of Toronto, Toronto, Canada. Toronto, Toronto, Canada. Toronto, Canada. University ofToronto, Toronto, Canada. California, San Francisco,CA,USA. California, Basler, 2001).IntheabsenceofaHhsignal, a singlezinc-fingerprotein,Cubitusinterruptus( Drosophila embryogenesis (reviewed byIngham andMcMahon,2001).In determination,proliferationandtissuepatterningduring fate, hedgehog (HH)family ofsecretedproteins,SHHcontrols cell hedgehog (SHH)signalingpathway. Amongthemembersof unknown. that controlthesedeleteriousGLI3-dependenteffects are observed inPHS(Boseetal.,2002).Themolecularmechanisms numerous malformations,includingrenalaplasia/dysplasiaas generates a699aminoacidN-terminalGLI3proteinexhibit 1997). Moreover, micehomozygousforatargeted mutationthat hypothalamic harmartomaandrenaldysplasia/aplasia(Kangetal., and malformationsincludingpolydactyly, imperforateanus, repressor arefoundinhumanswithPallister-Hall syndrome(PHS) generate aputative truncatedproteinsimilarinsizetoGLI3 crucial duringmammalianmorphogenesis. Regulation ofGLI3repressorformationviacleavage ofGLI3is INTRODUCTION KEY WORDS:Kidneydevelopment,SonicHedgehog,GLI3 renal morphogenesis,theexpressionofSHHtargetgenesandGLIbindingto humans andmice.Yet,thepathogenicmechanismsareundefined.Here,wereporteffectofdecreasedSHH-SMOsignalingon Truncating mutationsin Ming ChangHu GLI3-dependent transcriptionalrepressionof Development 133,569-578doi:10.1242/dev.02220 DEVELOPMENT ANDDISEASE Accepted 24November 2005 1 Genetics, UniversityofToronto, Toronto, Canada. *Author forcorrespondence (e-mail:[email protected]) University ofToronto, Toronto, Canada. Pax2 Norman D.Rosenblum of GLI3repressorrelativetoactivator. mediated SMOinhibitiondisruptedrenalorganogenesis,decreasedexpressionofGLI1andGLI2proteins,butincreased region andactstorepressgenetranscription(Aza-Blancetal., proteolysis intoanN-terminalfragmentthatincludesthezincfinger of kidney tissue.Together,theseresultsdemonstratethatSHH-SMOsignalingcontrolsrenalmorphogenesisviatranscriptional cont determined thebindingofGLIproteinsto5 cycle regulators(cyclinD1andMYCN).Eliminationof kidney patterninggenesdisruptsrenalmorphogenesis GLI3. However,cyclopaminefailedtodecrease contrast, treatmentofembryonickidneyexplantswithcyclopaminedecreasedGLI1and/orGLI2binding,andinducedbinding of using chromatinimmunoprecipitation.Innormalembryonickidneytissue,GLI1and/orGLI2wereboundtoeachtargetgene.By Program inDevelopmentalBiology, HospitalforSickChildren, UniversityofToronto, GLI3 isanintracellulartranscriptionaleffector inthesonic Gli , , renalpatterningandcellcycleregulatorgenesinamannerthatisopposedbyGLI3. Sall1 , cyclinD1,MYCN, , Hhsignalingismediated,atthetranscriptionallevel, by 2 Division ofNephrology, TheHospitalforSickChildren, University 1,2 7 Department ofLaboratoryMedicineand Pathobiology, , RongMo 3 Cardiovascular Research Institute,Universityof Gli3 1,2,5,6,7, 4 Department ofMedicalandMolecular , anintracellulareffectorintheSHH-SMO-GLIsignalingpathway,causerenalaplasia/dysplasia Gli1 6 Department ofPhysiology, Universityof 1 and * , SitaBhella 5 Gli2 Department ofPaediatrics, . TodefinemechanismsbywhichSHH-SMOsignalingcontrolsgeneexpression,we Shh Ј Gli3 flanking regionscontainingGLIconsensusbindingsequencesin Ci Gli1 deficiency decreasedexpressionofkidneypatterninggenes( Ci 1 is processedby , ChristopherW. Wilson mutations that ) (Methotand and Gli3 Gli2 in expression andbranchingmorphogenesisin Shh –/– mice rescuedkidneymalformationandrestoredexpressionof truncated form of GLI3(Boseetal.,2002)demonstrates acrucial the uretericbud lineage (Yu etal.,2002) andinmiceexpressing a 1997). Invertebrates, three presence ofrenalhypoplasia/dysplasia inmicedeficient performs crucialfunctions(reviewed byBouchard,2004).The is torepressexpression of analyses inmicedemonstratethatthepredominantactivity ofGLI3 explant cultures(Litingtungetal.,2002;Wang etal.,2000).Genetic can actasatranscriptionalrepressorinculturedcellsandlimb SHH signalprovokes processingofGLI3intoashortenedformthat embryogenesis (Baietal.,2002;Park etal.,2000).Absence ofa 1999) andactprimarilyastranscriptionalactivators duringmurine exist asfulllengthproteinsinculturedmammaliancells (Daietal., mediate Hhsignalsbycontrollinggeneexpression. GLI1 andGLI2 expression ofgenessuch as control cellularevents includingcellproliferationandtocontrolthe mesenchymal cellsactinaautocrine andparacrinemannerto ducts. Growth factors secretedbytheuretericbud andmetanephric proximal totheuretericbud branchesandtheirdaughter collecting metanephric mesenchymetothoseepithelialelementsthatexist as renalbranchingmorphogenesis,andconversion ofthe instigate growth andbranchingoftheuretericbud, aprocessknown structure, andthemetanephricmesenchyme,amesenchymaltissue, the kidney, interactions betweentheuretericbud, anepithelial epithelial-mesenchymal interactionsandgrowth factor signaling.In are largely undefined. formation. Themolecularmechanismsthatcontrolsuchinteractions suggesting adynamicinterplaybetweenGLIsignalsduringtissue and forebrain(Ralluetal.,2002),skin(Mill2005) (Litingtung andChiang,2000),limbetal.,2002),face phenotypes inembryonictissuesincludingtheneuraltube be critical.Geneticinactivation of Park etal.,2000).Integration ofoppositeGLIactivities appearsto The mammaliankidney isamodelwithwhichtostudyembryonic Shh 3 , Pao-TienChuang target genes. Shh Shh Ci Pax2 Gli1 homologs, GLI1,GLI2andGLI3, dependent genes(Baietal.,2002; deficiency orcyclopamine- 3 , , Chi-chungHui Sall1 RESEARCH ARTICLE Gli3 Gli3 , and Pax2 Gli2 can rescue -deficient embryonic Shh Mycn agtgenes target and , eachofwhich Sall1 and 1,4 Shh and ) andcell mutant Shh 569 rol in

