An auxin-inducible, GAL4-compatible, gene expression ... - eLife

AGES allows induction of GAL4 activity in the adult fat body · Auxin-inducible gene expression system (AGES) effectively induces GAL4 activity in ... Sharethisarticle Doi Copytoclipboard Citethisarticle ColinDMcClure AmiraHassan GabrielNAughey KhushbakhtButt AliciaEstacio-Gómez AneishaDuggal CheeYingSia AnnikaFBarber TonyDSouthall (2022) Anauxin-inducible,GAL4-compatible,geneexpressionsystemforDrosophila eLife11:e67598. https://doi.org/10.7554/eLife.67598 Copytoclipboard DownloadBibTeX Download.RIS Article Figuresanddata Abstract Editor'sevaluation Introduction Results Discussion Materialsandmethods Dataavailability References Decisionletter Authorresponse Articleandauthorinformation Metrics Theabilitytocontroltransgeneexpression,bothspatiallyandtemporally,isessentialforstudyingmodelorganisms.InDrosophila,spatialcontrolisprimarilyprovidedbytheGAL4/UASsystem,whilsttemporalcontrolreliesonatemperature-sensitiveGAL80(whichinhibitsGAL4)anddrug-induciblesystems.However,thesearenotideal.Shiftingtemperaturecanimpactonmanyphysiologicalandbehaviouraltraits,andthecurrentdrug-induciblesystemsareeitherleaky,toxic,incompatiblewithexistingGAL4-driverlines,ordonotgenerateeffectivelevelsofexpression.Here,wedescribetheauxin-induciblegeneexpressionsystem(AGES).AGESreliesontheauxin-dependentdegradationofaubiquitouslyexpressedGAL80,andtherefore,iscompatiblewithexistingGAL4-driverlines.Water-solubleauxinisaddedtoflyfoodatalow,non-lethal,concentration,whichinducesexpressioncomparabletouninhibitedGAL4expression.Thesystemworksinbothlarvaeandadults,providingastringent,non-lethal,cost-effective,andconvenientmethodfortemporallycontrollingGAL4activityinDrosophila. ThisworkwillbeofinteresttoDrosophilageneticistsinitsdevelopmentofanewtoolfortemporalcontrolofgeneinductionbythewidely-usedbipartiteGal4/UASsystem.Bytransferringproteinmodulesfromplantsthatmediateauxin-dependentproteindegradationtocontrolthestabilityofaGal4-inhibitor(Gal80),theauthorssuccessfullydemonstratetheabilitytocontrolGal4activityinfliesthroughtheprovisionofauxininthefoodsubstrateandprovideevidenceforthesensitivity,specificity,andnon-toxicityofthistool. https://doi.org/10.7554/eLife.67598.sa0 Decisionletter ReviewsonSciety eLife'sreviewprocess Theabilitytomanipulatetheexpressionofspecificgenesinmodelorganismshasbeenthecornerstoneofgeneticsresearchoverthelast50years.Itisfromsuchstudiesthatgeneticistshavebeenabletoelucidatefundamentalbiologicalprocess,suchasthoseunderlyingneurodegenerativediseases(LuandVogel,2009),oncogenicmechanisms(Villegas,2019),andneuralcircuitryinvolvedinmemoryformation(Cognignietal.,2018). Thefruitfly,Drosophilamelanogaster,hasbeenattheforefrontofgeneticssinceitscultivationinthelabintheearly1900s,bygeneticistMorgan,1910.Inthe100yearssince,aplethoraofgenetictoolshavebeendevelopedforthismodelorganism,enablinggeneticiststotightlycontrolandmanipulategeneexpression,allowingthemtostudytherolesofgenesindevelopment,physiology,andbehaviour.OneofthemostprominentdevelopmentsistheGAL4/UASsystem,whichhasbeenanincrediblypowerfultoolforthespatialcontroloftransgenesinDrosophila(BrandandPerrimon,1993).ThissystemreliesonaclonedorendogenouspromotertodrivetheexpressionoftheyeasttranscriptionfactorGAL4,whichcanthenbindtotheUAS(upstreamactivatingsequence)sitesupstreamofatransgeneandactivateitsexpression.Sincethedevelopmentofthissystem,Drosophilaresearchershavecreatedthousandsofgeneticallydistinctlinestoenableexpressionoftransgenesinspecificpopulationsofcells(e.g.Jenettetal.,2012;Robieetal.,2017).ThemajorityoftheselinesarepubliclyavailableattheBloomingtonDrosophilaStockCenter(BDSC)andViennaDrosophilaResourceCenter(VDRC). GAL4activitycanberegulatedbyGAL80,aproteinwhichantagonisesGAL4byinhibitingitsactivationdomain(Lueetal.,1987),andthustheabilityoftheGAL4toinducetranscriptexpression(LeeandLuo,1999;Susteretal.,2004).Thegenerationofaflylinethatubiquitouslyexpressedatemperature-sensitiveversionofGAL80(GAL80ts)(Matsumotoetal.,1978)wasasignificantadvanceforthefield,asitallowedtemporalcontrolofGAL4-inducedtransgeneexpression(McGuireetal.,2003).Fliescanbekeptattemperaturespermissiveoftranscriptexpression(29°C)ortemperaturesthatinhibitexpression(18°C),offeringresearcherstocontrolnotonlywhere,butwhentransgenesareexpressed.Theversatilityofthissystemenabledresearcherstodeterminetime-specificeffectsofgenefunction,andisasystemthatiswidelyemployedwithintheDrosophilacommunity. Althoughtheimpactofthistechnologyonthefieldcannotbeunderstated,itisbecomingincreasinglyevidentthatusingatemperatureshifttoinducegeneexpressionisfarfromoptimalforsomeexperiments,especiallyinfieldssuchasbehaviour,neuropathy,andageing.Flies,asectotherms,arehighlydependentontheirambienttemperature,whichaffectsawiderangeofphysiologicalandbehaviouraltraits(Abrametal.,2017).Theseincludesleep(BeckwithandFrench,2019),territorialsuccess(Zamudioetal.,1995),lifespan(McCabeandPartridge,1997),developmentrate(Danjumaetal.,2014),immunity(Huntetal.,2016),metabolism(Schouetal.,2017),andeventheirmicrobiome(Moghadametal.,2018).Therefore,theuseofasignificanttemperatureshifttoinducegeneexpressionshouldbeavoidedinthesecontexts. Asanalternativeapproach,arangeofdrug-induciblesystemshavebeendeveloped,wherethedrugcanbeadministeredtofliesintheirdiettoactivatetranscriptexpression(Barwelletal.,2017;KogenaruandIsalan,2018;Osterwalderetal.,2001;Potteretal.,2010;SethiandWang,2017).Thesesystemseachhaveadvantagesanddisadvantages(seeTable1)andarenotoftencompatiblewithavailableGAL4lines,thusrequiringthecreation(andoptimisation)oftheirowndrivers.Inaddition,thesystemsthatarecompatiblewithexistingGAL4linescanaffectthefly’sphysiologyorsurvival,therebylimitingtheiruse,andthecertaintyofanyfindingswhenemployed.Forexample,theGeneSwitchsystem(Osterwalderetal.,2001)isnotcompatiblewithexistingGAL4linesandrequirestheusertogenerateanewmodifiedGAL4line.Furthermore,thesystemshowsleakyexpressionintheabsenceofthedrug(RU-486)(Poirieretal.,2008;Scialoetal.,2016),andRU-486cancausebehaviouralchanges(LiandStavropoulos,2016)andisdangeroustohandleforpregnantwomenasitcancauseterminationofthepregnancy(Avrechetal.,1991).Anotherdrug-induciblesystemutilisedinDrosophilaistheQFsystem(Potteretal.,2010).Here,QFisatranscriptionalactivatorthatcanbeinhibitedbyQS(analogoustoGAL4andGAL80).QuinicacidinhibitstherepressiveactionofQS,therefore,canswitchontransgeneexpression.Quinicacidisnon-toxicforflies,however,thedrawbackofthissystemisthatitisnotcompatiblewiththevastcollectionofexistingGAL4lines. Table1 Mainadvantagesanddisadvantagesofthemostcommondrug-induciblegeneexpressionsystemscurrentlyavailabletotheDrosophilacommunity. ExpressionsystemReferenceAdvantagesDisadvantagesGeneSwitchOsterwalderetal.,2001Non-toxicforadultfliesNotcompatiblewithexistingGAL4linesLeakyexpression(Poirieretal.,2008;Scialoetal.,2016)Larvalexposureimpactsonadultsleeppatterns(LiandStavropoulos,2016)DrugisunsafetohandleforfemaleresearchersDrugisexpensiveQFSystemPotteretal.,2010Non-toxicforfliesNotcompatiblewithexistingGAL4linesTet-offGAL80Barwelletal.,2017GAL4/UAScompatibleLonginductiontime(>5days)Requiresx2copiesoftheGAL80transgeneAffectsmicrobiotaTMPINDUCIBLEKogenaruandIsalan,2018;SethiandWang,2017GAL4/UAScompatibleCurrentlynotcompatibleforgeneralusewithexistingGAL4linesTMPdrugisonlydissolvableinDMSO(affectssurvivaloflarvae)orhastobeaddedasadrypowdertofood