homeostasis. Anumberofreagentsknown toinhibitcholesterol by sensitivity oftheresponsetoperturbationcholesterol kinase A(PKA)(Hammerschmidtetal.,1996). regulated byprotein the activity oftheGliproteinisnegatively activation of signaling cascadebetweenthereliefofSmoinhibitionand the induce downstream target genes.Atpresent,thedetailsofHh expressed duringmidlinedevelopmentin activity istriggered, transducing theHhsignaltointeriorofcell.OnceSmo the seven-pass transmembraneproteinSmoothened(Smo),thereby Beachy, 2004).Thisbindingrelieves theinhibitoryeffect ofPtcon transmembrane protein(InghamandMcMahon,2001;Lum (Chen etal.,2004;Lewis etal.,2001;Zeng2001). essential toensureeffective potency andrangeforHhsignaling to productionofasolublemultimericproteincomplex thatis al., 2001;Pepinsky etal.,1998)andtheselipidmodifications lead also palmitoylated atahighly-conserved Cysresidue(Chamounet the CterminusofHhligand(Porteretal.,1996).ligandsare occurs concomitantlywiththecovalent attachmentofcholesterolto C-terminal cleavage product(Leeetal.,1994).Autoproteolysis generate two matureproteins,aN-terminal‘signaling’ligandand proproteins autoproteolyticallycleave their ~45 kDaprecursorto somite development anddigitformation.Aftertranslation,Hh variety ofprocesses,includingestablishmentmidlinestructures, The membersoftheHedgehog(Hh)family areinvolved inawide INTRODUCTION KEY WORDS: but mayalsoinvolvedefectsinDHCR7resultingderepressionofShhsignaling. human conditionSLOSiscausednotonlybydisruptionoftheenzymaticroleDHCR7asareductaseincholesterolbiosynthesis level ordownstreamofSmoothened(Smo)andaffects intracellularHhsignaling.Ouranalysisalsoraisesthepossibilitythatt cell. We presentgain-andloss-of-functionanalysessuggestingthatDHCR7functionsasanegativeregulatorofHhsignalingat toaffect Hhsignalingintherespondi regulator ofHhsignalingthatactstoregulatethecholesteroladductionligandor the preciseroleofDHCR7deficiencyinthisdiseaseremainsmurky. We reportthat developmental malformationsattributedtoSLOSoccurintissuesandorganswhereHhsignalingisrequiredfordevelopment,but by defectsin7-dehydrocholesterolreductase(DHCR7),anenzymecatalyzingthefinalstepofcholesterolbiosynthesis.Many Cholesterol regulatesHedgehog(Hh)signalingduringearlyvertebratedevelopment.Smith-Lemli-Opitzsyndrome(SLOS)iscaused Tetsuya Koide*,Tadayoshi Hayata cholesterogenic enzyme7-dehydrocholesterolreductase Negative regulationofHedgehogsignalingbythe Development 133,2395-2405(2006)doi:10.1242/dev.02393 DEVELOPMENT ANDDISEASE Accepted 4April2006 ‡ † Institute, 2-1Hirosawa, Wako-shi, Saitama351-0198,Japan *Present address: LaboratoryofNeurobiology ofSynapses,RIKENBrainScience Irvine,CA92697-2300,USA. University ofCalifornia, Department ofDevelopmentalandCellBiology, andDevelopmentalBiologyCenter, Tokyo 101-0062,Japan Institute, Tokyo MedicalandDentalUniversity, 2-3-10Kandasurugadai,Chiyoda-ku, Author forcorrespondence (e-mail:[email protected]) Present address: DepartmentofMolecularPharmacology, MedicalResearch Involvement ofcholesterolinHhsignalingwas furthersuggested Hh proteinsbindtotheirreceptorPatched (Ptc),a 12-pass Gli/Ci Xenopus are notwellunderstood.However, invertebrates, Gli/Ci , Midline,Cholesterol, Floorplate,Smith-Lemli-Opitzsyndrome (SLOS),Sonichedgehog transcription factors areactivated that † Xenopus and KenW. Y. Cho embryos. DHCR7haspreviouslybeenimplicatedtofunctionasapositive ‡ Guy, 2000).However, direct evidence demonstrating thatimpaired abnormalities seeninindividuals withSLOS(Cooperetal.,2003; deficiency istheroot causeofsomethedevelopmental interfere withnormalHhsignaling, andhave proposedthatthis embryogenesis. affect thedevelopment ofvarious tissuesand organs during sterol precursorsintissues,eitherofwhichareproposed,turn, to include (1)low levels ofcholesteroland/or(2)theaccumulation cholesterol biosyntheticpathway leadstothesemalformations (Kelley andHennekam,2000).Explanations ofhow blockadeofthe malformations inthelimbs,heart,kidneys, pancreasandgenitals addition tomidlinedefects,individuals withSLOSalsosuffer from that resultsfrommutationsin Syndrome (SLOS),whichisanautosomalrecessive humandisorder been observed in~5%ofindividuals with Smith-Lemli-Opitz al., 1998;Wassif etal., 1998).MildermanifestationsofHPEhave by reducing7-dehydrocholesterol(7-DHC)tocholesterol(Fitzky et catalyzes thefinalcholesterogenicstepinendoplasmicreticulum Aubry, 1966).DHCR7isanine-passmembraneproteinthat activity of7-dehydrocholesterolreductase(DHCR7)(Rouxand which inhibitscholesterolbiosynthesisbyinhibitingtheenzymatic observed intheoffspring ofpregnant ratstreatedwithAY-9944, halves oftheforebrainatmidline.Similarly, HPEisalso patterning defectcharacterizedbyincompleteseparationofthetwo holoprosencephaly (HPE)(InghamandMcMahon,2001).HPEisa congenital defects,includingbasalcellcarcinoma,polydactylyand associated withabnormalHhsignaling. could resultinthetypesofdevelopmental defectsknown tobe embryogenesis, withtheimplicationthatdisturbanceofthisprocess results pointtotheimportanceofcholesterolregulation during vitro (Cooperetal.,1998;Incardona1998).Together, these an inhibitorofcholesterolbiosynthesis,alsoblocksShhsignalingin specifically bindtoSmo(Chenetal.,2002).Additionally, AY-9944, 1998). Amongtheseagents,cyclopamine hasalsobeen shown to block Hhsignalinginchickneuralplateexplants (Cooper etal., transport, suchascyclopamine, jervine,progesteroneand U1866A, Several groups have shown thatcholesterol deficiency can Aberrant Hhsignalingunderliesanumberofhumandiseasesand DHCR7 and Sonic Hedgehog DHCR7 RESEARCH ARTICLE (Kelley etal.,1996).In ( Shh ) areco- he ng the 2395 ,