DEVELOPMENT rescued kidney expression of Shh Generation ofmice in a relative tofull-lengthGLI3protein.Homozygousinactivation of relative expression ofashortenedformGLI3was increased states lowered theexpression ofbothGLI1andGLI2protein,the (Chen etal.,2002),causedrenalaplasiaordysplasia.Althoughthese mice withcyclopamine, asteroidalalkaloidthatblocksSMOactivity Homozygous inactivation of signaling inmutantmiceandculturedembryonickidneys. renal organogenesis, wegeneratedmodelsofdeficientSHHorSMO embryogenesis areunknown. However, themolecularmechanismsbywhichGLI3controlsrenal role forSHH-GLIsignalingduringmammalianrenaldevelopment. 570 Immunohistochemistry inparaffin wax-embedded sections(4 Immunohistochemistry andimmunoblotting Minneapolis, MN).Apartialmouse Research Chemicals,Toronto, ON);andSHH-N(B&DSystems, D2 (SantaCruzBiotechnology, SantaCruzCA);cyclopamine (Toronto cyclin D1(BDPharMingen,SanDiego, CA);GLI3,MYCN,MYC,cyclin Antibodies andreagentswereasfollows: GLI1(Abcam,Cambridge,MA); Antibodies andspecializedreagents approved bytheethicscommitteeatTheHospitalforSickChildren. previously (Chiangetal.,1996; Moetal.,1997).Animalexperiments were for SickChildren(Toronto, Canada)andgenotypedbyPCRasdescribed et al.,1992)CD1/129micewerehousedintheAnimalFacility ofTheHospital (1:250 dilution)andanti- MYCN (1:250dilution),anti-cyclin D1(1:250dilution),anti-cyclin D2 GLI3 (1:250dilution,SantaCruzBiotech), anti-MYC(1:250dilution),anti- antibodies, anti-GLI1(1:3300dilution),anti-GLI2(1:250 anti- transferred tonylon membranewas performedusingthefollowing assay (Vector laboratories,Burlingame,CA).Immunoblottingofproteins Biotinylated secondaryantibodieswereusedinabiotin-avidin complex anti-GLI1 (1:10),rabbitanti-GLI2(1:700)andanti-GLI3(1:50). tissue was performed(Huetal.,2003)withthefollowing antibodies:mouse protein was expressed in acids 327-442was clonedintothepGEX1 vector (Amersham).Fusion of GLI1/GLI2 anddenovo associationswithGLI3. Geneticinactivation cultured kidneys, weobserved decreasedassociationswith flanking region. Bycontrast,incyclopamine-treated malformed bound aGLI-bindingconsensusregion withineachtarget gene5 immunoprecipitation. Innormalkidney tissue,GLI1and/orGLI2 controls expression ortheseSHHtargets usingchromatin well asGLI1andGLI2.We definedmechanismsbywhichGLI3 MATERIALS ANDMETHODS target genescrucialtoembryonickidney development. repressor, which,inturn,controls in whichSHH-SMOsignalingcontrolsgenerationofaGLI3 renal branchingmorphogenesisbycyclopamine. We proposeamodel Piscataway, NJ). Amersham PharmaciaBiotech, commercially available reagents(ECLkit; was performedusing dilution, respectively. Chemiluminescence Pharmacia Biotech,Piscataway, NJ) were usedata1:3000and1:5000 Secondary anti-mouseIgGandanti-rabbit IgGconjugatedHRP(Amersham injection. Shh-Nwas dissolved in1%FBSPBS. cyclodextrin (HBC,Sigma-Aldrich,StLouis,MO) inPBSforintraperitoneal for cultureofkidney explants anddissolved in45%2-hydroxypropyl-beta- with His-taggedGLI2antigen.Cyclopaminewas dissolved in100%ethanol affinity purifiedusingacolumnofAffi-gel 10beads(BioRad)conjugated (Amersham) accordingtothemanufacturer’s instructions.Antibodieswere To identifymechanismsbywhichSHH-GLIsignalingcontrols Gli3 +/– Shh (Chiang etal.,1996), RESEARCH ARTICLE ln lce niiino L1andGLI2expression, and alone blocked inhibitionofGLI1 -deficient backgroundnormalizedtherenalphenotypeand E. coli ␤ Gli2 -actin (1:3000dilution,Sigma,St Louis, MO). (BL21) andpurifiedonglutathionesepharose +/– Shh Pax2 (Mo etal.,1997)and or treatmentofpregnant wild-type Gli2 , Gli1 Sall1 cDNA correspondingtoamino and , cyclin D1andMYCN,as Gli2 as wellnon- Gli3 +/– ␮ (Schimmang m) ofkidney Gli3 Gli Ј The following primerswereused. random hexamers (SuperScriptFirstStrand SynthesisSystem,Invitrogen). (Conlon andHerrmann,1993). paraformaldehyde at4°Candprocessedaccordingtopublishedmethods whole-mount insituhybridization,embryoswerefixed in4% provided byDrRyuichiNishinakamura(Nishinakamuraetal.,2001)].For provided byDrPeterGruss(Stoykova andGruss,1994)] sections asdescribed(Moetal.,1997)usingprobesencoding In situhybridizationwas performedinparaffin wax-embedded tissue In situmRNAhybridization Pregnant wild-typeor Effect ofcyclopamineonkidneydevelopment Ham’s F-12nutrientmixture(DMEM-F12)containing50 the presenceofRichter’s modifiedDulbecco’s modifiedEagle’s medium- mm polyethyleneterephthalatemembranes(Corning)inmultiwellplates Kidneys weresurgically dissectedfrommouseembryosandculturedon0.45 Embryonic kidneyorganculture protocol. First-strandcDNA was generatedfrom2 kit (Qiagen,Mississauga,ON,Canada) accordingtothemanufacture’s Total RNA fromfrozentissuesampleswas extracted usingtheRNeasyMini Reverse transcriptase-PCR 5 cyclin D1promoter(GenBankAccessionNumberAF212040):forward, primers weredesignedtoencompassthesebindingsitesasfollows. Mouse TGGGTGGTC orGACCACCCA (Laietal.,2004).Oligodeoxynucleotide (Hu andRosenblum,2005).GLIconsensusbindingsiteswereidentifiedas Chromatin immunoprecipitationwas carried outusingpublishedmethods Chromatin immunoprecipitation described previously (Cano-Gauci etal.,1999). Identification ofBrdU-positive cellswas performedbyimmunostainingas BrdU (100 Germany). Pregnant femalemicereceived anintraperitonealinjectionof deoxyuridine (BrdU,RocheMolecularBiochemicals,Mannheim, byincorporationof5-bromo-2 Cell proliferationinkidneys was assayed In situcellproliferation assay culture mediumatfinalconcentrationof1.0 concentration of10 kidney explants, cyclopamine was addedtoculturemediumatafinal paraformaldehyde-PBS. Alternatively, fortreatmentofculturedembryonic days. Embryoswerethenisolatedsurgically andfixed in4% with cyclopamine (6mg/kgbodyweight)onceperdayfor4consecutive ENSMUSG00000025407): forward 5 45seconds),Mouse for GTAGCAATCC-3 5 Pax2 (299 bpPCRproductatannealingtemperature54°Cfor45seconds).Mouse CGGTGCCTAGGGTCT, reverse 5 promoter (GenBankAccessionNumberAC147558): forward 5 PCR productatannealingtemperature56°Cfor30seconds).Mouse GAGCTTCAA-3 (GenBank AccessionNumberX06993):forward 5 GGCTCCTTCC-3 two connectedbranches. theintersectionbetween 1997). Auretericbud branchpointwas definedas conjugated DBA (20 (FITC)- in whole-mountkidney specimenswithfluoresceinisothiocyanate (Sigma) (Picheletal.,1996).Uretericbud-derived structureswereidentified AACAG-3 reverse 5 TGAGGCTCCTGACAAT-3 (ENSEMBL geneIDENSMUSG00000051835):forward 5 seconds). annealing temperature55°Cfor40seconds).Mouse Ј Ј -AATTCTAAAGGTGGGGGAACA-3 -GGGCTTTGCAGCTTTTAGAG-3 promoter (GenBankAccessionNumberMMU13975):forward Ј -TCGGTCCGAAGGAAGGATATA-3 Ј ␮ (124 bpPCRproductatannealingtemperature55°Cfor40 g/g bodyweight)4hourspriortoscarifyingthesemice. Ј , reverse 5 Ј Ј (256 bpPCRproductatannealingtemperature54°C ␮ ␮ (148 bpPCRproduct).Mouse Gli3 M. Shh-Nwas dissolved in1%FBSandaddedto /l etrLb,Brigo,ON)(Piscioneetal., g/ml; Vector Labs,Burlington, +/– Ј mice wereinjectedviatheperitoneumatE9.5 -AGCTTCGCAAGTACCGCTTC-3 Ј and reverse 5 Gli1 Gli1 Ј -CTGAAGTTTTCGGGAGAAGC-3 Ј promoter (ENSEMBLgeneID -AGCTTCAGTGCCACCCCAC-3 : forward 5 Ј , reverse 5 Ј , reverse 5 ␮ Ј g/ml for4days. -CAGCAGAGCAAGGTG- Ј Ј Development 133(3) (197 bpPCRproductat -ATCACCTGTTGGG- Ј Ј -TTGGCAGAGAA- ␮ -GAGACACGATA- Ј -TAATATCCCCC- g totalRNA using ␮ Mycn g/ml transferrin Gli2 Sall1 Pax2 Pax2 Ј promoter promoter Ј -GTGC- Ј (254 bp [kindly [kindly -AGG- Sall1 Ј Ј - Ј ,

