebredwithC57BL/6Jfemales.E14.1 resistant E14.1EScloneswerecorrectlytargeted andtwo wereusedto we usedtodelete (see Fig.S1inthesupplementarymaterial).The 3 Sall4-deficient mice( shown inFig.4D,E.Thisstrain showed phenotypesidenticaltotheoriginal EcoRI 4.2kbfragment,the The Targeted disruptionofbothalleles of Sall4and␤ domains ofSall4 (pMC1DTA) intandem.Thisconstructdeletes alltheeightzinc-finger HindIII 3.5kbfragment,the The Generation ofaSall4 another strainofmice(Sall4- IRES 5 genotyping wereasfollows: 5 and experiments describedinthispaper. Theprimersequencesusedfor them was furthertransfectedwiththe motifs. Five out of 170cloneswerecorrectlytargeted (flox/+)andoneof were excised uponCretreatment,resultingindisruptionofallzinc-finger supplementary material).LoxPsequenceswereplacedsothatexon 2and3 flanked byFrtandloxPsequences pMC1DTA (seeFig.S1inthe ApaLI-Eco allele and379bpinthemutatedallele).As CCAG-3 del) lacked properlacZ IRES- Ј Ј -GTGCCCAGCTTCTTCAAGTC-3 ApaLI-EcoRV 6.0kbfragmentintoavector thatcontainedpMC1DTA Sall4- Sall4-flox vector was constructed byincorporatingthe5 - ␤ Hyg -geo Ј (the lengthoftheamplifiedsegment was 272bpinthewild-type IRES vector. The RV 6.0kbfragmentintoavector thatcontainedpGK-Neo vector describedbelow. -galactosidase (Fig.1A).Thisstrategy isidenticaltotheone ␤ and resultsinthefusionof39aminoacidsatNterminal Sall1 -geo IRES Sall4-del). vector was constructedbyincorporatingthe5 (Nishinakamura etal.,2001).Five outof114G418- expression, wealsogeneratedmiceusingthe Sall4- floxed allele - ␤ - EcoRI-SalI 6.0kbIRES- geo IRES HindIII ␤ embryos. and righttwocomulnsare from different embryos are shown.Leftthree columns two wild-typeand embryonic ectoderm.Serialsectionsfrom epiblast; whitearrowhead, extra- +/+ and–/–embryos.Blackarrowhead, signal isabsentintheepiblastofboth Sall4-deficient (–/–)embryosatE5.8.H19 ectoderm (H19)inwild-type(+/+)and for trophectoderm andextra-embryonic ( hybridization ofSall4,epiblastmarkers indicates epiblast.( deficient (–/–)embryosatE6.5.Arrow staining ofwild-type(+/+)and Fig. 1A.(C analysis usingtheprobes describedin Fig. 1.Embryoniclethalityof domains. E,EcoRI.( Sall4. Ovalsrepresent thezinc-finger deficient mice. vector andat66.7%(8/12)withtheSall4- Fgf4, geo Sall4 Ј -GAGGACTCCATACCGGTGAA-3 - ) formonitoringSall4 3 ␤ Sall4- U/ml) andseruminalltheprocedures -geo Nodal -ApaLI 4.7kbfragmentandthe3Ј Ј and 5Ј (thus facilitating theisolationof ) HematoxylinandEosin Sall4 IRES- clones wereusedtogenerate and Oct3/4),amarker -CCTCTTCGCTATTACG- Sall4-deficient mice( Sall4 Hyg ( Development 133(15) A D ) Targeting strategyof ␤ B - ) Insitu -geo ) Southern blot ) Southern vector. Bothresulting IRES- Sall4-deficient fragment andthe Hyg expression, as Sall4- vector was Ј Sall4- HindIII Ј Sall4- Sall4- Pac I- Ј - ,

DEVELOPMENT selected onpuromycin-resistantembryonicfibroblastsandexpanded. introduced intoSall4 ( incorporation ofBrdU.For rescueanalysis,the (BD Biosciences)was usedforcellcycle analysisofEScellsafter2hour days for16todeterminethecumulative cellnumber. ABrdUFlow Kit media containingLIFandserum,passagedatthesamedensityevery 4 plates intriplicateonmitomycinC-treatedprimaryembryonicfibroblasts treated embryonicfibroblasts. cells weredilutedandplatedonto6-wellplatescoatedwithmitomycinC- 2002) atam.o.i(multiplicityofinfection)50.Afterincubationfor1hour, provided byRIKENBioresourceCenter)(Niwa etal.,1991;Kim with adenovirus expressing CreundertheCAG promoter(AxCANCre affect Sall4expression asshown inFig.3C.TheseEScellswereinfected to analysisbyconfocalmicroscopy. Construction of of theplasmidsusingFuGENE6(Roche) andculturedfor48hoursprior per well1daypriortotransfection. Thecellsweretransfectedwith3 For theproliferationassay, 1ϫ Proliferation andrescue analysisofEScells IRES Two independentSall4-deficientclonesweretransfectedwithpCAG- Chimera formation proliferation oftheblastocysts upon30minutesofBrdUincorporation. uridine (BrdU)labelinganddetectionkitI(Roche)was usedtoexamine sequences usedforRT-PCR areavailable