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]

Voelker

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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.

Sujets

• Biologie

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2007
• Nombre de lectures: 22
• Poids de l'ouvrage: 2 Mo

Extrait

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 of differential expression in embryos exposed to the model compound 3,4-dichloroaniline

Doris Völker

PhD Dissertation 06 /2007

ISSN 1860-0387

C HEMICAL ‐S ENSITIVE G ENES I N Z EBRAFISH

(D ANIO R ERIO )E ARLY D EVELOPMENT –

I DENTIFICATIONAND C HARACTERISATION O F

D IFFERENTIAL E XPRESSION I N E MBRYOS E XPOSED T O T HE

M ODEL C OMPOUND 3,4‐D ICHLOROANILINE

Dissertation zur ErlangungdesakademischenGrades doctorrerumnaturalium(Dr.rer.nat.) derFakultätMathematikundNaturwissenschaften derTechnischenUniversitätDresden vorgelegtvon DorisMariaVölker,geb.am10.03.1978inHaselünne, am30.09.2006 verteidigtam 14.03.2007 Gutachter:Prof.Dr.GünterVollmer,TUDresden Prof.Dr.Roland Nagel,TUDresden Prof.Dr.AndrewCossins,UniversitätLiverpool i

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AnfertigungderDoktorarbeitam UFZ–UmweltforschungszentrumLeipzig‐HalleGmbH inderHelmholtzGemeinschaft, DepartmentZelltoxikologie unterBetreuungvonDr.StefanScholz undBetreuungvonProf.Dr.GünterVollmer, InstitutfürZoologie, ProfessurfürMolekulareZellphysiologieundEndokrinologie, TechnischeUniversitätDresden

DieseArbeitwurdealsTeildesVerbundprojekts „EntwicklungeinesFischembryotestsalsAlternativefürverlängerteund chronischeFischtests:AnalysetoxischerWirkungenaufderBasisveränderter GenexpressionimDaniorerio‐Embryotest(Gen‐DarT)“ „Teilprojekt1:SensitiveMarkergene“ durchgeführt.Gen‐DarTwurdedurchdasBundesministeriumfürBildungund Forschung(BMBF)imRahmenderFörderrichtlinie„Ersatzmethodenzum Tierversuch“,imProgrammderBundesregierung„Biotechnologie–Chancen nutzenundgestalten“(FKZ0313016)gefördert.

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DieWeisheitderSchöpfung erkenntmandaran, dassdieFischestummsind. Wasgäbeessonst füreinenLärm, wennsieüberjedesEi gackernwürden. FritzKortner(1892‐1970)

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T ABLEOFCONTENTS

T ABLEOFCONTENTS L ISTOFTABLES ....................................................................................................................................... viii L ISTOFFIGURES ........................................................................................................................................ ix A BBREVIATIONS ........................................................................................................................................ xi S UMMARY ............................................................................................................................................... xiii Z USAMMENFASSUNG ............................................................................................................................... xv C HAPTER I I NTRODUCTION .......................................................................................................................................... 1 1.1 Scopeofthestudy.................................................................................................................. 2 1.2 Alternativetestingmethodsforfishtoxicitytests............................................................. 3 1.3 Themodelorganismzebrafish(Daniorerio)....................................................................... 4 1.4 Themodelsubstance3,4‐dichloroaniline(3,4‐DCA) ........................................................ 5 1.5 Potentialapplicationsofgeneexpressioninalternativetestingstrategies.................... 7 1.6 Identificationoftoxicant‐sensitivegenes ........................................................................... 7 Biotransformation.......................................................................................................................... 8 Multixenobioticresistance ........................................................................................................... 9 Oxidative9stress ............................................................................................................................. Stressresponse ............................................................................................................................ 10 DNArepair ................................................................................................................................. 10 Apoptosis ..................................................................................................................................... 11 1.7Identificationofdifferentiallyexpressedgenesbymicroarraytechnology................... 12 1.8 Analysisofgenefunction ................................................................................................... 15 1.9 Specificobjectivesofthethesis .......................................................................................... 18 C HAPTER II M ATERIALS &M ETHODS ........................................................................................................................ 20 2.1Daniorerioembryotest(DarT)............................................................................................ 21 2.2 Earlylifestagetest(ELST)usingDaniorerio.................................................................... 22 2.3Chemicalanalysisof3,4‐22dichloroaniline .......................................................................... 2.4RNAExtraction .................................................................................................................... 23 2.5ConventionalandquantitativeRT‐PCR ........................................................................... 24 2.5.1Procedureanddataacquisition................................................................................ 24 2.5.2RT‐PCRdataanalysisandstatistics......................................................................... 25 2.6Microarrayexperiments...................................................................................................... 26 2.6.1Procedureanddataaquisition ........................................................................................... 26 2.6.2Microarraydataanalysisandstatistics ................................................................... 28 2.7Functionalmanipulationstudies ....................................................................................... 31

