DevelopmentoftechniquesforprimarycultureofC.elegansembryonicneurons LairdBloom MIT fromPh.D.thesis,MassachusettsInstituteofTechnology,1993 Introduction OneofthemajorlimitationsofthestudyofaxonaloutgrowthinC. elegansisthatdirectmanipulationoftheenvironmentsofspecific neuronsisnotpossIBLe,eitherinpartially-dissectedpreparationsor inculture.Inexperimentalsystemsinwhichcultureisavailable, detailedstudiesoftheinteractionsofgrowingaxonswiththeir substratesarepossible,includingantibodyperturbationstudiesof cell-surfacemolecules,directobservationofgrowthconebehavior,and pharmacologicalmanipulationofgrowingaxons"electricalactivity, secondmessengers,orCytoskeleton.InDrosophila,primarycultureof neuronsfrommutantstrainshasenabledstudyofmembranecyclingin shibiremutantsandtheelectrophysiologicaldefectsinnapmutants(Wu etal.,1983).TheavailABIlityofatechniqueforthecultureofC. elegansneuronswouldprovideanewmethodfortheanalysisofmutants defectiveinneuronaldevelopmentandfunction.Inparticular,several genesrequiredfornormalaxonaloutgrowtharebelievedtoencode moleculesthataffectthecytoskeleton,membranestructure,interaction withtheextracellularmatrix,andsignaltransduction(Leung-Hagesteijn etal.,1992;T.Otsukaetal.,inpreparation;Oshima,R.Steven,A. Ruiz,J.Mancillas,andJ.Culotti,personalcommunication).A techniqueforstudyingtheseprocessesinmutantcellslackingspecific moleculesinadefinedenvironmentmightprovideinformationapplicable tothestudyofaxonaloutgrowthinavarietyofspecies.. NotechniqueforculturingC.elegansneuronshasbeenpublished. Hedgecocketal.(1987)citedunpublishedobservationsthatembryonic cellsplatedonanadhesivesubstratumsendoutsingle,unbranched processes,butgavenoindicationoftheconditionsused.Conditions thatallownormalcelldivisiontooccurafterembryosarepermeabilized orpartiallydissociatedandreassembledhavebeenreported.L.Edgar (personalcommunication)hasdevelopedatechniqueforremovingthe eggshellsofembryosasyoungasthe1-cellstagebyacombinationof enzymaticdigestionandpipettingthroughanarrowaperture.These embryos,whichremainsurroundedbyamembrane,continuetodivideto produceupto500cells,andnormaldifferentiationofthemajor lineagesoccurs(asjudgedbyMarkersforgut,muscle,andgermline). Occasionalneuronalprocesseshavebeenobservedinthesepermeabilized embryos(L.Edgar,personalcommunication).Blastomeresthatare separated,reassociated,andculturedundertheseconditionscontinueto divideanddifferentiatenormally(Goldstein,1992). Theexperimentsdescribedbelowweredesignedtoextendthese techniquestothegrowthoflargenumbersofdissociatedembryonic cells.Thegoalwastodefinemedia,substrates,andcellisolation techniquesthatwouldpermitneuronaldifferentiationandaxonal outgrowthinshort-termcultures.Becauselong-termculturewasnot anticipated,effortstomaintainsterilityweremadeonlyinpreparation ofmedia.Bacterialandfungalcontaminationwassometimesevidentin three-daycultures,butnotbeforethis.Cellswerekeptoniceorat roomtemperatureduringinitialexperimentswithcellisolation procedures.Asthisappearedtomakelittledifferenceinthehealth ofthecells,experimentswithdifferentsubstrataandmediawere conductedwithcellskeptatroomtemperaturethroughouttheprocedure. ExperimentalProcedures Detailsoftheproceduresusedarediscussedinthetext. CultureswereobservedunderNomarskiopticsusinga100xPlanapo