DEVELOPMENT and tubules intherenal cortex of wild-typemice(B),thesinglekidneyformedin~50% ectopic kidneylocatedinthepelvis(D).( and thepresence ofdilated tubules.Scalebar:100 gonad; K,kidney. Dolichos BiflorusAgglutinin.Incontrast tovehicle,cyclopaminedecreased thenumberofureteric budbranchesformed.Scaleba in thepresence ofdrugvehicle (J,100%ethanolinculture medium)orcyclopamine(K) for4days.Ureteric budbrancheswere i deficient miceandinorkidney explants treatedwith kidney development byanalyzing therenalphenotypeinSHH- We determinedtherequirementforSHHsignalingduringmurine deficient andcyclopaminetreated mice Disruption ofkidneydevelopmentinSHH RESULTS significance was taken atavalue of 4.01; AbacusConcepts,Berkeley, CA)formorethantwo groups.Statistical tailed) orbyANOVA program(version usingStat-View statisticalanalysis Mean differences betweengroupswereanalyzedusingStudent’s Data analysis GATGCTGGAT-3 GLI3-dependent control ofrenal development i.1 DeficientSHH-SMOsignalingdisruptskidneydevelopment. Fig. 1. ␮ in cyclopamine-treated mice (H,I)demonstratedamarkeddecrease inureteric bud branchesandepithelialmetanephricderivative starting atE9.5forfourconsecutive daysblockedrenal development.Incontrasttokidneysfrom micetreated withdrugvehicl temperature 56°Cfor45seconds). AGCAGTGGAGCACGGACAT-3 forward 5 (317 bpproductatannealingtemperature56°Cfor45seconds). ␤ agarose gelelectrophoresisandstainedwithethidiumbromide. using HotStarTag. TheRT-PCR amplifiedproductswereseparatedby CTCTCAGCTGTGGTGGTGAA-3 GCAA-3 GAGCCTGAAGTCAT-3 -actin: forward 5 m. ( Shh J , K –/– rtrcbdbacigi mroi iny sltda 1. rmwl-yemice.Kidneys from thesamemousewere cultured a ) Ureteric budbranchingin embryonickidneysisolatedatE11.5from wild-type Ј Ј (270 bpproductatannealingtemperature56°Cfor40seconds). mice atE18.5.Incontrasttowild-type(A), -GTTCCAAGGCCTACTCTCGCCTG-3 Ј , reverse 5 Ј -TGTTACCAACTGGGACGACA-3 Ј , reverse 5 Ј -GGCGTGAATAGGACTTCCGACAG-3 Ј P< Ј (393 bpproduct).PCRwas performed (304 bpproductatannealing 0.05. Gli3 Ј -GTCTTGAGTAGGCTTTTGT- B , : forward 5 E ) RenalhistologicalphenotypeinE18.5mice.Incontrasttotheorganized appearanceofglomeruliand Ј ␮ , reverse 5 m. ( Ј -AGCAACCAG- F-I Ј , reverse 5 Shh ) Effect ofcyclopamineon renal development.Treatment ofmicewithcyclopamine t -test (two- –/– Ј -CTTG- mice exhibitedeitherabsenceofbothkidneys(C)orthepresence ofasingle Gli2 Ј Ј - : ( A , bud branchpointsby40%(branchpointnumber–drugvehicle number, showed thatcyclopamine inhibitedformationofureteric Quantitation ofthiseffect, performedbycountingbranchpoint by DolichosBiflorusAgglutinin(compareFig.1Kwith1J). demonstrated marked inhibitionofuretericbud branchingidentified cyclopamine for4consecutive days.Qualitative analysis kidneys wereharvested atE11.5andculturedinthepresenceof functions aftertheonsetofmesenchymal-epithelialinteractions, metanephric-derived epithelialstructures.To determineSMO primordial branchingoftheuretericbud andapaucityof 1F,G), thekidneys incyclopamine treatedembryosdemonstrated 1H,I). Comparedwithembryostreateddrugvehicle alone(Fig. exerted amajordeleteriouseffect onkidney development (Fig. of 6mg/kgcyclopamine dailyfor4consecutive daysstartingatE9.5 signaling duringrenalmorphogenesis.Anintraperitonealinjection SMO functionandinvestigate thefunctionofSHH-SMO Using acomplementarystrategy, weusedcyclopamine toblock and cystic renaltubules (PiscioneandRosenblum,1999)(Fig.1E). including disorganization ofstructureswithintherenalparenchyma kidneys demonstratedhistologicalfeaturesofrenaldysplasia single ectopickidney locatedinthepelvis(Fig.1D).Theseectopic renal aplasiain50%(Fig.1C).Theremainingexhibited a cyclopamine. Analysisof20 C , D ) Gross anatomicalfeatures ofkidneydevelopmentin wild-type Shh –/– mice (E)wascharacterizedbyapaucity ofglomeruli Shh –/– E14.5 embryosrevealed bilateral RESEARCH ARTICLE e alone(F,G), kidneys dentified with r: 100 s. Scalebar:100 ␮ m. G, s pairs 571

DEVELOPMENT decreased in expression inkidneywasnot A,E). Bycontrast,totalGLI3 compared withwildtype(D,Hversus decreased GLI1andGLI2expression (GLI3 activator:GLI3 repressor=1.43). on GLIprotein expression. Bycontrast,cyclopaminedecreased GLI1andGLI2 therelative expression ofGLI3activatorversu relative expression of GLI3activatorversusrepressor (GLI3activator:GLI3 repressor=11.9). Treatment withdrugvehicle h activator (190kDa)inexcessofGLI3 repressor (89kDa)(GLI3activator:GLI3repressor=3.68). Treatment withSHN-Nincreased expressed GLI1,GLI2andGLI3 culture medium,embryonic kidneys cyclopamine. Inthepresence of vehicle (100%ethanol),SHH-Nor medium supplementedwithdrug culture mediumaloneorwithculture mice andtreated for4dayswith kidneys isolatedfrom E11.5wild-type generated from cultured embryonic ureteric budbranches. derived epithelialstructures and were eachdetectedinmetanephric- mice(A,E,I),GLI1,GLI2andGLI3 type (blue). Intissuegeneratedfrom wild- counterstained withHematoxylin generated ared color. was Tissue antibodies. Thesubstratereaction kidney tissueusingspecificanti-GLI tissue sectionsgeneratedfrom E14.5 ( ( expression. SMO inhibitiononGLIprotein Fig. 2.Effects of deficiency andSMOinactivity onGLIexpression. Thespecificity specific fordistinctGLIproteinstoinvestigate theeffects ofSHH in responsetoSHHisnotwellunderstood.We usedantibodies SHH-SMO signaling.Yet theregulation ofGLIproteinexpression In mammals,GLI1,GLI2andGLI3aretheintracellulareffectors of repressor and formationofGLI3 SHH andSMOcontrol GLI1andGLI2expression nephrogenesis. uretericbud andduringbranchingmorphogenesis the signaling duringinductionofthemetanephricblastemaby SHH-SMO signalingdemonstrateacrucialrolefor together, theseresultsintwo complementarymodelsofdisrupted elements (seeFig.S1inthesupplementarymaterial).Taken these kidneys revealed apaucityofmetanephric-derived epithelial