identified asregulators ofTh2development, suchasc-Maf(Maf) (Ouyang etal.,2000).Thereareotherfactors thathave been (Takemoto etal., 1998). Thiscanoccureven intheabsenceofStat6 Institute ofMedicalResearch, 3050,Australia. 1GRoyalParade,Parkville,Victoria differentiation ofnaïve CD4 the immunesystem, playcentralrolesindetermining the Cytokines performcrucialfunctionsincellfate decisions.Within INTRODUCTION Accepted 30May 2007 † 3 1 KEY WORDS:Th2cells,Cytokines,Mammarygland,Signalling,Mouse peptide hormonesaretheprimaryregulatorsofmammaryglanddevelopment. epithelial cellfateandfunction,addsanunexpectedtierofcomplexitytothepreviouslyheldparadigmthatsteroid and IL-4/IL-13doublydeficientmiceduringpregnancy. Thisunexpecteddiscoverydemonstratesaroleforimmunecellcytokines crucial roleinmammaryglanddevelopmentvivo,becausedifferentiation andalveolarmorphogenesisarereducedinbothStat6 cytokines IL-4,IL-13andIL-5(Il5)asepithelialcellscommittotheluminallineage.Moreover, weshowthatTh2cytokinespl interferon gamma(INF undergo aswitchfromTh1toTh2cytokineproductionupontheinductionofdifferentiation. Thus,theTh1cytokinesIL-12(Il12 mediators ofTh2development.We showhere,forthefirsttime,thatthisparadigmappliesalsotomammaryepithelialcells,wh targets. TheTh1/Th2cytokinemilieuisakeyparadigminlineagecommitment,andIL-4(Il4),IL-13(Il13)Stat6areimport Naïve Thelpercellsdifferentiate intoTh1andTh2subsets,whichhaveuniquecytokinesignatures,activatorstranscriptio Walid T. Khaled mammary epithelialcelldevelopment The IL-4/IL-13/Stat6signallingpathwaypromotesluminal (2007)doi:10.1242/dev.003194 Development 134,2739-2750 Naomi Sprigg Manchester, M139PT, UK Oxford Road, *Present address: FacultyofLifeSciences,UniversityManchester, UK. CB2 1QP, UK. which facilitates transcriptionofthe because itisrequiredforchromatinremodellingattheIL-4locus, 1997). Gata3hasbeenshown tobeessentialforTh2development turn, activates thetranscriptionfactor Gata3(ZhengandFlavell, that Th2cellsactivate Stat6inresponse toIL-4/IL-13,which,in development andreducedtype-2immunity inmice.Itisnow clear (McKenzie etal.,1998)expression leadstoperturbedTh2cell et al.,1996;ShimodaTakeda etal.,1996)orIL-13 Reiner, 2002).AblationofIL-4(Kuhn etal.,1991),Stat6(Kaplan (reviewed inAnseletal.,2006;Farrar etal.,2002;Murphyand identified factors involved intheregulation ofTh2development 2004; Mossetal.,2004). with diseasessuchasasthma,autoimmunityandcancer(Chatila, the developmental controlofTh1andTh2cellshasbeenassociated (Il4/Il13), whichactivate Stat6(Kaplanetal.,1996).Disruptionin andIL-4/IL-13 (Jacobson etal.,1995;Thierfelder1996), regulated, respectively, byIL-12(Il12),whichactivates Stat4 1986). ThepolarizationofThcellsintoeitherTh1orTh2is lineages; Thelper1(Th1)or2(Th2)(Mosmannetal., Author forcorrespondence (e-mail:[email protected]) Medical Research CouncilLaboratoryofMolecularBiology, CambridgeCB22QH, Department ofPathology, UniversityofCambridge,Tennis CourtRoad,Cambridge Elegant experiments usingvarious transgenic mousemodelshave 2 Division ofCancerandHaematology, TheWalter andElizaHall 2 , Andrew N.J.McKenzie 1 , EliotK.C.Read ␥ ; alsoknownasIfng)andTnf + T helper(Th)cellsintoeitheroftwo IL-4 , 1 IL-13 , SandraE.Nicholson and 3 and ChristineJ.Watson IL-5 ␣ ( Il5 are downregulatedconcomitantlywiththeupregulationofTh2 ) 2 , FionaO.Baxter be expressed inTh1cells,whereitinteractswiththeIL-4R et al.,1999;Seki2003).Conversely, Socs5hasbeenshown to 12, respectively (Egwuaguetal.,2002;FujimotoMarine implicated innegatively regulating theTh1cytokines IFN- (Alexander andHilton,2004).Socs1Socs3 have been containing proteinsthatnegatively regulate signalling arecytokine-inducible Srchomology2(SH2)-domain- suppresser ofcytokine signalling(SOCS)family ofproteins.SOCS differentiation iscomplicatedfurtherbytheinvolvement ofthe (Xanthoudakis etal.,1996).Theregulation of Th1/Th2 ablation ofitsexpression leadstomarked Th2development been shown tobeanegative regulator ofTh2development, because transcription of that isexpressed predominantlyinTh2cellsandinduces the and NFAT1 (alsoknown asNfatc2). c-Mafisatranscriptionfactor Examination ofmammarygland development in in vivo and inmammaryepithelialcells(MECs)culture. are involved intheStat6signallingpathway inboth mammarygland characterized thecytokines, receptorsandtranscriptionfactors that in different mammary developmental processes.Therefore, we the rolesofStat5(Liuetal.,1997) andStat3(Chapmanetal.,1999) pathway isinvolved inmammaryglanddevelopment inaddition to 2004). Thus,wesoughttoaddresswhethertheStat6signalling expressed inmammaryglandsduringdevelopment (Clarksonetal., commitment totheselineageshave notbeenwelldefined. by contractionofthemyoepithelialcells.Thefactors that control cells; milkisproducedbytheluminalcellsandexpelled intoducts Progenitor cellsdifferentiate intoeitherluminalormyoepithelial prolactin (Prl)(HennighausenandRobinson,2001;Rosen,2004). primarily underthecontrolofestrogen(E),progesterone(P) and is thatproliferationanddifferentiation oftheseepithelialcellsis during pregnancy. Thecurrentparadigminmammaryglandbiology Th2 differentiation (Sekietal.,2002). and therebyattenuatesIL-4signalling,thusnegatively regulating 4 –/– Our previous microarraydataindicatedthatStat6was abundantly Mammary epithelialcellsundergo amassive expansion innumber /IL-13 1,† –/– animals revealed aroleforthe Stat6signallingpathway IL-4 1 , AmeliaJ.Brennan (Ho etal.,1998).Ontheotherhand,NFAT1 has RESEARCH ARTICLE 1 , PaulJ.Came Stat6 –/– ay a ␥ ant ␣ and and IL- nal chain 2739 1, ), in ich *, IL-