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2.7.1Preparationofsense‐RNAforoverexpressionstudies.......................................... 31 2.7.2Preparationofantisense‐RNAforknockdownstudies........................................ 35 2.7.3Productionofone‐cell‐stageembryos .................................................................... 40 2.7.4Microinjectionandexposureofzebrafishembryos ............................................... 40 2.7.5Dataanalysisandstatistics........................................................................................ 44 C HAPTER III R ESULTS .................................................................................................................................................... 45 3.1Toxicityof3,4‐dichloroanilineinDaniorerioembryos.................................................... 46 3.2Identificationofdifferentiallyexpressedgenesinembryosexposedto3,4‐DCA ...... 47 3.2.1Microarrayexperiments ............................................................................................ 47 3.2.2ConfirmationofdifferentiallyexpressedgenesbyquantitativeRT‐PCR .......... 51 3.2.3Expressionanalysisofgenesnotincludedinthemicroarray .............................. 53 3.3Geneexpressioninzebrafishearlylarvaeandjuvenilefishexposedto3,4‐DCA . 57 β 3.4Effectof ‐naphthoflavoneexposureongeneexpression ......................................... 60 3.5Characterisationoftherelevanceofgeneexpressionfor3,4‐DCAtoxicity................. 61 3.5.1mRNAoverexpression............................................................................................... 62 3.5.2mRNAsilencing.......................................................................................................... 66 EstablishmentofthesiRNA‐technique ....................................................................... 66 mRNArepressionbyRNAi........................................................................................ 67 C HAPTER IV D ISCUSSION .............................................................................................................................................. 72 4.1Identificationof3,4‐DCA‐sensitivegenes ........................................................................ 73 4.1.1Microarrayexperiments ............................................................................................ 74 4.1.2Expressionanalysisofgenesnotincludedinthearray ........................................ 78 4.1.3Sensitivityofgeneexpressionatdifferentlifestagesandwithrespecttotoxic endpoints...................................................................................................................... 79 4.1.4Mechanismof3,4‐DCAeffectsdeducedfromgeneexpressionanalysis............ 81 4.2Relevanceofgeneexpressionfortoxicity......................................................................... 84 mRNAmanipulationof3,4‐DCAsensitivegenes ........................................................ 85 Controlinjections .......................................................................................................... 88 C HAPTER V C ONCLUDING R EMARKS &F UTURE D IRECTIONS ................................................................................. 90 C HAPTER VI R EFERENCES .............................................................................................................................................. 94

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C HAPTER VII A NNEX .......................................................................................................................................................... I TableA.1‐I.I..............................................................................................3.A................................ FigureA.1…………………………………………………………………………………….…IV Buffers,ReagentsandMedia .................................................................................................. IV A.1Embryotestmedium .............................................................................................................V A.2 Stocksolutionof3,4dichloraniline(Fluka,50mg/L) .......................................................V A.3 ReversetranscriptionofRNAtofirststrandcDNA .........................................................V A.4 Polymerasechainreaction(RT‐PCR) ................................................................................ VI A.5 Quantitativepolymerasechainreaction(qRT‐PCR)...................................................... VII A.6 Microarraysolutions .......................................................................................................... VII A.7Cloning................................................................................................................................VIII A.8 DenaturingRNAagarosegelelectrophoresis....................................................................X A.9 Non‐denaturingpolyacrylamidegelelectrophoresis(PAGE)....................................... XI A.10 Tricainemethanesulfonate(MS222)stocksolutionforanesthetisingembryos ........ XII A.11 Fluoresceinisothiocyanatedextransolution................................................................... XII A CKNOWLEDGEMENT ........................................................................................................................... XIII V ERSICHERUNGGEMÄSS §5 A ...............................................................................................................V.X E RKLÄRUNGGEMÄSS §5 B ......................................X.........V....................................................................... C URRICULUM V ITAE …………………………………………………………………………………...XVI

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L ISTOFTABLES

L ISTOFTABLES Table2.1Invitrotranscriptionoffull‐lengthcDNAs……………………………………….. Table2.2SequenceinformationforsiRNAs………………………………………................. Table2.3Sequencesofmismatch‐siRNAs…………………………………………………… Table2.4InjectionvolumeandfinallyinjectedconcentrationsofmRNAandsiRNA… Table2.5Typesofinjectionsthatwereusedtostudytheeffectofgenemanipulation ontoxicityof3,4‐DCAinzebrafishembryos…………………………………….. Table3.1Acutetoxicityof3,4‐DCAinzebrafishembryos……………………..................... Table3.2Significantdifferentiallyexpressedgenesin50hpfembryosexposedfor48 hoursto12.4μM3,4‐DCA……………………………………………...................... Table3.3Candidategenesforpotential3,4‐DCA‐sensitiveexpression………………….. Table3.4Chronictoxicitydataof3,4‐DCAofanearlylifestagetest(ELST)using zebrafish………………………………………………………………………………