uponrequest.5-Bromo-2 SuperScript IIICellsDirectcDNA SynthesisSystem(Invitrogen). Primer described earlier(Nicholsetal.,1998).cDNA was synthesizedusing protocols. Blastocyst cultureandimmunosurgery werealsoperformedas an automatedDiscovery System(Ventana) accordingtothemanufacturer’s et al.,2001).Insituhybridizationwas performedusingtheAmpMapKitand Histological examination was performedasdescribedearlier(Nishinakamura Histology andblastocystculture clones, flox/–and+/–,proliferatednormally. Neo Sall4 NIH 3T3cellswereplatedontosix-well platesatadensityof1ϫ cysteine residueswerereplaced byglycinewereproducedusingPCR. (BioGenex). GFP-fusedSall4 antisera againstOct3/4(Niwa etal.,2005)andmonoclonalanti-Cdx2 blastocysts andEScells:monoclonalanti-Oct3/4(Santa Cruz), rabbit 95-216. Thefollowing additionalantibodieswereusedforstaining Sall4 antibodywas generatedusing corresponding toaminoacids1050-1064wereused.Amonoclonal anti- polyclonal antibodyraisedagainstpolypeptide(MAKHQFPHFLEENKI) The anti-Sall1monoclonalantibody(Satoetal.,2004)andananti-Sall4 Immunocytochemistry andconfocalmicroscopy the presenceofLIF. D3cellsatadensityof1 maintenance ofD3cells,whichwereculturedongelatin-coatedplatesin (Invitrogen) according tothemanufacturer’s instruction,except forthe transfected intoD3embryonicstemcellsusingLipofectamine2000 Sall4-siRNA orcontrolsiRNA containingthesameGCcontentwas Sall4 totarget thecodingregion ofmouse The siRNA duplexes weredesigned siRNA transfection non-transfected cells. morphological impairmentcomparedwiththemock-transfectedor the counted every day. Thenegative controlsiRNA showed nogrowth or well platesweretransfectedintriplicatewith25pmolsiRNA, and 2004). confocal microscopy were performedasdescribedpreviously (Satoetal., Sall1 (GFP) expression fromeach (Tucker etal.,1997).Cellsretainingubiquitousgreenfluorescentprotein detected usingImmunoPuremetalenhancedDAB substratekit(Pierce). chimeras werestainedbyananti-GFPantibody(MolecularProbes)and blastocysts, andconsistentresultswereobtained.Frozensectionsofthe Sall4 - 1-435 in EScellsandorgandevelopment puro in cDNA atnucleotide2761-2785andsynthesizedbyInvitrogen. pCAG- - DsRed, immunoprecipitation,immunocytochemistry and and selectedonpuromycin-resistantembryonicfibroblasts IRES- -deficient cellsbyelectroporation.Multiplecloneswere puro) orGFP and Sall4-deficient clonewereinjectedinto 10 4 expression vector (negative control)was Sall1 cells wereplatedperwellin24-well Sall4 zinc-finger mutantsinwhichthe cDNA encodingaminoacids ϫ r 10 placed inintron2didnot Sall4 4 cells perwellin24- expression vector Sall1- GFP 10 Ј -deoxy- 5 GFP- cells and ␮ g spheroid structurewithaprimitive endoderm surroundingit,andall trophectoderm abnormality. Of54ICMs,41(76%)grew intoa removed byimmunosurgery toruleoutsecondaryeffects of is requiredforICMproliferation.Next, thetrophectodermwas was notdetectable(Fig.2D;datashown), indicatingthat an apparentincreaseinapoptosis,asdeterminedbyTUNELassay, deficient ICMwhencomparedwithwildtypeorheterozygote,while 3 ofculture,BrdUincorporationwas significantlyreduced in commitment doesoccurintheabsenceofSall4.Bycontrast,atday H19 (ICM), lineage markers examined byRT-PCR was notimpaired: determined byimmunostaining(Fig.2B),andexpression ofthe becoming apparent,Sall4-deficinetICMwas positive forOct3/4,as ( outgrowth ofICMcomparedwithwild-typeandheterozygote culture, allhomozygotes( and thetrophectodermgrew inanidenticalmanner. Byday5in The embryoshatchedfromtheirzonaeandattachedtotheplates, Next, culturedblastocysts wereinvestigated forphenotypicchanges. lineage commitmentbetweenthesetwo lineagesoccursnormally. (trophectoderm marker) was notaltered(Fig.2A),indicatingthat examination, blastocysts from domains ofSall4.As Fig. 1A,Bdepictthestrategy usedtodeletealltheeightzinc-finger Sall4-deficient micedieshortlyafterimplantation RESULTS 08 600166 0 26 0 27 24 28 4 36 86 3 9 5 0 80 2 0 0 0 12 8 14 n.d., notdetermined. Onlyimplantationsiteswere detected. 