DEVELOPMENT in 1:1DMEM:F12supplementedwith10%FCS. were usedattheindicatedconcentrations(Fig.6).EpH4cellsgrown media changepriortothestartofcytokine treatment.IL-4andIL-13(R&D) and 17mMLinoleicacid.Zerotime-pointswerecollected24hourspost mM Insulin,0.2mMProlactin(Sigma),1Dexamethasone (Sigma) 0.8 differentiation mediawas addedcomprising1:1DMEM:F12,10%FCS, For differentiation induction,cellsweregrown toconfluency andthen Insulin (Sigma),0.8mMEGF(Sigma)and17Linoleicacid(Sigma). DMEM:F12 (Invitrogen) mediacontaining10%FCS(Sigma),0.8mM and eachtimepointwereanalysed. mortem toavoid pseudo-pregnancies. Atleastthreemiceofeachgenotype checked toconfirm timingofmatingandpregnancy was confirmed post- Office guidelines.Virgin femalemiceat8-to14-weeksoldweremated,plug animals weretreatedaccordingtolocalethicalcommitteeandUKHome Anne Cooke (DepartmentofPathology, University ofCambridge,UK).All immune deficiency (NOD-SCID)mice(Balb/cbackground)werefrom (NOD) andNOD-severe combined Victoria, Australia).Non-obesediabetic Douglas Hilton(Walter andEliza HallInstituteofMedicalResearch, corresponding wild-typeBL/6strain-matchedcontrolswereprovided by A.N.J.M. (co-author). corresponding wild-typeBalb/cstrain-matchedcontrolswereobtainedfrom Harlan Laboratories. within anSPFanimalfacility. Wild-type Balb/cmicewerepurchasedfrom Laboratories andweremaintainedbredinapositive pressureisolator reverse transcription from2 Technologies). cDNA was synthesizedbyrandomhexanucleotide-primed and integrity was determined usingaNanoDropND-1000(NanoDrop kit (Qiagen)accordingtothemanufacturer’s instructions.TheRNA quantity Tri-reagent (Sigma).RNA extraction was performedusinganRNeasymini powder usingamortarandpestle. Tissue (100mg)was dissolved in1mlof Mammary tissuewas snapfrozeninliquid nitrogenandgroundtoafine RNA extractionandPCRprimers Stat6 Mice andcelllines MATERIALS ANDMETHODS to theluminallineage. cytokine productioncoincidentwiththeinductionofdifferentiation analysis ofMECsinculturerevealed aswitchfromTh1toTh2 regulator ofStat6,resultedinaccelerateddevelopment. Furthermore, was foundtobedelayed.Conversely, deletionofSocs5,anegative in branchingmorphogenesis,becausedevelopment duringgestation 2740 positive pressureisolator. ACTGCATC, Rev, CCATCCTTTTGCCAGTTCCTC; GCAGAGTCTCGCCATTATGATTC; GCAAGAACA; Il13r TCGATTTTGCTTTTGG, Rev, GTGCTGGGGTGGGAATCTGGTC; were used(allprimersareshown 5 performed byPCRusingTaq polymerase(Qiagen).Thefollowing primers Il4ra transcription cDNA synthesiskit(Roche).Semi-quantitative detectionof GTCTAGCCCCTG; Il5 CCTGGCTCTTGCTTGCCTT, Rev, GGTCTTGTGTGATGTTGCTCA; CCCAGCTAGT, Rev, GCCGATGATCTCTCTCAAGTGAT; (http://pga.mgh.harvard.edu/primerbank/): PCR wereobtainedusingthePrimerBank (Wang andSeed,2003)website (BioRad) intriplicate.Sequencesofthefollowing primersusedforreal-time recommendations. Thereal-timePCRreactionswereruninaniCycler fluorescein (BioRad)andSYBR-green(Sigma)accordingtothesupplier’s cDNA was performedusingiCycler supermix (BioRad)withtheadditionof CACCCTGGCACATGAATCCTG. Quantitative real-timedetectionof and CAACGGCACAGTCAA, Rev, CTTTCCAGAGGGGCCATCCACAG; ACCGGGTCCCCAT TAGCGTTCCT; KIM-2 cells(Gordonetal.,2000)weregrown toconfluency in1:1 Fwd, CTCTGTTGACAAGCAATGAGACG, Rev, TCTTCAGTAT - , ␣ –/– Cyclophillin A 1 Il13ra1 RESEARCH ARTICLE mice (Kaplanetal.,1996)werepurchasedfromJackson Fwd, GGCCATC CTGCAAAATAGTG, Rev, ACAGCGTC G - , Gata3 Gata3 Il12a IL-4 IL-4 , Gapdh Fwd, TGGGTGGGGCCTCATCCTCAG, Rev, Fwd, CCTTGGGCCGCGTCTCCTT, Rev, –/– –/– Fwd, CTGTGCCTTGGTAGCATCTATG, Rev, ␮ Socs5 /IL-13 /IL-13 g oftotalRNA usingtheTranscriptor reverse n ylpilnA( and cyclophillin A –/– –/– –/– mice weremaintainedandbredina mice (Brenderetal.,2004)and mice (McKenzie etal.,1999)and Ifng Ј Gapdh -3 Ј ): Il4 w, ATGAACGCT ACAC - Fwd, Il4ra Fwd, GGTCTCAACC - Fwd, CGGCAAATT - w, TGGGCTG- Fwd, CypA , Tnfa Ppia Il13 )was Fwd, Fwd, HRP-conjugated antibodieswerefromDako Cytomation. rabbit anti-Stat6(M-20),anti-Gata3andanti-c-Maf.Secondary and goatanti-IL-4r Laboratories), mouseanti-pStat6(Y641)(Abcam)ratanti-tubulin (Abcam) Other commercialantibodiesusedweremouseanti-ERK1/2(Transduction Akt1)/PKB (Ser473),rabbitanti-totalAkt/PKBandmouseanti-pERK1/2. Cell SignalingTechnology wereused:rabbitanti-pAkt(Aktisalsoknown as as describedpreviously (Abell etal.,2005).Thefollowing antibodiesfrom using enhancedchemiluminescence(ECL,GEHealthcare)wereperformed separated oncriteriongels(BioRad).Immunoblottingandantibodydetection determined withtheBCAcolorimetricassay(Pierce)andsampleswere specificity was confirmed usingBLAST. System Software (BioRad).Allreal-timePCRproductsweresequencedand Aldrich. Analysiswas performedusingiCycleriQReal-Time Detection ACCTCTGCACCGTAGC. AllprimerswerepurchasedfromSigma- (Roche), 1mMNa deoxycholate, 150mMNaCl,1EDTA, 1 a lysisbuffer containing50mMTris-HCl pH7.4,1%NP-40,0.25%sodium Lymph node-freeabdominalmammaryglandsandcellswereextracted with Immunoblotting 2.5 galuminumpotassiumsulphateand500mlofdH slide was washed inwater andplacedincarminealumstain(1gCarmine, ethanol, 3partschloroformand1partglacialaceticacid)overnight. The forceps onaglassslideandincubatedinCarnoy’s fixative (6parts100% For whole-mountanalysis,abdominalglands(no.4)werespreadoutusing Whole mountsandH&Estaining CAG; GCTACGACGTGG GCTA- Rev, CCCTCACACTCAGATCATCTTCT, hyperfilm (GEHealthcare). manufacturer’s protocol.Chemiluminescencewas detectedusingECL- differentiation was analysedusing aCytokinearrayI(RayBio)following the Medium (1ml)fromculturesof KIM-2cellsatday0and8of Cytokine array Software). analysis was performedusingtheSigmaStat3.5software package(Sysstat using NationalInstitutesofHealth(NIH)ImageJsoftware. Statistical microscope. Quantification ofimagesandimmunoblotswas performed gland wholemountswerevisualisedusingtheLEICAMZ75 light stains werevisualizedonaLEICAlightmicroscopy. Themousemammary Acquisition Managersoftware (KineticImaging).TheDAB IHCandH&E with imagesvisualizedandmanipulatedusingAQM 6Advanced Kinetic microscope equippedwithaHamamatsuC4742-95ORCA1digitalcamera, Fluorescence microscopy was carriedoutusingaZeissAxiovert S100TV Microscopy andstatisticalanalysis and stainedwithantibodiesagainstIL-4(1:50;R&D)orIL-13R&D). fixing in 1:1acetone:methanol.Slideswereblocked in10%normalgoatserum (NUNC) andtreatedwithBrefeldin-A10 intracellular KIM-2cytokine staining,cellsweregrown onchamberslides antibodies (Sigma)andbisbenzimide-Hoechst33342(Sigma).For isotype controlovernight at4°CanddetectedusingCy3-conjugatedsecondary phosphorylated Stat6(pStat6);at1:50forGata3,c-MafandIL-4R Sections wereeitherincubatedwithprimaryantibodyat1:100for rabbit seruminthecaseofIL-4r 6.0, for10minutes.Sectionswereblocked in10%normalgoatserum(normal retrieval was performedusingboiling10mMTri-sodium citratebuffer, pH Paraffin-embedded mammarysectionswerede-paraffinized andantigen Immunohistochemistry being stainedwithhaematoxylinandEosin(H&E). sectioning. Alltissueswereembeddedinwax andsectionedat5 transferred to70%ethanolandstoredat–20°Cuntilembedding 4% formaldehydeinPBSfor24hoursatroomtemperature.Theglandswere documentation. For histologicalanalysis,abdominalglandswerefixed in slide was washed withethanolandclearedinxylenefor1daybefore was a kind gift from Bert Binas (Texas A & M University, Texas, University, TX). M was akindgiftfromBertBinas(Texas& A Gata3 Fwd, CTCGGCCATTCGTACATGGAA, Rev: GGAT - ␣ (R&D systems).AntibodiespurchasedfromSCBTwere: 3 VO 4 , and1mMNaF. Proteinconcentrationwas ␣ ) (Dako) for1houratroomtemperature. ␮ g/ml (Sigma)for4hourspriorto ϫ Cocktail proteaseinhibitors Development 134(15) 2 O) overnight. The ␤ -casein antibody ␮ ␣ m before or with

