Additionally, ectopic the anteriorlimbbud (Masuyaetal.,1995;Büscher etal.,1997). Gli3 function indigitformation(Chiangetal.,1996).Bycontrast,loss of loss ofalldigitsexcept one,reflectingtherequirementofShh gradient toformfromanteriorposterior. Lossof within thezoneofpolarizingactivity (ZPA), causesaGli3repressor (Niswander, 2003).Shhexpression intheposteriormesenchyme, anteroposterior identitiesandthenumberofdigitstobeformed Shh regulates processingofGli3tolaythefoundation for patterning ofthelimbbuds andtheneuraltube.Inearlylimbbud, Briscoe, 2003). signalling (Wang etal.,2000;BriscoeandEricson,2001;Jacob between Gliactivators andrepressorsrealizesHh-mediated appears tobeanobligateactivator (Daietal.,1999).Theratio mainly actsasactivator (Dingetal.,1998;Matise1998).Gli1 seems toactpredominantlyastranscriptionalrepressor, whileGli2 efficient (Daietal.,1999;Wang etal.,2000;Pan etal., 2006).Gli3 processed similarlytoCi,Gli2processinginvivo ismuchless homologues (Gli1,Gli2andGli3).WhileGli3isknown tobe more complex, involving threeHh(Shh,IhhandDhh)Ci acts asatranscriptionalrepressor. Hh signallinginvertebrates is transcriptional activator. IntheabsenceofHh,Ciisprocessedand interruptus (Ci)isinhibitedandCientersthenucleustoactasa transmembrane .Asaconsequence,processingofCubitus (Ptc) andtherebyactivates Smoothened(Smo),another In invertebrates, suchas and morphogenesisofmany tissues(InghamandMcMahon, 2001). role duringembryonicdevelopment inregulating growth, patterning The Hedgehog(Hh)family ofsecretedproteinsplaysanimportant INTRODUCTION Accepted 14May 2007 † 1 KEY WORDS:Basalbody, Left-rightasymmetry, Limbdevelopment,Mousemutant,Neuraltube Furthermore, theabsenceofFtminarthropodsunderlinesdivergencebetweenvertebrateand essential forciliaassemblybutfullShhresponse,Ftmcanbeconsideredasanovelcomponentcilium-relatedHhsignal loss ofFtmaffects theratioofGli3activatortorepressor, suggestinganinvolvementofFtminShhsignalling.Asi developmental processessuchastheestablishmentofleft-rightasymmetryandpatterningneuraltubelimbs.The In thisstudyweshowinmicethatFtm(Rpgrip1l)islocatedattheciliarybasalbody. OurdatarevealthatFtmisnecessaryf Jeanette Vierkotten signalling Ftm isanovelbasalbodyproteinofciliainvolvedinShh (2007)doi:10.1242/dev.003715 Development 134,2569-2577 Universität, 40225Düsseldorf,Germany. *Present address: Epidauros,Germany Biotechnologie AG,82347Bernried, University,Medical CollegeofCornell New York, NY10021,USA. 1998; Wang etal.,2000). function hasalsobeencorrelatedwithpolydactyly(Yang etal., Author forcorrespondence (e-mail:[email protected]) (EMT),Heinrich-Heine- Institut fürEntwicklungs-undMolekularbiologie derTiere During vertebrate development, Shh playsacrucialrolein leads topolydactylyandanectopicShhexpression domainin Shh 1 expression orareductionofGli3repressor , RenateDildrop 2 Department ofGeneticMedicine,Weill , HhbindsitsreceptorPatched 1 , ThomasPeters Shh leads tothe 1, *, BaolinWang in thepresence of Hhligand(Corbitetal.,2005; Mayetal.,2005). cilia (Haycraftetal.,2005).Furthermore, Smoisenrichedincilia Hh signalling,andGliproteins arelocalizedatthedistaltipof cilia structure.suppressoroffused (Sufu),anegative regulator of involved inHh-signaltransductionarelocalized withinthe compartment forsignaltransduction hasbeenshown. Several (Kim etal.,2005). organization processesrelating tociliaryandcentrosomalactivities that theseproteinsassistmicrotubule-related transport andcellular body ofciliaandcentrosomes(duringcellcycle), anditisproposed al., 2005;Stoetzelet2006).BBSproteinsarelocatedatthebasal result inclinicallyrelatedphenotypes(Katsanis,2004;Nishimura et in acommoncellularprocess,given thatmutations inany BBSgene to causeBBS,andthereisevidence thatallBBSproteinsparticipate diabetes andpolydactyly. Mutationsinanumberofgenesareknown pigmentosa, renalmalformations,situsinversus, cardiomyopathy, Biedl syndrome(BBS).Thisdisorderischaracterizedbyretinitis such asBardet- cilia isassociatedwithseveral humandisorders, role ofciliainembryonicdevelopment iseminent.Dysfunctionof establishment ofleft-rightasymmetry(Hirokawa etal.,2006).The of ciliaandthedirectionnodalflow arenecessaryforthe inversion ofembryonicturning suchas different mousemutations, an asymmetricdistribution ofsignallingmolecules.Analyses within thenodebyproducingaleftward flow andtherebycausing embryonic development. They are involved insymmetrybreaking between classIandIIproteins(Briscoeetal.,2000). subtypes arerefined andmaintainedbycross-repressive interactions expression. Additionally, thedistinctprogenitordomainsofneuronal Mouse GenomeInformatics)and whereas classIIgenessuchas classes: ClassIgenessuchas regulation thesetranscriptionfactors canbesubdivided intotwo interneurons (BriscoeandEricson,2001).Basedontheirmodeof neuronal subtypes:motoneurons(MN),V3,V2,V1andV0 of transcriptionfactors areregulated, whichdefine thefive ventral ventral todorsal.Inresponsedifferent Shhconcentrations,subsets neural tube.ThefloorplateinturnestablishesaShhgradientfrom notochord inducesthefloorplate,ventral signalling centre ofthe axial mesodermalcellsfromthenotochord.TheShhsignal Recently, thecrucialfunctionofciliaasaspecialized Cilia arespecializedstructureswithseveral functionsduring Shhisinitiallyproducedby During neuraltubedevelopment, 2 and UlrichRüther Pax6 ( inv Drosophila Nkx2 ,revealed thatboththemotility ), RESEARCH ARTICLE and Olig2 . inversus viscerum 2 1,† Irx3 (also known as require Shhfortheir Hh pathways. are repressedbyShh, s not or Nkx2-2 ( iv ling. ) and 2569 –