10 P0 16 E14.5-15.5 10 16 E11.5-13.5 10 E8.5-10.5 5 E6.5-7.5 7 E3.5 10 Stage Table 1.Genotypingofheterozygous crosses heterozygotes, andexpression of Homozygotes were,however, indistinguishablefromwild-typeor inner cellmass(ICM)andtrophectoderminthewild-type(Fig.2A). examined. Atthisstage,theSall4proteinwas expressed bothinthe present inSall4-nullembryos(Fig.1Danddatanotshown). These ectoderm marker ( embryonic ectodermmarker (H19),andtheextra-embryonic markers (Oct3/4 embryos (Fig.1D).Thoughtheareasoccupiedbyepiblast visceral endodermandextra-embryonic ectoderminthewild-type expression was observed intheepiblast,andlessabundantly in identified asSall4-nullbyinsituhybridization,while retained astructurethatresembledtheepiblast.Theseembryoswere embryos (26.9%)derived fromtheheterozygousintercrossbarely egg cylinder withacentralproaminioticcavity, seven outofthe26 E5.5-6.5 (Fig.1C,D).Althoughwild-typeembryosdeveloped an embryonic day(E)6.5(Table 1),theembryoswereexamined at As thedrasticimpairmentofSall4 in blastocystsvitro Sall4 epiblast orextra-embryonic lineages. during theperi-implantationperiod,but notforcommitmenttothe data demonstratethatSall4 n =43) (Fig.2B).Atday3ofculture,whenthephenotypewas (trophectoderm) (Fig.2C).Theseresultssuggestthatlineage is required forinnercellmassproliferation Gata6 and / / / ..Total n.d. –/– +/– +/+ , Fgf4 Bmp4) weresignificantlyreduced,they werestill Gata4 and Sall4-null micedidnotsurvive beyond (primitive endoderm),eomesoderminand is essentialforembryonicdevelopment Nodal), thetrophectodermandextra- n =14) showed significantlyreduced Sall4 Oct3/4 RESEARCH ARTICLE –/– +/– embryos hinderedfurther intercrosses (E3.5)were (ICM marker) andCdx2 Oct3/4 Sall4- 3007 Sall4 Sall4

DEVELOPMENT trophectoderm. reduced in expressed. (D endoderm; I,innercellmass;T, trophectoderm. ( and Sall4.E,primitive immunostaining ofOct3/4 Lower tworows show contrast at3dayofculture. Second row showsphase photo at5dayofculture. row showsphase-contrast cultured invitro. Uppermost mass in 3 ( middle andrightcolumns). homozygotes (compare occurs normallyintheSall4 mass andtrophectoderm commitment totheinnercell (green) stainingshowsthat Oct3/4 (red) andCdx2 trophectoderm (leftcolumn). inner cellmassand 3.5). Sall4isexpressed inthe Sall4-null (–/–)blastocysts(E (+/+), heterozygous (+/–)and development ofwild-type 2 promoterless we constructedotherversions ofthevectors thatcontained isolated atavery low frequency (Table 2).To confirmtheseresults, retargeted bythesecondvector. However, isolation ofheterozygouscellsinwhichtheinitialtargeted allelewas reverse order, andbothexperiments resultedinarelatively frequent (see Fig.S1inthesupplementarymaterial).We alsoattemptedthe in vitro. proliferation inblastocysts Sall4 Fig. 2.Requirement of 3008 xeietFrtrudScn on eodrudo ooisRno eagtn Secondallele Retargeting Random ofcolonies round second *Two out ofthree clonesinExperiment 2andonecloneinExperiment3 were finallydominatedby contaminatingheterozygous cells. Two rounds oftargetingwere carriedouttodisruptboththeallelesof Secondround Firstround 1 Experiment Table 2.Generationof was introducedintoheterozygouscellscontainingthe ES cells,avector containingthehygromycinresistance gene( cells, asthesecellsarederived fromtheICM.To obtain The above datapromptedustoinvestigate Sall4functionsinES Sall4-null EScellsshowreduced proliferation Sall4 rightmost column),suggestingthat,independentoftrophectoderm, 13 outof54(24%)ICMsshowed very few signsofgrowth (Fig.2C the ICMsweregenotypedaswild-typeorheterozygote.Bycontrast, Therefore, theabsenceofSall4 and homologous recombinantsshowed retargeting ofthemutatedallele, material). However, whenintroducedintoheterozygouscells,most homologous recombinants(seeFig.S1inthesupplementary B ) Reductionofinnercell Sall4-null cellswereobtainedatalow frequency (Table 2). for innercellmass is essentialforICMoutgrowth. RESEARCH ARTICLE Sall4-null blastocysts ( A Sall4-null blastocysts(arrow), compared withwildtype(arrowhead). ( ) Normal ) Reducedproliferation oftheinnercellmass ␤ -geo G e pGKHyg pGK Neo G y pGKNeo pGK Hyg RSHgIRES IRES Hyg RSHgIRES IRES Hyg or Sall4 Hyg, thusfacilitating theisolationof Vectors -null EScells is disadvantageous toEScells. ␤ ␤ -geo -geo Sall4 C ) RT-PCR analysisofmarkersinblastocystscultured for3days.Allthelineagemarkersare -null EScellswere eeto t Number Selection at e+y 31 1* 0 12 13 Neo+Hyg e 1 84 3* 44 68 115 Neo e 01 0 8 12 20 Neo y 3 1 62 16 116 134 Hyg Sall4 Neo Sall4-null Sall4-null blastocystscultured for3days.BrdU incorporationoftheinnercellmassis r in EScells. allele Hyg ) Indeed, whencellsweresubsequentlyreplatedandsingleclones served asanegative control, asdeterminedbywesternblot(Fig.3C). flox/– cellsbecamealmostSall4 two typesofcells.Uponinfectionwithadenovirus expressing Cre, expression, andtherewas nodifference inproliferationbetweenthe reduced oligonucleotides intoEScells.siRNA against Sall4 supplementary material).Whenthe of suggest thatSall4 were notalteredintheabsenceofSall4(datashown). Thesedata effect (Fig.3B).Bycontrast,colony morphologyandOct3/4staining kinetics totheSall4 siRNA-treated cellsshowed transientreducedgrowth withsimilar by Sall4immunostainingonEScolonies(datanotshown). expression recovered byday4(Fig.3A),whichwas alsoconfirmed blot, two typesofcells:flox/–and+/–(Fig.3C).Asshown bywestern frequency (wild-typeallele,10/23;floxed allele, 11/23),resultingin introduced intothisclone,bothallelesweretargeted withasimilar We next triedinducible knockdown of Next, heterozygouscellsweregeneratedcontainingafloxed allele Sall4 E ) Failure ofgrowth ofSall4-nullinnercellmassfree from the Neo Sall4 r by homologousrecombination(seeFig.S1inthe in intron2ofthefloxed alleledidnotaffect Sall4 expression byday2aftertransfection,but may berequiredforproliferationofEScells. expression, whilenegative controlsiRNA hadno Integration event -null byday3,while+/–cells Sall4-IRES-Hyg Sall4 Development 133(15) by introducingsiRNA vector was efficiently Sall4- Sall4

DEVELOPMENT Sall4-deficient EScells.Two heterozygous (flox/–),andtwo anti-GFP antibodyanddetectedbyDAB. cells transfectedwithGFP. (left)HighcontributionofGFP-expressing wasstainedbyan cellsintheE7.5embryo.(Right)Asectionofchimera the representative dataisshown.( using three Sall4-nullcells,and independent experiments data were obtainedfrom two Sall4-null EScells.Consistent and increased G1phaseinthe triplicate. (E Analysis wascarriedoutin upon thesameCre treatment. versus flox/–cellsobtained shown, usingSall4-null(–/–) expansion rateover16daysis Sall4-null EScells.Cell ( black triangle,loxP. panels). Whitetriangle,Frt; blot(lowest by western Sall4-null byday3,determined flox/– cellsbecamealmost adenovirus expressing Cre, +/–. Uponinfectionwith two typesofcells:flox/–and similar frequency, resulting in alleles were targetedwitha allele of heterozygous forafloxed was introduced intocells When aSall4-IRES-Hyg disruption ofSall4 in triplicate.(C treatment. Cellswere counted cells uponSall4-siRNA reduced proliferation ofES control siRNA.(B (negative control), treated with treated withSall4-siRNA;NC treatment. KD(knockdown), of Sall4 analysis showingthereduction dependent. Cellcycle analysisrevealed thatSall4-nullcellsshowed (data notshown), confirmingthatthisphenotypewas under aubiquitouspromoterin Sall4 showed impairedproliferation,andre-expression of from theothergroups(flox/–withoutCre,+/–withor Cre) a prolongedperiodoftime(morethan1month).Nonetheclones despite beingtreatedidentically(Fig.3D),but could be culturedfor null clonesgrew significantlyslower than theclonesretainingSall4 were picked, 44outof47cloneswere cells. proliferation ofSall4 Fig. 3.Reduced heterozygous cells(Fig.3E).Thesedatasuggestthat a decreasedS-phaseandincreasedG1-phasecompared with in vivo. explain thephenotypes observed inblastocyst cultureandembryos in EScellsleadstoinefficient G1/Stransition,whichmaypossibly upregulation betweenwild-typeand induced STAT3 phosphorylationorBMP4-induced Idfamily cells (Fig.3F).Nosignificant differences wereobserved inLIF- morphologically indistinguishable fromheterozygousorwild-type D Reduced proliferation of ) These in EScellsandorgandevelopment ( A upon Sall4-siRNA ) Northern blot ) Northern Sall4 Sall4-null EScellsformedcompact coloniesandwere ) ReducedSphase (flox/+), both ) Conditional ) Transiently in EScells. -null ES vector Sall4-null cellsrestoredproliferation F ) NormalmorphologyandpositivestainingofOct3/4a Sall4-null EScells,suggesting Sall4 null, andallthese Sall4 Sall4 Sall4-null ES(–/–)clonesare shown.