DEVELOPMENT by Petalidisandco-workers (Petalidisetal.,2003),except that5 protocols. cDNA target preparationwas amplified andlabelledasdescribed RNeasy columns(Qiagen),inaccordancewithtothemanufacturer’s 5 ofgestationwas isolatedusingTRIreagent(Sigma)andcleaned number of14cycles was used.For thelabellingstep,2 was addedtothesecond-strandamplification reactionmix.Aconstant cDNA from Microarray Stat6 controls mammaryglanddevelopment (Sykacek etal.,2005). using BlueFuse3.2(BlueGnome). Analysiswas performedusingFSPMA Axon 4100A(AxonInstruments)and signalquantification was performed biological replicates.Total RNA fromwild-typeand independent labellingofthesamestartingRNA preparations)andwiththree for technicalreplicate(repeathybridizationsonseparateslidesusing Resources, Cambridge,UK).Eachexperiment was repeatedwithadyeswap (MEEBO) arraywas used(Pathology Department,CentreforMicroarray hybridization tothearrays.Amouseexonic evidence basedoligonucleotide (8 and hybridizedfor16hoursat50°C with4 instructions (AmershamBiosciences UK).Labelledsampleswerecombined products werepurified usingG50columns, accordingtothemanufacturer’s Cy5-dCTP was usedwith22 ␮ g/ ␮ l) and1 Stat6 ␮ –/– l yeasttRNA (4 mice was comparedtowild-typecDNA viacompetitive ␮ l ofsecond-strandcDNA. The labelled ␮ g/ ␮ l). Arrayswerescannedusingan ␮ l ofCot-1DNA, 1 Stat6 ␮ l ofCy3-dCTPor –/– glands atday ␮ l ofMgCl ␮ l PolyA 2 1B,C). Interestingly, IL-4R gestation, mostductal/luminal cells exhibited nuclearpStat6(Fig. minority ofvirgin ductalepithelialcells,whereas,byday 5of (Fig. 1B-I).Thisstudyrevealed thatpStat6was expressed ina Maf andIL-4R immunohistochemistry forphosphorylatedStat6(pStat6),Gata3, c- gestation. To determine whichcellsexpress thesefactors, gestation, whereasc-Mafwas increasedlater, fromday15of Interestingly, levels ofIL-4R at day5ofgestationandmaintaineduntillactation(Fig. 1A). involved inStat6signalling.Phosphorylationofwas induced supplementary material)andimmunoblotsfortheprimaryfactors performed reverse transcriptase(RT)-PCR (seeFig.S1inthe having aroleinmammaryglanddevelopment. Therefore,we The Stat6signallingpathway hasnotbeendescribedpreviously as Stat6 signallinginMECsvivo RESULTS nuclei ofepithelial cells(Fig.1F-I). the transcriptionfactors Gata3 andc-Mafwerelocalisedinthe surface ofluminalcellsduring gestationonly(Fig.1D,E),whereas ␣ was carriedoutonsectionsofmammarytissue phosphorylated Stat6(pStat6),Stat6,IL-4R Immunoblotting wasperformedfor of birth),5and10lactation. 15 ofgestation;andfrom miceatday0(day from virginmice;from miceatday5,10and extracted from inguinalmammaryglands gestation. control stainingonwild-typemiceatday5of mice atday5ofgestation.(I)Rabbitisotype pStat6stainingin gestation (G).(H) gestation (F);andc-Mafinmiceatday15of gestation (E);Gata3inmiceatday5of virgin mice(D)andatday15of and miceatday5ofgestation(C);IL-4R wild-type micefor:pStat6invirgin(B) performed onmammaryglandsectionsfrom was left panels,mergedwithDAPIstaining) ( software andplottedonahistogram(right). immunoblots were quantifiedusingImageJ Gata3, c-Mafand at theonsetofpregnancy. Fig. 1.Stat6isupregulated andactivated B-I Immunohistochemistry(IHC,rightpanels; ) ␣ ␣ expression was localised totheapical and Gata3increasedfromday10of RESEARCH ARTICLE ␣ -tubulin. pStat6andStat6 ( A was ) Stat6 ␣ 2741 –/– in ␣ ,