kidneys/group). Analysisofhistologicalsectionsgeneratedfrom versus cyclopamine: 61.6±2.4versus 37.6±1.6; 572 from wild-typeand E14.5 kidneytissuelysatesgenerated 100 deficient mice(B,C,F,G,J,K). Scalebar: of stainingincorresponding specificity wasdemonstratedbylack type to0.23in markedly decreased from 3.25inwild to GLI3repressor inrenal tissuewas wild type.TheratioofGLI3activator kDa) wasunaffected compared with was decreased, GLI3repressor (89 Although GLI3activator(190kDa) deficiency decreased GLI1andGLI2. N A Westernanalysisoftissuelysates ) - L Immunohistochemistry of4 ) ␮ m. ( RESEARCH ARTICLE M ) Westernanalysisof Shh Shh –/– Shh –/– mice (L).Antibody Shh mice. Shh deficiency or –/– mice. deficiency Gli - Shh ␮ m P <0.0001. n =5 activator toGLI3repressorintissueisolatedfrom kDa). Thisresultedinamarked decreaseintheratioofGLI3 with preserved expression oftheshortrepressorformGLI3(89 marked decreaseinthefull-lengthactivator formofGLI3(190kDa) additional insightintotheexpression ofGLI3demonstratinga immunohistochemistry (Fig.2M).Western analysisprovided confirmed thedecreaseinGLI1andGLI2proteinsobserved by affected bySHHdeficiency (Fig.2L). in dysplastickidneys (Fig.2C,D,G,H).GLI3expression was not decreased GLI1andGLI2proteinexpression toundetectablelevels 2A,E,I). Bycontrast,homozygousinactivation of ureteric bud ofkidneys isolatedfromE14.5wild-typemice(Fig. was detectedinboththemetanephric-derived epitheliumand (Fig. 2K)mice,respectively. ExpressionofGLI1,GLI2andGLI3 GLI2 andGLI3inkidneys isolatedfrom of anti-GLIantibodieswas demonstratedbylackofdetection Analysis ofGLIproteinexpression inkidney tissuelysates Gli2 –/– Development 133(3) ad nosignificanteffect (Fig. 2F)and GLI1, GLI2andthe s GLI3repressor Shh Shh –/– but not mice (GLI3 Gli3 Gli3 –/–

DEVELOPMENT wild-type (P), expression inE14.5mouse kidneys. Pax2 bar: 200 development, weanalyzed To investigate theimpactofincreasedGLI3repressoronkidney SHH targetgeneexpression Gli3 increases therelative expression ofGLI3repressor. SHH deficiency orSMOinhibitiondecreasesGLI1andGLI2 versus 1.43,respectively). Together, thesedatademonstratethat versus SHH-Nversus cyclopamine, 3.68versus 3.8versus 11.9 activator:GLI3 repressor–culturemediumaloneversus drugvehicle decreased theratioofGLI3activator toGLI3repressor(GLI3 levels inall Pax2 mRNA expression wasmarkedlydiminished butwasrescued in rescued in kidney numberandhistologywas in ectopic, dysplastickidneywasobserved normally positionedkidneys.Asingle, Gli2 (F), stained withHematoxylin.Wild-type sections(4 embryos. Tissue examined. ( mice (Fig.3B).Intheremaining activator:GLI3 repressor:wildtypeversus GLI3-dependent control ofrenal development stage whenbranchingmorphogenesisandmetanephric 3E). Next, weinvestigated theroleof Fig. 3. of detectable intheWolffian ductin~50% (short arrow). duct (longarrow) andmetanephros Pax2 it restored function alonehadnoeffect on detected inthemetanephros(Fig.3C).Althoughlossof In modest degree thanthatobserved in repressor anddecreasedlevels ofGLI3activator, albeittoamore expression. Second,cyclopamine increasedformationofGLI3 2N). First,cyclopamine decreasedGLI1andGLI2protein cyclopamine-treated wild-typeembryonickidney explants (Fig. We observed similarchangesinGLIproteinexpression in 3A). Consistentwiththefindingofrenalaplasiain50% Pax2 (Dressler etal.,1990;Torres etal.,1995)(Fig.3A).Thus,weused metanephric blastemaandisrequiredfordevelopment expressed intheWolffian Duct,uretericbud andinduced memberofthepairedboxfamily oftranscription factors, is a developmental stages.Duringmurinekidney development, idtp (K), wild-type expression inE14.5mouse kidneys. rbs nwl-yeand probes. Inwild-type hybridization usingdigoxigenin-labeled identified bywhole-mountinsitu mouse embryos. ( during kidneydevelopment. control A-E Shh Shh Shh –/– ) mRNA wasrescued towild-type mRNA isexpressed intheWolffian Pax2 mRNA expression asamarker ofearlyinductive events (Fig. mRNA expression was barelydetectablein~50%of –/– inactivation rescues kidneydysplasiaand (H) and –/– deficient mice(G).Bycontrast, Shh Pax2 mice (L), embryos (compare BwithA). ␮ m. ( Shh mRNA expression inE11.5 Shh F-J Pax2 and Gli2 Gli2 Pax2 and Gli3 KidneyhistologyinE14.5 ) –/– K-O –/– ;Gli3 Gli3 ;Gli3 Pax2 Pax2 –/– –/– mRNA expression inall –/– ) Sall1 RAwasbarely mRNA Pax2 (R), and (M) and (I) miceexhibittwo –/– –/– interact to mRNA was mRNA expression wasmarkedly diminishedbutwasrescued in mice (J).Scale embryos Gli3 expression mRNA ␮ Gli3 –/– Gli3 m) were embryos, –/– –/– Shh Pax2 (S) kidneys, (N) kidneys, Shh Sall1 Pax2 Shh –/– –/– mRNA expression (Fig.3D), ;Gli3 Gli3 mRNA wasdetectedusingadigoxigenin-labeled probe andinsituhybridization4 mRNA wasdetectedusingadigoxigenin-labeled probe andinsituhybridization4 –/– embryos, mice. Third,cyclopamine Shh Shh at alaterdevelopmental –/– Sall1 Pax2 –/– –/– mice atdifferent , 3.25versus 0.23). ;Gli3 mRNA wasexpressed inmetanephric-derived epithelialstructures. In mRNA wasexpressed inureteric budbranchesandmetanephric-derivedepithelialstructures. Pax2 –/– Shh mRNA was mice (Fig. –/– Shh Shh Pax2 mice, Gli3 –/– ;Gli3 –/– , –/– with expression of kidney tissuefrom the uretericbud andmetanephricmesenchyme,respectively, in approximate twofold andfourfolddiminutionofcellproliferationin (Grisaru etal.,2001),andinmesenchymalcellsdemonstratedan in uretericbud branches,identifiedbyDolichosBiflorusAgglutinin By contrast,inkidneys isolatedfrom of kidneys isolatedfromwild-type, epithelial andmesenchymalderivatives locatedontheouterregions anti-BrdU antibody(Fig.4A-D)revealed incorporationofBrdUin kidneys (Fig.4).Qualitative analysisofkidney sectionsstainedwith (BrdU), asurrogatemarker ofcellproliferation,inembryonic proliferation bymeasuringincorporationof5-bromodeoxyuridine We determinedtheimpactofalteredSHHsignalingoncell proliferation resultsinrenaldysplasia(MichaelandDavies, 2004). regulated duringkidney development. Dysregulation ofcell deficiency onkidney development isdependentonGLI3. together, thesedatademonstratethatthedeleteriouseffect ofSHH 3G,L,Q). Interestingly, deficiency ofneither in kidneys. Bycontrast,BrdUincorporationwas markedly diminished the metanephricmesenchyme(Fig.3P).Both bud branches(Fig.3K).Inaddition, expressed inboththeinducedmetanephricmesenchymeandureteric