DEVELOPMENT EcoR h mutationDHCR7 the animal model.Inthefirst,exon 8was removed, generating embryogenesis, two DHCR7mutantshave beengeneratedinthis (Cooper etal.,1998;Cooper2003). fibroblasts hasproven effective inblockingsteroladductionofShh of cellswithAY-9944 nordeletionofDHCR7inmouseembryonic DHC (Witsch-Baumgartner etal.,2000).Finally, neithertreatment phenotype andtheplasmalevels ofcholesterolanditsprecursor7- there arealsoinconsistenciesbetweentheacutenessofSLOS cholesterol andincreasedlevels intheclinicalseverity ofSLOS, although somecorrelationsexist betweendecreasedlevels ofplasma of DHCR7activity duringearlydevelopment. Additionally, to earlyembryosmaternally, it isdifficult toassesstherequirement Hh signalingisnotablylacking.Becausecholesterolcanbesupplied DHCR7 functionleadstocholesteroldeficiency that,inturn,blocks 2396 probes weregenerated basedonaT7-basedRNA amplificationmethod mesodermal tissue fragmentsfromearlyneurulae. Fluorescentlabeled Total RNA was isolatedfrommicrodissected notochordalandpresomitic Microarray analysis and embryosaloneorwithcombinationsof into two-cell stage assays, dissected andculturedasdescribed(Choetal.,1991).For luciferase reporter using anAmbionmMessageMachinekit.Animalcapexplants were from theblastulastage(Perronetal.,2003).CappedmRNAs wereprepared 5-100 and Cho,1995).Embryosweremicroinjectedmaintainedwithorwithout Xenopus Embryo manipulations et al.,2001).Anothermutant,DHCR7 mutation frequentlyencounteredinindividuals with SLOS(Fitzky MATERIALS ANDMETHODS Hh signalingpathway atthelevel ofsmoothened(Smo). embryogenesis, anditsinhibitoryaffects appeartoimpingeonthe negative regulator oftheresponsetoShhsignalingduringearly functions contrarytopreviously heldnotions.DHCR7actsasa function analysesinearly vertebrate development, wehave performedbothloss-andgain-of- gastrulation. To characterizetheroleofDHCR7duringearly uncover genesinvolved inchordamesodermalspecificationduring activity, HhsignalingandSLOSneedtobeclarified. crucial roles.Therefore,thepreciserelationshipsbetweenDHCR7 central nervous systemorlimbs,tissueswhereShhisknown toplay neither mutantdisplayedmorphologicaldefectsinthedeveloping similar changeshave beenobserved inhumanSLOS.However, precursors andreductionintissuecholesterollevels; importantly, mutant micealsorevealed anoverall accumulationofsterol conversion of7-DHCtocholesterol. Furthercharacterizationofthe inactivate thereductaseactivity ofDHCR7,resultinginimpaired spanning region (Wassif etal.,2001).Bothofthesealterations exons 3,4and5,thuseliminatingmostofthetransmembrane- Mprl Rce assubstrate.Insituprobesfor BM purple(Roche) cultured andassayedatindicatedstages. caps expressing indicatedmRNAs andareportergene.Theconjugateswere cap conjugationexperiments wereconductedbyrecombining two animal reporter geneexperiments wererepeatedaminimumofthree times.Animal control embryosreachedstages12-18andsubjectedtoluciferaseassays. All HNF3 Whole-mount insituhybridization(Harland,1991)was performedusing In ordertouncover thefunctionofDHCR7duringmouse We identified DHCR7 I andtranscribingwithT7RNA polymerase. ␤ ␮ Gli /FoxA2 M AY-9944. Embryosweretreatedwithcyclopamine continuously RESEARCH ARTICLE embryos werefertilizedinvitroandprocessedasdescribed(Blitz -luciferase reporter(0.2ng)(Sasakietal.,1997)was microinjected mRNAs. Animalcapsweredissected(stage8),cultureduntil , Ptc1 Xenopus and ⌬ Gli1 EX8 Xenopus DHCR7 inamicroarray-basedscreento , whichresemblesDHCR7 eeprepared bydigestingtheDNAs with were embryos. We reportthatDHCR7 ⌬ 3-5 , was producedbydeleting Shh , DHCR7 Chordin IVS8-1G>C , Pax6 , , Shh-N Pax2 , a , CATTCTGT-3 CAACCGCTGTTTAG-3 GGCAGTTAGAGGCGCATAAG-3 Shh primers were5 GACCCTGCAAG-3 CTCTTCCGACGCACTA-3 previously (BlitzandCho, 1995). on (5 DHCR7-MO.ControlMO equimolar mixtureoftheseMOsisreferredas DHCR7b-MO (5 GTGGTGCGCCAATCTC-3 GAGAAGAGCGAATCTGTGGTGCGCCAATCTC-3 synthesized asdescribed(Blitzetal.,2000). templates weretranscribedwithSP6polymerase. Moon, 1996)andpCS2+XSmoM2-FLAG (Koebernick etal.,2003) templates (Ekker etal.,1995a). polymerase using polymerase. Full-length All DHCR7templatesweredigestedwith 1994) togenerate Xenopus DHCR7 Plasmids used forarraydataanalyses. image acquisitionprogramandExpressionistsoftware (GeneData)were scanning wereasdescribedpreviously (Shinetal.,2005).AGenePixPro (Amersham). Generationofmicroarrayslides,slidehybridizationand subsequently fluorescentlabeledusingaCy3-andCy5labelingkit transcribed inthepresenceofamine-modifiedrandomhexamers, and (Wang etal.,2000).Four microgramsofamplifiedRNA werereverse including al., 2005).Hierarchicalclusteringanalysisidentifiedseveral genes, consisting of21,000clonesrepresenting~8500transcripts(Shin et stage embryos(stage14-15)werehybridizedtocDNA microarrays from notochord,andleftrightpresomiticmesodermofneurula genes specificallyexpressed in A large-scale cDNA microarrayscreenwas conductedtoidentify microarrays Isolation of RESULTS DHCR7a-MO(5 were Morpholino oligos(GeneTools) usedtoinhibitDHCRfunction Morpholinos andRT-PCR was createdbyinsertingaPCRfragmentbetweenthe DHCR7wt-F andDHCR7 DHCR7 TACTACTGTAGGGTATAGAGGTAGGG-3 n DHCR7 and for DHCR7 DHCR sites ofpCS2AT+ (TsujiandHashimoto,2005).Primersusedwere DHCR7 hybridizationconfirmedthe expression of Whole-mount insitu interesting asthisenzymehas apresumedroleinHhsignaling. dehydrocholesterol reductase, EC1.3.1.21)was particularly developing notochord(Fig.1).Amongthese,DHCR7(7- FoxA4A CA-3 3 wt-R, DHCR7 were:DHCR7wt-F, DHCR7 the primersusedtogenerateDHCR7mutants using thefull-lengthDHCR7cDNA asatemplate.Thecombinationsof Ј Ј ) andDHCR7wt-R(5 RT-PCR primersusedtoamplify -CCTCTTACCTCAGTTACAATTTATA-3 Xenopus Ј primers were5 ). DHCR7mutantsweregeneratedbyPCR-basedmutagenesis 7 R350W wt-F (5 in themidlineof earlyneurulastageembryos(Fig. 1). and Pentraxin W149X development. R350W ; DHCR7wt-FandDHCR7 Ј DHCR7 and 5 Ј Ј R350W -GGAATTCCGCACCATGGGAGAGCGGAGAAGAG- -TGCAAGGACTGCAAGATACG-3 ; andDHCR7wt-F, DHCR7wt-R,DHCR7 Xenopus DHCR7 -R (5 was subclonedintopCS2+(Turner andWeintraub, Ј -GTCCCCAGCAGCTCTCCCCATGTAG-3 Ј Eco Ј , -F (5 -ATGGGGGACCCGTAACTAGA-3 Caveolin-1 and 5 Ј Ј , whicharedifferentially expressed inthe Shh -TCTGCTCTTTGTGTTCTGCTTATCT-3 Ј -GCTGACACGTAAAAACATTCGATG-3 Ј RI-linearized XshhTST7orXshhTST7-N -CAGCGACTTCCTCATGTTCA-3 Ј ; -GGCGCGCCAAAGGTGAGGCGGTAAAA- W149X mRNA transcripts.A FoxA2 Ј and Ј Ј Ј ) andDHCR7 -TACATCTTCTGGATGACCAATCAC-3 and 5 -CATCGGGACCTGCTGTTTCC-3 DHCR7 usingcDNA Not -R (5 Shh-N Xenopus I-linearized pCS2+dnPKA(Ungarand , primers were5 Ј Ј ; -CCAGGGACCCCATTAAATCT-3 FoxA1 Gli1 Ј DHCR7 -GGCGCGCCGCAGCCCATT-3 mRNA weresynthesizedwithT7 histone H4 IVS8-1G>C No primers were5 , ⌬ Ј notochord. RNAs isolated Chordin N2 Ј tI andtranscribedusingSP6 ) forDHCR7 ) hasnoobservable effect -F (5 primers were5 Development 133(12) DHCR7 -R (5 Ј Ј -CTTTCCCAGCAC- -ATGGGAGAGCG- Ј were asdescribed , chordin Ј Xnot and 5 Ј ) forDHCR7 -GGCGCGCCC- Eco rescue construct Ј Ј -GAGCTAGT- . ⌬ , N1 RI and Ј Ј FK506BP -ATCCAC- -F (5 RNA was Ј IVS8-1G>C and 5 Ј -AGAC- ). An Ј ; Ј ) and Ј Ј -CT- ) for Ptc1 Asc ⌬ N Ј Ј Ј Ј - ) ) I ; , ; .

DEVELOPMENT a DHCR7 expression in that isrelatedtoSSDsinotherproteinscurrentlyunknown. 181-362). ThesignificanceofthepossessionanSSDinDHCR7 179-360) thatis86%identicaltoofhumanDHCR7(residues a conserved 180aminoacidsterol-sensingdomain(SSD)(residues Fig. S1inthesupplementarymaterial). against itsmouse,human,ratandzebrafishcounterpartsis77%(see and thepercentageofidentityataminoacidsequencelevel The predicted Negative regulation ofHhsignalingbyDHCR7 Xenopus laevis duplicated formsof Therefore, weconcludethatthesesimilarcDNAs represent assaysperformed(T.K., T.H. andK.W.Y.C., unpublished). the activities ofthesegeneproductsappearedtobeidenticalinall of these in thesupplementarymaterial).Thepredictedaminoacidsequences databases(seeFig.S1 EST (http://Xenopus.nibb.ac.jp/) andTIGR in situhybridizationrevealing theexpression of notochord; RPM,rightpresomitic mesoderm.( decreased geneexpression. LPM,leftpresomitic mesoderm;NTC, colors rangingfrom red togreen correspond toincreased and represents asinglehybridizationandeachrow asingleclone.The ( Fig. 1.Expression of Shh (stage 10)(Ekker etal.,1995a), (stage 9).As Temporally, to itsrelationshipwithShhduring As DHCR7hasbeenimplicatedinHhsignaling,welooked forclues neurulation proceeds, expression ofboth restricted tothedorsalmidline of thegastrula(Fig.2B,parte).As Shh extends broadlyalongthe marginal zone(Fig.2B,part a),whereas onset ofgastrulation, during earlyembryogenesisatleast untiltadpolestages(Fig.2A). Top Xenopus Two related Whole-mount insituhybridization revealed thatshortlyafterthe . Both Hierarchical clusteringanalysisofmicroarray data.Eachcolumn ) is firstdetectedweaklyinthe organizer withexpression Xenopus DHCR7 erl tg embryo. neurula stage DHCR7 DHCR7 Shh Xenopus , andarehencedesignatedas DHCR7 is initiallytranscribedattheearlygastrulastage and DHCR7 transcripts arefirstdetectedattheblastulastage DHCR7 DHCR7 DHCR7 proteinconsistsof473aminoacids cDNAs wereidentifiedfromthe Shh Xenopus cDNAs were95%identical,andthe in developingnotochord. arising fromthepseudotetraploidyof transcripts continuetobeexpressed DHCR7 is expressed intheorganizer and Xenopus expression precedesthatof embryos Xenopus DHCR7 Bottom DHCR7a DHCR7 DHCR7 possesses ) Whole-mount embryogenesis. and in themidlineof and Shh DHCR7b Xenopus mRNA . terminal Shhligand producedfromrecombinant ShhmRNA lacking effect ofDHCR7on aShh-N-mediatedHhresponse.Shh-N,N- is independentofthecholesterol adductionofShh,weexamined the signaling. To examine whetherthisinhibitory behavior ofDHCR7 3C, lane3)consistentwithan inhibitory roleforDHCR7inShh late neuralstages(stage20), extend alongtheembryonicdorsalmidline(Fig.2B,partsb,f).By significantly reducedtheexpression of et al.,2002)(Fig.3C,lane2). However, expression ofDHCR7 presence ofShheffectively inducesShhtargets suchas to aneuralfate (Sasaietal.,1994).Chordinexpression inthe expressed inanimalcapectodermalexplants, the capsareconverted system: animalcapassays.WhentheBMPantagonistChordin is by co-expression ofDHCR7mRNA. and DHCR7 expression, whichisinhibitedbyShh,rescuedco-injectionof et al.,1996;Macdonald1995;UngarandMoon,1996). the expense of enhancement ofShhsignalingpromotestheexpression of genesinfluencedbyShh.Previous work hasshown that of or togetherwith overexpression. To accomplishthis,weinjected whether DHCR7iscapableofreversing phenotypesinducedbyShh as anegative regulator ofHhsignaling.We thereforeexamined cells (Dakuboetal.,2003)andsuggeststhatDHCR7mayfunction resembles thatreportedinmicedeficientShhretinalganglion 63%, DHCR7 developed ectopicpigmentationintheopticstalk(Fig.3A; At theswimmingtadpolestage,however, embryosoverexpressing to develop relatively normallyuntiltailbud stages(datanotshown). DHCR7 and notochord(Fig.2B,partsg,h,n).Atthetailbud stage,both parts c,d,j),while expression intheneuralplate,epidermisandnotochord(Fig.2B, telencephalon andanteriorventral endomesoderm(ave) withweak overexpression. Embryosoverexpressing during earlyembryogenesis,weexamined theeffects ofDHCR7 influencing theactivity ofShh.To explore thefunctionofDHCR7 suggesting thatDHCR7isanimportantenzymepositively signaling intheprocessingofprecursortoproduceHhligand, Sterol modificationofShhappearstobeobligatoryforproper inhibitor ofShhsignaling Gain-of-function analysissuggestsDHCR7isan embryogenesis. regulation