DEVELOPMENT available uponrequest. manufacturer’s instructions.Detailedinformationandprimersequencesare synthesized withtheExpandRT system(Roche)according tothe (GibcoBRL) followed byDNaseIdigestion.Firststrand cDNA was described (Büscheretal.,1997). Total RNA was isolatedusingTrizol neonates ortheyolksacsofembryos. blot analysisorPCRofDNA extracted fromtheorgans ortailtipsof embryos bytimedmating.Genotyping was carriedoutbyeitherSouthern animals wereintercrossedtoderive homozygousmutantF2newborns and used tocreatechimerasthatpassedthe all cloneswerescreenedbySouthernblotanalysis.Targeted ES cellswere andafterG418selection electroporated intoR1embryonicstem(ES)cells, replace 4and5ofthe targeting constructwas designedtointroduceaPGKneocassetteand thereby We usedhomologousrecombinationtoproducean targeting,genotypingandPCR MATERIALS ANDMETHODS that Ftmisanovel componentofHhsignalling. cilia andthatitisessentialforGliproteinfunction.We conclude The presentstudyshows thatFtmislocalizedatthebasalbodyof involvement ofotheroradditional genesinthese homozygous embryos,have notbeenobserved, suggesting phenotypes, suchasfusionofphalangesorearlylethalityin majority ofdefectsobserved in reveal thattheabsenceofFtmseemstobecause started togeneratesingleknockout experiments inmice.Ourdata which ofthesixgenesmightbeacomponentHhsignalling,we in the signalling, leadingtotheassumptionthatoneofgenesdeleted Interestingly, themajorityofthesedefectspointtoadisturbedHh maintenance isaffected (Heymer etal.,1997;Götz2005). is disturbed,andventral neuraltubepatterningandfloor plate Rüther, 2002).Inaddition,establishmentofleft-rightasymmetry fore limbsandsyndactylyinfore-hind(Grotewold and der Hoeven etal.,1994).Furthermore,they reveal polydactylyin severe malformationsofcraniofacial andforebrainstructures(van die betweenembryonicday(E)10.5and14.5.Theembryosshow embryos homozygousforthe ( , 8, affecting threemembersoftheIroquoisfamily ofhomeobox mutation iscausedbyadeletionof1.6Mbonmousechromosome toes; alsoknown as gene was originallyidentified inthemousemutation understood. between ciliaandHhsignallingisstillonlybeginning tobe several proteinsareessentialforciliafunction,theconnection to form(Pazour etal.,2000;Cole,2003).Despitethefinding that maintenance. IntheabsenceofIFTproteins,ciliastructurefails anterograde andretrogradetransport,whichisessentialforcilia Anderson, 2006).Additionally, IFTproteinsarenecessaryfor signalling, andarerequiredforprocessingofGli3(Scholey and factors. They arerequiredtogenerateactive Gli2inresponsetoHh bound proteinSmoothenedandupstreamofGlitranscription analysis, IFTproteinsseemtoactdownstream of themembrane- IFT88 andIFT172].Basedonphenotypicbiochemical (Dync2h1 –MouseGenomeInformatics)andIFTparticlesubunits [e.g. intraflagellartransport(IFT)motorskinesin-2andDnchc2 these genesarerequiredforproperciliaformationandfunction signalling andthegenerationofventral neuralcelltypes.Several of Genetic screenshave identified additionalgenesrequiredforHh 2570 AJ237917 The Ft Irx3 RESEARCH ARTICLE Ftm mutation mightbeinvolved inHhsignalling.To clarify , ) and Irx5 (fantom; and Ftm Irx6 Fts Rpgrip1l Ftm (Peters etal.,2002).Development of , andthreeothergenes,named – MouseGenomeInformatics).This coding sequence.Thelinearizedvector was Ft mutation isdelayedandembryos – MouseGenomeInformatics) Ft/Ft Gli3 Ftm mutation ontotheirprogeny. F1 mutant miceweregenotypedas embryos. However, some Ftm Ft -null allele.The phenotypes. Ft Fts (fused , Fto interacting protein1)(Boylan andWright,2000;Hongetal.,2001). related proteinRPGRIP1(retinitis pigmentosaGTPase regulator called RIDdomain.Thisdomain was characterizedintheFtm- intermediate filament proteins(Gallicanoetal.,1998),andaso- domain, whichisproposedtoplayaroleinthebinding of involved inhomopolymerformation(Zhaoetal.,2003),oneC2 1A). Thesecomprisethreecoiled-coildomains,whicharepossibly which containsseveral protein-proteininteraction domains(Fig. BRL) at37°Cin5%CO and 1/100(v/v)L-glutamine(GibcoBRL)pen/strep in DMEM(GibcoBRL)complementedwith1%fetalcalve serum(FCS) BRL) at37°Cin5%CO 1/100 (v/v)L-glutamine(GibcoBRL)andpen/strep DMEM (GibcoBRL)supplementedwith10%fetalcalve serum(FCS)and stageE12.5.Bothweremaintainedin generated fromisolatedlimbbuds, andmesenchymallimbcells(MLCs)were stage E12.5(Ftmlittermates), Mouse embryonicfibroblasts (MEFs)weregeneratedfromsingleembryos, culture cartilage andbonesusingstandardAlcianBlueAlizarinRedstaining. performed withHematoxylinandEosinembryoswerestainedfor as described(Timmer etal.,2001).Morphologicalstainingsonsectionswere embryos andimmunohistochemistrystudyonfrozentissuewas performed Whole-mountRNA insituhybridizationon (Moormann etal.,2001). hybridizations onparaffin sectionswerecarriedoutaspreviously described 1-butanol overnight andtransferredintoparaffin forembedding.Insitu incubated in70,80,90and100%ethanol,respectively, for2hours,andthen paraformaldehyde for2to4hoursandwashed