objectivelensonaZeissAxiovert10invertedmicroscopeoraZeiss Axiophotmicroscope.Forantibodystaining,cultureswerefixedfor30 minatroomtemperaturein4%paraformaldehydeinPBS,followedbythree washesinPBSpH7.2containing1%TritonX-100and1%BSA.Cultures wereblockedfor30min.with10%BSAinPBS,followedbyovernight incubationinprimaryantibodyat4"C,threeroom-temperaturewashesin PBS,anda1hrincubationinsecondaryantibodyat37"C.Following threewashesinPBS,theculturesweremountedinMowiolcontaining1 mg/mlp-phenylenediamineandobserved. Ascitesfluidfrommonoclonalantibody611B1(G.Pipierno)wasusedat a1:10dilution,theanti-tubulinmonoclonalantibodyYL1/2wasusedat a1:50dilution.Rhodamine-conjugatedgoat-anti-mouse(Cappell)and fluorescein-conjugatedgoat-anti-ratsecondaryantibodies(Jackson ImmunoResearch)wereusedata1:400dilution.Allantibodieswere dilutedinPBS. Results Dissociationmethods Dissociationofcellsforprimaryculturefromotherorganismsis usuallyachievedbyacombinationofdissectionofneuronsawayfrom othertissue,mildproteolyticdigestionofbasementmembranesandother connectivetissues,andphysicalseparationofcells.Thesmallsizeof C.elegansembryosmakesdissectionimpossible,andsoanadditional necessarystepistheremovaloftheeggshell.Krasnowetal.(1991) dissociatedwholeDrosophilaembryossimplybygentleDounce homogenization.Edgar"stechniqueforremovaloftheeggshellfrom singleembryosinvolvedabriefdigestionofintactembryosinamixture ofchitinaseandchymotrypsintobeginbreakdownoftheeggshell followedbypassageoftheembryothroughadrawn-outmicropipetwitha diameterslightlysmallerthanthatofanembryo. BothDouncehomogenizationandenzymaticdigestionwereusedtofreeC. elegansembryoniccellsfromtheeggshell.Inallcases,populationsof mixed-stageembryoswereobtainedfrommixed-stageC.elegans populationsbywashinginM9followedbytreatmentwith20%sodium hypochloritesolutionin0.5MNaOHuntilalllarvaeandadultswere dissolved.EmbryoswerewashedseveraltimesinM9toremove hypochloriteandthenresUSPendedineggbufferfor chitinase/chymotrypsindigestionorCa/Mg-freemediumforDounce homogenization(8g/lNaCl,200mg/lKCl,50mg/lNaH2PO4.H2O,1g/l NaHCO3,1g/lglucose;Wuetal.,1983).Douncehomogenizationwas performedona1mlcellsuspensionina15mlglasshomogenizer.A dropofthesupernatantwasinspectedunderthedissectingmicroscope every10-20strokes,andhomogenizationwasstoppedwhenalargenumber ofindividualcellswerevisible(60-100)strokes.Thecellsand embryoswerethenincubatedinacocktailofCollagenasetypeIA,IV, andVII(Sigma;0.1mg/mleachinCa/Mg-freemedium)for60minatroom temperature.Insomepreparations,collagenase-digestedembryoswere suckedupanddownrepeatedly(triturated)inadrawn-outpasteurpipet toseparatethecellsmechanically.YieldsfromtheDounceprocedure wereusuallylow,regardlessofthenumberofDouncestrokes,the collagenasemixtureused,andtheinclusionofatriturationstep. Embryostobedissociatedbyenzymaticdigestionwerepreparedby hypochloritetreatmentasabove.Theywerethenincubatedatroom temperatureinamixtureof5-10mg/mleachchitinase(Sigma)and alpha-chymotrypsin(ICN)withgentleagitationuntiltheembryosinasample observedunderthedissectingmicroscopebegantoroundupandthe outlinesofindividualcellsattheedgesoftheembryosbegantobecome moredistinct(usually5-6minutes).Thereactionwasstoppedby severalwashesinculturemedium(seebelow)containingfetalbovine