La lecture en ligne est gratuite
Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres

Partagez cette publication

Helmholtz Centre for Environmental Research – UFZ Permoserstraße 15 I 04318 Leipzig I Germany Internet: www.ufz.de
PhD Dissertation 06 / 2007
Chemical-sensitive genes in zebraIsh (Danio rerio) early development - identiIcation and characterisation ofdifferential expression in embryos exposed to the modelcompound 3,4-dichloroaniline
Doris Völker
PhD Dissertation 06 /2007
ISSN 1860-0387
CHEMICALSENSITIVEGENESINZEBRAFISH
(DANIORERIO)EARLYDEVELOPMENT
IDENTIFICATIONANDCHARACTERISATIONOF
DIFFERENTIALEXPRESSIONINEMBRYOSEXPOSEDTOTHE
MODELCOMPOUND3,4DICHLOROANILINE
DissertationzurErlangungdesakademischenGradesdoctorrerumnaturalium(Dr.rer.nat.)derFakultätMathematikundNaturwissenschaftenderTechnischenUniversitätDresdenvorgelegtvonDorisMariaVölker,geb.am10.03.1978inHaselünne,am30.09.2006verteidigtam14.03.2007Gutachter:Prof.Dr.GünterVollmer,TUDresdenProf.Dr.RolandNagel,TUDresdenProf.Dr.AndrewCossins,UniversitätLiverpooli
ii
AnfertigungderDoktorarbeitamUFZUmweltforschungszentrumLeipzigHalleGmbHinderHelmholtzGemeinschaft,DepartmentZelltoxikologieunterBetreuungvonDr.StefanScholzundBetreuungvonProf.Dr.GünterVollmer,InstitutfürZoologie,ProfessurfürMolekulareZellphysiologieundEndokrinologie,TechnischeUniversitätDresden
DieseArbeitwurdealsTeildesVerbundprojekts„EntwicklungeinesFischembryotestsalsAlternativefürverlängerteundchronischeFischtests:AnalysetoxischerWirkungenaufderBasisveränderterGenexpressionimDaniorerioEmbryotest(GenDarT)“„Teilprojekt1:SensitiveMarkergene“durchgeführt.GenDarTwurdedurchdasBundesministeriumfürBildungundForschung(BMBF)imRahmenderFörderrichtlinie„ErsatzmethodenzumTierversuch“,imProgrammderBundesregierung„BiotechnologieChancennutzenundgestalten“(FKZ0313016)gefördert.
iii
DieWeisheitderSchöpfungerkenntmandaran,dassdieFischestummsind.WasgäbeessonstfüreinenLärm,wennsieüberjedesEigackernwürden.FritzKortner(18921970)
iv
TABLEOFCONTENTS
TABLEOFCONTENTSLISTOFTABLES....................................................................................................................................... viii LISTOFFIGURES........................................................................................................................................ ix ABBREVIATIONS........................................................................................................................................ xi SUMMARY............................................................................................................................................... xiii ZUSAMMENFASSUNG............................................................................................................................... xv CHAPTERIINTRODUCTION.......................................................................................................................................... 1 1.1 Scopeofthestudy.................................................................................................................. 2 1.2 Alternativetestingmethodsforfishtoxicitytests............................................................. 3 1.3 Themodelorganismzebrafish(Daniorerio)....................................................................... 4 1.4 Themodelsubstance3,4dichloroaniline(3,4DCA) ........................................................ 5 1.5 Potentialapplicationsofgeneexpressioninalternativetestingstrategies.................... 7 1.6 Identificationoftoxicantsensitivegenes ........................................................................... 7 Biotransformation.......................................................................................................................... 8 Multixenobioticresistance ........................................................................................................... 9 Oxidative9stress ............................................................................................................................. Stressresponse ............................................................................................................................ 10 DNArepair ................................................................................................................................. 10 Apoptosis ..................................................................................................................................... 11 1.7Identificationofdifferentiallyexpressedgenesbymicroarraytechnology................... 12 1.8 Analysisofgenefunction ................................................................................................... 15 1.9 Specificobjectivesofthethesis .......................................................................................... 18 CHAPTERII MATERIALS&METHODS........................................................................................................................ 20 2.1Daniorerioembryotest(DarT)............................................................................................ 21 2.2 Earlylifestagetest(ELST)usingDaniorerio.................................................................... 22 2.3Chemicalanalysisof3,422dichloroaniline .......................................................................... 2.4RNAExtraction .................................................................................................................... 232.5ConventionalandquantitativeRTPCR ........................................................................... 242.5.1Procedureanddataacquisition................................................................................ 242.5.2RTPCRdataanalysisandstatistics......................................................................... 252.6Microarrayexperiments...................................................................................................... 262.6.1Procedureanddataaquisition ........................................................................................... 262.6.2Microarraydataanalysisandstatistics ................................................................... 282.7Functionalmanipulationstudies ....................................................................................... 31
v
TABLEOFCONTENTS
