XenopusPax-6andRetinalDevelopment NicolasHirsch,WilliamA.Harris DepartmentofBiology,UniversityofCalifornia,SanDiego,9500GilmanDrive, LaJolla,California92093 Received3June1996;accepted30July1996 ABSTRACT:WehaveclonedahomologofPax- eyephenotypeisobserved.Targetedmisexpression 6inXenopuslaevis.Itsdeducedaminoacidsequence intheretinaatlaterstagesdoesnotresultinany hasa95%overallidentitywithPax-6homologsin signi®cantbiastowardformationofamacrineorgan- othervertebrates.Itisexpressedearlyindevelop- glioncellsorawayfromphotoreceptors.Ectopicex- mentincellsfatedtoformtheeyeandpartsofthe pressionoftheproneuralgeneNeuroDaltersthe forebrain,hindbrain,andspinalcord.Ithastwo patternofPax-6,substantiallyreducingitsexpres- phasesofexpressionintheeye.Intheearlyphase, sionintheeye®eldandlaterreducingoreliminating fromstage12.5tostage33/34,XenopusPax-6isex- theeyeitself.OurresultsshowthatPax-6expression pressedthroughoutthedevelopingretina.Inthelate appearstobenecessary,butnotsuf®cient,foreye phase,afterstage33/34,itisexcludedfrommature formationinXenopus.q1997JohnWiley&Sons,Inc. cellsintheouterhalfoftheretinaandfromcellsin JNeurobiol32,45±61,1997. theciliarymarginalzone,remainingonlyinama- Keywords:eye;homeobox;misexpression;NeuroD; crineandganglioncells.MisexpressionofPax-6early paired indevelopmentresultsinaxialdefects,butnospeci®c INTRODUCTION occursintheheterozygote,albeittoalesserextent, implyingthatthephenotypeisduetoaninadequate ThePaxfamilyoftranscriptionfactorsisexpressed genedosageofPax-6.Themutationiscausedeither throughoutthedevelopingnervoussystem(Jostes bydeletionoftheentirePax-6locusorbysingle etal.,1990;Nornesetal.,1990;Plachovetal., basepairchangesresultinginprematuretermination 1990;Gouldingetal.,1991;Adamsetal.,1992; ofthepolypeptidechain(Hilletal.,1991;Tonet AsanoandGruss,1992).MutationsofPaxfamily al.,1992).Aniridia,ananalogoushumancondition memberPax-6resultinspeci®cdefectsinthedevel- characterizedbyirishypoplasiaandcornealopaci- opmentofboththemurineandhumannervoussys- ®cation,iscausedbysimilarmutationsofPAX6, tems.Smalleye(Sey),anautosomaldominantmu- thehumanhomologofPax-6(Tonetal.,1991; tationinmousePax-6,ischaracterizedinthehomo- Glaseretal.,1992;Jordanetal.,1992).TheDro- zygotebysubstantialreductionoreliminationofthe sophilaeyeless(ey)mutationhasalsobeenidenti- retinaandlens,lossoftheolfactoryepitheliumand ®edasadefectinPax-6(Quiringetal.,1994). olfactorybulb,andabnormalmigrationofcortical Therefore,substantialgeneticevidenceexiststhat platecells(Hoganetal.,1986;Schmahletal., Pax-6isnecessaryforcorrectretinaldevelopment 1993).Thelossofeyeandnasalstructuresalso inbothvertebratesandinvertebrates. Pax-6homologshavebeenidenti®edinmany oftheMetazoa,includingCaenorhabditiselegans Correspondenceto:W.A.Harris(e-mail:[email protected] (ChisolmandHorvitz,1995;ZhangandEmmons, cd.edu). Contractgrantsponsor:NationalInstitutesofHealth;contract 1995),Drosophila(Quiringetal.,1994),zebra®sh grantnumber:EY10422 (Kraussetal.,1991;Puscheletal.,1992),chick q1997JohnWiley&Sons,Inc. CCC0022-3034/97/010045-17 (Gouldingetal.,1993),mouse(WaltherandGruss, 45 46 HirschandHarris 1991),andhuman(Tonetal.,1991).Thesequence retinahasbeenextensivelystudiedwithrespectto