serum,whichcontainsproteaseinhibitors.Treatmentwithtwowashesof soybeantrypsininhibitorbeforetheserumwashesdidnotimprovethe apparenthealthofthecells.Cellswerethenmechanicallydissociated bytriturationinapasteurpipetwithaslightlydrawn-outtip, followedbyaperiodofseveralminutesinwhichwholeembryosandlarge clumpsofcellswereallowedtosettleoutofthesuspension.The mechanicaldissociationandsettlingstepswererepeatedwithmaterial thatsettledoutofsuspensionuntilfewintactembryosremained.High yieldsofcellscouldbeobtainedwiththistechniqueifthe dissociationwassufficientlygentle,aconditionaidedbykeepingthe tipofthepipetonlyslightlysmallerthanthenormalpasteurpipettip andbykeepingtheamountofpipettingtoaminimum.Inaddition, overdigestionwiththechitinase/chymotrypsinappeareddetrimentalto thehealthofthecells. Followingdissociation,cellsuspensionswerefilteredthroughtwo layersoffinenylonmeshstretchedovertheendofa3mlplastic syringe.Thiseffectivelyremovedallofthewholeembryosandmostof theL1larvaethatwerereleasedfromtheireggshellsduringthe dissociationprocedure,butitallowedlargeclumpsofcellstopass through.Filtrateswerethensubjectedtotworoundsoflow-speed centrifugation(750xg)toseparateintactcellsfromparticulate materialproducedduringdissociation.Cellswereresuspendedina volumeofmediumequivalentto50mlpersampletobeplated (approximatelythreesamplesper9cmplateofworms).Thisyieldeda dropofcellsthatwasconfluentinthecenterbutallowedobservation ofindividualcellsattheedges. Substrates Dissociatedcellsfromavarietyofspeciesgenerallyattachtoglass ortissuecultureplasticcoatedwithnonspecificchargedmoleculessuch aspoly-L-lysineorpolyornithine,relativelynonspecificadhesive proteinssuchasthelectinconcanavalinA(ChiquetandAcklin,1986), orspecies-specificextracellularmatrixmoleculessuchaslaminin, fibronectin,orcollagen(BankerandGoslin,1991).Becausenearly allneuronsarereportedtoshowsomeadhesionandaxonaloutgrowthon polylysine,initialexperimentsweredonewithglasscoatedwith0.01-1 mg/mlpolylysine.C.elegansembryoniccellsfromsomepreparations adheredwelltoPLL-coatedglasscoverslips,butoftentheyfailedto remainadhered,orwhentheysentoutaxons,theaxonsseemedvery looselyattachedandappearedtofloatinthemedium.Becauseamore adhesivesubstrateappearedtobenecessary,thesilanederivativeTESPA (3-aminopropyl-triethoxysilane;Sigma),oftenusedforattachingtissue sectionstoslides,wastestedforitsabilitytosupportC.elegans cellattachmentandaxonaloutgrowth.Initialexperiments(usingcells preparedbychitinase/collagenasetreatmentandtriturationandgrownin modifiedEdgar"smedium;seebelow)showedthatTESPA-coatedcoverslips allowedmoreextensiveaxonaloutgrowththandidPLL-coatedcoverslips, butthis,too,wasvariable.Reactivealdehydegroupscanbeaddedto TESPAbybrieftreatmentwithparaformaldehyde;coverslipscoveredwith 1%TESPAandactivatedwith4%paraformaldehydeproducedthemost consistentaxonoutgrowth(Fig.4-9).Coverslipspreparedlessthan twodaysbeforeuseappearedtobemorereliablethanoldercoverslips. Cellsgrownonuncoatedcleanglassfailedtoadhere. Poly-L-lysineappliedtoactivatedTESPA-coatedcoverslipsappearedto benobetterthanactivatedTESPAalone.Initialexperimentswiththe vertebrateextracellularmatrixproteinslaminin,fibronectin, thrombospondin,collagen(typesI,III,andIV)appliedtoPLL-coated