inPBS.Afterwards they were After dissectioninice-coldPBS,embryoswerefixed in4% Tissue processing andhistochemistry on scannedimages. loading. NIHImagewas usetodigitizeandnormalizethebandintensities incubated withanti-actinoranti-tubulin antibody(SantaCruz)ascontrolfor antibody (Wang etal.,2000)oranti-Ftm antibody. Themembranewas then Western blotanalysiswas doneessentiallyasdescribedusinganti-Gli3 Western blotting construct was organized suchthatthe mutationresultedina in murineEScellsbyhomologous recombination.Thetargeting The Targeted mutationofFtm RESULTS including partialgenepredictionsand/orESTsequences. derived fromDNA contigsgeneratedfromraw sequencedata(traces), NP_056087; NP_076368).SequencesofallotherFtmorthologues were were obtainedfrompublicdatabases(Accessionnumber:CAC87257; Protein sequencesofmouseandhuman In silicodata information isavailable uponrequest. with anFtmproteinfragmentcontainingtheRIDdomain.Detailed services). Antibodieswerepurified withNHSsepharose(Roche)conjugated fusion proteinencompassingtheFtm-RIDdomain(Pinedaantibody We generatedpolyclonalantibodiesbyimmunizingrabbitswithaGST-Ftm Antibodies 1994). Scanning electronmicroscopy was performedasdescribed(Suliketal., Scanning electron microscopy Olig2 (Chemicon). the NICHDandmaintainedbyTheUniversity ofIowa), Pax2 (Zymed) and Developmental Studies HybridomaBankdeveloped undertheauspicesof Informatics), Shh,Pax6 (monoclonalantibodieswereobtainedfromthe –MouseGenome (Sigma), actinNkx2.2,MNR2(Hlxb9 We alsousedantibodiesagainstacetylated To analysethefunctionof Ftm gene encodesaproteinof1264aminoacidsinlength, 2 2 . For ShhresponseanalysisMLCswereincubated and 1 ␮ Ftm /ml recombinantN-Shhprotein(Sigma). , wegeneratedatargeted mutation Ftm genes andofmurineRpgrip1 ␣ -tubulin (Sigma), Development 134(14) ␥ -tubulin

DEVELOPMENT antibody raisedagainsttheC-terminusofFtm.Ftmisabsentin Analyses of embryos Left-right asymmetrydefectsinFtm-deficient is requiredforseveral processesduringembryonic development. in theestablishmentofleft-rightasymmetry(Fig.1E,F).Thus, found anenlargement ofthe pericard, exencephaly anddisturbances unfused maxilliaryandnasaltissues(Fig.1D).Atearlierstages we includingmalformedmandibular structuresand were affected, fore andhindlimbs(Fig.1C).Additionally, craniofacial structures microphthalmia (eyes reducedinsize)andapreaxialpolydactyly homozygous forthe antibody. ( (D) Craniofacialabnormalitiesin arrowhead), reduction ofeyestructures andcraniofacial malformations. Ftm in comparisonwiththeirwild-typelittermates(leftside).(C)AtE18.5, Ftm embryos usingantibodiesraisedagainsttheC-terminus(Fig.1B). first coiled-coil-domain(Fig.1A).Ftmwas notdetectablein truncated proteinlackingallfunctionaldomainsofFtmexcept the Ftm isessentialforShhsignalling the right(Fig. 2C,D). Additionally, 100% of while inseveral position). Normally, thestomachdevelops intheleftbodyhalf, histological analysisrevealed heterotaxia(randomizedorgan of analysedembryos(Fig.1Fand datanotshown). Atlaterstages, inversed tailturningin39%andrandomizedheartlooping19% embryos ( the mutationin arrowhead marksthepositionwhere theprotein istruncateddueto domain (aa792-887)andanRID(aa1076-1264).Thered amino acids(aa)43-85,CC2aa264-375,CC3aa491-576],oneC2 embryos. Fig. 1.TheFtmprotein structure andthephenotypesof part ofthehearttubeafterloopingismarkedbywhitearrowhead. normal loopingdirection totheleft.Thefinalpositionofventricular dextrocardia (heartloopingtotherightbodyside)insteadof analysed. (F)AtE9.5,19%ofanalysedmutantembryosdisplayed exencephaly (blackarrowhead) are foundinmostoftheembryos tissues. (E)AtE10.5,pericard enlargement(whitearrowhead) and reduction ofmandibularstructures andunfusedmaxilliarynasal mutant embryosreveal polydactylyinfore andhindlimbs(white +/– mice show noobvious phenotype.However, embryos –/– C-F ( A ). Thelowerlanesshowtheloadingcontrols withanti-actin ) TheFtmprotein containsthree coiled-coildomains[CC1 ) Ftm Ftm Ftm Ftm –/– –/– mutant mice.( embryos (rightside)are shownatdifferent stages mutant embryosthestomachwas positionedto embryos betweenE9.5andE11.5 revealed Ftm mutation diedaroundbirthshowing Ftm B –/– ) Westernblotanalysisusingan embryos are shown,includinga Ftm –/– Ftm embryos Ftm Ftm –/– –/– Ftm –/– (Fig. 2E;datanotshown). In and eue I.f,fr ib l idlm;st,stomach;wt,wildtype. reduced (I).fl,fore limb;hl,hind unaffected in marker genes.Inwild-typeembryos(2-5somitestages),both hybridization andanalysedtheexpression ofleft-right-specific at themolecularlevel, weperformed whole-mountinsitu 1999; Liuetal.,2001).To confirm themorphologicalobservations misexpression (Menoetal.,1998;Lin1999;Tsukui stages (E).In left lateralplatemesoderminwild-typeembryosatthe2-to5-somite ofleft-rightmarkergene. pattern Loss of proper establishmentofleft-rightasymmetry. determination (Fig.2F;datanotshown). Thus, which indicatesdisturbancesinearlyevents ofleft-right both geneswereexpressed inabilateralfashion, expression was lost; digit inthehindlimb.( mutant limbs,visiblebytwoextradigitsinthefore limbandoneextra (F). ( Lefty1 pulmonary isomerismisknown fromleft-rightmutantssuchas developed onlytwo symmetricallunglobes(Fig.2A,B).Thisleft are affected in Fig. 2.Left-rightasymmetryspecificationandlimbdevelopment stomach waspositionedontheright(heterotaxia) (D).