( absence cDNA Sall4- Sall4 ES cells(datanotshown). Inthe major extrinsic signalsinvolved inmaintainingthepluripotency of that theabsenceofSall4 Utf1 and immunostaining(Fig.3F,G). Althoughexpression ofOct3/4 continued tobeexpressed, whichwas confirmedbynorthernblot which areimportantforthegrowth propertiesoftheEScells, Nanog, whichareessentialforpluripotency, and Sall4 pluripotency ofEScells but proliferation.To furtherconfirmthat not shown), again suggestingthat than didheterozygouscellswhen transplantedintonudemice(data ES cells,and Embryoid bodieswereformed,though weresmaller, from absenceofSall4isnotsecondarytoaberrant differentiation. the shown), whichsuggeststhattheproliferationdefectobserved in mesoderm (T discussion. Expressionofmarkers forprimitive endoderm(Gata6), and Eras was unalteredintheabsenceof -null cellsareundifferentiated, we injectedthesecellsinto was slightlydecreased,andthiswas detailedin the Sall4-deficient cellsproducedmarkedly smallertumors ), andtrophectoderm( Sall4-null EScolony. ( H ) Chimericembryoformationfrom Sall4-nullES does notaffect theresponsetotwo Sall4-null EScells,Oct3/4 Cdx2) was notdetected(data G RESEARCH ARTICLE Sall4 Sall4, theexpression ofNanog ) Northern blotanalysisof ) Northern absence doesnotaffect Eras Sall4 and 3009 Utf1, -null and and

DEVELOPMENT blastocysts togeneratechimeras.AtE7.5, 21/86(24.4) weeks afterbirth. 1/80(1.3) Dead pupsfrom theheterozygous crosses (describedinTable 1)were scored for6 Total: dead/born(%) 3-6 weeks 0-3 weeks Age birth Table 3.Increased deathrateof 3010 by humans, and,thus,anorectalandheartanomaliescouldbecaused mimic theOkihirosyndromecausedby heterozygotes (Fig.4BandTable 4).Thesephenotypespartially ventricular septumdefectswerealsoobserved insomeofthe secondary effect ofanaldysplasia.Whenexamined atE17.5-18.5, (data notshown); hence,gastrointestinaldilationislikely tobea number ofentericgangliaintheseheterozygoteswas notaffected bowels andapparentanalstenosis(Fig.4A,openarrowhead). The the next 3weeksandsixofthesemicehadsignificantly dilated their motherswithin3weeks(Table 3).Eightmorediedwithin born heterozygousmice,13wereruntanddiedoreatenby the Genotyping fromheterozygouscrossesshowed thatnearlyhalf heart anomaliesandexencephaly Sall4 not pluripotency ofEScells. together, ourdataindicatethatSall4isessentialforproliferationbut with GFPcontributed highlytotheembryos(Fig.3H).Taken data notshown). InnerearstructuresatE17.5( nuclei, eye andocularmuscleswerealsounaffected (Fig.4Dand mice (n Kohlhase etal.,2002),theabducensnucleiin adultheterozygous movements inOkihirosyndrome(Al-Baradieetal.,2002; and nucleiarereportedtoberesponsiblefortheabnormaleye metatarsus formation( newborns didnotshow any anomaliesindigit,metacarpusor normalappearingextremities andcloserexamination of had All theheterozygotesthatsurvived beyond 3weeks( Okihiro syndromewerenotdetectedinSall4-heterozygousmice. SALL4 Sall4 haploinsufficiency results inanorectal and RESEARCH ARTICLE =6) wereproperlyformed,andoculomotortrochlear heterozygous micediedinutero(Table 1).Outof86 haploinsufficiency. Otherphenotypesobserved in n =6) (Fig.4C).Thoughabducensnerves +/+ 0 1 Sall4 heterozygous miceafter Sall4 +/– SALL4 13 8 n -null cellstagged =9) andinadults mutations in n =73) Sall2/4 heterodimers inthedeveloping brain,heartandanorectalregions. to Sall1(Fig.5G).Therefore,thesetwo genesprobablyform using lysatesfromEScellsshowed thatendogenousSall4binds heterochromatin andoverlapped withSall4.Immunoprecipitation heterochromatin. EndogenousSall1was