Theauxin-degronsystem,firstidentifiedinArabidopsis,involvestheauxin-dependentubiquitination,andsubsequentdegradation,ofproteinsthataretaggedwithaspecificauxin-inducibledegron(AID)sequence(Dharmasirietal.,2005;DharmasiriandEstelle,2002;Lietal.,2019).ItsnaturalfunctionallowsfortheprompteliminationofAux/IAAtranscriptionfactorsinplantsandhasbeenadaptedforuseinotherspecies,mostnotablyCaenorhabditiselegans,toartificiallytargetproteinsforrapiddegradation(Zhangetal.,2015).Inthissystem,theF-boxproteinTIR1,anauxinreceptor,bindswithconservedproteinsSkp1andCullintoformaSkp1–Cul1–F-box(SCF)E3ligasecomplexwhichubiquitinatesproteinstaggedwithanAIDsequenceinanauxin-dependentmanner.Thespecificityandversatilityofthesystemhasmadeitaneffectivetoolinmultiplesystemsincludingmice(Yesbolatovaetal.,2020)andhumancellculture(Lietal.,2019). Importantly,theauxin-degronsystemhasbeensuccessfullyemployedinDrosophila(Chenetal.,2018;Trostetal.,2016).TrostandcolleaguesshowedthatAID-taggedEYFPandAID-taggedRuxproteinlevelscouldbeknockeddowninS2celllinesandtransgenicDrosophila,respectively.WhileChenandcolleaguesknockeddownAID-taggedPERIODproteinintheadultbrain.Inspiredbythesestudies,wehaveappliedtheauxin-degronsystemtoenablebothtemporalandspatialcontroloftransgeneexpressionwhencombinedwiththeGAL4/UASsysteminDrosophila.ThiswasachievedbycreatingaubiquitouslyexpressedGAL80fusedwithAIDtags,suchthatGAL80isdegradedinthepresenceoftheauxinphytohormone(Figure1).Wedemonstratethatthissystemworksinbothlarvae(5mMauxin)andadultflies(10mM),whichareconcentrationsthatdonotaffectflysurvival,development,orlocomotion(larvalcrawlingandadultclimbing).Thisauxin-induciblegeneexpressionsystem(AGES)issafetohandle,cheap,easytoprepare,providestuneableandstringent(i.e.notleaky)expression,andmostimportantly,iscompatiblewiththemajorityofexistingGAL4-driverlinesdevelopedbytheDrosophilacommunity. Figure1with1supplementseeall Downloadasset Openasset Designandactionofauxin-induciblegeneexpressionsystem(AGES). AtubulinpromoterubiquitouslyexpressesthebicistronicTIR1andAGEScassette.Auxin-inducibledegron(AID)-taggedGAL80iscleavedfromtheTIR1duetothepresenceoftheT2Asequence.Intheabsenceofauxin,GAL80caninhibitGAL4activity,whilstthepresenceofauxininducesdegradationofGAL80,allowingGAL4todrivetheexpressionofUAS-transgene(s). Togenerateanauxin-induciblesystemforcontrolofGAL4activityinDrosophila,wedesignedatransgenethatwouldubiquitouslyexpresstwoproteins,TIR1andAID-taggedGAL80.(Figure1).WeincludedshorterAIDsequences(minimaldegronofIAA17)thanpreviouslyusedinDrosophila(Trostetal.,2016).FusionofthisminimalAIDtoluciferaseresultedinafusionproteinwithshorthalf-lifeinplants(~10min)(Dreheretal.,2006).WealsoinsteadusedArabidopsisthalianaTIR1(AtTIR1),whichhastwopointmutations(improvingaffinityandauxinsensitivity)thatcandepletenuclearandcytoplasmicproteinsinC.elegans(Zhangetal.,2015).TheP2AsequencebetweenAtTIR1andAID-taggedGAL80encodesaself-cleavingpeptidethatisknowntoworkefficientlyinDrosophilacellculture(Danielsetal.,2014).TheAtTIR1-T2A-AID-GAL80-AIDsequencewascodonoptimised,synthesised,andcloneddownstreamoftheαTub84BpromoterinpattB(Figure1andFigure1—figuresupplement1).TheαTub84B(tubulin)promoterdrivesexpressionofthebicistronicsequenceinallcells,allofthetime,and(intheabsenceofauxin)willinhibitanyGAL4activity.Whenpresent,auxinwilltethertheTIR1totheAIDsequences,triggeringthedegradationofGAL80andthereleaseofGAL4inhibition(Figure1). WecombinedtheAGESwithc564-GAL4toseeifwecouldgetinducibleexpressioninfatbody.AnuclearlocalisedGFP(nls-GFP)wasusedasareporterofGAL4activity.Wefirstexaminedadultfemalefliesthatwereplacedonfoodcontainingdifferentconcentrationsof1-naphthaleneaceticacidpotassiumsalt(K-NAA)auxin,whichhasshowntobemorewatersolublethanNAA(Martinezetal.,2020).Anegativecontrolconsistingofflyfoodwithoutauxin(0mMauxin)wasincludedtoinvestigatewhethertherewasanyleakyGAL4activityinabsenceofauxin.After24hr,GFPfluorescenceintheabdomenwasassayed(notethatthereissomenaturallyoccurringautofluorescenceintheflyabdomen).At5and10mM,GFPfluorescenceisclearlydetectableintheabdomenoflivefemaleflies(Figure2A).Basedonthisassay,10mMfor24hrresultsin~60%oftheGFPfluorescencelevelsseeninthepositivecontrol(Figure2B).At1mMthereisnodetectableGFPabovebackgroundfluorescence,andthelevelsareindistinguishablefromboththe0mMandthenegativecontrol(lackingboththeGAL4driverandtheUAS-nls-GFP).Toprovideanalternativeandmoredirectassayoftransgeneinduction,quantitativePCR(qPCR)wasusedtomeasurethelevelsofGFPmRNA(Figure2C).Here,5and10mMshowlevelsofmRNAequivalenttothepositivecontrol,while0mMshowednosignificantdifferencewhencomparedwiththenegativecontrol. Figure2with2supplementsseeall Downloadasset Openasset Auxin-induciblegeneexpressionsystem(AGES)effectivelyinducesGAL4activityinDrosophilaadults. (A)VentralimagesoflivefemalesthatexpressGAL4infatbodytissue.Ingestionoffoodcontainingauxin(≥5mMfor24hr)inducesGAL4activityandtheexpressionofGFP.(B)QuantificationofGFPlevels.Pixelintensitythresholdingwasperformedtoisolateabdomensasregionsofinterest.TheaveragepixelintensitiesfromsixreplicateswerequantifiedandanalysedusingKruskal-WallistestwithDunnpair-wisecomparison(***,p<0.001and**,p<0.01).(C)QuantitativePCR(qPCR)dataforGFPmRNAlevelsusingdifferentconcentrationsofauxin(threebiologicalreplicates).ValueswerenormalisedtohousekeepinggeneRpL4(RibosomalProteinL4)andrelativeexpressionlevels(comparedtothenegativecontrol)werecalculatedusingtheΔΔCtmethod.Y-axisdisplayingΔΔCtvaluesandstatisticsdoneusingKruskal-WallistestwithDunnpair-wisecomparison(*,p<0.05).SeeFigure2—sourcedata1forrawdata. Figure2—sourcedata1 GFPintensityandqPCRvaluesforFigure2B,C. https://cdn.elifesciences.org/articles/67598/elife-67598-fig2-data1-v1.xlsx Downloadelife-67598-fig2-data1-v1.xlsx Adultmaleseatlessthanfemales(Wongetal.,2009),therefore,thiswillimpactonauxiningestionandpossiblyontransgeneinduction.Aswithfemales,weexaminedbothGFPfluorescencelevelsintheabdomen,andGFPmRNAlevelsinmaleflies(Figure2—figuresupplement1).Althoughreducedcomparedtofemales,thereisstillarobustinductionofGFPexpression. Toassesshowquicklytransgeneexpressionisturnedoffafterremovalofauxinfromthefood,weperformedatimecourseexperimentusingadultfemalesand10mMauxin.After24hr,flieswereshiftedtofoodwithoutauxinandGFPfluorescencelevelsintheabdomenmeasuredevery24hr.At48hr(24hrwithoutauxin),GFPlevelswerereduced(Figure2—figuresupplement2A).At72and96hr,thelevelswerenotsignificantlydifferentto0hr.Furthermore,wetestedhowstableauxinisinpreparedfoodkeptat4°C.Evenafter15weeks,theauxinfoodcouldinduceGFPlevelstothatofnewlypreparedfood(Figure2—figuresupplement2B). InadditiontotheneedfortemporalcontrolofGAL4activityintheadult,researchersmightalsowanttoinduceexpressionatspecificpointsduringlarvaldevelopment.TotestifAGESworksinlarvae,weusedthec564-GAL4fatbodydriverandtestedmultipleauxinconcentrations(Figure3AandC).Here,weobservedthatexpressioncouldbeinducedatlowerconcentrationsofauxin.Whenusingfoodcontainingnoauxin,thereisnodetectableGFP,whereaswith1mMauxin,GFPisexpressed,althoughatlowerlevelsthanwithhigherconcentrationsofauxin(Figure3AandC).FivemMauxinisanoptimalconcentration,asconcentrationsabovethatdonotincreaseGFPlevels.FivemMauxincaninducelow-levelGFPexpressionafter6hr(Figure3BandD).However,18hrisrequiredtoprovidemaximumlevelsofexpression. Figure3 Downloadasset Openasset Auxin-induciblegeneexpressionsystem(AGES)allowsinductionofGAL4activityinDrosophilalarvae. (A)GFPfluorescencequantificationinfatbodytissueafterinduction(for24hr)onfoodwithdifferentconcentrationsofauxin.TheaveragepixelintensitiesfromsixlarvaewerequantifiedandanalysedusingKruskal-WallistestwithDunnpair-wisecomparison(***,p<0.001and**,p<0.01).