( in theleftbodyhalf(C),whilesome formed (R1andL1).(C,D)Inwild-typeembryosthestomachdevelops lobe (L1),whileinmutantembryos(B)onlytwosymmetriclobesare embryos lungsbranchintofourrightlobes(R1-R4)andoneleft mouse stainings atE15.5ontransversesections.(A)Inwild-type stainings revealed onetotwo extra digitsinfore limbsandoneextra characterized aspreaxialpolydactyly (Fig.2G).Bonecartilage polydactylous phenotype.Atlater stages,thisphenotypecouldbe andhind-limbbuds (datanot shown), suggestinga of fore- From E12.5onwards, disturbances inShhsignalling G Lefty ) Bone-cartilagestainingsatE18.5reveal apreaxial polydactylyin –/– 2 ( Ftm ( Ebaf Leftb Ftm Ftm Ftm –/– results inpreaxial polydactyly and ) and ) wereexpressed intheleftlateralplatemesoderm mutant limbbuds(H),whereas embryos, –/– H embryos. Ftm , I Shh ) AtE11.5,expression of –/– Pitx2 –/– embryos displayedabroadened shape , anddependsonbilateral Ftm Pitx2 ( shows abilateralexpression pattern A-D –/– RESEARCH ARTICLE Ftm ) HematoxylinandEosin is exclusivelyexpressed inthe embryos, thelateralityofthis mutant embryosthe Ptc1 Shh Ftm expression is E appears tobe is requiredfor , F ) Expression Pitx 2571 Pitx 2 2

DEVELOPMENT Foxa2 (green arrowhead) andinthefloorplate(bluearrowhead) (A),and (F). In expressed withinthewholeneuraltubeexceptmostventralregion and themesenchymesourrounding thenotochord (E),and lost (D).Inwild-typeembryos, restricted tothedorsalneuraltube.In located intwolateraldomains(M),andPax6-expressing cellsare cells are greatly reduced (L).Inthewildtype,MNR2-positivecellsare show alossofShhprotein inthefloorplate(K),andNkx2.2-expressing tube (expression borders are markedwithwhitebars)(P). expressing cellsare expandedintothemostventralpart oftheneural MNR2-postive cellsare foundinaspottedfashion(O),andPax6- are ectopicexpression of digit inhindlimbs(Fig.2G).Known possiblecausesofpolydactyly affected in is Fig. 3.Floorplateinductionandventralneuraltubepatterning 2572 stage E11.5.Inthewildtypemouse, found adjacenttothefloorplate(J;whitearrowhead). and notochord (green arrowhead) (I),andNkx2.2-expressing cellsare embryos, Shhprotein isdetectableinthefloorplate(whitearrowhead) bars) (H).( includingthemidline(expression borders are markedwithred tube, positive cellsare expandedintothemostventralpartofneural Shh Ptc1 Joyner, 1993;Yang etal.,1998).We therefore examined limbs forectopic expression of expression inthefloorplateisabsent(C),and and Ftm expression isrestricted tothefloorplate(B).In RESEARCH ARTICLE Gli1 –/– I-P Ftm mro,expression of embryos, ) ImmunohistochemistryatstageE11.5.Inwild-type were alsopreviously foundtobeupregulated (Huiand –/– embryos. Shh ( Ptc1 A-H and/or ) Insituhybridizationsonsectionsat Shh is expressed withintheneuraltube Ptc1 Shh Ftm Hh and is strongly reduced (G). is expressed inthenotochord –/– activity, and Ptc1 mro,onlyafew embryos, . Foxa2 Shh Ftm Ftm Hh was expressed –/– expression is –/– target genes Irx3 Ftm embryos, embryos is mutant Irx3 - 2001). Accordingly, in a functionin neural tubepatterning(Persson etal.,2002). differentiation, whereas unprocessedGli3doesnotseemtohave antagonize floorplateinduction andtoblockmotoneuron of theneuraltube(Briscoeand Ericson,2001).Gli3Risknown to (Gli3R) toGliactivator (Gli1,Gli2),whichleadstothepatterning signalling isrealizedbythe relative amountofGlirepressor transcriptional repressorsor activators. TheoutcomeofHh Gli proteinsaremediatorsofShhsignallingandact as found toberepressedbyhighamountsof expressed inmotoneuronprogenitors(Fig.3F,N). Bothgeneswere expressed inV0,V1andV2progenitorsPax6 isadditionally plate andgive risetoV3interneurons(Fig.3J).In Nkx2.2-positive cellsarenormallypositioneddorsaltothefloor was furtherinvestigated byanalysesofventral neuraltubemarkers. in thesupplementarymaterial). within theneuraltubewerealsodetectablebyRT-PCR (seeFig.S1 reduction ofShhsignalling.ReducedamountsPtc1expression the mesenchymesurroundingnotochord,thusconfirming the found areductionofPtc1expression intheneuraltube as wellin notochord, weanalysedPtc1expression (Fig.3G).Interestingly, we failure mightbetheresultofalteredShhsignallingfrom successful inductionofthefloorplate(Fig.3D).To testwhetherthis addition, absenceofFoxa2 expression indicatedafailure of able todetectany Shhproteinintheventral neuraltube(Fig.3K).In Fig. S2inthesupplementarymaterial).Nevertheless, wewerenot asdemonstratedbywhole-mountinsituhybridizations(see axis, tube (Fig.3C).Thislosswas onlypartialalongtheanteroposterior expression ofShhinthenotochord,but alossintheventral neural 1998). Insituhybridizationsoncrosssectionsrevealed normal and thefloorplate(Fig.3A,I)(Roelinketal.,1995;Dodd in two ventral signallingcentresalongthemidline, thenotochord Shhisexpressed precise measurementofeffects onShhsignalling. we analysedpatterningoftheneuraltube,astructureallowing To confirm thatlossofFtmleadstoareductionShhsignalling, Ftm isnecessaryforfloorplateinduction detectable. Likewise, (Fig. 2H;datanotshown), andnoectopicexpression domainwas normally intheposteriormesenchymeboth,atE10.5andE11.5 Ftm entire neuraltubecannotbedifferentiated bythisanalysis. plate (Shhsource)orduetoimpairmentofHhsignallingwithin