DEVELOPMENT gestation, development progressed but was delayed,andthe matched wild-typecontrols(Fig. 2A).Atdays10and15of buds intheabsence ofStat6atday5comparedwithstrain- a strikingreductioninthenumber ofsidebranchesandalveolar material). However, analysisofthegestational timepointsshowed compared towild-typemice(see Fig.S2inthesupplementary old virgin 5-week- histological analysisofmammaryglandscollectedfrom tissue harvested atvarious timepoints. Whole-mountand deficient forStat6(Kaplanetal.,1996)werematedand mammary important fornormalmammaryglanddevelopment. Mice We hypothesized,based ontheabove data,thatStat6would be development Stat6-deficient mammaryglandsexhibitdelayed 2742 RESEARCH ARTICLE Stat6 –/– mice displayednoapparent abnormality points andtheability of for themoresubtlephenotype observed atthelategestationtime the absenceofStat6.This‘catch-up’ proliferation would account by day15ofgestation,suggesting acompensatorymechanismfor gestation. Interestingly, thisreductioninproliferationwas reversed approximately 50%intheStat6-deficient glandsatday5of that thenumberofKi67positively stainingcellswas reducedby immunostaining forKi67,amarker ofproliferation.Fig.2E shows suggests adefectinproliferation,andthiswas confirmed by respectively (Fig.2D).Thereducednumber ofepithelialcells approximately 70,50and30%lessatdays5,10 15, Eosin (H&E)-stainedsectionswherethenumberofalveoli was 2B,C). Thiswas clearlydemonstratedinthehaematoxylin and density ofthelobuloalveolar structureswas diminished(Fig. Stat6 –/– dams tonursetheir pups. respectively. P significance with and ***indicatestatistical test wasperformedand*,** independent mice.Student’s represent s.d.ofthree magnification. Error bars different fieldsat400 67-positive cellsfrom three was measured byscoringforKi- respectively. ( and gestation from wild-type(WT) day 5(A),10(B)and15(C)of panels) ofmammaryglandsat (H&E)-stained sections(right and haematoxylinEosin ( alveoli/mm ImageJ software. Thenumberof the fatpadwasmeasured using magnification andthearea of P significance with and ***indicatestatistical test wasperformedand*,** type wasplotted.Student’s the percentage relative towild development in Fig. 2.Delayinlobuloalveolar Stat6 images from wild-typeand alveoli wasscored inH&E ( three mice. are representative ofatleast A-C <0.001, <0.001, Stat6 ) Wholemounts(leftpanels) –/– mice at40 Development 134(15) –/– P P 2 <0.01 and =0.011 and was calculatedand mice. Imagesshown D E ) Thenumberof ) Proliferation rate P P Stat6 ϫ values of values of ϫ P <0.003, P –/– =0.052, mice. t - t -