oftheShhsignalcascadeduringearly These resultsimplicateapotentialroleforTGF presence oftheproteinsynthesisinhibitorcycloheximide (Fig.2C). and thisinductionwas directas a memberoftheTGF DHCR7 Overexpression ofShh,BMPorWntinanimalcapsdidnotinduce examine how theexpression of to of afunctionallinkbetweentheiractivities. patterns aresimilarfor there aredifferences inthedetailsoftheirexpression, theoverall region oftheave (Fig.2B,partsk,l,o,p).Inconclusion,although region, andpresumptive anteriorgutendoderm,andinavery limited central nervous system,floorplate,branchialarches,frontonasal Next, weexamined thebehavior ofDHCR7 inamoredefined We thenusedassaysinanimalcap(ectodermal)tissueexplants Ptc1 n =46). Thisisasurprisingobservation asthisphenotype , whichisstimulatedbyShhoverexpression, canberescued (data notshown). However, treatmentwithactivin protein, mRNA (Fig.3B).Likewise, theexpression of and Shh Pax6 Shh are similarlyexpressed inthenotochord,ventral DHCR7 expression inopticvesicles (Hammerschmidt is expressed inthefloorplate,prechordalplate ␤ Shh superfamily, induced mRNA, andexamined theexpression and DHCR7 DHCR7 DHCR7 RESEARCH ARTICLE is expressed inthepresumptive Gli1 was upregulated even inthe , allowing fortheexistence DHCR7 DHCR7 , Ptc1 DHCR7 ␤ Shh mRNA appeared and signaling inthe is controlled. mRNA alone Gli1 FoxA2 expression, Pax2 Xenopus Pax2 (Tsuda , 2397 Pax6 (Fig. Gli1 at

DEVELOPMENT cycloheximide; WE,wholeembryos. reporter activation is due toHhsignaling,asmGli-BS (areporter activation isblocked inthepresenceofDHCR7(Fig.3D).This Gli activated inanimalcapsexpressing ChordinandShh-N, this was shown to respondtoHhsignalingdirectly. TheGlireporteris vesicle. ( age, presumptive anteriorgutendoderm;nd,notochord; ov, otic anterior ventralendoderm;ba,branchialarches; fnr, frontonasal region; dorsal lip;fp,floorplate;arc, archenteron; tel, telencephalon;ave, Lateral viewswithanteriortowards theleftanddorsalupwards. dl, (k,l,o,p) stages.(a,b,d,e,f,h)Dorsalviews.(c,g)Anterior(i-p) used aGlireportergene(8 of Shhligand. Shh signalingdoesnotoccuratthelevel of cholesterolmodification (data notshown), suggestingthattheroleofDHCR7inantagonizing expression inbothanimalcaps(Fig.3C,lanes4and5)embryos DHCR7 development. 2398 DHCR7 efficiently blocked Shh-N-induced overexpression studies(Ekker etal.,1995a;Lai1995). its activity, but isasactive asfull-lengthShhconstructin the C-terminaldomain,doesnotrequirecholesteroladduction for ( Fig. 2.Developmentalexpression profiles of A RT-PCR analysisof ) To determinewhetherDHCR7impingesonHhsignaling, we and RESEARCH ARTICLE C ) Inductionof Shh H4 at gastrula(a,e),neurula(b-d,f-h,i,j,m,n),tadpole , histone H4 DHCR7 DHCR7 ϫ . ( and 3 B by activininanimalcapexplants.CHX, Ј ) Whole-mountinsituhybridizationof -Gli-BS) (Sasakietal.,1997),which Shh expression during Gli1 DHCR7 , Ptc1 Xenopus and and Shh. FoxA2 regulated byShhsignaling.Expressionof examined theexpression patternsofmarker genesknown tobe effects, weinjectedDHCR7-MOinto effect ofDHCR7onShhsignaling,ratherthanbyotherindirect address whetherthedefectsinopticvesicle patterningare duetothe a negative, ratherthanapositive, regulator ofShhsignaling.To phenotypes areconsistentwiththenotionthatDHCR7functionsas Eggenschwiler etal.,2001).Thesimilaritiesbetweenthese signaling withintheopticvesicle (Bulgakov etal.,2004; FKBP8, whichresultfromaninappropriateactivation ofShh of-function phenotypesfortheknown HhantagonistsRab23and not shown). Interestingly, thisDHCR7phenotypeissimilartoloss- stronger phenotypesthaninjectionoftheindividual MOsalone(data injection ofMOsdesignedagainstboth the MO-bindingsequence(Fig.4C,F;78%, was efficiently rescuedbyco-expression ofDHCR7mRNA lacking in theventral halfoftheeye (Fig.4B,E;90%, developed smalleyes withreduced retinasandpigmentedepithelia developing recognize both Antisense morpholinooligonucleotides(MOs)designedto Shh signaling Loss-of-function analysis:DHCR7isaninhibitorof of theligand. signaling inHhrespondingcellsandnotatthelevel ofproduction blocked byDHCR7(Fig.3F),indicatingthatinhibits assay. We foundthatinductionofGli-BSreporterwas effectively recombined capswerethenincubatedandsubjectedtoaluciferase injected withGli-reporterinthepresenceorabsenceofDHCR7.The were usedtoprovide theShhproteintoconjugatedanimalcaps 3E). Animalcapsexpressing Chordinwithorwithout To addressthisissue,weusedananimalcapconjugationassay(Fig. signaling byblockingrespondingcellsfromreactingtothesignal. stage 12embryos. N. We observed theeffect ofShh-NonGlitranscriptionasearly gene withmutationsinGli-bindingsites)was notinducedbyShh- pattering. cellular responsetowards Hhsignalingduringopticvesicle et al.,2005),suggestingthat the lossofDHCR7enhances similar tothoseofembryosreceiving high n DHCR7 MO-injectedembryos(compareFig.4GwithH;70%, weakly altertheexpression of DHCR7-MO withlow dosesof responsiveness towards Hhsignaling.Therefore,weco-injected signaling, weexamined whetherlossofDHCR7enhances al., 2003). and zebrafish(Ekker etal.,1995b;Macdonald1995; Perronet observed afterShhoverexpression orPKAinhibitionin the DHCR7loss-of-functioneffects resemblethephenotypes upregulated inMO-injectedembryos(Fig.4O,P;58%, ventral eye (Fig.4K,L;60%, entire opticvesicle (Fig. 4N;80%, Stimulation ofHhsignalingbyinjecting50pg Pax6 DHCR7-MO and ventral halfoftheeye. Importantly, embryosco-injectedwith concomitant expansion of alone resultedinareductionof =30), accompaniedbyslightlyexpanded expression of We alsowishedtodeterminewhetherDHCR7inhibitsHh As loss-of-functionsuggeststhatDHCR7isaninhibitorofShh expression (Fig.4J;80%, Xenopus DHCR7a Shh-N embryos. mRNA showed completesuppressionof Pax2 and DHCR7 n Pax2, Pax6 =16) andexpansion of Pax6 n Shh-N DHCR7b (Fig. 4M;80%, =28). n DHCR7a =16). -MO-injected embryoshad (Fig. 4I;80%, mRNA that,alone,only Gli1 Xenopus Development 133(12) n Pax6 and =48). Thisphenotype These effects were were injectedinto hsgaig(Lupo Hh signaling was alsobroadly n and =40). Combined Gli1 was reducedin Shh-N DHCR7b Shh-N embryos and n n =22) inthe expression. n =22). Thus, Pax2 Pax2 =16) with Xenopus mRNA mRNA in the in the gave