the of theneuraltubeisonlyaconsequenceabsencefloor loi h eta iln Fg O.Px and also intheventral midline(Fig.3O).Pax6 embryos. MNR2-expressing cellswerefoundinaspotted fashion Like Nkx2.2,MNR2expression was strongly reduced in positioned dorsolateraltotheNkx2.2expression domains (Fig.3M). expression isindicative formotoneurons, whicharenormally single scatteredcellswerefound(Fig.3L,datanotshown). MNR2 Nkx2.2-expressing cellswerestronglyreduced.Onlyin afew cases, signalling isimpairedin (Fig. 3H,P).Thus,theseresultsstrengthentheassumptionthat Hh domains wereexpanded intothemostventral part oftheneuraltube suggesting alackofincreased in limbbuds of material). ThesedataindicateareductionofShhsignaltransduction be confirmed byRT-PCR analysis(seeFig.S1inthesupplementary reduced numberof expression in Absence ofafunctionalfloorplateandthereforeShhsignalling acts upstream ofGli3 Ftm Ftm Ptc1 mutant limbbuds was reduced(Fig.2I).The –/– Ptc1 embryos. Ftm Ftm transcripts in was notectopicallyexpressed (Fig.2I), –/– –/– embryos. Whetheralteredpatterning embryos, Pax6 and Hh signalling. Moreover, Ftm Shh mutant limbbuds could Development 134(14) (Briscoe andEricson, Ftm Irx3 Irx3 –/– Ftm expression embryos, are both mutant Ptc1

DEVELOPMENT cells (L)seemtobecompletelyrestored. midline andlocatedintwolateraldistinctdomains.Pax2-expressing expressing (J)andNkx2.2-expressing (K)cellsare absentfrom the fewer cellscrossing themidline(I).( expanded intomore ventralpartswithintheneuraltube,butthere are increased, butthesecellsstillcross themidline.Pax2-expressing cellsare the numberofMNR2-positive(G)andNkx2.2-positive(H)cellsis the dorsalregion oftheneuraltube(C).( expressed adjacenttothefloorplate.Furthermore, Pax2isexpressed in wild-type embryos,MNR2(A)isexpressed lateraltoNkx2.2(B),whichis stage E11.5mouseembryosfrom fourdifferent genotypes.( abrogating Gli3function. interestingly, Ftm 1998). Ftm carry anullalleleofGli3(HuiandJoyner, 1993;Büscheretal., tube. Therefore,wecrossedFtmmutantmicewithXtmice,which mispatterningoftheneural embryos shouldrescuetheobserved of Ftmaffects Shhsignalling,areductionofGli3inFtmmutant combined mutantembryos(JacobandBriscoe,2003).Ifthe loss alleviated byabrogatingGli3function,asshown inShh neurons (motoneuronsandseveral classes ofinterneurons)canbe activator). Thus,inShhmutantembryos thelossofventral secondly, Gli1andGli2activation isdecreased(lessGli processing ofGli3isnotinhibited(morerepressor);and Absence ofShhsignallinghastwo consequences:firstly, Ftm isessentialforShhsignalling including themidline(Fig.4C,F). Interestingly, combinedFtm Shh, was foundtobeexpanded intothemostventral part, In addition,Pax2 expression, whichisnegatively regulated by (MNR2-positive cells)andNkx2.2-expressing cells(Fig.4D,E). most ventralpartoftheneuraltube(F).( Nkx2.2-expressing cells(E).Expression ofPax2isexpandedintothe embryos showastrong reduction ofMNR2-positivecells(D)and in Fig. 4.Rescueofneuraltubepatterning was reducedincomparison withFtm type (Fig.4G,H).Inaddition, the expansion ofthePax2 domain cells increased,but were stillreducedincomparisonwiththewild ventral neuraltube.The numbersofMNR2-andNkx2.2-positive Xt/+ embryosrevealed apartialrescueofthemispatterningin –/– embryos show astrongreductionofmotoneurons –/– ; Xt/Xtembryos showed anearly complete Shown are immunohistochemistrydataat J-L ) In G-I D-F –/– Ftm ) In ) Bycontrast, embryos (Fig.4I).Most –/– Ftm Ftm ; Xt/Xt –/– –/– ; embryos by Xt/+ embryos, MNR2- Ftm embryos, A-C –/– –/– ; Gli3 ) In –/– –/– ; contrast, inFtm Gli3-83 andlow amountsoffull-lengthGli3-190(Fig.5A).By Gli3-190 was processedintoGli3-83,leadingtohighamounts of be increasedinFtm to wild-typelevels. Thus,thetotalGli3protein amountseemsto 1 (Fig.5A,B).However, theamountofGli3-83seemstobeequal dramatically increased,leadingtoaGli-190:Gli-83ratioclose to in Gli3-190 isincreased. Notethatthere isanincrease intotalGli3protein the amountofGli-83isequaltowild-typelevels,but Gli3 tubulin antibody. ( in wild-type, normalization. ( in comparisonwiththewildtype.ThelowerpartshowsHprt wild-type and 2000). Inbothwild-typeandFtm activator (Gli3-190)andrepressorform (Gli3-83)(Wang etal., against theN-terminusofGli3thatrecognizesboth function, weanalysedGli3processing.We usedanantibody data alsoconfirm thatFtmisacomponentofShhsignalling. 4L). Theseresultsshow thatFtmactsupstreamofGli3,andthe dorsalregions (Fig. midline andtheexpression was restrictedto seemed tobefullyrestored,positive cellswereabsentfromthe distinct lateraldomains(Fig.4J,K).Furthermore,Pax2 expression expressing cellswereabsentfromthemidlineandlocated intwo rescue ofventral neuronalsubtypes.MNR2-and Nkx2.2- and onlysmallamountsofGli3-190are detectable.In themajorityofGli3protein wasprocessed toformGli3-83, embryos, Gli3-190 andGli3-83inE11.5embryos.Inwild-type 190/Gli3-83 ratioinmice. Fig. 5.Ftmisnecessaryfortheestablishmentofaproper Gli3- RNA level was increasedinFtm RNA levels. Asshown byRT-PCR analysis(Fig.5C), theGli3- increase ofGli3proteincanbe correlatedwithincreasedGli3 of Gli3RNA in mutantandwild-typeembryostoseewhetherthe is negatively regulated byShh (Schweitzeretal., amount ofGli3transcriptsinFtm relative Gli3-RNA levels revealed anearlytwofold increaseinthe embryos. Theamountof genotype havebeenanalysed.( Ftm To directlyaddressthe questionofhow Ftmmayeffect Gli3 transcripts isincreased nearlytwofold. –/– embryos. Thelowerpartshowstheloadingcontrol withanti- Ftm Ftm D +/– ) Comparisonofrelative –/– B ) GraphicalevaluationoftheGli3-190/Gli3-83ratio and –/– embryos ( –/– embryos, theamountofGli3-190was Ftm embryos. We therefore analysedthelevels Gli3 –/– ( A transcripts isincreased in ) Immunoblotsshowtheamountsof n embryos. Atleastthree embryosofeach C =3). In ) RT-PCR analysisofRNAfrom E11.5 +/– –/– –/– RESEARCH ARTICLE Ftm embryos ofstageE11.5,most embryos. Quantification of embryos (Fig.5D).AsGli3 Gli3 –/– mro,thenumberof embryos, transcription levelsin Ftm Ftm Ftm –/– –/– embryos, +/– embryos 2573