DEVELOPMENT differentiated luminalcells,because levels of theshortandlong al., 2003).Thesechangesmight reflectthereducednumberoffully development (Liuetal.,1997)andTh2celldifferentiation (Zhu et of Stat5,whichhasbeenshown tobeessentialforlobuloalveolar expression ofthemilkprotein was reflectedatthemolecularlevel bythereduced mammary glands control tissue. (Akt isalsoknown asAkt1)/PKBlevels in signalling revealed areductionofapproximately 50%inpAkt A molecularanalysisofthedifferences inproliferation/survival absence ofStat6 Multiple signallingpathwaysare perturbedinthe Stat6 controls mammaryglanddevelopment target, werefoundtobecomparable betweenthe lactation. However, theproteinlevels ofGata3,another Stat6 15 ofgestation,whereasthisdifference was lessstrikingduring detected atlower levels inStat6-deficient miceatdays5,10and exhibited intheseglands.Inaddition,theStat6target IL-4R Pten (Fig.3A,B).Thisisconsistentwiththereducedproliferation 5 and10ofgestation,withnosignificant changein thelevels of The morphologicaldelayin development inStat6-deficient ␤ -casein andthedelayedactivation Stat6 –/– glands atdays Stat6 –/– ␣ was and gestation (Fig. 3A). expression of Despite reducedlevels ofpStat5andthe absenceofStat6,the proliferation and differentiation in theabsenceofStat6. microarray dataprovide furtherevidence foradelayin longer detectable(Shillingford etal.,2003).Therefore,these however, duringpregnancy, expression ofthese proteinsisno are known tobeexpressed intheductalepitheliumofvirgin mice; ␣ in selectedtranscriptsareshown inFig.3C.Inadditiontocaseins Stat6 aquaporins 4,5and11wereall expressed athigherlevels inthe for theinductionofmitosis(DoreeandHunt,2002).Interestingly, be expressed individing cellsduringMphase andareresponsible reduced byapproximately2-fold.CyclinB1andB2areknown to glands, cyclins B1andB2theproliferationindicatorKi67are Stat6 forms oftheprolactinreceptor, PrlR,werecomparablebetween and Global expression profiling ofmammarytissuefromday5 –/– –/– ␤ and controlmice(seeFig.S3inthesupplementarymaterial). glands comparedwithwildtype. Thesewater transporters , whichweresix-toeight-foldreducedinthe Stat6 ␤ –/– -casein was relatively unperturbedduring lactation and wild-typeglandswas carriedout.Changes type (WT)and tubulin were performedonwild- of <0.01. biological replicates witha significant changeacross three Genes listeddisplayedastatistically Stat5, Gata3,Pten,IL-4R pERK1/2, ERK1/2, Stat6, phosphorylated(p)Akt,Akt, Stat6 differentiation markersin and inthelevelsof Fig. 3.Reductioninproliferation type and day 5ofgestationcomparingwild- of differentially regulated genesat histogram. ( software andplottedona were quantifiedusingImageJ points. ( extracts attheindicatedtime RESEARCH ARTICLE –/– B mice. Stat6 ) Immunoblotsfrom A C ) Amicroarray analysis –/– Stat6 ( A ) Immunoblotsfor mammary glands. ␤ -casein, pStat5, –/– tissue ␣ and P Stat6 value 2743 ␣ - –/–

DEVELOPMENT 0(C)and15(D)ofgestation.( 10 aes n &-tie etos(right panels)ofinguinalmammaryglandsfrom wild-type(strain matched)and panels) andH&E-stainedsections and15ofgestation.Error bar represents s.d.ofthree samplesandis representative ofatleasttwoexperiments.( 5 day Fig. 4.IL-4/IL-13expression isreduced in 2744 type wasplotted.( ( and thearea ofthefatpad wasmeasured usingImageJsoftware. Thenumber of alveoli/mm The densityofthe bandswasquantifiedusingImageJ software andtheratio ofpStat6toStat6plottedonahistogram. A ) Quantitativereal-time-PCR wasperformedusingspecificprimersfor RESEARCH ARTICLE F ) Immunoblotsforphosphorylated (p)Stat6andStat6were performedonwild-typeand E ) Thenumberofalveoliwasscored inH&Eimagesfrom wild-typeand Stat6 –/– mammary glandsandthesecytokines are required formammaryglanddevelopment. Il4 , Il13 , Il5 , Il12a and 2 was calculatedandthepercentage relative towild- Gata3 on wild-type(WT)and IL-4 –/– IL-4 /IL-13 –/– IL-4 /13 –/– –/– –/– mice at40 /IL-13 B-D mice atday5ofgestation. ) Wholemounts(left Development 134(15) Stat6 –/– mice atday5(B), ϫ –/– magnification glands at

DEVELOPMENT glands from wild-type(WT)and and Stat6 Th1 cytokine IL-12awas expressed atelevated levels inthe samples comparedwithcontrol mice(Fig.4A).Bycontrast,the approximately 90( of gestation,allthreecytokines werereducedbybetween points byquantitative reverse transcriptase(QRT)-PCR. Atday5 three Th2cytokines –IL-4,IL-13andIL-5atgestationaltime not welldefined. We determined,therefore,expression levels of expression profiles oftheseTh2cytokines inmammaryglandsare IL-13, whicharealsodownstream targets ofStat6.However, the Stat6 isactivated byanumberofcytokines –particularlyIL-4and exhibit delayeddevelopment IL-4/IL-13 doublydeficientmammaryglands Stat6 controls mammaryglanddevelopment Fig. 5. NOD-SCID miceatday5ofgestation.( calculated andthepercentage relative towild-typewasplotted.( the absenceofStat6, whichcouldbeattributed toGata3because control mice(Fig.4A).Thisreflects compensatorysignallingin and, infact, gestation were plottedonahistogramforcomparison. 15, cytokine levels werenolongerdiminishedin Socs5 –/– Socs5 samples comparedwithcontrol mice(Fig.4A).Byday –/– mice at40 –/– Il4 mice exhibitacceleratedmammaryglanddevelopment. was detectedathigherlevels comparedwith Il13 ϫ magnification andthearea ofthefatpadwasmeasured usingImageJsoftware. Thenumberofalveoli/mm ), 60( Socs5 Il5 ) and50%( –/– D mice atday10and15ofgestation.( ) Thenumberofalveoli/mm Il4 ) inthe Stat6 –/– Stat6 C tissue ) WholemountsandH&Estainingofinguinalmammaryglandsfrom NODand 2 –/– from wild-type,NOD,NOD-SCID, Stat6 intheabsence ofIL-4andIL-13(Fig.4F). Stat6 (Fig. 4E).Thephenotypeobserved was lesssevere thanthat of the approximately 60,60and16%less atdays5,10and15,respectively H&E-stained sections,where thenumberofalveoli was structures andofsidebranches. Thisisclearlydemonstratedinthe in thesupplementarymaterial). IL-4 continuedtobeexpressed throughoutlactation(seeFig.S4 this proteinisstillexpressed inStat6-deficient mammaryglands. et al.,1999)wereanalysed.Asexpected, To addressthisdirectly, micedeficient for both cytokines (McKenzie and IL-13areinvolved inthedevelopment ofthemammarygland. observed inStat6 histological analysis(Fig.4B-D).Thedelaywas similar tothat strain-matched controls,asindicatedbywhole-mount and exhibited adelayinlobuloalveolar development comparedwith B ( A The mRNA expression datafrom ) Thenumberofalveoliwasscored inH&Eimagesfrom wild-type ) WholemountsandH&E-stainedsectionsofinguinalmammary –/– mice, whichcouldbeexplained bythepresenceofactive –/– mice, withareducednumberof lobuloalveolar Stat6 –/– and RESEARCH ARTICLE Stat6 IL-4 –/– –/– /IL-13 mice suggestthatIL-4 IL-4 –/– –/– mice atday5of /IL-13 2 was –/– 2745 mice