2.7.1PreparationofsenseRNAforoverexpressionstudies.......................................... 312.7.2PreparationofantisenseRNAforknockdownstudies........................................ 352.7.3Productionofonecellstageembryos .................................................................... 402.7.4Microinjectionandexposureofzebrafishembryos ............................................... 402.7.5Dataanalysisandstatistics........................................................................................ 44CHAPTERIII RESULTS.................................................................................................................................................... 45 3.1Toxicityof3,4dichloroanilineinDaniorerioembryos.................................................... 463.2Identificationofdifferentiallyexpressedgenesinembryosexposedto3,4DCA ...... 473.2.1Microarrayexperiments ............................................................................................ 473.2.2ConfirmationofdifferentiallyexpressedgenesbyquantitativeRTPCR .......... 513.2.3Expressionanalysisofgenesnotincludedinthemicroarray .............................. 533.3Geneexpressioninzebrafishearlylarvaeandjuvenilefishexposedto3,4DCA . 57β 3.4Effectof ‐naphthoflavoneexposureongeneexpression ......................................... 603.5Characterisationoftherelevanceofgeneexpressionfor3,4DCAtoxicity................. 613.5.1mRNAoverexpression............................................................................................... 623.5.2mRNAsilencing.......................................................................................................... 66EstablishmentofthesiRNAtechnique ....................................................................... 66mRNArepressionbyRNAi........................................................................................ 67CHAPTERIV DISCUSSION.............................................................................................................................................. 72 4.1Identificationof3,4DCAsensitivegenes ........................................................................ 734.1.1Microarrayexperiments ............................................................................................ 744.1.2Expressionanalysisofgenesnotincludedinthearray ........................................ 784.1.3Sensitivityofgeneexpressionatdifferentlifestagesandwithrespecttotoxicendpoints...................................................................................................................... 794.1.4Mechanismof3,4DCAeffectsdeducedfromgeneexpressionanalysis............ 814.2Relevanceofgeneexpressionfortoxicity......................................................................... 84mRNAmanipulationof3,4DCAsensitivegenes ........................................................ 85Controlinjections .......................................................................................................... 88CHAPTERV CONCLUDINGREMARKS&FUTUREDIRECTIONS................................................................................. 90 CHAPTERVI REFERENCES.............................................................................................................................................. 94
vi
TABLEOFCONTENTS
CHAPTERVII ANNEX.......................................................................................................................................................... I TableA.1I.I..............................................................................................3.A................................FigureA.1…………………………………………………………………………………….…IVBuffers,ReagentsandMedia .................................................................................................. IVA.1Embryotestmedium .............................................................................................................V A.2 Stocksolutionof3,4dichloraniline(Fluka,50mg/L) .......................................................V A.3 ReversetranscriptionofRNAtofirststrandcDNA .........................................................V A.4 Polymerasechainreaction(RTPCR) ................................................................................ VI A.5 Quantitativepolymerasechainreaction(qRTPCR)...................................................... VII A.6 Microarraysolutions .......................................................................................................... VII A.7Cloning................................................................................................................................VIII A.8 DenaturingRNAagarosegelelectrophoresis....................................................................X A.9 Nondenaturingpolyacrylamidegelelectrophoresis(PAGE)....................................... XI A.10 Tricainemethanesulfonate(MS222)stocksolutionforanesthetisingembryos ........ XII A.11 Fluoresceinisothiocyanatedextransolution................................................................... XII ACKNOWLEDGEMENT........................................................................................................................... XIII VERSICHERUNGGEMÄSS§5A...............................................................................................................V.X ERKLÄRUNGGEMÄSS§5B......................................X.........V....................................................................... CURRICULUMVITAE…………………………………………………………………………………...XVI
vii
LISTOFTABLES
LISTOFTABLESTable2.1InvitrotranscriptionoffulllengthcDNAs………………………………………..Table2.2SequenceinformationforsiRNAs……………………………………….................Table2.3SequencesofmismatchsiRNAs……………………………………………………Table2.4InjectionvolumeandfinallyinjectedconcentrationsofmRNAandsiRNA…Table2.5Typesofinjectionsthatwereusedtostudytheeffectofgenemanipulationontoxicityof3,4DCAinzebrafishembryos……………………………………..Table3.1Acutetoxicityof3,4DCAinzebrafishembryos…………………….....................Table3.2Significantdifferentiallyexpressedgenesin50hpfembryosexposedfor48hoursto12.4μM3,4DCA……………………………………………......................Table3.3Candidategenesforpotential3,4DCAsensitiveexpression…………………..Table3.4Chronictoxicitydataof3,4DCAofanearlylifestagetest(ELST)usingzebrafish………………………………………………………………………………
343839424347505458TableA.1PrimersequencesforRTPCR……………………………………………………...IIIIIIV
TableA.2PrimersusedfortheidentificationofsuccessfullytransformedE.coli(“colonyPCR”)……………………………………………………………………….TableA.3PrimersusedforconfirmationofsiRNAmediatedgeneknockdownbyRTPCR.…………………………………………………………………..........................