(B)TimecourseofGFPexpressioninfatbodytissuewhenusing5mMauxin.TheaveragepixelintensitiesfromsixlarvaewerequantifiedandanalysedusingKruskal-WallistestwithDunnpair-wisecomparison(**,p<0.01).(C)Representativeimagesoflarvaefedondifferentconcentrationsofauxin.(D)Representativeimagesoflarvaeimagedateachtimeintervalsinceinduction.SeeFigure3—sourcedata1forrawdata. Figure3—sourcedata1 GFPintensityvaluesforFigure3. https://cdn.elifesciences.org/articles/67598/elife-67598-fig3-data1-v1.xlsx Downloadelife-67598-fig3-data1-v1.xlsx ForAGEStoworkwithGAL4linesexpressedinthecentralnervoussystem,auxinmustbeabletopasstheselectively-permeableglialmembrane,akintothemammalianblood-brainbarrier(Limmeretal.,2014).NAAcancrosstheblood-brainbarrierinDrosophila(Chenetal.,2018).ToverifythisusingK-NAA,wefirstusedelav-GAL4(expressedinallneurons)andexaminedtheexpressionofGFPintheadultbrain.Similartothefatbodyc564-GAL4driver,weobserverobustexpressionofGFP,whenfliesarefedauxinfood(10mM)for24hr(Figure4—figuresupplement1).ThisdemonstratesthatAGESworksinthecentralnervoussystemandthatK-NAAauxincancrosstheblood-brainbarrierinDrosophila. WethentestedmorerestrictedGAL4-driverlinesinthelarvalventralnervecord(grh-GAL4)andintheadult(Or85a-GAL4).InbothcasestherewasnodetectableGAL4activityintheabsenceofauxin,whileinthepresenceofauxin(10mMforadultsand5mMforlarvae)GFPinapatternconsistentwiththeGAL4driverisobserved(Figure4AandC)atlevelssignificantlyabovebackgroundfluorescencelevels(Figure4BandD). Figure4with1supplementseeall Downloadasset Openasset Auxin-induciblegeneexpressionsystem(AGES)allowsinductionofGAL4activityintheDrosophilaadultandlarvalbrain. (A)GFPfluorescencedrivenbyOr85a-GAL4intheantennallobe,withandwithoutauxin.(B)GFPfluorescencequantificationintheantennallobe24hrafterinduction(fourtosixreplicates).Statisticsperformedusingordinaryone-wayANOVA(*,p<0.05).(C)GFPfluorescencedrivenbygrh-GAL4inthelarvalventralnervecord,withandwithoutauxin.(D)GFPfluorescencequantificationintheventralnervecord24hrafterinduction(sixreplicates).Statisticsperformedusingordinaryone-wayANOVA(*,p<0.05).SeeFigure4—sourcedata1forrawdata. Figure4—sourcedata1 GFPintensityvaluesforFigure4. https://cdn.elifesciences.org/articles/67598/elife-67598-fig4-data1-v1.xlsx Downloadelife-67598-fig4-data1-v1.xlsx Forauniversallyapplicabledrug-induciblesystem,thedrugshouldnotimpactondevelopmentormortalityoftheflies.Wetestedwhethercontinuousexposuretodifferentconcentrationsofauxin(K-NAA)wouldimpactondevelopmentaltiming.Timetopupation(fromegg-laying)isunaffectedforconcentrationsupto5mM,however,10mMcausedadelayofapproximately1day(Figure5A).TenmMauxincausessomedevelopmentaldelay,althoughithasnoimpactonthesurvivalofthefliesthroughthesedevelopmentalstages(comparedtoanoauxincontrol)(Figure5B).Thesedataindicatethat10mMauxinshouldbeavoidedforlarvalinduction,however,thisisnotanissueas>1mMissufficientforrobustinductionofexpression(Figure5A)and5mMdoesnotcauseanydevelopmentaldelay.Concentrationsof5and10mMauxincaninduceexpressioninadults(Figure2andFigure2—figuresupplement1).Survivalassaysonbothmaleandfemaleadultsshowthatconcentrationsof5and10mMhavenoeffectonsurvivalandlifespan(Figure5DandE).Therefore,insummary,upto5mMauxinisoptimalforuseinlarvae,whilstupto10mMauxinisbestforuseinadults.Wealsotestedwhetherauxinaffectslocomotionoflarvae(crawlingassay)andadults(climbingassay)andseenosignificanteffectswith5and10mMauxin,respectively(Figure5—figuresupplement1). Figure5with1supplementseeall Downloadasset Openasset Impactofauxinondevelopmentaltimingandsurvival. (A)Timetakenfromegg-layingtowanderingL3larvae.(B)Timetakenfromegg-layingtopupation.(C)Survivalacrossdevelopmentalstages(larvaltopupalandpupaltoadult)with10mMauxin.(D)Survivalofadultfemalesduringcontinuousexposuretoauxin.(E)Survivalofadultmalesduringcontinuousexposuretoauxin.LogranktestandweightedGehan-Breslow-Wilcoxonmodel(ns)wereusedfortheadultsurvivalassays.SeeFigure5—sourcedata1forrawdata. Figure5—sourcedata1 Rawdataforlarvalcrawlingandadultclimbingassays. https://cdn.elifesciences.org/articles/67598/elife-67598-fig5-data1-v1.xlsx Downloadelife-67598-fig5-data1-v1.xlsx TodeterminewhetherAGESissuitableforacuteadultbehaviouralmanipulation,werecapitulatedaclassicGeneSwitchmanipulationofclockneuronexcitabilityusingAGES(Depetris-Chauvinetal.,2011).AkeycircuitforcontrolofDrosophilarhythmicbehaviouristheventrolateralneuron(LNv)cluster,ofwhichthesmallventrolateralneurons(sLNvs)arebothnecessaryandsufficientformaintenanceoffree-runninglocomotorrhythms(Grimaetal.,2004;Rennetal.,1999;Stoleruetal.,2004).ThefourlargeLNvsandfouroutoffivesLNvsexpresstheneuropeptidepigmentdispersingfactor(PDF)(Helfrich-Förster,1995;Rennetal.,1999).PDFisakeyclockoutputneuropeotiderequiredforsynchronisationofclockneurongroups,andlossofPDFfunctionresultsinarrythmicity,desynchronisationofclockoscillators,andalteredperiodlength(Learetal.,2009;Linetal.,2004;Pengetal.,2003;Sheebaetal.,2008).Adult-specificsilencingofPDF+LNvsbyexpressionoftheinwardlyrectifyingpotassiumchannelKir2.1usingPDF-GAL4-GeneSwitchissufficienttonearlyablatecircadianlocomotorrhythmsinconstantconditionswithoutresettingthemolecularclock(Depetris-Chauvinetal.,2011). Torecapitulatethisbehaviouralexperiment,wecombinedthePDF-GAL4linewiththeAGEStoallowauxin-induciblecontrolofKir2.1expressioninPDF+neurons.WesimultaneouslyreplicatedGeneSwitch-drivenKir2.1expressioninPDF+neurons.Aftereclosionandentrainmenttoa12hrlight:12hrdark(12:12LD)scheduleonstandardflyfood,flieswereindividuallyloadedintoactivitytubesandplacedintheDrosophilaactivitymonitoring(DAM)system(Trikinetics)for3daysin12:12LD,followedby8daysinconstantdarkness(DD).ControlactivitytubescontainedstandardDAMfood(2%agar,5%sucrose)whileexperimentalfoodcontainedRU-486(200mg/mL)orNAA(2or10mM).Incontrasttothepreviousexperiments,NAAauxinwasusedhereinsteadofK-NAAauxin.AnalysisoftheamplitudeofthebehaviourallocomotorrhythminconstantdarknessbyfastFouriertransform(FFT)(Plautzetal.,1997)showedrobustinhibitionoflocomotorrhythmsinbothmaleandfemaleexperimentalPDF-GAL4;AGES>UAS-Kir2.1flieson2or10mMNAA(Figure6A-D,Figure6—figuresupplement1A,B).TwomMNAAwassufficienttocompletelyablatecircadianlocomotorrhythmsinbothmaleandfemaleexperimentalflies(Figure6CandD),withoutasignificanteffectonaverage24hrlocomotoractivityofparentalcontrols(Figure6—figuresupplement2A-D).TwomMNAAfeedingsignificantlylengthenedthebehaviouralperiodofmalePDF-GAL4/+;AGES/+parentalcontrolsby19min,withnoeffectonfemalesofthesamegenotype.InagreementwiththeclassicstudybyDepetris-Chauvinetal.,2011,200μg/mLRU-486feedingalsoreducedcircadianlocomotorrhythmsinbothsexesofflieswithPDF-GAL4-GeneSwitch-drivenexpressionofKir2.1(Figure6EandF),thoughtheeffectwasnotasstrongas2mMNAAfeedingintheAGESflies.Inaddition,RU-486feedingresultedinabroaderdistributionoflocomotorrhythmstrengthsandanetincreaseinrhythmicityinmalePDF-GAL4-GeneSwitchparentalcontrolflies(Figure6E),whichwasaccompaniedbya51minincreasedperiodlengthandincreasedaverage24hrlocomotoractivity(Figure6—figuresupplement2G).RU-486alsoincreasedtheperiodoffemalePDF-Gal4-GeneSwitchparentalcontrolsby46min,withnoeffectontotalactivityorrhythmstrength(Figure6F,Figure6—figuresupplement2F,H).Insummary,wefindthat2mMNAAfeedingissufficientforAGES-inducedexpressionofKir2.1inadultPDF+clockneuronstoablatecircadianlocomotorbehaviour.Indeed,weobservedastrongerablationoflocomotorrhythmicityintheAGEScomparedtoGeneSwitch,withfeweroff-targetbehaviouraleffectsofNAAfeedingofparentalcontrolflies. Figure6with2supplementsseeall