DEVELOPMENT staining marksthe nucleibyDAPI. localized with By contrast,in not overlapwithacetylated mesenchymal cellexhibitsciliastained byacetylated each on sectionsoflimbmesenchymeat E11.5.Inthewildtype(left), test thishypothesis,weinvestigated ciliain onitssurface,whereas in eachcellpossessesasingle images atE7.5.Inthewild-typenode(left), ( cilia isreduced andthecilia are malformed.( normal cilianumbersinmice. Fig. 6.Ftmislocatedatthebasalbodyandnecessaryfor observed in Anderson, 2006).Therefore,weconsideredthephenotypes during embryonicdevelopment (Hirokawa etal.,2006;Scholey and formation affect symmetrybreakingandShhsignal transduction different developmental processes. Defectsinciliafunctionand Cilia have beenidentified asspecializedstructuresnecessaryfor Loss ofFtmaffects ciliafunction embryos. transcripts confirmed theimpairmentofShhsignallinginFtm 2000; Wang etal.,2000),theincreaseofnumberGli3 2574 to thewildtype,numberofciliain single ciliaprotrudesfromtheapicalsurface ofeachcell.Incontrast we examined themotileprimaryciliawithinnode.Normally, a (Huangfu andAnderson,2005;Haycraftetal.,2005).Staining cells ofthelimbbuds andonepithelialcellsintheneuraltube found tobereduced(Fig.6A).Ciliaarealsopresentinmesenchymal C ImmunostainingsonMEFs.(i)Ftm (red) islocatedatcilia,butdoes ) RESEARCH ARTICLE Ftm ␥ Ftm -tubulin (green) inthebasal bodyofthecilia.Theblue mutant embryostobecausedbyciliadefects.To –/– mro rgt,thenumberofciliais reduced. embryos (right), ␣ -tubulin (green). (ii)Ftm(red) isco- Ftm ( A ) Scanningelectron microscopy –/– mro rgt,thenumberof embryos (right), Ftm B ) Immunohistochemistry Ftm mutant embryoswas –/– ␣ -tubulin antibody. embryos. First, –/– cilia lengthin left part;datanotshown). Notably, wefoundaslightenhancementof developed oneciliainbothwild-typeand response in of theHhtarget genes MLCs wereincubatedwithShhproteinandanalysedfortheactivation os prp*35 *Ftm-related protein. the supplementarymaterial. from aa250to930wasused.Forthemultiplesequencealignment, seeFig.S3in For thepairwisesequencecomparisons, thecentralpartofmouseFtmprotein Mouse Rpgrip1* Hydra magnipapillata Nematostella vectensis Lottia gigantea Capitalla Strongylocentrotus purpuratus Branchiostoma floridae Danio rerio Homo sapiens Mus musculus Table 1.HomologyofFtmproteins against phase. Thecomparisonofwild-typeand maintenance in tube (datanotshown), suggestingdisturbedcilia assembly and/or indicated fewer cilia,bothinlimbbuds (Fig.6B)andintheneural whether Hhsignallingwas affected in MEFs) inG issue, weisolatedMLCsandMEFsofwild-type indirect consequenceofalteringtheciliastructure.To addressthis affected Hhsignalling(e.g.processingornucleartransport)isan and Anderson,2005).ThequestionarisesastowhetherthelossofFtm affect ciliastructureandthereforeHhsignallinginmammals(Huangfu As shown byrecentstudies,lossofseveral cilia-associatedproteins Ftm regulates Hhsignalling realization ofthisfundamentaldifference. Anderson, 2006),Ftmcouldbeoneoftheproteinsimportantfor arthropod memberisnotassociatedwithcilia(Huangfuand nematodes. Asitwas shown for material). Strikingly, wewerenotabletofind Ftminarthropodsand only inhighervertebrates (Table 1;seeFig.S3inthesupplementary toRPGRIP1,acilium-relatedprotein,whichisfound see Fig.S3inthesupplementarymaterial).Furthermore,Ftmshows revealed thatFtmisconserved fromcnidarianstohumans(Table 1; whether Ftmcanalsobefoundinotherspecies.Insilicoanalyses basal bodyofcilia. found tobeco-localized(Fig.6Cii).Thus,Ftmisacomponentofthe the basalbody, suchas basal bodyofthecilia,weanalysedproteinsthatareindicative for clarify whetherthislocalizationisatthedistaltiporproximal localizes tooneendoftheciliaryaxonemeinMEFs(Fig.6Ci).To Interestingly, useofanantibody againstFtmrevealed thatFtm Ftm isabasalbodyprotein, highlyconservedin and measuredtheirabilitytogeneratecilia,whicharegeneratedinG responsiveness inG Ptc1 analysed Shhtarget geneinduction inproliferating(non-ciliated)wild- To testwhethertheShh responseisexclusively dependent oncilia,we As Ftmseemstobeessentialforciliafunctioninmice,weasked and ␣ Gli1 p 47 sp. -tubulin, whichmarkstheciliaryaxoneme,clearly Ftm 0 ess%identity versus phase revealed nodifferences inciliogenesis:eachcell expression (Fig.7A,lower leftpart;Fig.7B, panels). –/– Ftm Ftm MLCs was stronglyreduced,indicatedbydecreased –/– 0 –/– phase ofwild-typeand Ptc1 MLCs andMEFs(datanotshown). To test embryos. ␥ -tubulin. Indeed,Ftmand and Gli1 Drosophila . Incontrasttowildtype,theShh Ftm Ftm Ftm –/– that Hhsignallinginthis –/– Development 134(14) cells, wemeasuredShh –/– Ftm cells (bothMLCsand cells (Fig.7A,upper –/– MLCs. Ciliated Ftm ␥ -tubulin were –/– embryos 43 47 46 48 52 54 89 0