DEVELOPMENT for normaldevelopment of themammarygland. results suggestthatIL-4andIL-13 regulate Stat6andarerequired regulation ofmammary glanddevelopment. Taken together, these development, furthersupportingaroleforStat6signallinginthe mammary epitheliumof This suggeststhatenhanced IL-4/IL-13signallinginthe with increasednumbersoflobuloalveolar structures(Fig.5B). mammary glanddevelopment comparedwith controls(Fig.5A), Interestingly, wefoundthat that Socs5issuperfluousinthesecelltypes(Brenderetal.,2004). mice, thereisnoeffect onBandTcelldevelopment, suggesting Socs5 2746 IL-4/IL-13 signallingbybindingtoIL-4R enhanced IL-4/IL-13signalling.Socs5hasbeenshown toinhibit gland development further, weinvestigated amodel with To confirm the notionthatStat6signallinghasaroleinmammary development expressed inTh1cells(Sekietal.,2002).However, in RESEARCH ARTICLE –/– mice exhibitacceleratedmammarygland Socs5 Socs5 –/– mice resultsinprecocious –/– mice have accelerated ␣ and itisprimarily Socs5 –/– high numberofCD4 mice. NODmicearepronetoauto-immunediseasebecauseof the and inNOD-severe combinedimmunedeficiency (NOD-SCID) mammary glanddevelopment innon-obese diabetic(NOD)mice of IL-4/IL-13onlymphocyte development, weinvestigated To determinewhether thephenotypeobserved isduetotheeffects affect mammarygland development Perturbed lymphocytedevelopmentdoesnot that thephenotypeobserved in lymphocytes have littleeffect onlobuloalveolar development and development (Guptaetal.,2007).Theseresultssuggestthat suggested thatNOD-SCIDmice have normalmammarygland although wedidnotexamine latertimepoints,otherresearchhas the NODorNOD-SCIDmiceat day5ofgestation(Fig.5C,D)and, 1992). We foundnodefects inlobuloalveolar development ineither mutation thatblocksTandBcelldevelopment (Prochazkaetal., 2005; Prochazkaetal.,1992),whereasNOD-SCIDmiceharbour a due toepithelial cell-autonomouseffects. + and CD8 (F) antibodiesornoantibody(G). stained withanti-IL-4(E)oranti-IL-13 (10 were treated withbrefeldin A Stat6 and time coursewere performedforpStat6, <0.02. ( denotes Student’s performed for real-time-PCR was Quantitative recombinant murineIL-13(rmIL-13). treatment withorwithout40ng/mlof harvested atvarioustimepointsafter was changedandthencellswere Stat6 antibodies.( using phosphorylated(p)Stat6and manner, confirmedbyimmunoblotting ( KIM-2 cells. Fig. 6.Stat6signallingisintactin A Stat6 + ) andIL-13( T cells(AndersonandBluestone, ␮ g/ml) for4hours,fixedand –/– D ) Immunoblotsforthesame ␣ and -tubulin. ( KIM-2 cellsrespond toIL-4 B Il4r Development 134(15) IL-4 ) inadose-dependent ␣ C t -test 1 ) KIM-2medium –/– and E-G /IL-13 P ) KIM-2cells Gata3 value of –/– ; * mice is

