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