coverslipsshowednoobviousimprovementincellattachmentoraxonal outgrowthoverthatobservedwithPLLalone. Theapparentadvantageofparaformaldehyde-activatedTESPAoverother, lessadhesive,substratessuggestedthatcellspreparedby chitinase/chymotrypsinandtriturationwerenotparticularlyadhesive. Thismightbecausedbylossofcellsurfaceadhesionmoleculesthrough excessiveproteasetreatment.Theadhesivityoftheculturesubstratum hasbeenshowninotherorganismstoaffecttheamountofneurite outgrowthandcellspreADIng(BrayandChapman,1985).Itispossible thatlessadhesivesubstratawouldhavepromoteddifferentbehaviorof C.eleganscells,suchascelldivisionratherthandifferentiation. Media Cellculturemediatypicallycontainsalts,abufferingagent, vitamins,precursorsforaminoacidandnucleicacidbiosynthesis, antibiotics,andasourceofgrowthfactors.Whilesomeinvertebrate cellculturesystemsuseinvertebratetissuesasasourceofgrowth factors(e.g.,AplysiaorHelisomahemolymph),manyinvertebratecell typeshavebeensuccessfullyculturedinfetalbovineserum(Beadleet al.,1988).Becauseinvertebratehemolymphisnotcommercially available,fetalbovineserumwasusedtodevelopmediaforC.elegans cellculture.(Coelomicfluidisolatedfromearthworms(ArlingtonBait andTackle,Arlington,MA)showedconsiderabletoxicitytoC.elegans cellsinaninitialexperimentandwasnottestedfurther.) Mostinvertebratecellsareculturedinairincubatorsratherthanin theenvironmentofCO2inairusedformostmammaliancells.Several mediadesignedforuseinairincubatorsweretestedfortheirability tosupportC.eleganscelladhesion,differentiation,andsurvival.A mediumsimilartothatusedbyWuandco-workersforthecultureof Drosophilalarvalneurons(Wuetal.,1983)wastestedininitial experimentswithcellsisolatedbyDouncehomogenizationandplatedon PLL-coatedcoverslips,andsubsequentexperimentswereconductedwith modificationsofthemediumdesignedbyEdgarforusewithpermeabilized C.elegansembryos.Inallexperiments,adropofdissociatedcells (approximately50ul)wasplacedinthecenterofa22x22mmor24x 50mmglasscoversliprecentlycoatedwithPLLorTESPA.Insome experiments,thedropwasheldinplacewithbyathicklinemadewitha greasepencil,butthiscouldbeomittedwithoutseriousdifficulty. Coverslipswereplacedcell-sideupontoParafilm-coveredmicroscope slidesinamoisturechambermadefromaplasticboxlinedwith water-soakedWhatmanfilterpaper.Thechamberswerecoveredin aluminumfoiltoprotectthecellsfromlightandplacedinthesame 20"Cincubatorusedforgrowingworms.CellswereviewedwithNomarski opticsanda100xoil-immersionobjectivelens.24x50mmcoverslips couldbeplaceddirectlyontothestageofaninvertedmicroscopefor observationwithoutdisturbingthedropofmedium.Smallercoverslips wereviewedwithaconventionalmicroscopeafterbeinginvertedonto viewingchambersmadefrommicroscopeslidestowhichtwocoverslipshad beenglued,separatedbya5mmgap.Thisconfigurationallowedthe cellstoremaininculturemediumduringobservationwithoutbeing squashed,butwaspronetodrying.Culturesthatweretobeobserved multipletimeswerekeptonlargercoverslips. ThefirstmediumtestedwassimilartothatusedbyWuandco-workers (1983),whichcontained25%L-15medium(Gibco),66%modifiedSchneider saline,and9%fetalcalfserum,heat-treatedtoinactivatecomplement. AfterpreparationbyDouncehomogenization,C.eleganscellswerewashed inthismediumandplatedonfreshly-preparedPLL-coatedcoverslips