DEVELOPMENT demonstrated that thissignallingpathway isfunctionalinthese with eitherIL-4orIL-13(Fig. 6A,B). ImmunoblottingforpStat6 13 andactivate Stat6,weperformedadose-responsetreatment S5C inthesupplementarymaterial). constant levels inKIM-2cellsthroughoutdifferentiation (see Fig. addition, thetranscriptionfactors Gata3andc-Mafweredetectedat differentiation (seeFig.S5Binthesupplementarymaterial).In and theexpression ofIL-4R IL-4R RT-PCR analysis showed thattheIL-4/IL-13receptorcomponents, response tolactogenichormone-induceddifferentiation ofthesecells. morphology ofconfluentKIM-2cellsandthedomesformed in S5A inthesupplementarymaterialshows thecuboidalepithelial days thatisanalogoustoMECsinvivo (Gordonetal.,2000).Fig. 2000). KIM-2cellsundergo a programmeofdifferentiation over 10 accurately mimicsmammaryglanddevelopment (Gordonetal., we studiedthemousemammaryepithelialcelllineKIM-2,which To furtherstudyandconfirm theStat6signallingobserved invivo, Stat6 signallinginMECs Stat6 controls mammaryglanddevelopment To determinewhetherKIM-2cellscanrespondtoIL-4 orIL- ␣ and IL-13R ␣ 1, areexpressed inundifferentiated KIM-2 cells ␣ is slightlyincreasedafter2days was suggested atthetime. IL-4 treatment(Moriggletal., 1997);however, nomechanism have previously beenshown toproduce cells synthesizethesecytokines. Itisworth notingthatHC11cells cytoplasm ofKIM-2cells (Fig.6E-G),confirming thatKIM-2 Importantly, wefoundsignificant levels ofIL-4andIL-13inthe stained thecellsusinganti-IL-4 oranti-IL-13antibodies. with BrefeldinAfor2hourstoblockcytokine secretionandthen exogenous (Fig.6D).Therefore,wetreatedKIM-2cells Stat6 becametyrosinephosphorylatedintheabsence of into themedium.Inaddition,immunoblotanalysisshowed that was induced,suggestingthatKIM-2cellssecreteIL-13 orIL-4 absence ofrecombinantIL-13,expression ofGata3andIL-4R elevated by7.5-fold24hours.Itis noteworthy thateven in the significantly higherinthetreatedcellsafter4hours andwas after 30minutesoftreatment,whereasIL-4R in responsetoIL-13.Gata3expression was significantly induced expressed atlow levels inuntreatedcellsandareinducedfurther cells byQRT-PCR. Asshown inFig.6C,Gata3andIL-4R cells. We alsoexamined theexpression ofStat6targets inKIM-2 granulocyte-colony stimulatingfactor. macrophage colony-stimulatingfactor;GCSF, negative control; GM-CSF, granulocyte- differentiation. Pos,positivecontrol; Neg, elevated levelsofIL-4secretion atday8of indicated inred. Green arrows indicate compared today0(left).Th2cytokinesare by differentiated KIM-2cellsatday8(right) array demonstratesTh2cytokineproduction at leasttwoexperiments.( s.d. ofthree samplesandisrepresentative of days ofdifferentiation). Error barrepresents cells across adifferentiation timecourse(DD, Th2 cytokines cytokines performed usingspecificprimersfortheTh1 cells. and secreted bydifferentiating KIM-2 Fig. 7.Th-2cytokinesare upregulated ( A ) Quantitativereal-time-PCR was Il12a RESEARCH ARTICLE Il4 , Tnfa , Il13 andIl5 ␤ and -casein inresponseto Ifng ␣ B ) Cytokineprotein expression was (left) onKIM-2 (right) andthe ␣ 2747 are ␣

DEVELOPMENT

differentiation progresses.Bycontrast,levels ofINF between neighbouringcellsandthusdelaysdevelopment. presence ofGata3.We propose thatthisaffects paracrinesignalling the absenceofStat6,IL-4/IL-13/IL-5production isreduced eveninthe cells. Gata3isalsoactivatedbyotherpathways,suchasNotch.( cytokines thattriggerthedifferentiation process inundifferentiated facilitating thebindingofStat6,Stat5andc-Maf,whichproduces induces achromatin conformationalchangeattheIL-4/IL-13/IL-5locus, orchestrated byStat6-mediatedGata3genetranscription. differentiation (Fig.7A).To confirm thatthese cytokines arenot differentiation time course,whereasIL-5peaked atday2of expression ofIL-4and IL-13continuedtoincreaseduringthe IL-5 wereimmediatelyupregulated. Itisnoteworthy thatthe (Gordon etal.,2000).Strikingly, theTh2cytokines IL-4, IL-13and not decreaseuntilday8,when thecellsarefullydifferentiated as Ifng–MouseGenomeInformatics), anotherTh1cytokine, do milk protein are significantly downregulated at day2ofdifferentiation, whenthe differentiation (Fig.7A).Thus,theTh1cytokines IL-12aandTnf cytokine expression thatcoincidedwiththe inductionof (Gordon etal.,2000).We foundastrikingswitchfromTh1toTh2 cells duringdifferentiation totheluminal(milk-producing)lineage We theninvestigated theexpression profile ofcytokines inKIM-2 from Th1toTh2cytokine synthesis Differentiation ofKIM-2cellsinducesaswitch on mammaryglanddevelopment. Fig. 8.Modelexplainingtheeffect ofStat6/IL-4/IL-13deficiency 2748

B A

NOTCH

Reducedexpansion/delayed development differentiation Paracrine signallingpromotingfurther NOTCH

PRLR RESEARCH ARTICLE PRLR

Differentiated cell Differentiated cell Stat5

IL-5/2R Stat5 ␤ IL-5/2R GATA3

-a c-Maf c-Maf Stat5 -casein isfirst expressed, andlevels declineas GATA3

-a c-Maf c-Maf Stat5 Stat6

IL4Ra IL4Ra Stat6 IL-4 IL-4 IL-4 IL-13 IL-13 IL-5 IL-5 IL-13 ( A ) Th2cytokineproduction is Undifferentiated cell Undifferentiated cell