DEVELOPMENT that lackciliaand thereforefunctionalShhsignalling (Huangfuet E10.5 onwards, aphenomenon alsodescribedforIFTmutantmice development wewerenotabletodetectShhintheneuraltubefrom Although Shh function(BriscoeandEricson, 2001;JacobandBriscoe,2003). structure extensively usedforquantitative and qualitative studiesof interpretation, weinvestigated patterningoftheneuraltube,a Tsukui etal.,1999).To provide detailedevidence forthis Impaired Hhsignalling inFtm All observed midlinedefectsin target geneswas reducedto30%inthese able togenerateciliaonevery cell.However, theinductionofShh of cilia.Nevertheless, intheabsenceofFtm,primarycellswerestill In thisstudyweshow thatFtmisaproteinlocatedatthebasalbody DISCUSSION necessary fornormalcilium-relatedShhsignalling. interferes withtheefficiency ofsignaltransduction.Thus,Ftmis completely abolishShhsignalling.However, lossofFtmseverely Furthermore, lossofFtmdoesnotaffect ciliogenesisanddoesnot to thephenotypesdescribedfor similar can beexplained byareductionorabsenceofHhsignalling, heart looping,left-lungisomerism,dorsalizationoftheneuraltube) remaining inductionofShhtargets in that Shhsignalsareexclusively transduced byciliaandthatthe Shh (Fig.7A,rightpanels;Fig.7B,panels).Thesedataconfirm MLCs showed noinductionofShhtarget genesafterstimulationwith type and Ftm isessentialforShhsignalling changes detectablein This relationofFtmtoShhsignallingisinlinewiththephenotypic transduction cascadenecessarytoelicitaquantitative Shhresponse. we concludethatFtmisanintegral partofthecilia-associatedsignal of Ptc1 and Ftm Shh Gli1 –/– MLCs (Fig.7A,Bright).Bothwild-typeand was absolutelydependentonthepresenceofcilia, was expressed in thenotochordthroughout Ftm –/– embryos. Shh Ftm –/– Ftm –/– embryos (Chiangetal.,1996; –/– –/– embryos (e.g.randomized Ftm embryos MLCs iscilia-dependent. –/– cells. Asinduction Ftm –/– be aconsequence ofthechangeinratioGli3-190 toGli3-83, 2005). Thepolydactylousphenotype ofIFTmutantmiceappearsto MLCs. Therefore,in in to acompletelossofdigitidentity (Niswander, 2003).Thedefects 83 causesasevere polydactyly(two to threeextra digits)andleads patterning isGli3-83(Niswander, 2003). AcompletelossofGli3- changes digitnumberandidentity. Themajormediatorinlimb whereaslossofGli3dramatically no effect onlimbpatterning, indifferent toGliactivator function.MutationsinGli1andGli2have tube asthatinIFTmutantmice. Gli3 repressorfunction,leadstothesamemispatterningofneural to areductioninShhresponsiveness, incombination withremaining Ftm identity, andinIFTmutantembryosapolydactylycomparable to limbs onlyonetotwo extra digitswereformed, withacleardigit responsiveness isstronglyreducedin (Haycraft etal.,2005).Similarly, wecouldshow thatShh responsiveness andafailure ofHhtarget geneactivation byGli2 of MLCspolarismutantmiceshowed acompletelossofShh (Haycraft etal.,2005;LiuMay2005).Analysis addition, Gliactivator functionandShhresponsewas impaired in IFTgenes(e.g.polaris)negatively influenceGli3processing.In similar mechanism.Biochemicalanalysesrevealed thatmutations the neuraltubeof al., 2003;Haycraftet2005;Liu2005).Asaconsequence, found in embryos (Huangfuetal.,2003).Thesamepartialrescuewas also partially berescuedbydepletionofGli3incombinedmutant neural tubeinpolaris( Furthermore, ithadbeenshown previously thatmispatterningofthe consequently motoneuronsandV3neuronswerereduced. embryos:thefloorplatewas lost,and to thatseeninIFTmutant Ftm In contrasttoneuraltubepatterning,limbdevelopment israther –/– –/– embryos was described(Haycraft etal.,2005;Liu panels) andproliferating (rightpanels)wild-typeand and each wild-typeand ( induction ofShhtargetgenesafterstimulation. response inG Ftm antibodieswere notusedinthisstaining.Lowerpart:Shh by acetylated the cytoplasmisenrichedwithcytoskeletaltubulins,marked treatment withShh(wild-typeMLCs1.1; changes intherelative expression levelsof Shh protein for24hours.InG induction of mice. proliferating (non-ciliated)wild-typeand induction of induction reveals thatG MLCs afterShhstimulation.Thequantificationof Fig. 7.CiliogenesisandShhsignallingin Proliferating (non-ciliated)wild-typeand increase of proliferating wild-typeand in red) ispresent onlyinwild-typeMLCs.(Rightpanels)In by acetylated (left) andproliferating (right)MLCs.(Leftpanels)InG experiments. wt,wildtype. Data represent thequantificationofthree independent phase B and IFTmutantembryosweremuch lesssevere. In Ftm Quantificationof ) Ftm –/– ( Ftm A ; –/– ) Upperpart:immunohistochemistryonG Gli3 Ftm –/– MLCs. G Ptc1 Gli1 Gli1 MLCs showanincrease ofonlyafactor3.1. Ftm 0 ␣ ␣ –/– –/– Ift88 -phase (left)andproliferating (right)wild-type -tubulin antibody(green staining).Notethat -tubulin antibody(green staining).Ftm(stained rncit yafco f94 whereas G transcripts byafactorof9.4, embryos was mispatternedinasimilarway –/– and and combined mutantembryos,suggestinga 0 ) andwimple( Ftm embryos, reducedactivator functiondue -phase wild-typeMLCsshowastrong Ptc1 Ptc1 Ptc1 –/– 0 -phase wild-typeMLCsshowan -expression levelsinG cell exhibitsasinglecilium,detected after incubationwithrecombinant is strongly reduced. Both, Ftm RESEARCH ARTICLE Ftm 0 –/– -phase Ls ciliaare absentand MLCs, –/– Ift172 embryos andincultured Ftm Ftm Ftm ) mutantmicecould Ftm –/– Ptc1 –/– Ftm –/– MLCs, the –/– 0 MLCs showno MLCs showno upon -phase (left MLCs 0.8). –/– Ptc1 0 cells in phase 0 Ftm phase, Ftm 0 –/– 2575 - –/–