DEVELOPMENT Shh signaling. partial ventralization of theneuraltubeowing toupregulation of are consistentwiththenotionthat DHCR7loss-of-functioncauses DHCR7 phenotypes wereeffectively rescuedbyoverexpression of expression (Pieranietal., 1999;Briscoeetal.,2000).Allthese previous findingthat highlevels ofShhsignalingrepress cord,isalsonarrowed (Fig.6H,I),inagreement withthe spinal Negative regulation ofHhsignalingbyDHCR7 significant increasesintheexpression levels ofboth hybridization analysisof negative regulator ofShhin thatregion. Whole-mountinsitu Therefore, wewishedtoknow whetherDHCR7alsoactsasa Shh playsanimportantroleinpatterningtheventral neural tube. patterning DHCR7 functionsasaShhinhibitorinneuraltube Expression ofventral neuraltubemarkers suchas performed unilateralinjectionofDHCR7MO(Fig.6A). effect ofDHCR7MOonventral neural pattering,wethen n (Fig. 5A,B;78%, Expression of Nkx2.2 =26), whicharethemarkers forthemidline.To testfurtherthe mRNA (Fig. 5C,F,I,L,O; Fig.6D,G,J). Thesephenotypes expanded dorsallyinMO-injectedside(Fig.6). Dbx1 n , whichmarkstheintermediateregion ofthe =28) and DHCR7 Shh MO-injected embryosshowed (Fig. 5D,E,J,K,M,N;92%, Shh, FoxA2 Hnf3 ␤ /FoxA2 Dbx1 and shortened bodyaxis whencomparedwithDHCR7 aloneorwithAY- 9944. Theembryosdeveloped noticeablemicrocephalyanda of DHCR7isresponsibleforits inhibition ofShhactivity. signaling, weconsideredthepossibility thatanon-enzymaticactivity the knockdown of DHCR7clearlyshows itsrequirementinShh sufficient toinhibitShhsignalingin the enzymaticactivity ofDHCR7byAY-9944 alonemaynotbe largely unaffected (Fig.7B).Theseresultssuggestthatinactivation of (Cooper etal.,1998),inductionoftheGli-reporterbyShh-N was completely AY-9944, afivefold higherconcentrationthanthatnecessarytoinhibit 7D). Similarly, whenanimalcapexplants weretreatedwith10 no obvious morphologicaldefectswerenoted(compareFig.7Cwith with AY-9944 (upto100 and Aubry, 1966).When pharmacological inhibitoroftheenzymaticactivity ofDHCR7(Roux inhibitor ofcholesterolbiosynthesis,inparticularAY-9944, a We next examined how DHCR7mightfunctiontogether withan embryogenesis Shh signalingduring De novocholesterol synthesisisnotessentialfor Therefore, wetreated FoxA2 induction byShh-Ninchickneuralplateexplants DHCR7 Xenopus ␮ M) fromtheearlygastrulastageonwards, Xenopus conjugates. reporter geneassayusinganimalcap conjugation experiment.( animal capassay. ( 1). ( equivalent animalcapcontrol (lane (lane 7)mRNA.Uninjectedstage28 plus pg of of (lane 2), Chordin from animalscapsinjectedwith ( ( Pax2 efficiency was: type level(toprow). Therescue expression wasrestored tothewild- with When 35 (tailbud)usingindicatedprobes. mount insituhybridizationatstage Embryos were subjectedtowhole mRNA (bottomrow; Shh+DHCR7). together with500pgof Xenopus Shh were injectedwith250pgof embryo. ( one blastomere ofthetwo-cellstage was microinjected unilaterallyinto by DHCR7. Fig. 3.InhibitionofShhsignaling mRNA-injected embryoswithAY- C n embryos werecontinuouslytreated =34); RT-PCR analysisofRNAisolated ) DHCR7 RESEARCH ARTICLE Xenopus D Shh-N , 90%( DHCR7 ) Gli-reporter assaysusing Shh-N Shh Patched1 (50 pg)plus Chordin early B (lane 3), Two-cell stageembryos ) mRNA wasco-injected with 1ngof n ( (lane 4)and A mRNA, markergene =48); embryos. However, as mRNA (middlerow), or ) Pax6 DHCR7 , 93%( plus E Chordin Gli1 Schemaofa ) , 87%( Shh Shh , 91% mRNA (2ng) DHCR7 n DHCR7 Chordin (100 pg) F =28). with 1ng ) Gli- n plus 100 =46); 2399 ␮ M

DEVELOPMENT cyclopamine (100 (G-J), expandstheexpression of J,K-N,O-R). Gli1 mRNA (J,J alone (I,I 20ng ofDHCR7MOsalone(H,H mRNA (C,F). (B,E) orDHCR7MOstogetherwith125pgoffull-lengthrescue in allfourblastomeres with5ngofcontrol MO(A,D), DHCR7MOs eye defects.Four-cell stage signaling inopticvesicle. affects cellularresponse towards Hhsignaling. the swimmingtadpolestages(datanotshown).( activity otherthanitsreductaseactivity. antagonistic activity ofDHCR7towards Shhbyinfluencingan (Fig. 7B).ThesedatasupporttheideathatAY9944 augmentsthe treated withAY-9944 thanthatofeitherAY-9944 orDHCR7alone was suppressedmorestronglyinDHCR7-expressing embryos affects ofAY-9944 onGlireporteractivity. InductionofGlireporter 9944 alone(compareFig.7C-F;76%, 2400 Fig. 4. embryos were injectedwith 20ngofcontrol MO(G,G expression (O-R).(G Loss-of-DHCR7 affects cellularresponse toward Hh RESEARCH ARTICLE Ј ,M,M Ј ,N,N Circled areas markopticvesicles. Additional defectsdetectedwere intheheartandgutof Ј Ј ,Q,Q ,R,R ␮ Ј M) from the blastulastageblocksHhsignaling (G- Ј ). KnockdownofDHCR7reduces ) or20ngofDHCR7MOswith50 pgof Ј -R Ј Xenopus ( ) Continuouslytreatment of embryoswith A-F ) DHCR7knockdownbyMOsleadsto Pax2 Ј ,L,L Ј embryos were injectedmarginally ,P,P expression (K-N)andupregulates Ј ), 50pgof n =58). We alsoassessedthe Four-cell stage G-R ) LossofDHCR7 Shh-N Pax6 Ј ,K,K Ј mRNA expression ,O,O Xenopus Shh-N Ј ), DHCR7 is missinginthemutantprotein.Importantly, expression of cap conjugationassay(Fig.7L),despitethefact thattheentireSSD effectively blocked theinduction oftheGli-reporterinanimal microcephaly in expression patternsof Additionally, DHCR7 fhumanDHCR7 of DHCR7 DHCR7 Witsch-Baumgartner etal.,2000).Anothermutation, 