viii
LISTOFFIGURES
LISTOFFIGURESFigure1.1Chemicalstructureofthemodelsubstance3,4dichloroaniline.............Figure1.2Workflowschemeofthemicroarrayexperimentsconductedinthisthesis.…………………………………………………………………………Figure1.3PrincipleofRNAinterference……………………………………………..Figure2.1Relativedifferenced(i)ingeneexpression……………………………….Figure2.2ExampleofasiRNAtemplateoligonucleotide………………………......Figure2.3PrincipleofsiRNApreparation……………………………………...........Figure2.4Spawningapproachtoobtainembryosofidenticalstagesatdefinedtimepoints………………………………………………………………….. Figure3.1Developmentalaberrationscausedby3,4DCAinzebrafishembryos…………………………………………………………....................Figure3.2Significanceanalysisofmicroarrays……………………………………...Figure3.3RTPCRofgenesthathavebeenshowntobedifferentiallyexpressedbymicroarrayanalysis……………………………………………………...Figure3.4Geneexpressionofcyp1a,ahr2andfzr1inzebrafishembryosanalysedbyquantitativeRTPCR………………….....................................................Figure3.5RTPCRofcandidategenesfordifferentialexpressioninresponseto3,4DCA.……………………………………………………………………...Figure3.6Geneexpressionofthegenesnrf2,mafg1,maft,ho1andhsp70inzebrafishembryos……………………………..............................................Figure3.7Expressionofcyp1a,ahr2,fzr1,nrf2,maftandho1insacfryandjuvenilestagesofzebrafishexposedto3,4DCA………………………...Figure3.8Geneexpressionofcyp1a,nrf2,ho1andfzr1inzebrafishembryosβ exposedto ‐naphthoflavone……………………………………………..Figure3.9DenaturingagarosegelelectrophoresisofasynthesisedmRNA…………………………………...……………………………………Figure3.10Developmentaldisordersinducedbyoverexpression………………….Figure3.11ImpactofmRNAinjectionontranscriptabundance.……........................Figure3.12Overexpressionofthegenescyp1a,ho1andnrf2inembryosexposedto12.4μM3,4DCAcausedasignificantreductionofdevelopmentaldisorders.…………………………………………………………………….Figure3.13siRNAmediatedrepressionofntl………………………………..……....Figure3.14ComparisonofnotailmutantandsiRNAgeneratedphenotypesinzebrafish……………………………………………………………………...Figure3.15Nondenaturingpolyacrylamidegelelectrophoresisofsicyp1asynthesis………..……………………………………………………………Figure3.16ImpactofsiRNAinjectionsontranscriptabundance…………………....Figure3.17Injectionsofcyp1asiRNAorho1siRNAintozebrafishembryosincreasedseverityofmalformationscausedby3,4DCAexposure……………………………………………………………………...
61417303637404649515356575961626364656667686970ix
LISTOFFIGURES
LISTOFFIGURES(CONTINUED)Figure3.18Repressionofthegenescyp1aandho1inembryosexposedto12.4 μM3,4DCAcausedanincreaseinthefrequencyofdevelopmentaldisorders………………………………………………………………………..Figure4.1Illustrationofsignallingpathwaysthatmightbeinvolvedintheinductionofgenesinzebrafishembryosexposedto3,4DCA………….....Figure5.1RelativesensitivityofGeneDarT:LOECGeneDarT/LOECELST………...........................................................................................................βFigureA.1ChemicalstructureoftheAHRagonistnaphthoflavone(BNF)…………………………………………………………………………….FigureA.7.1MapofexpressionvectorpCMVSport6.1.………………………………….FigureA.7.2MapofexpressionvectorpME18SFL…………………………………….....
x
71
84
92
IVIXX