epithelialization areestablished.ByE13.5, proliferation inboththesetissuecompartmentswas rescuedtolevels rescue ofkidney number, histologyand markers ofuretericbud andmetanephricdevelopment, weobserved 2001; RothenpielerandDressler, 1993).Using required formetanephrogenesisatthisstage(Nishinakamuraetal., in mice (T).Scalebar: 100 Shh Shh Growth factor-dependent controlofcellproliferationistightly –/– –/– ;Gli3 kidneys. QuantitationofthenumberBrdU-positive cells Shh –/– –/– ;Gli3 mice (Fig.3J,O,T)comparedwith –/– Pax2 Shh mice (O).Scalebar:100 ␮ –/– m. (Fig. 3M,R)and mice comparedwithwildtype(Fig.4E). RESEARCH ARTICLE Sall1 Gli3 Pax2 Shh expression islocalizedto Shh Sall1 Gli2 ␮ –/– –/– –/– m. ( and ␮ ␮ Pax2 ;Gli3 mice (Q), m tissuesections.In m tissuesections.In (Fig. 3N,S).Taken Pax2 and nor Pax2 P-T Sall1 Shh Gli3 ) –/– and Shh Sall1 –/– and is normally expression mice, cell mice (Fig. Sall1 interfered –/– Sall1 mRNA Sall1 ;Gli3 573 are as –/–

DEVELOPMENT Gli3 Hematoxylin (blue).Inwild-type(A), were counterstainedwith Tissues stained nucleiare BrdU positive. situ BrdU incorporationassay. Red- incorporation wasdetectedbyanin sacrificed 4hourslater. BrdU mice were injectedwithBrdU and BrdU incorporationatE14.5.Pregnant proteins. expression ofcellcycleregulatory control cellproliferation and MYC wasnotaffected by decreased in greater thanthatobservedinwildtype.( significantly different thanthoseobservedinwildtype.Removalof diminished inkidneys isolatedfrom the expression ofbothcyclin D1andMYCNwas greatly products isexpressed intheembryonickidney inwild-typemice, and westernanalysis(Fig.4F).Althougheachofthesegene MYC inthekidney ofSHH-deficientmiceusingspecific antibodies deficiency ontheexpression ofcyclin D1,cyclin D2,MYCNand 2004; Milletal.,2005).Thus,wedeterminedtheeffect ofSHH regulate cellproliferationinaSHH-dependentmanner (Lietal., with wildtype. bud cellproliferationwas observed in tissue.Interestingly, ahigherrateofureteric observed inwild-type Fig. 4. 574 branches orinthemetanephricmesenchyme(blackbars). BrdU incorporationatE14.5.BrdU incorporationisexpressed asafractionofthetotalnumbercellsinureteric bud(wh 100 was markedlydecreased. Scalebar: Shh structures andureteric budtips.In mesenchyme-derived epithelial metanephric mesenchymecells, BrdU incorporationwasobservedin detectable. Strikingly, weobserved de novo binding ofGLI3with the bindingofGLI1andGLI2 with binding withthesetarget promoters(Fig.5B).Mostremarkably, explants withcyclopamine inducedchangesinGLIprotein the supplementarymaterial). Treatment ofembryonickidney (Fig. 5B)orprocessedimmediately afterisolation(seeFig.S2in promoter elementsinwild-type kidneys eitherculturedinvitro indicate thatGLI1and/orGLI2,but notGLI3, bindthese GLI3 withregions encompassingthesesequences.Ourresults immunoprecipitation toanalyzetheassociationofGLI1,GLI2 or cyclin D1and 1990; Sasakietal.,1997)inthe5 consensus sequences(Ikrametal.,2004;KinzlerandVogelstein, genes inthekidney. First, weidentifiedputative GLI-binding by SHH-SMOsignalingcontrolstheexpression ofSHHtarget We determinedhow differential controlofGLI1,GLI2andGLI3 within SHHtargetgenes proteins withGLI-binding consensusregions Inhibition ofSMOchangestheassociationGLI expression. deficiency atthelevel ofcell proliferation andtarget gene a crucialroleforGLI3incontrollingtheresponsetoSHH the kidneys of derived epithelialstructures significantlycompared withwildtype.Removalof In nonrenaltissues,membersofthecyclin andMYC families –/– –/– ␮ m. ( mice (B),BrdU incorporation (C) and Shh RESEARCH ARTICLE E ( A-D ) Quantitativeanalysisof and Shh Shh ) Insituanalysisof Shh Gli3 –/– –/– Mycn compared withwildtypeandwasrescued towild-typelevelsin –/– ;Gli3 ;Gli3 interact to –/– Shh (Fig. 5A).Next, weusedchromatin –/– (D) mice, mice. Together, thesedatademonstrate deficiency. Ј flanking regions of Shh Gli3 F Pax2 ) WesternanalysisofE14.5kidneytissuelysates.Expression ofcyclinD1andMYCNwasmarkedly –/– deficient micecompared mice but was rescuedin and Sall1 Pax2 Shh was barely deficiency decreased BrdU incorporationinbothureteric budandmesenchyme- , Sall1 Gli3 , increased BrdU incorporationinureteric budderived cellstoalevel Gli3 controlsGli1andGli2in primary roleinorchestratingtheseevents, weinvestigated whether expression of target genes.Bycontrast,removal ofboth deficiency decreasesGLI1andGLI2aswellexpression of these mRNAs wererescuedtowild-typelevels in encoding levels. Our concurrentfindingthatcyclopamine decreasedmRNAs the associationswithGLI2were decreasedtoalmostundetectable promoter andbetweenGLI3 the Gli2 observed betweenGLI1 and GLI2 boundboth (see Fig.S3inthesupplementarymaterial)demonstrated that cultured invitro(Fig.6D)orprocessedimmediatelyafterisolation encoding GLIbindingsequencesinwild-typekidneys either by chromatinimmunoprecipitation.Analysisoftheregions weexamined theassociationofGLIproteinswiththesesites Next, binding sitesinthe5 controls mice. Theseresultsprovided abasisfordetermininghow GLI3 mice usingreverse transcriptasePCR(Fig. 6B).Although Our geneticanalysesin in SHHdeficientmice GLI3 controls decreased GLI1andGLI2expression promoters. and decreasestheassociationofGLI1GLI2withtarget SMO signalingchangesinducesthedenovo associationofGLI3 each ofthesepromoters.Theseresultsdemonstratethatdecreased mRNA inkidney tissueisolatedfrom wild-typeand GLI1 andGLI2expression atatranscriptionallevel, weexamined in wild-typemice(Fig.6A).To determinewhetherGLI3controls GLI1 andGLI2levels in protein expression inkidney tissuelysatesdemonstratedrescueof Gli2 Gli3 . cyclopamine enhanced bindingbetweenGLI3andthe mRNA levels weredecreasedin in Shh Gli1 Gli1 Shh –/– ;Gli3 –/– Shh and and mice rescued BrdU incorporationtolevelsnot –/– target genes.To determinewhetherGli3playsa Gli2 mice. Bycontrast,expression ofcyclinD2and Gli2 Gli1 Ј flanking regions of and Shh transcription. First,weidentifiedGLI and Shh Pax2 –/– Gli1 Shh ;Gli3 Gli2 –/– showed thattheseeffects invitro –/– or mice demonstratedthat . Aweaker association was ;Gli3 –/– Gli2 Gli2 mice tothelevels observed Shh –/– Gli1 and betweenGLI3 promoter. Bycontrast, Gli3 –/– mice. AnalysisofGLI Development 133(3) ite bars)andits mice, thelevels of and and Gli2 Shh Shh Shh (Fig. 6C) –/– –/– Gli1 restored ;Gli3 ;Gli3 Gli1 Shh Shh and –/– –/– .