2001; abrogationof95-97%enzymeactivity (Fitzky etal., in (corresponding toR350Win in In lightofourfindingsboththatDHCR7canactasaShhinhibitor of thesterol-sensing domainforShhinhibition SLOS-associated mutationsreveal dispensability on nSO (Fig.7A).OnehumanmutationisDHCR7 found inSLOS Xenopus DHCR7 isdispensable,wewishedtoexamine theeffects of of DHCR7 examined whetherthecyclopic phenotypecausedbytheexpression DHCR7 acts,weperformed epistasis experiments. First,we ligand. To determinewhereinthesignaltransduction pathway in Hhrespondingcellsandnotat thelevel ofproductiontheShh conjugation assay, wealsoshowed thatDHCR7 inhibitssignaling modified, isalsoinhibitedbyDHCR7.Usingananimal cap not appeartobeessentialandasShh-N,whichischolesterol cholesterol adductionoftheligand,aslossreductaseactivity does DHCR7 ofHhsignalingdoesnotappeartobeatthelevel of At whatlevel doesDHCR7inhibitHhsignaling? Theinhibitionby Smoothened Epistasis analysis:DHCR7actsatthelevelof domain ofDHCR7. inhibition ofHhsignalingbyDHCR7ismediatedviatheN-terminal reporter gene(Fig.7L).Altogether, these resultsshow thatthe n ihSLOS.Overexpression ofeitherDHCR7 with SSD have alsobeenidentifiedamongsomeindividuals (Fig. 7A).Several missensemutationsmappedwithintheDHCR7 471), andisalsosignificantlyreducedinitsreductaseactivity part ofthesterolsensingdomain(SSD;aminoacidresidues318- splicing ofexon 8,generatesatruncatedproteinthatismissing mutation inindividuals withSLOS.Thisdefectcauses aberrant was generated.WhenDHCR7 signaling, wegenerateda reductase activity. still capableofinhibitingShhsignalingdespitetheirlack suggest thatmutantDHCR7proteinsassociatedwithSLOSare Shiota, 2002).Theexperiments presentedinthecurrent study with holoprosencephaly(HPE)(Chiangetal.,1996;Cohen,Jrand observed inmouseembryosdeficientShhandindividuals detected (Fig.7K).Itisnoteworthy thatcyclopia isfrequently for theeye marker, respectively). Whensuchseverely affected embryoswerestained and ashortenedbodyaxis(Fig.7G,H;90%, DHCR7 reductase activity. Additionally, embryosexpressing either the mutantproteinshave presumablylostessentially alloftheir in theanimalcapconjugationassay(Fig.7L),despitefact that =30) but insteadmoderatelyupregulated theexpression oftheGli- Next, inordertodeterminethecontribution of theSSDonHh Xenopus ⌬ IVS8-1G>C IVS8-1G>C IVS8-1G>C N DHCR7 mutantscorrespondingtohumancounterparts did notaffect cephalicdevelopment (Fig.7J;100%, R350W early embryos,andthatthereductaseactivity of , isthemostfrequentlyencounteredDHCR7 or DHCR7 resulted inblockadeofinductionGlireporter Xenopus was duetoadefectin Shh signaling.The Pax6 W151X ⌬ FoxA2 N lacking mostoftheNterminusDHCR7 , asinglesmall‘cyclopic’ eyespot was , whichcompletelylackstheSSD. Xenopus embryos (Fig.7I;70%, R350W , Shh Xenopus W149X and displayed severe microcephaly counterpart (DHCR7 Ptc1 was expressed, itcaused DHCR7), whichresults Development 133(12) in theseembryos were n =44 and78%, n =20), and R350W W149X n R352W =44, or )

DEVELOPMENT ectopic Shhexpression. age, presumptive anteriorgutendoderm.Whitearrowheads indicate branchial arches; fl,floorplate(arrowheads); fnr, frontonasal region; injected embryos(40%, expression ofShhwasdetectedinthenotochord region ofDHCR7MO Transverse sectionsofembryosindicatedinD,E.(K)Ectopic (M-O) nasal region, branchialarches andanteriorgutendoderm. injected embryos. injected embryosexpand phenotype causedbyDHCR7 Negative regulation ofHhsignalingbyDHCR7 of examined. Injectedembryos(Fig.8)exhibited significant reductions marked by microinjected withDHCR7-MOsshowsexpansionoffloorplateas Whole-mount insituhybridizationofthetailbudstageembryos alone (B,E,H,K,N)orwith500pgof with 20ngofcontrol MO(A,D,G,J,M),with20ngofDHCR7MOs Fig. 5.Expansionof DHCR7-MO or both theinhibitionof effects oneye development wereexamined. Cyclopamine blocked injected withDHCR7MOwere treatedwithcyclopamine andits DHCR7 functionsupstreamor downstream ofSmo,embryos function ofSmo(Perronetal.,2003). Inordertodeterminewhether Hh signalinginhibitorcyclopamine, whichisknown toinhibitthe vesicle patterningwas inhibitedin acting upstreamofPKA. demonstrating thatDHCR7actsasaninhibitsShhsignaling by yDHCR7 by Kinase A(dnPKA)couldblockthemidlinedefectsinduced transcripts. We alsoexamined whetherdominant-negative Protein Previous studieshave demonstrated thattheHhdependentoptic FoxA2 (100%, FoxA2 R350W Shh-N ( A-C . dnPKAreversed themicrocephalic/cyclopic n Four-cell stage =20), Shh ) and n Pax6 overexpression (Fig.4G FoxA2 =28). ave,anteriorventralendoderm;ba, and Shh Shh FoxA2 (100%, and inductionof and ( D-O Xenopus R350W DHCR7 Xenopus Shh ) expression. (B,E)DHCR7MOs expressing domainsinMO- n expression domainsinfrontal =34) and (Fig. 8G-J;96%, rescue mRNA(C,F,I,L,O). embryos were injected embryos treatedwiththe Pax2 Ј Ptc1 -J Ј ,K (92%, and Ј -N Ј ,O Gli1 n n Ј =46), -R =38) by Ј ). of neuraltubebylossDHCR7. FoxA2 expression limitoftheindicatedmarkers.( ventralization ofneuraltubeasmarkedby of thetailbudstageembryosmicroinjected withDHCR7-MOsshows mRNA (D,G,J).Transverse sectionofwhole-mountinsituhybridization or 20ngofDHCR7MOsalone(C,F,I) orwith500pgof unilaterally (rightside)microinjected with20ngofcontrol MO(B,E,H) unilateral injectionexperiment.Eight-cellstage animal capassays revealed thatSmo-M2partiallyrescued the the rescuewas onlypartially successful(datanotshown). Similarly, experiments asshown inFig.8G-JwereperformedusingSmo-M2, using anactivated formofSmo(Smo-M2).Whenembryonicrescue Smo. inhibit Hhsignalingbyactingeither atthelevel of,orupstream One interpretationoftheseobservations isthatDHCR7functionsto Fig. 6. Neural patterning modulatedbyDHCR7. Fig. 6.Neuralpatterning To confirmthispossibility, weperformedanepistasisexperiment and Dbx1 ( H-J ) expression. Arrowheads indicatethedorsal RESEARCH ARTICLE K Shh ) Summaryofventralization Xenopus ( B-D ), ( A Nkx2.2 ) Schemaof DHCR7 embryos were ( E-G rescue ), 2401

