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Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system

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Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system
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  MOLECULARBRAINRESEARCH MolecularBrainResearch41(1996)111-120 Researchreport CellularexpressionoftheimmediateearlytranscriptionfactorsNurrlandNGFI-Bsuggestsageneregulatoryroleinseveralbrainregionsincludingthenigrostriataldopaminesystem RolfH.Zetterstroma’”,RegWilliamsb,ThomasPerlmannc,LarsOlsonaa DepartmentofNeuroscience,KarolinskaInstitute,S-17177Stockholm,SwedenbLaboratoryofDevelopmentalBiology,DepartmentofCellandMolecularBiology,KarolinskaInstitute,S-17177Stockholm,Sweden‘LudwigInstituteforCancerResearch,Box240,KarolinskaInstitute,S-17177Stockholm,Sweden Accepted27February1996 Abstract NurrlandNGFI-Barecloselyrelatedorphanmembersofthesteroid-thyroidhormonereceptorfamilyinvolvedinimmediateearlyresponsestostimulisuchasgrowthfactors.In-situhybridizationnthedevelopingandadultmouseandratdemonstratedNurrlmRNAinseveralregionsduringearlycentralnervoussystem(CNS)development.Expressionpersistedthroughthepre-andpostnatalperiodsandwasalsofoundinseveralareasintheadultCNS.Positiveareasincludetheolfactorybulb,partsofthecortex,thehippocampalformationandsubstantialnigrawhereNurrlandtyrosinehydroxylasemRNAswereco-expressed.6-Hydroxydopamine-inducedegenerationofmesencephalicdopamineneuronsledtoacorrespondingossofNurrlmRNA,demonstratingalinkbetweenNurrlanddopaminergicneurons.NGFI-BmRNAwasnotfoundintheprenatalCNSbutwashighlyexpressedintheadultbraininmanyareasincludingtheolfactorybulb,cortex,basalgangliaandhippocampus.ThespatiotemporaldistributionofNurrlandNGFI-BmRNAssuggeststhatthesetranscriptionfactorsareinvolvedinthedevelopmentandmaturationofspecificsetsofCNSneurons.Theexperimentaldataimplythatoneofthesefunctionsmaybetocontrolgeneregulato~eventsimportantfordevelopmentandfunctionofthoseneuronsthatdegenerateinpatientswithParkinson’sdisease. Keywords: Nurrl;NGFI-B;In-situhybridization;Immediateearlygene;Transcriptionfactor;Orphanreceptor 1.Introduction Nurrl(nur-relatedfactor1,alsoknownasRNR1,NOT,HZF-3)andNGFI-B(nervegrowthfactor-inducibleB,alsoknownasNur77,NAK1/TR3,NIO)aretwocloselyrelatedmembersofthesteroid-thyroidhormonereceptorfamilyofligand-activatedtranscriptionfactors[6,11,15,22].SinceNurrlandNGFI-Blackidentifiedligandstheyarealsoreferredtoas‘orphanreceptors’.AdditionalreceptorswithsimilaritytoNurrlandNGFI-Bwererecentlyidenti-fiedinrat,C. elegans and Drosophila [10,16,23].Thelatterinvertebrateorphanreceptorsdemonstratearemark-ableevolutionaryconservationoftheNurrl/NGFI-Bsub-classofsteroid-thyroidhormonereceptors.TheNurrl/NGFI-Bsubgroupisuniquewithinthelargefamilyofnuclearreceptorsbybeingencodedbyimmedi- “Correspondingauthor.Fax:(46)(8)32-3742;E-mail:rolf.zetterstrom@neuro.ki.se0169-328X/96/ 15.00PublishedbyElsevierScienceB.V. PZISO 