DEVELOPMENT GLI3-dependent control ofrenal development Gli3 cyclopamine-treated embryonic kidney explants isolatedfrom Gli1 inhibited. the material). Together, theseresults demonstratethatGLI3bindsto paralleled ourfindingsinvivo (seeFig.S4inthesupplementary Pax2 consensus bindingsequencesinthe5 5 cyclopaminechangesbindingofGLIprotein speciesto Fig. 5. cross-linked DNAbefore immunoprecipitation. using genomicDNAandspecificprimers;Input,amplifiedfrom immunoprecipitation withnon-immuneserum;Pos,DNAamplified GLI3 toeachpromoter region. Neg,DNAamplifiedafter binding mostmarkedlyin regions wasdetectable.Cyclopaminedecreased GLI1andGLI2 in bound 5 cyclopamine. Invehicle-treated embryonickidneys,GLI1and/orGLI2 tissue cultured for4daysinthepresence ofdrugvehicleor kidney after chromatin immunoprecipitation usingE11.5wild-type (see below).( segments amplifiedbyPCRduringchromatin immunoprecipitation consensus bindingsequences(seetext).Arrows indicatepromoter case are exactmatchestothoseidentifiedpreviously inGLI Ј Pax2 To investigate whetherGli3 isprimarilyinvolved in controlling flanking regions in , Gli1 –/– Sall1 and , mice. Ourprior results(Fig.6)predictedthatcyclopamine Ј Sall1 flanking regions containingGLIconsensusbindingregions , cyclinD1and and Gli2 , cyclinD1and B ) PCRproducts identifiedbyagarose electrophoresis Gli2 expression, weanalyzed theirexpression in promoters whenSHH-SMO signaling is Shh Pax2 Mycn Mycn target genes. and . Nucleotidesrepresented inupper . NobindingofGLI3withthese Sall1 Ј flanking region ofmouse , andinducedbindingof ( A ) IdentificationofGLI mRNA wasdecreased in RNA isolatedfrom E14.5embryonickidney. Expression of was decreased compared withwildtypeand amplified from cross-linked DNAbefore immunoprecipitation. DNA amplifiedusinggenomicandspecificprimers;Input, amplified afterimmunoprecipitation withnon-immuneserum;Pos, and inducedbindingofGLI3toeachpromoter region. Neg,DNA Gli1 binding regions in and/or GLI2bound5 drug vehicleorcyclopamine.Invehicle-treated embryonickidneys,GLI1 kidneytissuecultured for4daysinthepresence of E11.5 wild-type agarose electrophoresis afterchromatin immunoprecipitation using immunoprecipitation (seebelow).( Arrows indicatepromoter segmentsamplifiedbyPCRduringchromatin identified previously inGLIconsensusbindingsequences(seetext). Nucleotides represented inuppercaseare exact matchestothose binding sequencesinthe5 tissue isolatedfrom cyclopamine onGLI1 and GLI2oruretericbud branchingin morphogenesis (Fig.7B).By contrast, weobserved noeffect of decreased GLI1andGLI2 (Fig.7A)andbranching kidneys isolatedfrom wild-type mice,cyclopamine markedly would fail todecreaseGLI1andGLI2inGLI3deficient tissue.In ( mice, GLI1andGLI2expression wasrescued towild-typelevels. specific anti-GLIantibodies.In mechanisms. Fig. 6 is inhibited. GLI1 andGLI2expression andrenalmorphogenesiswhenSMO was rescued in B Agarose gelelectrophoresis ofproducts generatedbyRT-PCR using ) was detectable.Cyclopaminedecreased GLI1andGLI2binding . GLI3 controls GLI1andGLI2expression viatranscriptional ( Shh A ) WesternanalysisofE14.5kidneytissuelysatesusing Gli1 –/– Ј ;Gli3 Gli3 flanking regions containingGLIconsensus and Shh –/– Ј –/– Gli2 mice. ( flanking region ofmouse –/– mice (Fig.7A,B).Thus,GLI3controls Shh oprdwt idtp and compared withwildtype . NobindingofGLI3withthisregion in –/– C D ) IdentificationofGLIconsensus ) PCRproducts identifiedby mice, expression ofGLI1andGLI2 RESEARCH ARTICLE Gli3 –/– . In Gli1 Shh Gli1 –/– and ;Gli3 Gli3 and Gli2 –/– –/– Gli2 . and 575

DEVELOPMENT proliferation (cyclinD1and genes thatcontrol renal morphogenesis( repressor controls expression of treatment withcyclopamine increases formationofGLI3repressor. GLI3 inhibition. Decreased SMO activitygeneratedbySHHdeficiencyor Gli3 by DolichosBiflorusAgglutinin.This inhibitoryeffect wasabrogated in cyclopamine decreased the numberofureteric budbranchesidentified explants. Treatment ofkidneysisolatedfrom E11.5wild-typemicewith Gli3 decreased GLI1andGLI2.Thisinhibitoryeffect wastotally abrogated in kidneys isolatedfrom wild-typemicewithcyclopaminemarkedly presence ofdrugvehicleorcyclopaminefor4days.Treatment of finding thathomozygousGLI3deficiency in significance ofGLI3repressorformationwas demonstratedbyour length formandthatofGLI2GLI3isdecreased.The Expression oftherepressorformGLI3isincreased,whileitsfull- expression inamannerdistinctfromthatofGLI1andGLI2. SMO signalingdisruptskidney development andregulates GLI3 The experiments reportedheredemonstratethatdecreasedSHH- DISCUSSION 576 Fig. 7. harvested from wild-typeor tissue lysatesusingGLI-specificantibodies.Embryonickidneyswere expression andkidneydevelopment. rescued thedysplasticrenalphenotypeandexpression of -deficient mice.( -deficient mice.( Gli3 RESEARCH ARTICLE is required forSMO-dependentcontrol ofGLI C B ) ModelofGLI3activityinastate of SMO ) Ureteric budbranchingincultured kidney Mycn Gli3 Gli1 –/– ). mice atE11.5andcultured inthe and Pax2 Gli2 ( A ) Westernanalysisofkidney as wellSHHtarget and Sall1 Shh ) andcell deficient mice Pax2 , GLI activator functions. underlie thisrequirement?Onepossibilityislossofoneormore during kidney development. What arethemechanismsthat Our resultsdemonstratearequirementforSHH-SMOsignaling kidney development A requirement forSHH-SMOsignalingduring morphogenesis. signaling, GLI3repressesGLI1,GLI2andgenescrucialtorenal formation ofGLI3repressor. InastateofdecreasedSHH-SMO Together, ourresultsindicatethatSHH-SMOsignalingregulates the morphogenesis provided functionalsupportforourhypothesis. GLI1andGLI2expression andnormalizesbranching on deficiency abrogatesthedeleteriouseffects ofSMOinhibition SMO supportedthishypothesis.Ourdemonstrationthat to the which GLI3controlsgeneexpression. BindingofGLI1andGLI2 target genesprovides anovel insightintopotentialmechanismsby GLI consensusbindingregions inthe5 Sall1 of GLI3tothe possibility thatGLI3regulates GLI1andGLI2expression. Binding decreased bindingoftheseproteinstotheirtarget genesraisedthe represses thesegenes.Decreasedexpression ofGLI1andGLI2 regions inastateofSMOinactivity suggeststhatGLI3directly genes asdirecttargets ofSHH.Denovo GLI3bindingtothesesame Gli1 2003; McDermottetal.,2005).Yet, homozygousdeficiency of in acontext-dependent manner(Baietal.,2004;Buttitta etal., to functionasSHH-SMO-dependentactivators innonrenaltissues Gli1 repressor showing thatwhenSMOisinhibited,GLI3controls studies provide novel insightsintothepotentialactionsofGLI3 mice (Boseetal.,2002)alsodisruptkidney development. Our Hall syndrome(Kangetal.,1997)andingeneticallyengineered shortened repressor-like formofGLI3inhumanswithPallister- 1992). Bycontrast,mutationsthatgenerateexpression ofa development (Baietal.,2002;Kim2001;Schimmang GLI3 represses promoters. ThesefindingssuggestthatinPallister-Hall syndrome, Sall1 controls expression of invasion