ofthePax-6proteinishighlyconservedbetween themoleculargeneticsofcellfatedetermination thesespecies,includingitsthreefunctionaldo- (Holtetal.,1988;WettsandFraser,1988;Wetts mains:thepairedbox,thepairedhomeobox,and etal.,1989;Turneretal.,1990;HarrisandMesser- theproline,serine,andthreonine±rich(PST-rich) smith,1992;Dorskyetal.,1995).Thecloningand region.Thepairedbox,128aminoacidsinlength characterizationofPax-6areimportantadditionsto andlocatedattheamino-terminusoftheprotein, thestudyofeyedevelopmentinthissystem.We was®rstdescribedintheDrosophilagenespaired, areinterestedinexaminingtheroleofPax-6in gsb-p,andgsb-d(Boppetal.,1986;Frigerioetal., determiningcellfateduringdevelopment.Inpartic- 1986;Baumgartneretal.,1987),andischaracteris- ular,weaddresstheroleofectopicexpressionof ticofallmembersofthePaxgenefamily.The Pax-6indirectingectopiceyeformationandchang- pairedhomeoboxisapaired-classhomeodomain ingcellfateintheretina.Wealsoexaminethe foundinitsentiretyonlyinPax-6,Pax-3,andPax- effectsofmisexpressingtheproneuralgeneNeuroD 7.OtherPaxfamilymembershaveonlyapartial bothoneyedevelopmentandonthenormalexpres- homeodomainorlackahomeodomainentirely(re- sionpatternofPax-6. viewedinHillandHanson,1992).Boththepaired domainandthepairedhomeoboxhavebeenshown tobindDNA(Treismanetal.,1989;Chalepakiset MATERIALSANDMETHODS al.,1991;Gouldingetal.,1991;Treismanetal., 1991).SeveralPaxgeneshavebeenshowntofunc- LibraryScreeningandDNASequencing tionastranscriptionalactivatorsincludingpaired (Hanetal.,1989)andPax-5(Adamsetal.,1992). Twelveprimarycloneswereidenti®edfromaXenopus Atthecarboxy-terminusofthePax-6proteinisa neurulacDNAlibraryinlgt10(KintnerandMelton, PST-richdomain,whichissimilartoaregionof 1987)usinga700-bppolymerasechainreaction(PCR) theubiquitoustranscriptionfactorOct-1(Sturmet fragmentofXenopusPax-6.PCRampli®cationandpri- al.,1988).Ratandmousemutantswhicharemiss- marycDNAcloneisolationwascarriedoutaspreviously describedforchickenPax-6(Gouldingetal.,1993).Fur- ingonlythePST-richregionofPax-6stillexhibit therplaquepuri®cation,probesynthesis,andnucleicacid atypical``smalleye''phenotype(Hilletal.,1991; hybridizationwerecarriedoutusingstandardmethods Matsuo,1993),implyingthatthisregionisrequired (Sambrooketal.,1989),withthefollowingmodi®ca- forproperfunction. tions.Pax-6probewasmadefrom50ngofthePCR ThediscoverythatPax-6isinvolvedinthedevel- fragmentandlabeledwith 32PusingtheMultiprimeDNA opmentofeyesasdifferentasthelenseyeofverte- LabelingKit(Amersham,ArlingtonHeights,IL)follow- bratesandthecompoundeyeofDrosophilapoints ingthemanufacturer'sinstructions.Hybridizationwas toacommonevolutionaryoriginforalleyes carriedoutin43%formamide,51Denhardt's,50mM (Zuker,1994).Thisargument®ndssupportinthe NaPO4 ,0.1%sodiumdodecylsulfate(SDS),0.1mg/ 7 workofHalderandcolleagues(1995),whoshow mLdenaturedsalmonspermDNAfor16hat42 C.Mem- thatmisexpressionofthemurinePax-6homologin braneswerewashedtwicein21SSCand0.1%SDSfor 10minatroomtemperature(217C),thentwicein0.21 Drosophilawingandlegimaginaldiskscausesthe SSCand0.1%SDSfor40minat607C.Intactlambda productionofectopiceyesintheadultstructures;a DNAwasisolatedfrom11pureclonesusingstandard