alsolocalizedinthe indicating thatSall4islocalizedintheconstitutive colocalized with4,6-diamidino-2-phenylindole (DAPI) (Fig.5F), Endogenous Sall4was localizedinthepunctatenuclearfocithat determine ifSall1andSall4werecolocalizedinEScells. Sall4 myocardium but alsointheendocardium,thusoverlapping with arrowhead). AtE11.5, arrowhead) andinalltissuesofthetailregion (Fig.5C,open mesenchymeoftheanteriorportion(Fig.5C,filled the neural tubesclose,both overlapped intheaffected organs. AtE8.5,astageatwhichthe vivo. Next, wedeterminediftheexpression of (Table 4),suggestingageneticinteractionof significantly increasedincomparisonwithSall4 defects (datanotshown); theincidenceofthesephenotypes was shown), anorectalmalformations(Fig.5B)andventricular septum uni- orbilateralrenalagenesis(Fig.5A),exencephaly (datanot mice having othergeneticcombinations( Sall1/4 redundancy amongmembersoftheSall Compound heterozygotesweregeneratedtoinvestigate functional Sall4 event. the neuraltubetoclose, shown). Asexencephaly andakinked tailarecausedbyfailure of that survived upto3weeksexhibited tailflexion anomalies(datanot exencephaly (Fig.4F),andfouroutof73 heterozygotes (threeoutof26examined atE11.5-15.5)exhibited haploinsufficiency. notallOkihirophenotypeswerecausedbySall4 mice, ( interventricular septum,while was detectedinthemyocardium,includingdeveloping anorectal region (Fig.5D,arrowhead). IntheheartatE11.5, n =6) werenotimpaired(Fig.4E;datashown). Thus,in We alsofoundotherphenotypesnotreportedinhumans.Some in themyocardium(Fig.5E).We furtherchecked to genetically interactswith and compound heterozygotessurvived afterbirth,whereas Sall3/4) survived. TheSall1/4 haploinsufficiency. Fig. 4.Phenotypescausedby Blue andAlizarinRedS.(D new born formation ofdigits,metacarpusandmetatarsusin Hematoxylin andEosinstaining.(C ventricular septumdefectintheheterozygotes. ventricular septuminwild-type;openarrowhead, Sall4 heterozygotes. ( type; openarrowhead, imperforateanusinthe heterozygous mice.Blackarrowhead, anusinwild megacolon (below)of5-week-old ( sections were examinedby Klüver-Barrera staining. (arrow) in8-week-oldSall4 abducens nuclei(arrowhead) andfacialnerve heterozygous miceatE14.5. Eosin staining.( Sall4 E ) Normaldevelopmentofinnerear structure in Sall4 heterozygotes atE18.5.Blackarrowhead, heterozygotes atE17.5.Hematoxylin and Sall4 Sall4 may alsoplayanimportantroleinthis Sall4 Sall1 and F heterozygotes. StainedwithAlcian B and Exencephaly of ) ) Ventricular septumdefectin Sall1 was expressed notonlyinthe ( Sall1 A ) Analstenosis(above)and Sall1 Sall1/2, Sall4 family. Surprisingly, no heterozygotes exhibited were expressed inthe Development 133(15) ) Normalformationof heterozygotes. Serial were expressed in heterozygous mice Sall4 Sall4 Sall1/3, Sall4 Sall4- ) Normal Sall4- heterozygotes and and Sall1 Sall2/3, Sall1 Sall4 in

DEVELOPMENT Sall4- (arrowheads). Heterozygotes of the anorectal region atE11.5 expressed inmyocardium (arrowhead) andendocardium (arrow). ( neuroepithelium, respectively. mesenchyme and white arrowheads indicatethe (transverse section).Blackand anterior region oftheembryo E8.5. Theuppersideisthe hybridization ofSall4 bladder; r, rectum. (C rectum. a,anus;b,urinary arrowhead showsabsenceofthe rectoanal junction;white complete stenosisofthe panels). Blackarrowhead shows heterozygotes atE17.5(righttwo of Sall4andSall1, andcounterstainingwithDAPIin EScells.( stained usingX-gal.