169-328X(96)OO074-5 ateearlygenesthatarerapidlyinducedbyvariousstimuli,e.g.,growthfactors.Otherimmediateearlygenes,includ-ingc-~osand c-myc,are involvedincriticalprocessessuchasgrowth,differentiationandapoptosisinresponsetoexternalstimuli,suggestingthatalsoNurrlandNGFI-Bmightplaysimilarcentralrolesincellsignaling.Indeed,NGFI-BhasrecentlybeenimplicatedinTcellapoptosisandadenosteroidogenesis[14,25,26],eventhoughrecentresultsindicatethatthereisnoabsoluterequirementforthepresenceofNGFI-Bforapoptosistotakeplace[12].NofunctionhasyetbeendescribedforNurrl.NurrlandNGFI-BbindtospecificDNAsequences(responseelements)inthevicinityofgenesthattheyregulate[4,15,20].Severalnuclearreceptors,suchastheall-tramretinoicacid(RA)receptor(RAR),bindtosuchDNAsequencesasheterodimerswiththereceptorfor9-cisRA(RXR)[13].NurrlandNGFI-BhavebeenshowntointeractwithDNAasmonomers[25]butcanalsoformheterodimerswithRXRandbindtosimilarresponseele-  112 R.H.Zetterstrtimetal./MolecularBrainResearch41 1996)111-120 mentsasthoserecognizedbyRXR-RARheterodimers[20].However,whereasRARpreventsRXRfrombeingactivated,RXR-NtuTl/NGFI-BheterodimersarehighlyresponsivetotheRXRligand, 9-cis RA[3,20].Thus,heterodimerizationwithNurrlorNGFI-BshiftsRXRfromasilenttoanactiveheterodimerizationpartner.Beingencodedbygrowthfactor-inducibleimmediateearlygenes,theinteractionofNurrlandNGFI-BwithRXRthusprovidesamechanismforcross-talkbetweenvitaminAandgrowth-factorsignalingpathways.AsafirststeptowardsunderstandingthefunctionofNurrlandNGFI-Bwehaveusedin-situhybridizationtoanalyzethespatiotemporalcellulardistributionoftheirmRNAsduringembryonicandpostnataldevelopmentandintheadult.WefindthattheseimmediateearlygenesareexpressedinspecificregionsofthedevelopingandadultCNS.ThedatasuggestthatNurrlplaysaroleindevelop-ingandadultCNSneuronsincludingthedopaminergicneuronsofsubstantialnigra.2. Materialsandmethods2.1. Animals Atotalof22mice(NMRI,BomholtBreedingandResearchCenter,Copenhagen,Denmark)and33rats(Sprague-Dawley,BtkKLaboratories,Sollentuna,Swe-den)fromdifferentdevelopmentalstages(E13,E15,E18,PO,P7,P14andadult)wereused.Pregnantfemaleswerekilledbycervicaldislocationandembryoswereremoved,staged(E15andE18decapitated)andfixedimmediatelyin Fig.1,Bright-(A,C)anddark-field(B,D)photomicrographsoftransversesectionsshowing zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML urrlmRNAexpressioninthe E13mouseCNS.. B:Strong NurrlmRNAexpressionisseenwithinthemedialneuroepitheliumoftheposteriorneocortex,inventralnucleiofthehypothalamus(HT)andinthedifferentiatingfieldofthedevelopingpens.ThedistributioninthisregionsuggeststhattheNurrlmessageisspecificallyexcludedfromthetrigeminalandfacialmotornuclei.C,D:NurrlmRNAisfoundtobeheavilyexpressedinthespinalcord.Thedorsalrootganglion(G)isalsoincludedinthissection,andlikeallotherperipheralganglia,isnegative.Thalamus(T),3rdventricle(V),centralcanal(CC).Scalebar=350pm.  