Stat5 IL-5

GATA3 IL-5/2R

GATA3

Stat5 IL-5/2R Stat6

IL4Ra Stat6 IL4Ra ␥ (also known IL-4 B IL-13 ) In IL-5 ␣ signalling inMECsvivo. Mammaryglandsfrom signalling byTh2cytokines (Fig.8). differentiation oftheluminalepitheliallineagerequiresautocrine cytokines IL-4,IL-13andIL-5isinduced.Thesedatasuggest that induced differentiation ofMECs,whereasexpression oftheTh2 IFN of mammaryepithelialcells.ExpressiontheTh1cytokines for thefirst time,thatthisparadigmalsoappliestodifferentiation lineage suppresstheTh2andviceversa development. Cytokinesthatpromotedifferentiation oftheTh1 switch incytokine milieuisacrucialcomponentofThelpercell Cytokines have aprofoundroleincellfate determination.A DISCUSSION factor (GCSF)andIL-6was diminishedupondifferentiation. cytokine. Notably, secretionofgranulocyte-colony stimulating This ismostprobablyaconsequenceoftherapidturnover ofthis 3, IL-5,IL-9andIL-10.IL-13secretiondidnotappeartochange. undifferentiated cells,asaresomeotherTh2cytokines –IL-2,IL- secreted athigherlevels byday-8differentiated cellsthanin 7B, rightpanel)KIM-2cells.Thisassayshowed thatIL-4is undifferentiated (Fig.7B,leftpanel)or8-daydifferentiated (Fig. a cytokine arraywas usedwithmediaharvested from only synthesizedbymammaryepithelialcellsbut arealsosecreted, (Fig. 8). signal andfortheexpansion ofluminalmammaryepithelialcells Stat6 signallingisrequiredfor amplification oftheIL-4/IL-13 mammary gland.Ourresultssuggest that,similarlytoTh2cells, not addressthemechanismby whichGata3isregulated inthe Labat etal.,2007;Kouros-Mehr etal.,2006).Theseauthorsdid to maintainthedifferentiation ofluminalepithelialcells(Asselin- this work was underreview, itwas shown thatGata3isrequired Gata3 issufficient toultimatelypromotealveologenesis. While gland development andthat,intheabsence ofStat6signalling, of Gata3aresufficient tomediateTh2differentiation. al., 2004).Taken together, theseresultssuggestthateven low levels the Notchpathway inanIL-4/Stat6-independentmanner(Amsen et It isalsoworth notingthatGata3hasbeenshown toberegulated by cells have beenshown toautoregulate Gata3(Ouyangetal.,2000). theTh2 which in observed inTh2cellsfromStat6-deficient mice, promotes functionaldifferentiation. Thisscenarioissimilartothat mice, suggestingthatthereisacompensatorymechanism that 13 in thesupplementarymaterial)and phenotype was alsoobserved inmammaryglandsfrom reduced proliferationandmilkproteinproduction.Asimilar alveolar development duringgestation, whichwas accompaniedby displayed uptoa70%reductioninsidebranchinganddelayed signalling, lactationoccurrednormallyinboth Despite themarked developmental delayintheabsenceofStat6 system Compensatory mechanisms:insightsfrom theTh2 development. cytokine signallingisrequiredfornormalmammary gland together, theseresultsfurthersupportourcontentionthatTh2-biased Socs1/IFN development. Thisphenotypeissimilartothatobserved in and thelossofSocs5doesindeedresultinprecociousalveolar 4/IL-13 would beanticipatedtoresultinaccelerateddevelopment, –/– This was confirmed byinvestigating theinvolvement ofTh2 We suggestthatGata3 isanimportantregulator ofmammary ␥ doubly deficient mice.Deletionofanegative regulator ofIL- , IL-12andTnf ␥ doubly deficient mice(Lindemanetal.,2001).Taken ␣ is suppresseduponlactogenichormone- IL-4 –/– /IL-13 Development 134(15) Stat6 –/– . (data notshown) We show here, show We –/– Stat6 (see Fig.S6 IL-4 –/– –/– mice /IL-

DEVELOPMENT Alexander, W. S.andHilton,D.J. Abell, K.,Bilancio,A.,Clarkson,R.W., Tiffen, P. G., Altaparmakov, A.I., References http://dev.biologists.org/cgi/content/full/134/15/2739/DC1 Supplementary materialforthisarticleisavailableat Supplementary material funded byBBSRC,UKandtheNHMRC,Australia. Research CouncilBiomedicalCareer Developmentaward. Thisproject is studentship; andS.E.N.wassupportedbyaNationalHealthMedical CancerResearch;International P.J.C. was fundedbyaCambridgeUniversity Wellcome Trust PhDstudentship;F.O.B. wasfundedbytheAssociationfor Sciences Research Council(BBSRC)PhDstudentship;E.K.C.R.wasfundedbya biological services.W.T.K. wasfundedbyaBiotechnologyandBiological the Socs5mice;BarryPotterfortissuehistology;andCliveLebozer We wouldliketothankDouglasHiltonandWarren Alexanderforproviding are theprimaryregulators ofmammaryglanddevelopment. to thepreviously heldparadigmthatsteroidandpeptidehormones differentiation. Ourstudyreveals anadditionallayerofcomplexity exquisitely controlledcytokine network toensurefunctional suggests that,duringevolution, themammaryglandhijacked an association betweenadaptive immunityandmilkproduction differentiation oftheadultmammarygland.This unexpected hinder immunesurveillance andhencepromotetumourgrowth. pregnancy andcontinuedcytokine expression duringlactationcould explanation forthisapparentanomaly, becauseaTh2biasduring developing breastcancer(Schedin,2006).Ourresultsprovide an that arecentfull-termpregnancy transientlyincreasestheriskof cancer andreduceslife-timerisk(Medina,2004),itisalsoknown it isgenerallyacceptedthatpregnancy isprotective againstbreast shown toexpress highlevels ofGata3(Usaryetal.,2004).Although receptor-positive breasttumoursofaluminalsubtypehave been established tumourssecreteTh2cytokines. Significantly, estrogen rejection (Czarneskietal.,2001;Jensen2003)andthatmany growth. Ithasbeenshown thataTh1biasismoreeffective intumour Immune surveillance isanimportantfactor inpreventing tumour Implications forcancerprogression Stat6 controls mammaryglanddevelopment Brender, C.,Columbus, R.,Metcalf,D.,Handman,E.,Starr, R.,Huntington, Asselin-Labat, M.L.,Sutherland,K.D.,Barker, H.,Thomas,R., Shackleton, Ansel, K.M.,Djuretic, I.,Tanasa, B.andRao,A. Amsen, D.,Blander, J.M.,Lee,G.R.,Tanigaki, K.,Honjo,T. andFlavell,R.A. Chapman, R.S.,Lourenco, P. C.,Tonner, E.,Flint,D.J.,Selbert,S.,Takeda, K., Anderson, M.S.andBluestone,J.A. Czarneski, J.,Meyers,Peng,T.,Czarneski, Abraham,V., Mick,R.andRoss, S.R. Clarkson, R.W., Wayland, M.T., Lee,J.,Freeman, T. andWatson, C.J. Chatila, T. A. differentiation andIl4locusaccessibility. Immunol. signaling (SOCS)proteins inregulation oftheimmuneresponse. composition. Stat3-induced apoptosisrequires amolecularswitchinPI(3)K subunit Burdon, T. G.,Asano,T., Vanhaesebroeck, B.andWatson, C.J. lymphocyte production and function. 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