ofthemetanephricblastema.Theobservation that SHH andSMO,demonstratingthat Our resultsprovide new insightsintothetarget genesregulated by transcriptional targets genesareKidney patterning direct GLI dependent target genes. and requirement for severe dysgenesis (RothenpielerandDressler, 1993), indicatinga Homozygous inactivation of coloboma syndromeandrenalhypoplasia (Sanyanusin etal.,1995). Dressler, 1993). Mutationsin renal organogenesis (Nishinakamura etal.,2001;Rothenpielerand GLI3 repressordirectlycontrols Our resultssuggestthatinthesedifferent developmental contexts 2000; Litingtungetal.,2002;Mill2005;Rallu2002). mutant phenotypesinnonrenaltissues(LitingtungandChiang, observation thatgeneticinactivation of elements. Moregenerally, ourfindingsprovide insightintothe , Gli2and , cyclin D1and . Both and Mycn Pax2 Gli2 , Pax2 Sall1 via interactionswiththeirrespective promoter expression andassociateswiththe Gli1 Gli3 Gli1 and Pax2 , cyclin D1and Mycn and Sall1 does notnegatively impactmurine kidney and Gdnf during outgrowth of theuretericbud and Gli2 Gli1 . AnalysisofGLIproteinassociationwith Gli2 perform crucialfunctionsduringmurine (Brophy etal.,2001), whichencodesa Pax2 promoters intissuetreatedtoinhibit , Gli2 as well PAX2 Mycn Gli in miceresultsrenalaplasiaor Shh and transcription andthatof promoters establishedthese are associatedwithrenal Ј acts upstreamof Gli3 flanking regions ofthese Pax2 Gli3 Development 133(3) have allbeenshown , Sall1 can rescueShh Gli1 , cyclin D1 Pax2 and Pax2 Gli2 Gli3 Gli and -

DEVELOPMENT proliferation observed in In bothskinandkidney Gli2 Shh proliferation appeartodiffer betweenskinandkidney. Inskin,both which GLIproteinsmediatetheactionsofSHHtocontrolcell Sall1 (Nishinakamura etal.,2001).Ourresultsprovide novel insightinto include includes thosecrucialtorenalpatterning.Membersofthisclass Gli family, specifically which arecrucialtokidney development (Fig.7C).Thefirstisthe inhibiting thetranscriptionofseveral classes ofgenes,theactions disrupting kidney morphogenesis(Fig.7C).GLI3repressoractsby GLI3 repressororchestratestheeffects ofSMOinhibition,thereby morphogenesis byrestrictingtheactivity ofGLI3.Inourmodel, and predicts that The experiments reportedhereprovide abasisformodelthat SHH-SMO activity A modelofgeneregulation instatesofdecreased development requiresfurtherinvestigation. et al.,2005).Therelevance ofthismoderegulation tokidney proliferation. Thesefactors mayincludeGSK3 factors modulatetheactionsofGLIproteinstocontrolcell is muchgreaterinkidney comparedwithskin,suggesting thatother ureteric bud (Mooreetal.,1996),suggeststhat secreted growth factor essentialforoutgrowth andinvasion bythe GLI3-dependent control ofrenal development http://dev.biologists.org/cgi/content/full/133/3/569/DC1 Supplementary material forthisarticleisavailableat Supplementary material grant (HL67822).N.D.R.issupported byaCanadaResearch Chair. technical assistance.Work intheChuanglaboratorywassupported byanNIH Institutes ofHealthResearch (toN.D.R.andC.C.H.).We thankYa-Jun Lifor This workwassupportedbyoperating grantsawarded bytheCanadian of hair follicle(Milletal.,2005),cyclin D2expression isindependent Interestingly, whilecyclin D2isregulated bySHHinthedeveloping in non-renalembryonictissues(Longetal.,2001;Mill2005). expression ofcyclin D1andMYCNduringkidney development as Our resultsdemonstratethatSHHcontrolscellproliferationand dependent oncontext GLI transcriptionalcontrol oftargetgenesis state ofSHH-SMOsignaling. degrees andindifferent combinations,depending,inpart,onthe Pax2 genetic analysesanddemonstratethatpromoterelementsinboth inactivation of the metanephricmesenchymewhereitisessentialashomozygous During murinekidney development, syndrome andrenalaplasia/dysplasia(Kohlhase etal.,1998). SALL1 the GDNF-RETsignalingpathway duringrenalorganogenesis. SHH-SMO target genes. the interplayofGLI1,GLI2andGLI3inregulation ofthese provide abasisforinvestigating molecularmechanismsthatcontrol GLI3 repressoriscriticaltorepressionofthesegenes.Ourfindings cell proliferationandincludescyclin D1andMYCN.Formation of Our resultsusingchromatinimmunoprecipitationextend our Shh and Gli2 deficiency doesnotaffect cellproliferation(datanotshown). regulation showing thatitiscontrolledby and expression intheembryonickidney. Themechanismsby mutations arefoundinindividuals withTownes Brock Pax2 Gli2 in thedeveloping kidney andthat Sall1 Gli3 control cellproliferation(Milletal.,2005).Inkidney, and are boundbyGLI1,GLI2andGLI3tovariable Sall1 Sall1 acts downstream ofSMOandupstream . Thethirdclassofgenesisthatcontrolling abrogates outgrowth oftheuretericbud Gli1 Shh Gli3 –/– and mice. However, thedegree ofrescue deficiency rescuesdecreasedcell Gli2. Sall1 The secondclassofgenes expression isrestrictedto Shh ␤ Shh and Shh programs kidney acts upstreamof . ␤ -catenin (Mill Gli1 Long, F., Zhang,X.M.,Karp,S.,Yang, Y. andMcMahon,A.P. Litingtung, Y., Dahn, R.D.,Li,Y., Fallon,J.F. andChiang,C. Litingtung, Y. and Chiang,C. Kohlhase, J.,Wischermann,A.,Reichenbach,H.,Froster, U.andEngel,W. Kim, P. C.,Mo,R.andHui,C. Li, Y., Zhang,H.,Choi,S.C.,Litingtung,Y. andChiang,C. Lai, K.,Robertson,M.J.andSchaffer, D.V. Dai, P., Akimaru,H.,Tanaka, Y., Maekawa,T., Nakafuku,M.andIshii,S. Aza-Blanc, P., Ramirez-Weber, F. A.,Laget,M.P., Schwartz,C.andKornberg, References Kang, S.,Graham,J.M.,Jr, Olney, A.H.andBiesecker, L.G. Ingham, P. W. andMcMahon,A.P. Ikram, M.S.,Neill,G.W., Regl,G.,Eichberger, T., Frischauf,A.M.,Aberger, Kinzler, K.W. andVogelstein, B. Cano-Gauci, D.F., Song,H.H.,Yang, H.,McKerlie,C.,Choo,B.,Shi,W., Chiang, C.,Litingtung,Y., Lee,E.,Young, K.E.,Corden, J.L.,Westphal, H. Chen, J.K.,Taipale, J.,Cooper, M.K.andBeachy, P. A. Dressler, G.R.,Deutsch,U.,Chowdhury, H.O.andGruss,P. K.,Nornes, Conlon, R.A.andHerrmann,B.G. Bouchard, M. Brophy, P. D.,Ostrom, L.,Lang,K.M.andDressler, G.R. Bose, J.,Grotewold, L.andRuther, U. Bai, C.B.,Auerbach,W., Lee,J.S., Stephen, D.andJoyner, A.L. Buttitta, L.,Mo,R.,Hui,C.andFan,M. Bai, C.B.,Stephen,D.andJoyner, A.L. Hu, M.C.,Piscione,T. D.andRosenblum,N. Hu, M.C.andRosenblum,N.D. Grisaru, S.,Cano-Gauci,D.,Tee, J.,Filmus,J.andRosenblum, N.D. manipulation ofhedgehog signalingintheendochondralskeleton reveals a identity. Gli3 are dispensableforlimbskeletonformationbutregulate digitnumberand 979-985. mediated byanantagonisticinteraction betweenShhandGli3. differentiation during mouselungorganogenesis. hedgehog signalingregulates Gli3processing, mesenchymalproliferation, and signaling systemasabistablegeneticswitch. Genet. frameshift mutationscauseautosomaldominantPallister-Hall syndrome. development: paradigmsandprinciples. Brocks syndrome. (1998). MutationsintheSALL1putativetranscriptionfactorgenecauseTownes- 642. which bindsspecificsequencesinthehumangenome. Role ofsonichedgehogsignalingpathway. Dev. Hedgehog signalingbydirect