Downloadasset Openasset Auxin-induciblegeneexpressionsystem(AGES)inducedexpressionofKir2.1inPDF+clockneuronsablatescircadianlocomotorrhythms. (A,B)Representativedouble-plottedactogramsfor5-to10-day-oldmalefliesmaintainedonstandardfood(A)orfoodsupplementedwith2mmNAA(B)for3daysin12hrlight:12hrdark(12:12LD)and7daysinDD.Left:parentalcontrolUAS-Kir2.1,centre:parentalcontrolPDF-GAL4;AGES,right:experimentalPDF-GAL4/UAS-Kir2.1;AGES/+.BarsindicateLDcycle,greyshadeddaysindicateconstantdarkness.(C)Amplitudeofcircadianrest:activityrhythmsonDDdays2–8representedbyfastFouriertransform(FFT)powerat24hrformalePDF-GAL4;AGES>UAS-Kir2.1fliesandtheirparentalcontrolsonstandardfood(orange)andfoodsupplementedwith2mMNAA(blue).Pointsrepresentindividualflies,boxshows25–75%confidenceinterval,medianline,andoutliers.(D)Twenty-fourhrFFTpowerasin(C)forfemalePDF-GAL4;AGES>UAS-Kir2.1fliesandtheirparentalcontrols.(E)Twenty-fourhrFFTpowerforasin(C)malePDF-GAL4-Geneswitch>UAS-Kir2.1fliesandtheirparentalcontrolsmaintainedonvehiclecontrolfood(orange)andfoodsupplementedwith466mMRU-486(red).(F)Twenty-fourhrFFTpowerasin(E)forfemalePDF-GAL4-Geneswitch>UAS-Kir2.1fliesandtheirparentalcontrols.Forallpanels,meanswerecomparedbytwo-wayANOVAbygenotypeandfoodsubstrate,seeFigure6—sourcedata1forrawdata,p-values,andkeyresourcedata.MeanssharingthesameletterarenotsignificantlydifferentfromoneanotherbyTukey’sposthoctest(p>0.05). Figure6—sourcedata1 Rawdata,pvaluesandkeyresourcedataforcircadianandactivitybehaviourialexperiments. https://cdn.elifesciences.org/articles/67598/elife-67598-fig6-data1-v1.xlsx Downloadelife-67598-fig6-data1-v1.xlsx TheGAL4/UAS/GAL80tssystemsenableDrosophilaresearcherstocontroltheexpressionoftransgenesbothspatiallyandtemporally.However,theimpactoftherequiredtemperatureshiftonphysiologyandbehaviourtoactivate/deactivatethesystemcanaffecttheexperimentalresultsandsubsequentconclusions(Abrametal.,2017).Drug-induciblesystemshavebeendevelopedasanalternativetotheGAL80tsmethod,however,thesearenotideal,limitedbyissuessuchasleakyexpression,longinductiontimes,orincompatibilitywithexistingGAL4lines.Here,wepresentAGES,anauxin-induciblegeneexpressionsystem,whicheliminatestheneedfortemperatureshiftstomanipulatetemporalexpressionandlacksmanyofthedrawbacksofthecurrentdrug-induciblesystems. AsignificantbenefitofAGESoverexistingsystemsisitscompatibilitywithmostexistingGAL4-driverlines.Currently,themostprevalentdrug-inducibleexpressionsystems,thatis,theGeneSwitch(Osterwalderetal.,2001)andtheQ-system(Potteretal.,2010),requirethegenerationoftheirowndriverlines.AGESusesaGAL80fusionprotein,whichallowstheuseofthesystemalongsidethemajorityofexistingGAL4-driverlines,wherebysuppressionoftheGAL4-UASactivationisachievedinallcellswhereauxinisabsent.Notableexceptionsaresplit-GAL4linesthatdonotusetheGAL4activationdomain.AswithallGAL80-basedmethodsofGAL4control,theGAL4linemustcontainthestandardGAL4activationdomainforGAL80tobeaffective.Therearesplit-GAL4linesthatusetheGAL4activationdomain(Luanetal.,2006;Pfeifferetal.,2010),however,manyuseap65activatordomain(Dionneetal.,2018;TirianandDickson,2017),oraVP16activatordomain(Luanetal.,2006),whichwillnotworkwithAGES.Wehavedemonstratedthesystemusingmultipledriverlines,includingc564-GAL4,elav-GAL4,grh-GAL4,Or85a-GAL4,andPDF-GAL4.Thesewere,inpart,usedtoassesswhethertheauxinhormoneused(K-NAA)couldfreelyaccessvariouscelltypeswithinthefruitfly,particularlythecentralnervoussystemwhereauxinmustpasstheselectivelypermeableglialmembrane,akintothemammalianblood-brainbarrier(Limmeretal.,2014).Ourresults,usingonefatbodyGAL4driverandfourdifferentcentralnervoussystemGAL4drivers,demonstratethatauxincantransversetheblood-brainbarrier,andthatthissystemshould,intheory,becompatiblewithallotherGAL4linesthatcontainaGAL4activationdomain. Successfulgenedriversystemsrequirenegligibleexpressionwhenun-induced.Whilesomecurrentsystemsareknowntoexhibitleakyexpression(Scialoetal.,2016),AGESdemonstratesanalmostcompleterepressionofGAL4activitywhenun-induced.UsingGFPfluorescenceasareadout,0mMauxindoesnotshowanygreatersignalthanthenegativecontrol(Figures2Band4).Also,thelevelofGFPmRNAwithoutauxinshowednosignificantdifferencewhencomparedtothenegativecontrol(Figure2C).TheAIDtagsonGAL80allowtherapid,andtuneable,degradationoftheGAL80protein,providinguserswiththeoptiontodeterminethestrengthofexpressionbyadjustingtheconcentrationofauxininthefood,orbylimitingtheexposuretimeofthefliestoauxin. TemporalcontrolofgeneexpressionisessentialformanybehaviouralexperimentsinDrosophila.WedemonstratethatAGEScanelicitstrongbehaviouralchangesthroughactivatingexpressionofKir2.1(aninward-rectifierpotassiumionchannel)inPDF-GAL4expressingadultneurons(Figure6).Kir2.1inhibitstheactivityofPDFneuronsresultinginablationofcircadianrhythms.Thestronglossofcircadianrhythmwasobservedinbothmalesandfemales,andevenworkedatthelowerconcentrationof2mMauxin.ThisisanimportantdevelopmentfortheDrosophilabehaviourfield,asitallowsexperimentstobedonewithexistingGAL4lines,andwithouttheneedofatemperatureshiftorhavingtouseGeneSwitch(Osterwalderetal.,2001).While2mMauxinfoodhadnoeffectonthecircadianrhythmoffemaleflies(Figure6D)orthePDF-GAL4/+controlmaleflies(Figure6C),itdidimpactontheUAS-Kir2.1/+controlmaleflies.Wecurrentlyhavenoexplanationforthis.However,itdoesemphasisetherequirementtoalwaysperformtheappropriatecontrols.Also,inthiscontext,10mMauxinfooddidhaveaneffectonbothmalecontrollines(Figure6—figuresupplement1A).So,while10mMauxinhadnoimpactonadultclimbing(Figure5—figuresupplement1B),itdidimpactoncircadianrhythms.ItshouldbenotedthatNAAwasusedforthecircadianrhythmexperiment,whereasK-NAA(morewatersoluble)wasusedforalltheotherexperiments.Therefore,K-NAAmayhavelessofaneffectonthecontrolsaswasseenwithNAA. AGESisalsoasystemthatissafeforbothhumansandfliesalike.Auxinhormonecarriesnohazardousconcernsforhumanhandling,andnolethaleffectswereidentifiedfortheeffectiveconcentrationsof10mMorbelowforbothadultandlarvalstages(Figure5).Continuousexposureoflarvaeto10mMauxindoesresultinsomedevelopmentaldelay;however,itdoesnotaffectsurvival.Higherconcentrationsofauxinresultinincreasedmortalityrates(datanotshown)andshouldbeavoided.Usersareencouragedtoconsiderthisifattemptingtooptimiseexposureconditionsforspecificexperiments,orGAL4-driverlinesthathavenotbeenassessedinthispaper.Overall,basedonourdata,werecommendtheuseofupto5mMauxinandupto10mMforoptimalinductioninlarvaeandadults,respectively. Itisalsoparamountthatinduciblesystemsareeasyandcheaptoapply,tofacilitatetheirpracticaluseindiverseexperimentalsettings.WhileexpensesoftheGeneSwitchsystemaverage£0.31(GBP)pervial,AGEScosts~120×lessat£0.0025pervial(authors’estimate).Furthermore,thewater-solubleauxin(K-NAA)canbeeasilyaddedtothefoodimmediatelybeforethefoodispouredintovialsorbottles,requiringnoadditionalsafetymeasures,enablingeaseofproduction.Inourexperience,auxin-containingfoodcanbestored4°Cforupto15weeks(Figure2—figuresupplement2B)wherethehormone’spotencystillpersists.Furtherstudiesarerequiredtoassessthestabilityandeffectivenessofauxinwhenstoredforlongerperiodsoftime,orindifferingconditions. Thesensitivityofthesystemisdirectlyrelatedtotheamountofauxinpresentwithinthetissue,andthusconcernsareraisedregardingthefly’sageandsex,asthesefactorsimpactfeedingrates(Careyetal.,2006;Wongetal.,2009).Asallexperimentaladultflieswereagedto5dayspost-eclosion,theeffectsofageonthesystemareyettobedetermined,althoughlowerexpressionistobeexpectedinolderflies.Toaddresstheseconcerns,alterationstotheauxinapplicationcouldbemade,suchasalongerexposure(>24hr),whichaswehaveshowndoesnotimpactonsurvival(Figure5DandE). DespitethevariousadvantagesthatAGESofferstheflycommunity,therearestilllimitationstoconsider.Theseinclude(i)theresponsetimeofthesystem(18–24hr),(ii)auxincanhavesubtleeffectsonadultbehaviour(circadianrhythms),and(iii)inducedlevelsofexpressionarenotalwaysashighasthemaximumlevelsobtainedwithoutthesystem.Forexample,asmaleadultseatlessthantheirfemalecounterparts,lowerexpressionofthetransgeneisobservedinmales(Figure2—figuresupplement1).However,asGAL4-inducedexpressionisinherentlystrong,reducedtransgeneexpressionmaynotbeanissue.Thisisevidentinourcircadianrhythmbehaviouralexperiment,wherethereisarobustresponseinbothmalesandfemaleswhendrivingexpressionofUAS-Kir2.1(Figure6CandD).Whilea24hrinductiontimeisnotinstant,itisbothpracticalandconvenientforresearchersperformingexperimentsonadultfliesinthelab. ThereisscopeforAGEStobeoptimisedinthefuture.OneapproachwouldbetoutiliseamutatedversionofTIR1thatcanaccommodatebulkyanaloguesofauxin.Suchmutants,incombinationwithmodifiedauxins(5-Ph-IAAor5-adamantyl-IAA),allowthedegronsystemtoworkwithmuchlowerlevelsofauxin(>500-foldless)(Yesbolatovaetal.,2020;Zhangetal.,2022)anddemonstrateamorerapiddegradation.EffortstotestthesemodificationswithAGESareunderway. Insummary,AGESofferstheflycommunityacheap,safe,andeasysystemfortemporaltransgeneexpressionusingexistingGAL4lines.Furthermore,itdoesnotrequireashiftintemperaturechangesforinductionofgeneexpression.Itisaparticularlypromisingtoolforresearchfieldswhereresearcherswanttoavoidusingtemperatureshifts(e.g.ageing,behaviouralgenetics,andneuropathology),andwillundoubtedlyhavewide-rangingbenefitsformultiplefieldsofstudy. TheAtTIR1-T2A-AID-GAL80-AIDsequencewassynthesisedbyTwistBiosciences(twistbioscience.com)(Figure1—figuresupplement1).TheGAL80sequence(fromSaccharomycescerevisiae)andtheAtTIR1sequence(Zhangetal.,2015)werecodonoptimisedforDrosophilaformoreeffectivetranslation.TheαTub84Bpromoterwasamplifiedfromatubulin-eGFPplasmid(giftfromMDionne)usingthefollowingprimers:tub_FWD:GATATCAAGCTTGCACAGGTCCandtub-RV:GTACCTTCACGCTGTGGATGAGG.TheαTub84BandAtTIR1-T2A-AID-GAL80-AIDsequenceswereclonedintopattB(Bischofetal.,2007)usingGibsonassembly(Gibsonetal.,2009).Successfulclonesweresequenceverified.AnnotatedsequenceisinFigure1—figuresupplement1—sourcedata1.TheplasmidispublicallyavailableatDGRC:theDrosophilaGenomicsResourceCenter(DGRC:):https://dgrc.bio.indiana.edu/product/View?product=1568MicroinjectionwasperformedbyCambridgeFlyFacilityusingtheVK00040line,whichhasanattBsiteatlocationatt3B(87B10)onchromosomalarm3R.Injectedadultmaleswerecollectedandmatedtow1118virginfemalestoidentifytransgenics(orangeeyes).tub-TIR1-T2A-AID-GAL80-AIDisavailableattheBDSC(stock#92470)ortheVDRCStockCenter(stock#311020). Inthisstudy,c564-GAL4,UAS-nls-GFP/CyO-actin-GAL4-GFPflieswereusedtoperformfluorescentreporterexperimentsintheadultandlarvalfatbody,andforquantificationofGFPmRNAexpression.Moreover,theseflieswereusedinadultsurvivalassays.CantonSflieswereusedinthedevelopmentalsurvivalanddevelopmentaltimelineexperiments.Forfluorescentreporterexperimentsintheadultbrain,weusedelav-GAL4,UAS-nls-GFP.Forbehaviouralexperiments,Iso31flieswereusedasabackgroundstrain(Ryderetal.,2004),andweusedPDF-Gal4(Parketal.,2000)andPDF-GeneSwitch-GAL4(BDSC81116)todriveexpressionofUAS-Kir2.1(BDSC6597).Flieswerekeptat25°ConandstandardDrosophilafood(recipeinsupplementarymaterial)wassupplemented(justbeforebeingaliquotedintovials/bottles/plates)withauxin(K-NAAavailablefromPhytotech[#N610]orGlenthamLifeSciences[GK2088])atvaryingconcentrations.Forbehaviouralexperiments,flieswereraisedandentrainedonstandardfood,andtransferredtoactivitymonitoringtubescontaining2%agar,5%sucrose,supplementedwithindicatedconcentrationsofRU-486inethanol(Mifepristone,Sigma-Aldrich)orNAA(Phytotech,#N600). Adultandlarvalbrainsweredissectedin1×PBSandfixedfor25minatroomtemperaturein4%formaldehyde(methanolfree)in0.3%TritonX-100PBS(PBST).Theywerewashedfourtimesfor1hrwith0.3%PBST.Normalgoatserum(2%inPBST)wasusedfortissueblocking(RT,15minto1hr)andsubsequentovernightprimaryantibodyincubation.AlltissuewashesweredoneinPBST. Primaryantibodiesusedwerechickenanti-GFP(Abcam#13970,1:2000),guineapiganti-Dpn1:10,000(CaygillandBrand,2017)andratanti-Elav1:500(DevelopmentalStudiesHybridomaBank[DSHB]).SecondaryantibodiesusedincludeAlexaFluor488,545,and633ataconcentrationof1:200(LifeTechnologies)andtissuewasincubatedfor1.5hratroomtemperature.TissuewasmountedonstandardglassslidesinVectashieldMountingmedium(VectorLaboratories).BrainswereimagedusingaZeissLSM510microscope.AnalysisofacquiredimageswasdoneusingFiji(Schindelinetal.,2012). LiveimagingofGFPinadultfliesandlarvaewasperformedusingaNikonSMZ1500microscope.Inlarvae,toidentifyearliestauxininductioneffects,animalswerethenplacedon1mMauxin-supplementedfoodandimagedat0,1,5,10,and25hrintervals.Adultswereplacedonfoodcontainingvaryingauxinconcentrationsfor24hrpriortoimaging. LiveGFPlevelswerequantifiedinsixanimalspercondition(includingnegativeandpositivecontrols).Binaryimageswerecreatedbyusingathreshold(Outsu’sthresholding)andtheWandToolinFijiwasusedtotracetheGFP-positiveareaasregionofinterest(ROI).TheROIcorrespondedtoabdomensinadultsorwholelarvae.MeanpixelintensityperROIwascalculatedusingtheMeasureplugininFijiforatotalofsixbiologicalreplicates.Statisticalsignificanceanalysedusingone-wayANOVA(normallydistributed)orKruskal-WallistestwithDunnpair-wisecomparison(non-normallydistributed).Parametrict-testornon-parametricWilcoxontestswereusedtocompareindividualconditionswithrespectivecontrols.StatisticaltestswerecompletedinR(v3.6.3)andplotswereperformedusingthesoftwareGraphPadPrismversion9forWindows. Allanimalswerekeptat25°C.Todeterminetheeffectsofauxinonlarvaldevelopment,10matedCanton-Sflieswereallowedtolayeggsonflyfoodcontaining0,1,5,or10mMofauxinforamaximumof24hr,oruntil~50eggswerecounted.Fivereplicateswereobtainedforeachconcentration,exceptfor0mMwherefourreplicateswereused.DailyemergenceofL3larvaeandpresenceofpupaewererecordedtodeterminethetimerequiredforegg-to-L3,andegg-to-pupaedevelopmentforeachconcentration. Adultsurvivalassayswereperformedonc564-GAL4,UAS-nls-GFP/AGESfliesonthreereplicates,with20fliesperreplicate.Maleandfemaleanimalswereseparated5dayspost-eclosion.Eachreplicatewascontinuouslyexposedto0,5,and10mMauxinfood.Deathwasscoreddailyuntilallfliesweredeceased.StatisticaltestsandplotswereperformedusingthesoftwareGraphPadPrismversion9forWindows. InordertoquantifywhetherlevelsofGFPmRNAexpressionchangedinthepresenceofauxin,thec564-GAL4,UAS-nls-GFP/AGESflieswereplacedon0,5,and10mMauxin-supplementedfoodfor24hr.Thec564-GAL4,UAS-nls-GFP/+flieswereusedasapositivecontrolwhilsttheAGESflieswereusedasanegativecontrol(lackingboththeGAL4driverandtheUAS-nls-GFP).The0mMwasincludedtoinvestigatewhethertherewasanyleakyGAL4activityinabsenceofauxin. TotalRNAwasextractedfromwholeadultflies,threeperreplicatepercondition,usingastandardTRIzolextractionprotocol(Scientific,2016).DNAwasdegradedusingRNAse-freeDNAse(ThermoScientific)andcDNAsynthesiswasperformedusingtheiScriptcDNAsynthesiskit(Bio-Rad),followingmanufacturer’sinstructions.Real-timeqPCRwasperformedwithiTaqUniversalSYBRGreenSupermix(Bio-Rad)usingtheStepOnePlusReal-TimePCRSystem(AppliedBiosystems). ThegeneRpL4(RibosomalProteinL4)wasusedasreferencegene.TheGFPmRNAexpressionlevelswerecalculatedusingtheΔΔCtmethod(ΔCt=Ct[meanCtofreferencegene]–Ct[target];ΔΔCt=ΔCt[target]−meanΔCt[control])(LivakandSchmittgen,2001).Targetreferstotheauxinconcentrations(0,5,10mM)andcontrolreferstotheAGESflies.Primersused:RpL4_FW-5’-TCCACCTTGAAGAAGGGCTA-3’,RpL4_RV–5’-TTGCGGATCTCCTCAGACTT-3’,GFP_FW–5’-GAGCTGTACAAGAGCAGGCA-3’,GFP_RV–5’-GTTGACGGCGTTTTCGTTCA-3’.StatisticalsignificanceanalysedKruskal-WallistestwithDunnpair-wisecomparisoninR(v3.6.3). WeevaluatedcircadianlocomotorrhythmsusingtheDAMsystem(Trikinetics).Maleandfemalefliesoftheindicatedgenotypewereentrainedina12:12LDcyclepriortoloadingindividual5-to7-day-oldfliesintoglassDAMtubescontainingcontrolorexperimentaldiets.ForAGESflies,thecontroldietconsistedofstandardDAMfood.ForGeneSwitchflies,thecontroldietconsistedof1%ethanolvehicleinstandardDAMfood.FliesweremonitoredviaDAMassayfor3daysin12:12LDat25°C,followedby8daysinconstantdarkness(DD).CircadianlocomotorparameterswereanalysedusingClockLabsoftware(Actimetrics)fordatafromdays2to8ofDD.Periodlengthwasdeterminedbyχ2periodogramanalysis,andrelativerhythmpowerat24hrwasdeterminedusingFFT.FFTpowerandaveragedailyactivitycountsforeachsexwerecomparedbytwo-wayANOVAbygenotypeandfoodsubstrate,withTukey’sposthoccomparisons.Becauseperiodlengthcouldnotbeestimatedforarrhythmicflies,periodlengthwascomparedbyStudent’st-testforfliesofthesamegenotypeondifferentfoodsubstrates. 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Decisionletterafterpeerreview: Thankyouforsubmittingyourarticle"Anauxin-inducible,GAL4-compatible,geneexpressionsystemforDrosophila"forconsiderationbyeLife.Yourarticlehasbeenreviewedby2peerreviewers,andtheevaluationhasbeenoverseenbyaReviewingEditorandKVijayRaghavanastheSeniorEditor.Thereviewershaveoptedtoremainanonymous. Thereviewershavediscussedtheirreviewswithoneanother,andtheReviewingEditorhasdraftedthislettertohelpyoupreparearevisedsubmission. Essentialrevisions: Itisusefulforanewuserofthistooltohaveamorecompletedescription,ratherthanhavetoworkitoutthemselves.Currently,theanalysisofthetoolismodest.More'proof-of-concept'experimentscanbeusefulandcanmakethetoolmorelikelytobeused.Inadditiontosuchexperiments,itwillbeusefuliftheycouldhavebetterquantificationofthedataandprovidebettercontrolstoo. Pleasesee,fromtheaboveperspective,therecommendationsofboththereviewersbelowandthespecificsuggestionstheyhavemade.Couldtheauthors,whileembarkingonarevisedsubmission,pleaseoutlinealistofexperimentsthattheyplantodotoaddressthepointsintheprecedingparagraph,includinghowthecurrentstudysignificantlyadvancestheTrost2016work?GiventhatthisstudydevelopsonthatoftheTrost2016study,itsapplicationtoGal80needstogofurther. Reviewer#1(Recommendationsfortheauthors): Figure2B-C:thestatisticalcomparisonsbeingmadearenotveryclear:betweentheplotsattheendsofeachbracket?(ifso,whynotshowallcomparisons?).InpanelC,itisalsonotclearifthe****referstocomparisonof0mMwith1and5mMaswellas10mm. HowdotheauthorsexplainthedifferencebetweenGFPRNAandproteininduction?(presumablytranslationofGFPRNAshouldnotbeimpactedbytheAGESsystem). Thetitleisabitover-hyped:–AGEScomprisesamodificationofGal80control;geneexpressionitselfisstilldependentupontheGal4/UASsystem. Thenumberofanimalsuseindifferentexperimentsisnotclearlyindicated. Line71:"thataretagged"not"whicharetagged" Line78:missingwordbetweensystemsandmice Figure2B:panelB,"fluorescence"ismisspelt Figure2–figuresupp1:panelB,"fluorescence"ismisspelt Reviewer#2(Recommendationsfortheauthors): McClureetal.havetakenadvantageoftheArabidopsisauxin-degronsystem,whichhasbeenshowntofunctioninawiderangeoforganisms,includingDrosophila.Inthisstudy,theauthorscleverlyengineeraubiquitouslyexpressedbicistronictransgenethatcontainsallofthenecessarycomponentstoinhibitGal4-inducedexpressionintheabsenceofauxin.Theauthorsconvincinglyshowtheirsystem'sefficacy,showingtheadditionofauxintofoodenableGal4-inducedgeneexpressionofaGFPreporterinthefatbody,examiningbothexternalfluorescenceandmRNAlevels.GFPexpressioninthenervoussystemisanecessaryadditiondue,astheauthorsstate,totheblood-brainbarrier.AstherewasnothingquantitativeinFigure3,anegativecontrol(asusedinFigure2)wouldhavebeenhelpful,asitwashardtodetermineif0mMauxinhadnoGFPexpressionvisually.Asmanyresearcherswouldbeparticularlyinterestedinusingthissystemforbehavioralstudies,furtherinvestigationofthesystem'sefficacyforstudyingbehaviorswouldaddtotheinterestandimpactofthiswork;forexample,simplebehavioralcontrolswhenthefliesarerearedonauxin,e.g.,generallocomotion.AnexcellentadditioncouldbetoshowthataneuronaleffectorUAS(forexample,csChrimsonorTRPA1)doesnotinduceaGal4-drivenbehaviorintheabsenceofauxin,andperhapshowlongafterashifttoauxinbehaviorscanbeinduced.Lastly,itisnotablethatalltheshiftsinthispaperturnGal4-inducedexpressionfromanofftoanonstate.Itwouldbeinterestingtoinvestigatethetemporaldynamicsoftheopposite(turningthesystemfromontooff),whichwouldalsobeofgreatvaluetothebroadercommunity. –Thepaperlacksclarityinpartsthatmayimpactitsaccessibilitytoamoregeneralaudience,forexample,thedescriptionoftheshortcomingsofexistingsystemscouldbemoreclearlystated. –Line116"properinductionofGAL4activity"isquiteavaguestatement,perhapsthetimecourse(statedasdatanotshown)couldbeaddedtosupplemental. –Theauthorsstateinline173/174thattheAGESsystemiscompatiblewiththemajorityofexistingsplit-Gal4lines,howeverGal80itselfisnotcompatiblewithmostextantsplit-Gal4combinationsasmostactivationdomainlinesarenotderivedfromGal4. https://doi.org/10.7554/eLife.67598.sa1 Essentialrevisions: Itisusefulforanewuserofthistooltohaveamorecompletedescription,ratherthanhavetoworkitoutthemselves.Currently,theanalysisofthetoolismodest.More'proof-of-concept'experimentscanbeusefulandcanmakethetoolmorelikelytobeused.Inadditiontosuchexperiments,itwillbeusefuliftheycouldhavebetterquantificationofthedataandprovidebettercontrolstoo. WehaveincludedseveralnewexperimentsdemonstratingthesuccessfulapplicationofAGES.TheseincludeswitchingonGFPexpressioninasubsetoflarvalbraincells(grhGAL4)andasubsetofadultbraincells(Or85a-GAL4)asshowninournewFigure4. Furthermore,wehaveshownthatAGEScanbeusedtoelicitchangesinadultbehaviour(PDF-GAL4drivingUAS-Kir2.1).Hereauxin-supplementedfoodcandisruptcircadianbehaviourinbothmaleandfemaleflies(newFigure6). Regardingadditionalquantificationandcontrols,wehave: –QuantifiedGFPexpressionlevelsinlarvaeondifferentconcentrationsofauxin,andoveratimecourseusing5mM(newFigure3). –Quantifiedonandofftemporaldynamicsinadultfliesusing10mMauxin(Figure2—figuresupplement2A). –Testedhowlongauxinfoodlasts(Figure2—figuresupplement2B). –Confirmedthatauxinfooddoesnotaffectthebehaviouroflarvae(crawling)oradults(climbing)(Figure6—figuresupplement1). Pleasesee,fromtheaboveperspective,therecommendationsofboththereviewersbelowandthespecificsuggestionstheyhavemade.Couldtheauthors,whileembarkingonarevisedsubmission,pleaseoutlinealistofexperimentsthattheyplantodotoaddressthepointsintheprecedingparagraph,includinghowthecurrentstudysignificantlyadvancestheTrost2016work?GiventhatthisstudydevelopsonthatoftheTrost2016study,itsapplicationtoGal80needstogofurther. Weagreethatweshouldhaveintroduced/emphasisedtheTrostpapermoreinourmanuscript.However,webelievethatourstudyadvances/differsfromtheTrost2016paperinseveralimportantways: –Wehaveimproved(usingthehigheraffinityAtTIR1andcodonoptimisedallcomponents)andappliedthemethodforthespecifictaskoftemporallycontrollingGAL4activity. –Wehavedemonstratedthatthisnewlyengineeredsystemprovidesverytightcontrolofproteinlevels(throughthereadoutofGAL4activity). –WehaveinvestigatedandoptimisedtheconcentrationsofauxinrequiredtoactivateGAL4(throughthedegradationofGAL80)inbothlarvaeandadults. –Wehavetesteddifferentconcentrationsofauxinforitseffectonsurvivalanddevelopment. –Wehaveshownthatitcanbeusedtomanipulateadultbehaviour. Reviewer#1(Recommendationsfortheauthors): Figure2B-C:thestatisticalcomparisonsbeingmadearenotveryclear:betweentheplotsattheendsofeachbracket?(ifso,whynotshowallcomparisons?).InpanelC,itisalsonotclearifthe****referstocomparisonof0mMwith1and5mMaswellas10mm. WehaveincludedbracketsforalltherelevantcomparisonsinthenewFigure2tomakeitclearer. HowdotheauthorsexplainthedifferencebetweenGFPRNAandproteininduction?(PresumablytranslationofGFPRNAshouldnotbeimpactedbytheAGESsystem). WearenotsurewhythereisadifferencebetweenGFPRNAandGFPfluorescencerelativetothepositivecontrols.OnepossibilityisthatGFPfluorescenceisnotalinearreadoutofproteinlevels. Thetitleisabitover-hyped:–AGEScomprisesamodificationofGal80control;geneexpressionitselfisstilldependentupontheGal4/UASsystem. Wethinkthatthetitleisstillappropriate,andthatfurtherclarificationwouldmakethetitletoolong. Thenumberofanimalsuseindifferentexperimentsisnotclearlyindicated. Thankyou,thisisnowfixed. Line71:"thataretagged"not"whicharetagged" Thankyou,thisisnowfixed. Line78:missingwordbetweensystemsandmice Thankyou,thisisnowfixed. Figure2B:panelB,"fluorescence"ismisspelt Thankyou,thisisnowfixed. Figure2–figuresupp1:panelB,"fluorescence"ismisspelt Thankyou,thisisnowfixed. Reviewer#2(Recommendationsfortheauthors): McClureetal.havetakenadvantageoftheArabidopsisauxin-degronsystem,whichhasbeenshowntofunctioninawiderangeoforganisms,includingDrosophila.Inthisstudy,theauthorscleverlyengineeraubiquitouslyexpressedbicistronictransgenethatcontainsallofthenecessarycomponentstoinhibitGal4-inducedexpressionintheabsenceofauxin.Theauthorsconvincinglyshowtheirsystem'sefficacy,showingtheadditionofauxintofoodenableGal4-inducedgeneexpressionofaGFPreporterinthefatbody,examiningbothexternalfluorescenceandmRNAlevels.GFPexpressioninthenervoussystemisanecessaryadditiondue,astheauthorsstate,totheblood-brainbarrier.AstherewasnothingquantitativeinFigure3,anegativecontrol(asusedinFigure2)wouldhavebeenhelpful,asitwashardtodetermineif0mMauxinhadnoGFPexpressionvisually. WehavenowincludedanegativecontrolandquantifiedGFPexpressionlevelsinlarvaeondifferentconcentrationsofauxin,andoveratimecourseusing5mM(newFigure3). Asmanyresearcherswouldbeparticularlyinterestedinusingthissystemforbehavioralstudies,furtherinvestigationofthesystem'sefficacyforstudyingbehaviorswouldaddtotheinterestandimpactofthiswork;forexample,simplebehavioralcontrolswhenthefliesarerearedonauxin,e.g.,generallocomotion. Wehaveexaminedtheeffectofworkingconcentration(5mMforlarvaeand10mMforadults)onlarvalcrawlingandadultclimbing(Figure6—figuresupplement1).Hereweseenoimpactofauxinonthesebehaviours.Thecircadianexperimentdataalsoshowedthatauxindidnotaffectlocomotoractivity(Figure6–supplement2,A-D). AnexcellentadditioncouldbetoshowthataneuronaleffectorUAS(forexample,csChrimsonorTRPA1)doesnotinduceaGal4-drivenbehaviorintheabsenceofauxin,andperhapshowlongafterashifttoauxinbehaviorscanbeinduced. WearepleasedtoshowthatAGEScanbeusedtoelicitchangesinadultbehaviour(PDF-GAL4drivingUAS-Kir2.1).Hereauxin-supplementedfoodcandisruptcircadianbehaviourinbothmaleandfemaleflies(newFigure6). Lastly,itisnotablethatalltheshiftsinthispaperturnGal4-inducedexpressionfromanofftoanonstate.Itwouldbeinterestingtoinvestigatethetemporaldynamicsoftheopposite(turningthesystemfromontooff),whichwouldalsobeofgreatvaluetothebroadercommunity. Wehavequantifiedonandofftemporaldynamicsinadultfliesusing10mMauxin(Figure2—figuresupplement2A). –Thepaperlacksclarityinpartsthatmayimpactitsaccessibilitytoamoregeneralaudience,forexample,thedescriptionoftheshortcomingsofexistingsystemscouldbemoreclearlystated. Wehaveincludedthedescriptionsandshortcomingofthetwomainexistingsystems(GeneSwitchandQF-QS)intheintroduction(lines66-74). –Line116"properinductionofGAL4activity"isquiteavaguestatement,perhapsthetimecourse(statedasdatanotshown)couldbeaddedtosupplemental. DatanowinFigure2—figuresupplement2A. –Theauthorsstateinline173/174thattheAGESsystemiscompatiblewiththemajorityofexistingsplit-Gal4lines,howeverGal80itselfisnotcompatiblewithmostextantsplit-Gal4combinationsasmostactivationdomainlinesarenotderivedfromGal4. Thankyouforpointingthisout–wehavenowemphasisedthisinthepaper(lines268-273). https://doi.org/10.7554/eLife.67598.sa2 Authordetails ColinDMcClure DepartmentofLifeSciences,ImperialCollegeLondon,London,UnitedKingdom Presentaddress Queen’sUniversityBelfast,SchoolofBiologicalSciences,Belfast,UnitedKingdom Contribution Conceptualization,Datacuration,Fundingacquisition,Investigation,Methodology,Supervision,Writing–originaldraft,Writing–reviewandediting Contributedequallywith AmiraHassan Forcorrespondence [email protected] Competinginterests Nocompetinginterestsdeclared "ThisORCIDiDidentifiestheauthorofthisarticle:" 0000-0001-6298-5296 AmiraHassan DepartmentofLifeSciences,ImperialCollegeLondon,London,UnitedKingdom Contribution Datacuration,Investigation,Methodology,Visualization,Writing–reviewandediting Contributedequallywith ColinDMcClure Competinginterests Nocompetinginterestsdeclared "ThisORCIDiDidentifiestheauthorofthisarticle:" 0000-0003-1640-1602 GabrielNAughey DepartmentofLifeSciences,ImperialCollegeLondon,London,UnitedKingdom