Chemical-sensitive genes in zebrafish (Danio rerio) early development - identification and characterisation of differential expression in embryos exposed to the model compound 3,4-dichloroaniline [Elektronische Ressource] / Doris Völker. [Helmholtz Centre for Environmental Research, UFZ]

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PhD Dissertation 06 / 2007Chemical-sensitive genes in zebrafish (Danio rerio) early development - identification and characterisation of differential expression in embryos exposed to the model compound 3,4-dichloroanilineDoris VölkerHelmholtz Centre for Environmental Research – UFZPermoserstraße 15 I 04318 Leipzig I GermanyInternet: www.ufz.de ISSN 1860-0387 PhD Dissertation 06 / 2007CHEMICAL‐SENSITIVE GENES IN ZEBRAFISH  (DANIO RERIO) EARLY DEVELOPMENT –  IDENTIFICATION AND CHARACTERISATION OF DIFFERENTIAL EXPRESSION IN EMBRYOS EXPOSED TO THE MODEL COMPOUND 3,4‐DICHLOROANILINE   Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden  vorgelegt von Doris Maria Völker, geb. am 10.03.1978 in Haselünne, am 30.09.2006  verteidigt am 14.03.2007   Gutachter: Prof. Dr. Günter Vollmer, TU Dresden   Prof. Dr. Roland Nagel, TU  Prof. Dr. Andrew Cossins, Universität Liverpool                                                                                                                                                                    i                              ii  Anfertigung der Doktorarbeit am  UFZ – Umweltforschungszentrum Leipzig‐Halle GmbH  in der Helmholtz Gemeinschaft, Department Zelltoxikologie unter Betreuung von Dr. Stefan Scholz  und Betreuung von Prof.
Publié le : lundi 1 janvier 2007
Lecture(s) : 21
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Source : D-NB.INFO/1007282541/34
Nombre de pages : 142
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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
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AnfertigungderDoktorarbeitamUFZUmweltforschungszentrumLeipzigHalleGmbHinderHelmholtzGemeinschaft,DepartmentZelltoxikologieunterBetreuungvonDr.StefanScholzundBetreuungvonProf.Dr.GünterVollmer,InstitutfürZoologie,ProfessurfürMolekulareZellphysiologieundEndokrinologie,TechnischeUniversitätDresden
DieseArbeitwurdealsTeildesVerbundprojekts„EntwicklungeinesFischembryotestsalsAlternativefürverlängerteundchronischeFischtests:AnalysetoxischerWirkungenaufderBasisveränderterGenexpressionimDaniorerioEmbryotest(GenDarT)“„Teilprojekt1:SensitiveMarkergene“durchgeführt.GenDarTwurdedurchdasBundesministeriumfürBildungundForschung(BMBF)imRahmenderFörderrichtlinie„ErsatzmethodenzumTierversuch“,imProgrammderBundesregierung„BiotechnologieChancennutzenundgestalten“(FKZ0313016)gefördert.
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DieWeisheitderSchöpfungerkenntmandaran,dassdieFischestummsind.WasgäbeessonstfüreinenLärm,wennsieüberjedesEigackernwürden.FritzKortner(18921970)
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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
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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
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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
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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.…………………………………………………………………..........................
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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……………………………………………………………………...
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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…………………………………….....
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