phenotypeidenticaltothatproducedbymisexpres- methods(Sambrooketal.,1989).ThisDNAwasdi- sionoftheeyelessgeneitself.Themurinegene, gestedwithEcoR1andtheinsertsweresizedona0.8% however,causesthedevelopmentofcompound agarosegel.Thelongestclone,about2kb,wassubcloned ratherthanlenseyes,suggestingthatwhiletheearly intopBSII(SK/)(Stratagene,LaJolla,CA).Thiscon- stepsofeyedevelopmentareconserved,thelater structwasdesignatedpPax-6. speci®cationofstructureismodi®edbyspecies- NesteddeletionsofpPax-6weremadeusingthe speci®cfactors.Thisexperimentdemonstratesthat Erase-a-Basekit(Promega,Madison,WI)followingthe notonlyisPax-6necessaryforeyedevelopment manufacturer'sinstructions.Thesedeletionswerethen butthat,inDrosophilaatleast,itissuf®cientas sequencedinbothdirectionsbythedideoxychain-termi- nationmethod(Sangeretal.,1977)usingeitherthe well. Sequenase2.0kit(USBiochemical,Cleveland,OH)or Herewedescribethecloningandcharacteriza- theNEBThermalSequencingkit(NewEnglandBiolabs, tionofavertebratePax-6homologintheamphibian Beverly,MA)andfollowingthemanufacturer'sinstruc- Xenopuslaevis.TheXenopusembryoniceyeiseasy tions.SequenceanalysiswascarriedoutusingtheIntelli- toobservedinvivoanditsdevelopmentandstruc- geneticsSuite(IntelliGenetics,MountainView,CA)pro- turehavebeenwellcharacterized.Inaddition,the gram. XenopusPax-6andRetinalDevelopment 47 InSituHybridizationand myc-epitope±taggedvectorpCS2/MTinthesenseori- Immunohistochemistry entation.ThisconstructwasdesignatedP6mycS.The pCS2/MTvectorcontainsaCMVpromoter,apolyli- AlbinoX.laevisembryoswereobtainedfrompairmat- nkerincludingsixmyc-epitoperepeats,andanSV40con- ingsofanimalsraisedattheUniversityofCalifornia,San sensuspolyadenylationsite.Inaddition,itcontainsbind- Diego.Embryosweredejelliedwith2%Cysteine(pH8) ingsitesforSP6andT7RNApolymerases,whichallow in10%Holtfreter'ssolutionfor2±3minandthenwashed fortheproductionofmRNAfromthesamevectorused by10rapidchangesof10%Holtfreter's.Embryoswere forDNAmisexpression.ThepCS2vectorhasbeenused thenstainedwith0.5%NileBluein10%Holtfreter's previouslytomisexpresstranscriptionfactorsinXenopus for5minandwashedby®verapidchangesof10% (Ruppetal.,1994;TurnerandWeintraub,1994). Holtfreter's.Allembryoswerestagedaccordingto ForRNAinjections,cappedmRNAtranscriptswere NieuwkoopandFaber(1956)andmaintainedateither generatedusingtheSP6MessageMachineKits(Ambion, 147Cor217Cbefore®xation.Embryoswere®xedin0.1 Austin,TX)followingthemanufacturer'sinstructions. MMOPS,2mMEDTA,1mMMgSO4 ,3.7%formalde- PlasmidP6mycSwasusedasatemplatetomakesense hyde(MEMFA)for2hatroomtemperatureandstored mRNAforinjection.Asacontrol,Pax-6wasinserted 7 inethanolat020 C.Digoxygenin-labeledRNAprobes intopCS2/MTintheantisenseorientation.Messenger weremadefrompPax-6eitherbylinearizingtheplasmid RNAmadefromthisconstructencodesa40±aminoacid withXho1andtranscribingwithT3RNAPolymerase nonsensepolypeptidefusedtothesixmycepitopes.Either forsenseprobeorbylinearizingwithXba1andtranscrib- senseorantisensemRNA,50pgin5nLofwater,was ingwithT7RNApolymeraseforantisenseprobe(en- injectedintoeachofthetwopresumptivedorsoanterior zymessuppliedbyBoehringer-Mannheim,Indianapolis, blastomeresofafour-cellstage(NieuwkoopandFaber IN).Whole-mountinsituhybridizationwascarriedout stage3)Xenopusembryo.ForDNAinjections,approxi-