(E (Nishinakamura etal.,2001)were When various Sall4 negative manner localization ofSall4,andfunctionsinadominant- Truncated Sall1disturbsheterochromatin may exist inkidney development. (data notshown). Thus,heterodimer-independent mechanisms However, inthedeveloping kidney, thetwo genesdidnotoverlap Sall4 § ‡ † *Examined atE13.5-P0. Anorectal malformations Exencephaly* Renal agenesis* Table 4.Phenotypicexacerbationin ( bladder; c,colon;k,kidney. a, adrenal glands;b,urinary while 10hadunilateralagenesis. analyzed hadthisphenotype, compound heterozygotes heterozygotes. Sixoutofthe38 renal agenesisin Sall4 Fig. 5.Geneticinteractionsof transfection ofSall1 lacked theC-terminalheterochromatinlocalizationdomain.Co- throughout thecytoplasm andeuchromatin(Fig.6C),as thismutant Ventricular septumdefects localization domains.SALL1 these C-terminaldoublezincfingersconstituteheterochromatin and weresufficient forheterochromatinlocalization(Fig.6B).Thus, case ofSall1,two doublezincfingers(Zn4and5)wererequired, sufficient forlocalizationtotheheterochromatin(Fig.6A).In terminal doublezinc-fingerdomain(Zn4)ofSall4was essentialand finger domainsweregenerated,weobserved thatthemostC- Sall1 are likely toproduceC-terminallytruncatedproteins.Truncated D B Six outof16doubleheterozygotes hadnokidneysbilaterally, andtenhadunilateralkidneyagenesis. Two Sall1 Examined atE17.5-18.5. Anal stenosisin ) ) Sall4 ␤ and Sall1 in EScellsandorgandevelopment fused toDsRed geo and Sall1 heterozygotes hadunilateralkidneyagenesis. and Sall1-lacZ . Sall1/4 ( A expression in Sall1/4 ) Bilateral ) Insitu 1-435 and and Sall1 ) OverlapofSall4 ( Sall1 -DsRed Sall1 † † 1-435 mutations inTownes-Brocks syndrome at constructs withmutationsinthezinc- and -DsRed) was ubiquitouslylocated /3(.% 0/61(0%) 2/43 (4.6%) 0/43 (0%) /0(00)0/10(0%) 0/14(0%) 2/10 (20.0%) 4/14 (28.6%) Sall4-GFP and Sall1 Sall4+/– Sall4/Sall1 in thedevelopingheartatE11.5. showed disturbance double heterozygous mice 2/61 (3.3%) G ) BindingofSall4 andSall1shownbyimmunoprecipitation usingEScelllysates. Sall1+/– Sall4- ␤ geo ‡ technique identifiedtheimportanceof Blastocyst cultureanalysiscombinedwiththetrophectoderm removal embryogenesis andproliferationofEScellswas entirelyunexpected. abnormalities, theindispensableroleofmouseSall4 syndrome, characterizedbylimbdeformityandeye movement negative manner. AshumanSALL4 Brocks syndromecausedmislocalizationofSall4inadominant- Sall1 andrevealed thattheC-terminallytruncatedSall1inTownes- both invivo andinvitroevidence forthedimerizationofSall4and embryogenesis andforproliferationofEScells.We alsoprovided In thisstudy, weshowed thatmouse DISCUSSION be explained bythefunctionalreductionofSall4. heterozygotes, thephenotypesobserved inSall1truncationscould anal andheartanomaliesexencephaly in mislocalization ofSALL4intheheterochromatin.Considering dimerize withSALL4inadominant-negative manner, resultingin truncated SALL1proteinsinTownes-Brocks syndromeprobably in Sall4localizationtheheterochromatin.Thus,C-terminally and Sall1-lacZmicewere stainedusingX-gal.( 17/38 (44.7%) 11/16 (68.8%) 16/38 (42.1%) Sall4 al+–Sall1+/– Sall4+/– 7/10 (70.0%) is expressed inmyocardium (arrowhead), while § is acausative genefortheOkihiro RESEARCH ARTICLE Sall4 Sall4 in theinnercellmassthat F ) Immunocytochemistry is essentialforearly Sall1/4 Sall1 in early double is 3011

DEVELOPMENT heterochromatin. (C two clustersare notsufficient forproper localizationinthe Zn5 alsoshowadefectinheterochromatin localization, thoughthese heterochromatin localization(broken outline).MutationsinZn2and localization (rectangle). Zn4andZn5are alsosufficient for the C-terminalzincfingers(Zn4andZn5)ofSall1forheterochromatin categorized asZn1,Zn2,Zn3andZn4,shown.( fusion were expressed inNIH3T3cells.Zinc-fingerclustersare heterochromatin localization(broken outline). MutantsofSall4-GFP heterochromatin localization(rectangle). Zn4is alsosufficient for of Sall4isdisruptedbyC-terminallytruncatedSall1. DsRed) andSall4-GFP 3012 inner cellmass,itisunderstandablethat is theoriginofembryoproper. AsEScellsarederived fromthe ( truncated Sall1thatfunctionsinadominant-negativemanner. Fig. 6.MislocalizationofSall4from theheterochromatin bya methylating complexes. Centromeric andpericentromericregions, residues, followed by recruitmentofhistone-methylatingandDNA- also bindHDAC complexes anddeacetylate thehistonelysine RbAp46/48, MTA1 and MTA2 (Kieferetal.,2002)],Sall4could chromatin remodelingcomplexes [namelyHDAC1, HDAC2, localized intheheterochromatin andbindstothecomponentsof phenotypes observed inblastocyst cultureandembryos invivo. significantly