R,H.Zetterstrtimetal./MolecularBrainResearch41 1996)111-120 113Fig.2.FrontalsectionofanewbornratbrainshowingNurrlmRNAexpression.StrongexpressionofNurrlmRNAisseenindeeplayerVIneuronsincortexclosetothedevelopingcorpuscaflosum(arrowheads).MorelateraIIyincortexNurrlmRNA-positivecellsarealsoseeninthe other corticatlayers.Verystrongexpressionisalsoseeninsubiculum(S)andthemedialhabenukn’nucleus.Inthalamus(T)noexpressionofNrrrrlmRNAisseenbutagroupofcellsintheareaoftheperiventricularhypothrdamicnucleusinhypothalamus(HT)areNurrlmRNApositive.Additionally,Nurrl-expressingceilsarefoundinCA1ofthehippocampalformation(H)andscatteredNurrlmRNA-positivecellsareseeninthelateralhabenularnucleus(L)andtheamygdatoidcomplex(A).Scalebar=1mm. 4 paraformaldehyde,clearedovernightinphosphatebufferedsaline(PBS)andinfiltratedwith20 sucrosepriortocutting.Animalsfrompostnatalstageswerekilledbydecapitationandtissueswererapidlyfrozen.Tissuesweresectionedat10p,m(embryonic)or14Lm(post-natal)andthaw-mountedontoslides(ProbeOn,Fischer).bindingtotissuecomponents,AsapositivecontrolweusedthefactthathybridizationwithaTH-specificprobeinallcasesgeneratedtheexpectedpatternofmRNAdistribu-tion.2.3. In-situhybridization 2.2. Probepreparationandspecificitycontrols Oligonucleotideprobescomplementarytonucleotides1430–1477inNun-lmRNA[11],nucleotides1191-1238inNGFI-BmRNA[15]and1441–1478intyrosinehy-droxylase(TH)mRNA[5]havingnosimilaritiestoknownsequences,weresynthesized(ScandinavianGeneSynthesisAB)andradiolabeledwith[35S]deoxyadenosine5’-[a-thio]triphosphate(NEN)atthe3’-endusingterminalde-oxynucleotidyltransferase(Amersham).Specificitycon-trolsincluded:(1)useofasecondprobecomplementarytoanotherpartofthemRNAstrand(1334–1381forNurrlmRNAand1272–1319forNGFI-BmRNA).Thepatternsobservedwiththesecomplementaryprobeswerealwaysidentical;(2)Inclusionofa100-foldexcessofunlabeledprobeinthehybridizationsolution,inwhichcasethelabelingnormallyobtainedwascompletelyabsent;(3)useofa50bprandomprobe,uniquecomparedtoknownsequencesinGenbank,labeledwiththesameamountof35S.ThisprobedidnotshowanylocalizedorspecificIn-situhybridizationwasperformedaccordingtoDager-lindetal.[2].Inbrief,sectionswerehybridizedfor16–18hat42°Cinhumidifiedboxesandrinsed5X15minin1 X SSCat55or60”C.Sectionswerethendehydrated,air-dried,dippedinphotographicemulsion(KodakNTB-2,diluted1:1indeionizedwater),exposedfor4–6weeksat–20°C,developed4min(D-19,Kodak),fixed6min(mixtureofKodak3000Aand3000B)andrinsed30mininwaterbeforebeinglightlycounterstainedwithCresylVioletorToluidineBlueandmounted(Entellan,Merck).Sectionswereanalyzedusinglight-anddark-fieldmi-croscopy(Zeiss,Axiophot)andphotographed(KodakT-Max100).”TheidentificationofCNSareaswasguidedbypublishedatlases[1,9,18,19].Amountofspecificlabelingwassemiquantitivelyrecordedusingascalewith4steps:–,(+),+and++.Reliabilityandvalidityofthisscalewasascertainedbythepositivecorrelationobtainedbe-tweenindividualanimals,betweenratsandmiceaswellasbetweenobservers.  