Presentaddress UniversityCollegeLondon,DepartmentofClinicalandExperimentalEpilepsy,UCLQueenSquareInstituteofNeurology,London,UnitedKingdom Contribution Datacuration,Investigation,Methodology,Writing–reviewandediting Competinginterests Nocompetinginterestsdeclared KhushbakhtButt WaksmanInstituteandDepartmentofMolecularBiologyandBiochemistry,Rutgers,theStateUniversityofNewJersey,NewBrunswick,UnitedStates Contribution Formalanalysis,Investigation Competinginterests Nocompetinginterestsdeclared AliciaEstacio-Gómez DepartmentofLifeSciences,ImperialCollegeLondon,London,UnitedKingdom Contribution Investigation,Validation,Writing–reviewandediting Competinginterests Nocompetinginterestsdeclared AneishaDuggal DepartmentofLifeSciences,ImperialCollegeLondon,London,UnitedKingdom Contribution Investigation,Visualization Competinginterests Nocompetinginterestsdeclared CheeYingSia DepartmentofLifeSciences,ImperialCollegeLondon,London,UnitedKingdom Contribution Investigation,Writing–reviewandediting Competinginterests Nocompetinginterestsdeclared AnnikaFBarber WaksmanInstituteandDepartmentofMolecularBiologyandBiochemistry,Rutgers,theStateUniversityofNewJersey,NewBrunswick,UnitedStates Contribution Formalanalysis,Investigation,Validation,Writing–reviewandediting Competinginterests Nocompetinginterestsdeclared TonyDSouthall DepartmentofLifeSciences,ImperialCollegeLondon,London,UnitedKingdom Contribution Conceptualization,Fundingacquisition,Methodology,Projectadministration,Supervision,Visualization,Writing–originaldraft,Writing–reviewandediting Forcorrespondence [email protected] Competinginterests Nocompetinginterestsdeclared "ThisORCIDiDidentifiestheauthorofthisarticle:" 0000-0002-8645-4198 TonyDSouthall ColinDMcClure AnnikaFBarber Thefundershadnoroleinstudydesign,datacollectionandinterpretation,orthedecisiontosubmittheworkforpublication. ThankstoMarcDionne(ImperialCollegeLondon)forprovidingthetubulin-eGFPplasmid,andtoChristianLehner(UniversityofZurich)forprovidingthepMT-OsTIR1-P2A-H2B-aid-eYFPplasmidaswellinvaluableinsightsintothemolecularbiologyoftheauxin-degradationsysteminDrosophila.WethankAndreaBrandfortheanti-Dpnantibody.Furthermore,thankstotheentireSouthalllabforhelpfuldiscussionsandpre-readingthemanuscript.ThisworkwasfundedbyWellcomeTrustInvestigatorgrant104567toTDS,anInnovationGrantfromtheSocietyofDevelopmentalBiologytoCDMandanNIH-NationalInstituteofNeurologicalDisordersandStrokeGrantR00NS105942toAFB. KVijayRaghavan,NationalCentreforBiologicalSciences,TataInstituteofFundamentalResearch,India Preprintposted:January26,2021(viewpreprint) Received:February16,2021 Accepted:March27,2022 VersionofRecordpublished:April1,2022(version1) ©2022,McClureetal.ThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduseandredistributionprovidedthattheoriginalauthorandsourcearecredited. 3,173 Pageviews 300 Downloads 1 Citations Articlecitationcountgeneratedbypollingthehighestcountacrossthefollowingsources:Crossref,PubMedCentral,Scopus. Atwo-partlistoflinkstodownloadthearticle,orpartsofthearticle,invariousformats. Downloads(linktodownloadthearticleasPDF) ArticlePDF FiguresPDF Opencitations(linkstoopenthecitationsfromthisarticleinvariousonlinereferencemanagerservices) Mendeley Citethisarticle(linkstodownloadthecitationsfromthisarticleinformatscompatiblewithvariousreferencemanagertools) ColinDMcClure AmiraHassan GabrielNAughey KhushbakhtButt AliciaEstacio-Gómez AneishaDuggal CheeYingSia AnnikaFBarber TonyDSouthall (2022) Anauxin-inducible,GAL4-compatible,geneexpressionsystemforDrosophila eLife11:e67598. https://doi.org/10.7554/eLife.67598 DownloadBibTeX Download.RIS Categoriesandtags ResearchArticle GeneticsandGenomics auxin GAL4/UAS inducible temporal transgene geneexpression Researchorganism D.melanogaster Ofinterest Furtherreading Furtherreading Inhumansandothermammals,germlinemutationsaremorelikelytoariseinfathersthaninmothers.AlthoughthissexbiashaslongbeenattributedtoDNAreplicationerrorsinspermatogenesis,recentevidencefromhumanspointstotheimportanceofmutagenicprocessesthatdonotdependoncelldivision,callingintoquestionourunderstandingofthisbasicphenomenon.Here,weinfertheratioofpaternal-to-maternalmutations,α,in42speciesofamniotes,fromputativelyneutralsubstitutionratesofsexchromosomesandautosomes.Despitemarkeddifferencesingametogenesis,physiologiesandenvironmentsacrossspecies,fathersconsistentlycontributemoremutationsthanmothersinallthespeciesexamined,includingmammals,birds,andreptiles.Inmammals,αisashighas4andcorrelateswithgenerationtimes;inbirdsandsnakes,αappearsmorestablearound2.Theseobservationsareconsistentwithasimplemodel,inwhichmutationsaccrueatequalratesinbothsexesduringearlydevelopmentandatahigherrateinthemalegermlineaftersexualdifferentiation,withaconservedpaternal-to-maternalratioacrossspecies.Thus,αmayreflecttherelativecontributionsoftwoormoredevelopmentalphasestototalgermlinemutations,andisexpectedtodependongenerationtimeevenifmutationsdonottrackcelldivisions. Insmallholderfarmingsystems,traditionalfarmervarietiesofneglectedandunderutilizedspecies(NUS)supportthelivelihoodsofmillionsofgrowersandconsumers.NUScombineculturalandagronomicvaluewithlocaladaptation,andtransdisciplinarymethodsareneededtofullyevaluatetheirbreedingpotential.Here,weassembledandcharacterizedthegeneticdiversityofarepresentativecollectionof366Ethiopianteff(Eragrostistef)farmervarietiesandbreedingmaterials,describingtheirphylogeneticrelationsandlocaladaptationontheEthiopianlandscape.Wephenotypedthecollectionforitsagronomicperformance,involvinglocaltefffarmersinaparticipatoryvarietyevaluation.Ouranalysesrevealedenvironmentalpatternsofteffgeneticdiversityandallowedustoidentify10geneticclustersassociatedwithclimatevariationandwithunevenspatialdistribution.Agenome-wideassociationstudywasusedtoidentifylociandcandidategenesrelatedtophenology,yield,localadaptation,andfarmers’appreciation.TheestimatedteffgenomicoffsetunderclimatechangescenarioshighlightedanareaaroundlakeTanawhereteffcroppingmaybemostvulnerabletoclimatechange.OurresultsshowthattransdisciplinaryapproachesmayefficientlypropeluntappedNUSfarmervarietiesintomodernbreedingtofostermoreresilientandsustainablecroppingsystems. Cancergenomesexhibitsurprisinglyweaksignaturesofnegativeselection1,2.Thismaybebecauseselectivepressuresarerelaxedorbecausegenome-widelinkagepreventsdeleteriousmutationsfrombeingremoved(Hill-Robertsoninterference)3.Bystratifyingtumorsbytheirgenome-widemutationalburden,weobservenegativeselection(dN/dS~0.56)inlowmutationalburdentumors,whileremainingcancersexhibitdN/dSratios~1.Thissuggeststhatmosttumorsdonotremovedeleteriouspassengers.Tobufferagainstdeleteriouspassengers,tumorsupregulateheatshockpathwaysastheirmutationalburdenincreases.Finally,evolutionarymodelingfindsthatHill-Robertsoninterferencealonecanreproducepatternsofattenuatedselectionandestimatesthetotalfitnesscostofpassengerstobe46%percellonaverage.Collectively,ourfindingssuggestthatthelackofobservednegativeselectioninmosttumorsisnotduetorelaxedselectivepressures,butrathertheinabilityofselectiontoremovedeleteriousmutationsinthepresenceofgenome-widelinkage. 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