bindingofcyclopaminetoSmoothened. GLI3. (1999). SonicHedgehog-inducedactivationoftheGli1promoter ismediatedby 225 situ hybridizationtopostimplantationembryowholemounts. interruptus protein tothenucleusandconvertsitarepressor. T. B. Invest. Dermatol. epidermis andBCCinducesGLI1expression bybindingtoitspromoter. F., Quinn, A.andPhilpott,M. Development and theirrequirement inmediatingShh-dependentsclerotome induction. Simpson-Golabi-Behmel syndrome. deficient miceexhibittheovergrowth andrenal abnormalitiestypicalofthe Pullano, R.,Piscione,T. D.,Grisaru,S.,Soon,S.etal. lacking Sonichedgehoggenefunction. and Beachy, P. A. the developingexcretory system. (1990). Pax-2,anewmurinepaired-box-containing geneanditsexpression in Differentiation in micemutantforGli3. neurotrophic factorgene. ureteric budoutgrowth byPax2-dependentactivationoftheglialderived Gli3. byhedgehogisGlidependentandinvolvesanactivatorfunctionof patterning Shh pathway. but notGli1,isrequired forinitialShhsignalingandectopicactivationofthe 1053. transgenic mice. catenin molecularcomplexesandrenal 225. tissue andcooperatetocontrol Myctranscription. associate inamolecularcomplexwiththeMycpromoter indysplasticrenal Morphogenesis. Glypican-3 modulatesBMP-andFGF-MediatedEffects duringRenalBranching , 373-383. 16 (1997). Proteolysis thatisinhibitedbyhedgehogtargetsCubitus Dev. Cell J. Biol.Chem. 15 , 2743-2748. Nature , 266-268. (2004). Transcriptional control ofkidneydevelopment. 130 Development 6 72 , 103-115. 418 Dev. Biol. Development 122 , 295-306. , 6233-6243. Nat. Genet. (1996). Cyclopia and defective axial patterning inmice (1996). Cyclopiaanddefectiveaxialpatterning , 979-983. 274 , 1503-1509. , 8143-8152. Hum. Mol.Genet. Development 230 129 (2000). Specificationofventralneuron typesis 18 , 31-46. 130 (2001). MurinemodelsofVACTERL syndrome: , 4753-4761. (2004). GLI2isexpressed innormalhuman , 81-83. (1990). TheGLIgeneencodesanuclearprotein Development (2005). Smad1,{beta}-cateninandTcf4 , 2753-2766. (2001). Hedgehogsignalinginanimal (1993). DetectionofmessengerRNAbyin J. CellBiol. (2002). Pallister-Hall syndrome phenotype medullary cysticdysplasiainALK3 Nature 128 (2004). Allmouseventralspinalcord Genes Dev. RESEARCH ARTICLE 11 J. Pediatr. Surg. , 4747-4756. (2004). Thesonichedgehog Biophys. J. (2003). InterplaysofGli2andGli3 , 1129-1135. 146 383 109 (2003). ElevatedSmad1/b- Dev. Biol. , 255-264. , 407-413. , 787-795. Development 15 , 3059-3087. Mol. Cell.Biol. (2002). Inhibitionof 86 (1999). Glypican-3- (2001). Regulationof , 2748-2757. 36 270 (2004). Sonic Methods Enzymol. , 381-384. (2002). Shhand (1997). GLI3 , 214-231. Nat. Neurosci. (2001). Genetic Cell 132 (2002). Gli2, 89 , 215- (2001). 10 Genes , 1043- Nat. , 634- 577 J. 3 ,

DEVELOPMENT Piscione, T. D.andRosenblum,N. McDermott, A.,Gustafsson,M.,Elsam,T., Hui,C.C.,Emerson,P., Jrand 578 Pichel, J.G.,Shen,L.,Sheng,H.Z.,Granholm,A.-C.,Drago,J.,Grinberg,A., Park, H.L.,Bai,C.,Platt,K.A.,Matise,M.P., Beeghly, A.,Hui,C.C., Nishinakamura, R.,Matsumoto,Y., Nakao,K.,Nakamura,Sato,A., Moore, M.W., Klein,R.D.,Farinas,I.,Sauer, H.,Armanini,M.,Phillips, Mo, R.,Freer, A.M.,Zinyk,D.L.,Crackower, M.A.,Michaud,J.,Heng,H.H., Mill, P., Mo,R.,Hu,M.C.,Dagnino,L.,Rosenblum,N.D.andC.-C. Michael, L.andDavies,J.A. Methot, N.andBasler, K. 73-76. of glomerularandtubulardevelopment. during murineureteric buddevelopment. function inskeletalmuscleformation. Borycki, A.G. 5099-5108. direct role intheregulation ofchondrocyte proliferation. enteric innervationandkidneydevelopmentinmicelackingGDNF. Lee, E.J.,Huang,S.P., Saarmas,M.,Hoffer, B.J.etal. Development have defectsinSHHsignalingcombinationwithaGli2mutation. Nakashima, M.andJoyner, A.L. development. (2001). MurinehomologofSALL1isessentialforureteric budinvasioninkidney Copeland, N.G.,Gilbert,D.J.,Jenkins,A.,Scully, S.,Lacey, D.L.etal. Renal andneuronal abnormalitiesinmicelackingGDNF. Reichardt, L.F., Ryan,A.M.,Carver-Moore, K.andRosenthal,A. development. redundant and functionsofGli2andGli3zincfingergenesinskeletalpatterning Chik, K.W., Shi,X.M.,Tsui, L.C.,Cheng,S.H.etal. Developmental Cell dependent GLIactivatorandrepressor functionsintheembryonicepidermis. (2005). Transcriptional andpost-translationalproliferative control bySHH- interruptus inHedgehogsignaling. RESEARCH ARTICLE 127 Development Development (2005). Gli2andGli3haveredundant andcontext-dependent , 1593-1605. 9 , 293-303. (2001). Anabsoluterequirement forCubitus 128 124 (2004). Pattern andregulation(2004). Pattern ofcellproliferation , 3105-3115. , 113-123. Development (2000). MouseGli1mutantsare viablebut (1999). Themalformedkidney:disruption Development Clin. Genet. J. Anat. 128 204 132 , 733-742. 56 , 241-255. , 343-358. , 345-357. (1997). Specificand Nature Development (1996). Defectsin 382 Nature , 76-79. (1996). 128 382 , , Zhang, S.L.,Moini,B.andIngelfinger, J.R. Yu, J.,Carroll, T. J.andMcMahon,A.P. Wang, B.,Fallon,J.F. andBeachy, P. A. Rothenpieler, U.W. andDressler, G.R. Rallu, M.,Machold,R.,Gaiano,N.,Corbin,J.G.,McMahon,A.P. andFishell, Piscione, T. D.,Yager, T. D.,Gupta,I.R.,Grinfeld,B.,Pei,Y., Attisano,L., Sasaki, H.,Hui,C.,Nakafuku,M.andKondoh,H. Sanyanusin, P., Schimmenti,L.A.,McNoe,Ward, T. A.,Pierpont,M.E. Torres, M.,Gomez-Pardo, E.,Dressler, G.R.andGruss,P. Schimmang, T., Lemaistre, M.,Vortkamp, A.andRuther, U. Stoykova, A.andGruss,P. 15 Pax-2 expression infetalkidneycellsviatheAT(2) receptor. kidney. proliferation anddifferentiation ofmesenchymalcellsinthemousemetanephric vertebrate limb. of Gli3produces ananterior/posteriorrepressor gradientinthedeveloping Development mesenchyme-to-epithelium conversionduringkidneydevelopment. 4974. mutants lackingbothGli3andHedgehogsignaling. G. F975. opposite effects onrenal branchingmorphogenesis. Wrana, J.L.andRosenblum,N.D. and canrespond toShhinvitro. proteins isessentialforHNF-3betafloorplateenhanceractivityintransgenics vesicoureteral reflux. PAX2 M., Sullivan,M.J.,Dobyns,W. B.and Eccles,M.R. multiple stepsofurogenital development. brain assuggestedbyexpression patterns. extra-toes (Xt). of thezincfingergeneGli3isaffected inthemorphogeneticmousemutant , 1452-1465. (2002). Dorsoventral patterning isestablishedinthetelencephalonof (2002). Dorsoventralpatterning gene inafamilywithopticnervecolobomas,renal anomaliesand Development 119 Development Cell , 711-720. 100 Nat. Genet. 129 , 423-434. , 5301-5312. (1994). RolesofPax-genesindevelopingandadult 116 Development , 799-804. 9 , 358-363. (1997). BMP-2andOP-1exertdirect and (1993). Pax-2isrequired for (2000). Hedgehog-regulated processing (2002). Sonichedgehogregulates Development J. Neurosci. (2004). AngiotensinIIincreases 124 , 1313-1322. (1997). AbindingsiteforGli Development Am. J.Physiol. Development 133(3) 14 (1995). Mutationofthe 121 , 1395-1412. (1995). J. Am.Soc.Nephrol. , 4057-4065. (1992). Expression Pax-2 129 273 , 4963- , F961- controls

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