114 R.H,Zetterstrtimetal./MolecularBrainResearch41 1996)111-120 Table1DistributionofNurrlandNGFI-BmRNAinthepostnatalratCNSBrainareaNrrrrlmRNANGFI-BmRNAPoPIP14AdultPoPIP14AdultOlfactorybulb*Neocortex*ClaustmmCaudate/putamen*AccumbensnucleusOlfacto~tubercleSeptum+DiagonalbandiGlobuspallidusHippocampalformationCA1CA2CA3DentategymsSubiculumMedialhabenularnucleusLateralhabenularnucleusThakunusHypothalamus“AmygdalaVentraltegmentalarea*Substantialnigrap.compacta“Substantialnigrap.reticulateInterpedunculurnucleusRednucleusLocuscoeruleusMotornucleusofvagusnerveCerebellumtSpinalcord*  — ——— — — — ————————————————————— —— —— ; ;  + : ; (+)  + — — ———  + —   :+ ++  T+;—:+;++++———— ————————— ————————  :+ :+;  + — + +)— — —  + ++:+;—;+;++++—   :+ ;+;  + :+ ++:+;—;+;++++ — ; ;—: ; ——  + (+)— ——————— —— — — —— ,noexpression;(+)scatteredmRNA-positivecells;+,expression;++,strongexpression.*Seeresultsforafurtherdescription.+possiblystriatepatterningranularlayer.*individuallyvariableandscatteredexpreSSiOn. 2.4. 6-Hydroxydopaminelesions mg/kg,s.c.)rotationalbehaviortoassureacompletedopaminedenervation[24].Ratswereanesthetizedandplacedinastereotaxicframe.Eightmicrogramsof6-hydroxydopamine(6-OHDA)in4I.L1ofRinger’ssolutionwith80p,gof3. Results ascorbicacidwasunilaterallyinjectedintothenigro-striatalpathway[24].Tworatswerekilledeachafter1,3and7TheexpressionofNurrlmRNAwashighinmanydaysandanadditionalsixanimalsweretaken9monthsregionsofthedevelopingratandmouseCNSandtheaftersurgery.Theratstakenafter9monthswerealsoexpressionremainedhighinmostoftheseareasintotestedinarotometerforapomorphine-induced(0.05adulthood.Incontrast,onlylimitedexpressionofNGFI-B Fig. 3, Coronalsectionsofthenewborn(C,D)andadult(A,B)ratbrainatthelevelofcaudate/putamen.StrongNurrlmRNAexpressionisfoundincorticallayerVIandintheareaofclaustmmanddorsalendopiriformnucleus,bothinthenewborn(C)andadult(A)brain.In(C)alsonotethescatteredNrrrrlmRNA-positivecellsinseptum(S)anddiagonalband(DB).B,D:CorrespondingsectionslabeledwiththeNGITB-specificprobe.Incortex(CX),noNGFI-BmRNAexpressionisseeninthenewbornanimal(D)whereasastrongin-situsignalisfoundinmostcorticallayersintheadultratbrain(B).Thelabelingincaudate/putarnen(CPU)isalsostrikinglydifferentbetweenthenewbornandadultanimals.Withinthenewborncaudate/putamen,expressionisseeninclusters(arrowsinD)whileintheadultratstriatum,NGFI-B-positivecellsarescatteredthroughouttheentirearea.ScatteredNGFI-BmRNA-labeledcellsarealsoseeninthepirifonmcortex(Pir)inthenewbornratbrain.Duetothespecialobliquelightilluminator(Zeiss)usedinsteadofanordinarydark-fieldcondensortovisualizeNurrlandNFGI-BmRNAexpressioninlow-powermagnifications,areaswherecellsaredenselyarranged,asinthecaudate/putamenneuroepitheliumcpu,tendtorefractlightandappearlabeled.Thisisalsotrueforwhitematterlikecorpuscallosum(CC).However,noNurrlorNGFI-BmRNAexpressionwasobservedinthoseareas.Scalebar=500~m.  R.H.Zetterstrometal./MolecularBrainResearch41 1996)111–120115
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