DEVELOPMENT DHCR7 (with orwithout injected with by DHCR7mutantsusinganimalcap conjugation.Embryoswere 2402 8X3 with AY-9944. Embryos were injectedwiththereporter construct mutant constructs.( Fig. 7.Shhsignalingandcholesterol synthesis. with 10 mutants. ( DHCR7 (F). Embryonicphenotypescaused bymicroinjection of1ng caused minordefects,butacombination ofbothcausessevere defects overexpression of defective inreductase activitycausescyclopicphenotypes.The Ј Gli-BS Lucwithorwithout R350W R350W RESEARCH ARTICLE ␮ M AY-9944. ( K ) . Arrow marks‘cyclopic’eye. ( Pax6 (H), Chordin DHCR7 DHCR7 DHCR7 staining oftailbudstageembryos injected with B ) InhibitionHhsignalingisenhancedbytreatment (with orwithout C-J IVS8-1G>C mRNA). mRNA (E)ortreatment withAY-9944 (D) ) Microinjection ofDHCR7mutantsthatare Shh-N, DHCR7 (G), DHCR7 Shh-N L ) ModulationofGlireporter ⌬ N mRNA) orwithGli-BS Luc mRNA andtreatment (J) and ( A ) Various DHCR7 DHCR7 W149X (I) n McMahon, 2005).Defectsinthese componentsleadto and Goodrich etal.,1997;Hammerschmidt etal.,1996;Jeong Hip1 andFKBP8(Bulgakov etal.,2004;Eggenschwiler2001; negatively regulated bytheHhantagonistsPatched1, Rab23,PKA, transcription factors (Ericson etal.,1997).Shhsignalingisalso tube, therebycontrollingtheexpression ofspecifichomeodomain concentration gradientalongthe dorsoventral (DV) axisof the neural Shh secretedfromthenotochordandfloorplateforms a DHCR7 andShhsignalinginmidlinedevelopment phenotypes. content maycontribute tothedifference inDHCR7loss-of-function et al.,2001;Wassif et al., 2001)],differences incellularcholesterol where cholesterolissuppliedbyyolksac/placentaltransfer(Fitzky Xenopus As embryosfrommany non-placentalvertebrates, including impaired onlywhenresidualendogenouscholesterolwas depleted. note thattheShhresponseoffibroblastcellslackingDHCR7 was this discrepancy isunclearatpresent.However, itisimportantto knockdown promotesShhsignaling(Figs3,4),andthereasonfor (Cooper etal.,2003).ThisisincontrasttoourfindingthatDHCR7 suggested thatDHCR7isrequiredforcellularresponsetoShh enzymatic propertiesofDHCR7. observed effects ofDHCR7onShhsignalingareindependentthe to inhibitionofShhsignaling(Fig.7),suggestingthatsomethe mutant DHCR7proteinsdeficientinreductaseactivity, bothstilllead the pharmacologicalinhibitorAY-9944 orbyoverexpression of enzymatic activity ofDHCR7eitherbytreatmentembryoswith both theeye andneuraltube(Figs4-6).Importantly, inhibitionofthe antisense oligonucleotidesleadstoenhancementofShhactivity in 3, 7and8),whereasknockdown ofDHCR7bymorpholino ectopic expression ofShh,aswellendogenousShhactivity (Figs development. Overexpression ofDHCR7inhibitstheeffects of that DHCR7serves toinhibitHhsignalingduringearly function analysesdonotsupportthisview. Instead,ourdataindicate cholesterol. However, resultsofourcurrentloss-andgain-of- DHCR7 stimulatesHhsignalingthroughincreasedproductionof responsible forthefinalstepinprocess,would suggestthat The fact thatDHCR7isakey enzymeincholesterol biosynthesis, development Requirement forDHCR7activityduringearly exerts thisactionatthelevel ofSmo. signaling pathway duringearlyembryogenesis,andsuggestthatit evidence demonstratingthatDHCR7negatively modulatestheShh negative regulator ofShhsignaling.We provide multiplelinesof presented hereinsteadsuggestanovel functionforDHCR7asa functions asapositive regulator ofShhsignaltransduction,thedata embryogenesis. Contrarytotheprevailing view thatDHCR7 regulating chordamesodermaldevelopment duringvertebrate early We identified DISCUSSION signaling byimpingingonSmo. Gli reportergenebySmo-M2,suggestingthatDHCR7affects Hh DHCR7 potent inhibitorofHhsignaling,wenext addressedwhether upstream ofSmooratthelevel ofSmo.AsDHCR7 observations areconsistentwiththenotionthat DHCR7acts DHCR7-mediated suppressionofHhsignaling(Fig.8K).These Fig. 8L,DHCR7 Previous work usingDHCR7-deficientmouse fibroblastshas IVS8-1G>C stockpilecholesterolmaternally[incontrasttomammals, , Xenopus IVS8-1G>C could blocktheactivity ofSmo-M2.Asshown in DHCR7 inamicroarrayscreenforgenes effectively blocked theactivation ofthe Development 133(12) IVS8-1G>C Xenopus is a

DEVELOPMENT and thereporter geneactivitywasmeasured. ( inhibition. Embryoswere co-injectedwith8 nii h fet foeepeso fSh Second,theinhibition inhibit theeffects ofoverexpression ofShh. regulating thetranscription oftheShhbecauseDHCR7was ableto primary effects of DHCR7donotappeartooccuratthelevel of downstream ofSmo basedonthefollowing observations. First,the We proposethatDHCR7inhibitsHhsignalingat thelevel or inhibition byDHCR7? What isthemolecularmechanism underlyingShh negatively regulate levels ofShhsignalinginboththesestructures. in thesetissues(Fig.5),leadustosurmisethatDHCR7functions to that thelossofDHCR7enhancestranscription DHCR7 isexpressed bothinthenotochordandventral CNS,and Shh signalinginthattissueonceformed(Chiangetal.,1996). That required intheformationofnotochordandformaintenance of Baudet etal.,2003).InadditiontoitsroleintheCNS,Shhis also microinjected with1ngof Negative regulation ofHhsignalingbyDHCR7 neurons intheventral CNSofDHCR7 on highShhsignaling,showed athreefoldincreaseinthenumberof that theformationofserotonin(5-HT)neurons,whichisdependent al., 2001),but ourfindingsareconsistentwiththerecent observation the developing centralnervous system(Fitzky etal.,2001;Wassif et note thatmicedeficientinDHCR7donotdisplayobvious defectsin embryos whenDHCR7activity was inhibited (Figs5and6).We ventralized neuraltubephenotypewas alsoobserved in ventralization oftheneuraltube.Inpresentstudy, asimilarly Fig. 8.Theeffects ofDHCR7impingesonSmo. protein. PKAinhibitsGliactivation.DHCR7Shhsignalingeitheratthelevelofordownstream ofSmo. the role ofDHCR7inShhsignaling.inactivatesPtc,thuspermittingtheactivationSmo.ActivatedSmotransmitsas dnPKA inhibitsthecyclopicphenotypecausedbyDHCR7 Gli reporter togetherwith Chordin, DHCR7 DHCR7 R350W mRNA inhibitstheexpression of IVS8-1G>C ⌬ ϫ 3-5 3 L Ј Gli-BS Luctogetherwith ) DHCR7 mutant mice(Waage- or ( A-F Smo-M2 Microinjection of ) Shh IVS8-1G>C R350W and . ( Xenopus mRNA, orincombination,andreporter geneactivitywasmeasured. ( J blocks Glireporter activationmediatedbySmo-M2.Embryoswere co-injectedwith ) Control uninjectedembryo.