34 38 39 42 43 47 50 54 58 TableA.1PrimersequencesforRT‐PCR……………………………………………………... II III IV

TableA.2PrimersusedfortheidentificationofsuccessfullytransformedE.coli (“colonyPCR”)………………………………………………………………………. TableA.3PrimersusedforconfirmationofsiRNA‐mediatedgeneknockdownbyRT‐ PCR.…………………………………………………………………..........................

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L ISTOFFIGURES

L ISTOFFIGURES Figure1.1Chemicalstructureofthemodelsubstance3,4‐dichloroaniline............. Figure1.2Workflowschemeofthemicroarrayexperimentsconductedinthis thesis.………………………………………………………………………… Figure1.3PrincipleofRNAinterference…………………………………………….. Figure2.1Relativedifferenced(i)ingeneexpression………………………………. Figure2.2ExampleofasiRNAtemplateoligonucleotide………………………...... Figure2.3PrincipleofsiRNApreparation……………………………………........... Figure2.4Spawningapproachtoobtainembryosofidenticalstagesatdefined timepoints………………………………………………………………….. Figure3.1Developmentalaberrationscausedby3,4‐DCAinzebrafish embryos………………………………………………………….................... Figure3.2Significanceanalysisofmicroarrays……………………………………... Figure3.3RT‐PCRofgenesthathavebeenshowntobedifferentiallyexpressed bymicroarrayanalysis……………………………………………………... Figure3.4Geneexpressionofcyp1a,ahr2andfzr1inzebrafishembryosanalysed byquantitativeRT‐PCR…………………..................................................... Figure3.5RT‐PCRofcandidategenesfordifferentialexpressioninresponseto 3,4‐DCA.……………………………………………………………………... Figure3.6Geneexpressionofthe genesnrf2,mafg1,maft,ho‐1andhsp70in zebrafishembryos…………………………….............................................. Figure3.7Expressionofcyp1a,ahr2,fzr1,nrf2,maftandho‐1insacfryand juvenilestagesofzebrafishexposedto3,4‐DCA………………………... Figure3.8Geneexpressionofcyp1a,nrf2,ho‐1andfzr1inzebrafishembryos β exposedto ‐naphthoflavone…………………………………………….. Figure3.9Denaturingagarosegelelectrophoresisofasynthesised mRNA…………………………………...…………………………………… Figure3.10Developmentaldisordersinducedbyoverexpression…………………. Figure3.11ImpactofmRNAinjectionontranscriptabundance.……........................ Figure3.12Overexpressionofthegenescyp1a,ho‐1andnrf2inembryosexposed to12.4μM3,4‐DCAcausedasignificantreductionofdevelopmental disorders.……………………………………………………………………. Figure3.13siRNA‐mediatedrepressionofntl………………………………..…….... Figure3.14ComparisonofnotailmutantandsiRNA‐generatedphenotypesin zebrafish……………………………………………………………………... Figure3.15Non‐denaturingpolyacrylamidegelelectrophoresisofsi‐cyp1a synthesis………..…………………………………………………………… Figure3.16ImpactofsiRNAinjectionsontranscriptabundance………………….... Figure3.17Injectionsofcyp1asiRNAorho‐1siRNAintozebrafishembryos increasedseverityofmalformationscausedby3,4‐DCA exposure……………………………………………………………………...

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L ISTOFFIGURES

L ISTOFFIGURES ( CONTINUED ) Figure3.18Repressionofthegenescyp1aandho‐1inembryosexposedto12.4 μM 3,4‐DCAcausedanincreaseinthefrequencyofdevelopmental disorders……………………………………………………………………….. Figure4.1Illustrationofsignallingpathwaysthatmightbeinvolvedinthe inductionofgenesinzebrafishembryosexposedto3,4‐DCA…………..... Figure5.1RelativesensitivityofGene‐DarT:LOECGene‐DarT/LOEC ELST………........................................................................................................... β − FigureA.1ChemicalstructureoftheAHR‐agonistnaphthoflavone (BNF)……………………………………………………………………………. FigureA.7.1MapofexpressionvectorpCMVSport6.1.…………………………………. FigureA.7.2MapofexpressionvectorpME18SFL…………………………………….....

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