reducedproliferation,whichmaypossiblyexplain the A ) Requirement oftheC-terminalzincfinger(Zn4)Sall4for How canSall4regulate theproliferationofEScells?AsSall1is RESEARCH ARTICLE ) Co-transfectionoftruncated into NIH3T3cells.Heterochromatin localization Sall4-null EScellsshowed B Sall1 ) Requirement of ( Sall1 1-435 - Japan. Science, andTechnology; andbythe MinistryofHealth,Labor, andWelfare of work wassupportedinpartbytheMinistry ofEducation,Culture, Sports, technical assistance;andT. Haraforcriticalreading ofthemanuscript.This Takeda, Y. Kataoka,H.Meguro, S.Yamada, A.NakaneandR.Sakamoto for mice; H.Masai,M.Nakao,T. AotoandK.Okitafortechnicaladvice;N. antibody forimmunosurgery;A.P. Monaghanforproviding strains onthesamegeneticbackgroundwould testthispossibility. phenotypes arenotdescribed.Directcomparisonofthetwo mouse this trapallelehasmoresevere phenotypes thanours,althoughother If thisalleleserves asadominant-negative form,itispossiblethat exhibited digitandheartanomalies(Koshiba-Takeuchi etal.,2006). in theheterochromatinstructureSall4 facultative heterochromatinformation).Moredetailedexamination heterochromatin formationintheeuchromaticpromoters(known as downstream genes,includingcellcycle inhibitors,byinducing Sall4 affects thesestructures.Alternatively, Sall4couldsuppress spindle formationinmitosis,anditispossiblethattheabsenceof which compriseconstitutive heterochromatin,arerequiredfor We thankH.Niwaforproviding theplasmids( and H19)antiseraagainstOct3/4;H.Suemori, fortheanti-mouse gene trapalleleretainingtruncated negative manner, asisthecaseforSALL1truncations.Recently, a terminally truncatedSALL4proteinscouldfunctioninadominant- proliferation of 2003). ThoughEras deficient cellsdifferentiate intoprimitive endoderm(Mitsuietal., heterozygous cellsshow noreductioninproliferation, and and further elucidationofSall4functions.ItisinterestingthatNanog identification ofdownstream target genesofSall4,isrequiredfor not explained bySall4 not detectedinSall4 anomalies tolimbs,abducensnuclei,innerearsandkidneys were Sall1, but notbyhaploinsufficiency ofSall1.However, other could becausedbythedominant-negative effect ofthetruncated important becausemostphenotypesinTownes-Brocks syndrome these phenotypesarecausedby heterozygous micehadanalandheartanomalies,suggestingthat anorectal, ear, heart,andkidney anomalies.Some deformity, eye movement (abducensnerve) abnormalities,and disorder Okihirosyndrome,whichischaracterizedbylimb formation. from theinhibitionofSALL4functionsthatisduetoheterodimer of Townes-Brocks syndromecausedbySALL1truncationsresult functional reductionofSall4.Thus,weproposethatsomesymptoms phenotypes observed inSall1truncationscanbeexplained bythe exencephaly in SALL1 truncations.Consideringtheanalandheartanomalies unlikely tosurvive, exencephaly couldbeoneofthephenotypes well asexencephaly. Ashumanfamilies withsevere phenotypesare mice retainingtruncatedSall1proteinsshow similarphenotypesas mutations exhibits limb,anal,ear, kidney andheartanomalies, negative manner. Townes-Brocks syndromecausedby truncated Sall1proteinsalteredSall4localizationinadominant- stages ofdevelopment. function inEScellstothestudyoforgan formationduringlater approaches, weexpect toapplythedetailedanalysisofSall4 deficiency. AsEScellsaresuitableforquantitative biochemical that areductionofthesegenescouldexplain thephenotypesof abnormalities invivo (Takahashi etal.,2003).Thus,itisunlikely SALL4 We demonstratedthatSall4andSall1formheterodimers Eras were slightlydecreasedinSall4 mutations inhumanscausetheautosomaldominant Sall1/4 Sall4-null EScells, heterozygous mice;thus,thesephenotypesare could bepartlyresponsibleforimpaired haploinsufficiency inmice.Inhumans, C- double heterozygotes,atleastthesethree Sall4 Sall4 IRES- haploinsufficiency. Thisis was reportedandthisstrain mutant EScells,aswell Eras-null micehadno -deficient cells.Nanog ␤ Development 133(15) geo, IRES- Sall3-deficient Hyg, Oct3/4 Nanog- SALL1 Sall4 Sall4

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