( FoxA2 Chordin, Shh-N,DHCR7 FoxA2 DHCR7 absence ofHhligand (Denefetal.,2000),itistempting tospeculate membrane andcytoplasmic vesicles inresponsetothepresence/ and thatbothPtcSmoappear toshuttlebetweentheplasma largely detectedwithinintracellularvesicles (Incardona etal.,2000), sterol trafficking (Wassif etal., 2002).Given thatthePtcproteinis abnormal storageofsterols,presumably resultingfromdefectsin fibroblasts isolatedfrommany individuals withSLOSshow and NPC1Lareinvolved insteroltrafficking. Accordingly, implicated inthevesicular trafficking ofHh,whereasSCAP, NPC1 trafficking (Kuwabara andLabouesse, 2002).Ptch1andDisp1are found predominantlyamongproteinsinvolved invesicular Smo andpossessesaconserved sterol-sensingdomain(SSD)thatis signaling byactingupstreamofPKA. DHCR7 dnPKA reversed themicrocephalic/cyclopic phenotypecausedby orimpingesdownstream ofthe Smo.We alsoobserved that Smo notion thatDHCR7regulates Shhsignalingeitheratthelevel of analysis usingSmo-M2andDHCR7 and notatthelevel ofproductiontheligand.Fourth, epistasis demonstrated thatDHCR7inhibitssignalinginHh-respondingcells is alsoinhibitedbyDHCR7.Third,ananimalcapconjugationassay adduction oftheligand,asShh-N,whichisnotcholesterolmodified, of HhsignalingbyDHCR7appearstobeindependentcholesterol DHCR7 functionsintracellularlyatthelevel ordownstream of , Shh R350W and R350W mutant inhibitsendogenousShhsignaling.Embryos Ptc-1 , demonstratingthatDHCR7actsasaninhibitorofHh . ( K G-I ) Smo-M2rescues DHCR7-mediatedHhsignaling or ) Microinjection ofmRNA(500pg)encoding Smo-M2 mRNA (0.5ng),orincombination, RESEARCH ARTICLE IVS8-1G>C is consistentwiththe ignal toactivateGli M ) Modelfor 2403

DEVELOPMENT for normalmidlinedevelopment. the occurrenceofSLOSasproperexpression ofDHCR7 isessential address how DHCR7expression isregulated anditsrelationshipto relative toitsreductaseactivity. Additionally, itisimportantto SSD domainordeletionsaffect theantagonisticactivity ofShh Blitz, I.L.,Shimmi,O.,Wunnenberg-Stapleton, K.,O’Connor, M.B.andCho, Chen, M.H.,Li,Y. J.,Kawakami,T., Xu,S.M.andChuang,P. T. Chen, J.K.,Taipale, J.,Cooper, M.K.andBeachy, P. A. Chamoun, Z.,Mann,R.K.,Nellen,D.,vonKessler, D.P., Bellotto,M.,Beachy, Bulgakov, O.V., Eggenschwiler, J.T., Hong,D.H.,Anderson,K.V. andLi,T. Chiang, C.,Litingtung,Y., Lee,E.,Young, K.E.,Corden, J.L.,Westphal, H. nature ofthe cellular responsestoHhsignaling(Cooperetal.,2003). of alteringintracellularsterolconcentrations,whichmayaffect directionality. Additionally, mutationsin is oppositelymodulatedtobringphenotypesofopposing the precisenatureofspecificmutations,activity ofDHCR7 activity andareductaseenzymaticactivity, perhaps,dependingon DHCR7 appearstohave two distinctactivities, aHhantagonistic in asinglegenecausesuchseeminglydivergent phenotypes?As macrocephalic (Kelley andHennekam,2000).How canmutations (a singledigit);someshow holoprosencephaly, whileothersare individuals arepolydactyl(extra digits),whileothersaremonodactyl the sameunderlyinggeneticcondition.For example, some this disease.Individuals appeartodisplayopposing phenotypesfor One curiousaspectofSLOSisthewiderangesymptomsseenin The role ofDHCR7inthepathogenesisSLOS required tofullyunderstandthefunctionofeachdomainDHCR7. role ofthisN-terminalregion mightbe,andfurtherwork willbe to inhibittheactionofHhsignaling.Itiscurrentlyunclearwhat containing anintactNterminus(but lackingtheSSD)issufficient analyses confoundthissimpleinterpretation,asaDHCR7mutant Hh signalingcomponents.However, ourcurrentDHCR7mutational that DHCR7participatesintheregulation ofvesicular trafficking of 2404 Blitz, I.L.andCho,K.W. References http://dev.biologists.org/cgi/content/full/133/12/2395/DC1 Supplementary materialforthisarticleisavailableat Supplementary material wassupportbyaNIHgrant(HD29507)toK.W.Y.C. work contributions ofplasmids,andDrsWolf andRouxforproviding AY-9944. This useful discussion.We thankDrsH.Sasaki,S.Ekker, T. PielerandR.Moonfor Mr SouichiOgataforinitialwork,andthemembersofCholaboratory We thankDrsIraBlitzandTom Schillingforcriticalreading ofthemanuscript, C B h r Dev. Hedgehog signalingbydirect bindingofcyclopaminetoSmoothened. regulation. BMP1-related metalloproteases inchordin andBMP4autofeedbackloop K. W. Development during gastrulation:therole oftheXenopushomeoboxgeneorthodenticle. palmitoylation andactivityofthehedgehogsignal. P. A.andBasler, K. neural tissues. (2004). FKBP8isanegativeregulator ofmousesonichedgehogsignalingin lacking Sonichedgehoggenefunction. and Beachy, P. A. 659. protein complexandlong-rangesignalinginvertebrates. Palmitoylation isrequired fortheproduction of asolublemultimericHedgehog gene goosecoid. Molecular nature ofSpemann’s organizer:therole of the i in theventralneuraltube. homeodomain protein code specifiesprogenitor cellidentityandneuronal fate o An importanttopictoaddressinthefutureisbiochemical s c ,

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DEVELOPMENT