DEVELOPMENT mutant cells,100%of has beenshown onlyfor this expression (Schweitzeretal.,2000;Wang etal.,2000).Sofar impaired andwould beexpected toleadanincreaseof et al.,2005;Haycraft2005).InbothcasesHhsignallingis polydactylous phenotypeanddorsalizationoftheneuraltube(May contrast topolarismutantembryos(Haycraftetal.,2005), loss ofShhresponsiveness (Haycraftetal.,2005).Furthermore,in responsiveness inMLCs,whereasalossofpolariscausescomplete responsiveness. AlossofFtmleadstoastrongreductionShh but strikinglythey show quantitative differences inShh Ftm andIFTproteinsarebothnecessaryforproperHhsignalling, Ftm andcilium-related Hhsignalling 2007) supportsthisinterpretation. amount ofGli3-190.ArecentstudybyWang etal.(Wang etal., the ratioofGli3-190toGli3-83ischangeddueanincreased acetylated cells possesscilia,whicharedetectablebyantibodiesagainst revealed equallevels ofGli3-83in due toimpairedGli3processing.Althoughourwesternblotanalysis 2576 Gli3-190 toGli3-83inIFTmutantsand Nevertheless, theconsequencesaresame:changesinratioof amount ofGli3-190isincreaseddueto tissues, suchastracheaandolfactory epithelium,in that Ftmisnotessentialfor ciliogenesis. Interestingly, certain moderate malformationsoftheciliastructure,whichclearlyshows (MLCs andMEFs)showed noreductionincilianumberandonly maintenance inasimilarfashion. However isolatedprimarycells cilia number. Therefore,Ftmcouldbenecessaryforcilia in specific tissuessuchasthenode,andalsoleadsto areductionin (Pawlyk etal.,2005).ThelossofFtmcausesmalformations ofcilia maintaining theintegrity ofthisspecializedcilium-relatedstructure in retinitispigmentosa,suggestingafunctionofthisprotein in Zhao etal.,2003).AbsenceofRPGRIP1inmiceandhumansresults retroflagellar transport(Boylan andWright,2000;Hongetal., 2001; cilium. Thisinteractionisnecessaryforciliastability and pigmentosa GTPase regulator) tothebasalbody ofthisconnecting outer andinnersegment. RPGRIP1anchorsRPGR (retinitis cells intheretina,especiallyatconnectingciliumbetween 2002; Pawlyk etal.,2005).RPGRIP1functionsinphotoreceptor pigmentosa, adegeneration ofphotoreceptorcells(Mavlyutov etal., Mutations ofRPGRIP1causethehumandisorderretinitis Sequence analysisrevealed FtmtobehomologousRPGRIP1. Ftm anditsrelative RPGRIP1 reduced expression ofHhtarget genes(suchas slight changesinthearchitectureof allowing efficient cilia-dependentShhresponse.Aswenoticed Thus, Ftmisnotnecessaryforthegenerationofciliabut ratherfor for ciliamaintenance. that afullyfunctionalcilia-coupled signallingpathway isnecessary which morphogeneticprocesses take place.Thisfinding suggests unpublished). Thus,ciliaarereduced onlyinstructures/tissues show nochanges incilianumber(UlrichDirksandU.R., Ftm 83. AlossofFtmseemsnottoaffect theefficiency ofprocessing.In increases theamountofGli3-190anddecreasesGli3- experiments didnotgive any indicationforthishypothesis. consequences onthehalf-lifeofcilia.However, ourcellculture a roleinthestabilityorshapeofcilia,whichcouldhave IFT functionisnecessaryforproperGli3processingandthereby –/– RESEARCH ARTICLE embryos theGli3-83level appearstobenormalandthe ␣ -tubulin. Inaddition,andagainincontrasttopolaris Ftm Ftm –/– -negative embryos. MLCs inculturedogeneratecilia. Ftm Ftm –/– –/– Ftm cilia, Ftmmightalsoplay and wild-typeembryos, –/– embryos resultina Gli3 Ftm –/– expression. Ptc1 embryos Ftm Gli3 ), a –/– Dai, P., Akimaru,H.,Tanaka, Y., Maekawa,T., Nakafuku,M.andIshii,S. Corbit, K.C.,Aanstad,P., Singla,V., Norman,A.R.,Stainier, D.Y. andReiter, Ding, Q.,Motoyama,J.,Gasca,S.,Mo,R.,Sasaki,H.,Rossant,J.andHui, C. Grotewold, L.andRüther, U. Götz, K.,Briscoe,J.andRüther, U. Haycraft, C.J.,Banizs,B.,Aydin-Son, Y., Zhang, Q.,Michaud,E.J.and http://dev.biologists.org/cgi/content/full/134/14/2569/DC1 Supplementary materialforthisarticleisavailableat Supplementary material grant toB.W. (R01CA111673). imaging. ThisworkwassupportedbytheDFG(SFB590)toU.R.andanNIH the manuscript.Furthermore, wethankRüdigerRiehlforelectron microscope Hillforcriticalreading of We thankJürgenKnobloch,JuliaFischerandPatrick point toinvestigate vertebrate-specific Hhsignalling. andthusmaybeusedasanentry seems tobeoneoftheseproteins, to gaintheabilityelicitafullandrobust Shhresponsiveness. Ftm we speculatethatasubsetofbasalbodyproteinsisessentialforcilia loss ofFtmleadstoaquantitative impairmentoftheHhresponse, specific roleinHhsignaltransductionassociationwithcilia.As in arthropodshasyetnotbeenshown, suggesting thatFtmplaysa (Basto etal.,2006).TheconnectionbetweenHhsignallingandcilia are presentonlyinspecific neuronalsubtypesandinspermcells arthropods andnematodes.Insuchas Surprisingly, wewerenotable todetectFtmhomologuesin stressing theimportanceofFtmingeneralciliumfunction. expressed intheeye, whereasFtmisubiquitouslyexpressed, isexclusively (datanotshown). 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