Abstract

PreviousstudiesbyStachelandcolleaguesindicatethatlithiumchlorideinductionofpost-midblastularBrachydaniorerioembryosresultsindeficienciesinnormalanterior-posteriordevelopment.Todeterminewhetherornothigherconcentrationsoflithiumchlorideleadtosuccessivelygreaterdefectsinanterior-posteriordevelopment,D.rerioembryoswereinducedwiththefollowingvariableconcentrationsoflithiumchloride:0.45M,0.30M,0.15M,and0.00M.Embryosinducedwith0.15MofLiClexhibitedlittleornophenotypicdeviationfromthecontrolgroup,whereas0.30Mand0.45MLiCl-inducedembryosdidnotdevelopeyesandshowedsignsofperturbedtaildevelopment.Theresultsofthisexperimentsuggestthatthedegreeofinhibitionofnormalanterior-posteriordevelopmentincreasedinembryosinducedwithhigherconcentrationsoflithiumchloride.LithiumchlorideinductionmayberesponsIBLeforthereducedtranscriptionofgoosecoidintheanteriorregionsofD.rerioorthepreventionofcellmovementsallowingtheanterolateralaccumulationofgoosecoid,resultingintheperturbationofnormalanteriordevelopment(Stachelet.al.,1993).

Introduction

Lithiumchlorideisaknownteratogenwhichaltersdevelopmentinavarietyoforganismsincludingseaurchins,Xenopus(frogs),andBrachydaniorerio(Zebrafish)(Gilbert,2003).Inseaurchinembryos,lithiumchloridecausestheaccumulationofnuclearBeta-cateninineverycell,andtransformspresumptiveectodermintoendoderm(Gilbert,2003).Lithiumexposureincleavage-stageembryosofXenopusinhibitsdorsal/ventralaxisspecificationandresultsinrADIally-symmetric,dorsal-anteriorizedembryos(Stachelet.al.,2003).TheresearchconductedbyStachelandcolleaguessuggeststhatlithiuminductionofpre-midblastularZebrafishpreventsnormaldorsal/ventralaxispatterningbyactingasaninhibitortothephosphoinositolpathway,whichresultsingoosecoidandnogginexpressionoutsidetheregionofthepresumptiveembryonicshieldinsteadofthesegenesbeingconfinedtotheregionproximaltothedorsalblastoporelip.

ExperimentsbyStachelandcolleagueshaveshownthatthedevelopmentofanteriorstructuresisdependentonWntsignaling,especiallythetranscriptionofgoosecoid,whichcodesforadorsalizingproteinnecessaryfornormalanteriordevelopment(Stachelet.al.,2003).Theincreaseingeneexpressionoforganizer-specificproteinssuchasGoosecoidinthepresumptiveventralregionsoftheorganismproducesdifferentphenotypicresultswhentheinductionoccursatcertainstagesindevelopment(Stachelet.al.,1993).Forinstance,theexposureofembryostoLiClbeforethemidblastulartransition(2hourstage)resultsinhyperdorsalizationandtheinhibitionofnormaldorsal/ventralaxispatterning(Deitrich,1999).Incontrast,embryosexposedtoLiClatthefour-hourstageafterthemidblastulartransitionexperiencednormaldorsal/ventralaxisspecificationbutperturbeddevelopmentofanteriorstructuressuchaseyes(Stachelet.al.,1993).

Thepurposeofthisexperimentistostudytheeffectsoflithiumteratogenesisonpost-midblastularembryotoobservewhetherornottheinhibitionofanteriordevelopmentoccursalongagradient,withincreasingconcentrationsoflithiumchlorideleadingtoincrementallymoreseveredefectsinanteriordevelopment.Alternatively,theremaybeacertainthresholdatwhichlithiuminducesdefectsinanteriordevelopment.Indicationsoftheformerhypothesiswouldconsistoflithiumteratogenesisleadingto,forexample,partialformationofaneyeatoneconcentrationandthecompleteabsenceofeyeformationataslightlyhigherconcentration,thenitmaybepossiblethatlithiumteratogenesisaffectsdevelopmentalongagradient.Inaccordancewiththelattersituation,theobservableeffectsoflithiumteratogenesiswouldconsistinthecompleteabsenceoftheeyesatoneconcentrationandcompleteformationandpresenceofalleyestructuresincludingretinaandlensataslightlylowerconcentration.

Objective

ThepurposeofthisexperimentistomonitortheeffectsofdifferentconcentrationsofLiClonanteriordevelopmentinzebrafishembryos.Thiswillbecarriedoutbyassessingthedegreetowhichcertainamountsofthelithiumcholrideteratogenaffectthemorphologyofanteriorstructuressuchastheeye.

Materials

• 60-80Brachydaniorerioembryosatthesphere/dome-stageofdevelopment(seeprocedureformoredetails)
• Zebrafishembryomedium.
• LiCl(0.15M,0.30M,and0.45M)inZebrafishembryomedium
• distilledH₂O
• 960mmglasspetridishes
• wide-mouthpastuerPipette(withlargeenoughopeningforsuctioningchorionatedzebrafisheggs)
• dissectionscope

Procedure

1.Obtain15-20zebrafishembryosatthesphere/dome-stageofdevelopment(fourhourspost-fertilization)foreachofthefourtitrationsoflithiumchloridetobetested,inclusiveofthecontrolgroup.Therefore,about60-80sphere/dome-stageembryosmustbeisolatedintotal.Foradescriptionofsphere/dome-stageembryos,refertotheZebrafishInformationNetworkhomepage(www.zfin.org).

2.Placetheembryosintofourseparatepetridishes,eachcontaining10mLofZebrafishembryomedium.

3.Next,procurethreedishesandfillthemwiththefollowingconcentrationsofLiCl:

• 0.45M
• 0.30M
• 0.15M

AtableincludingthecalculationsforthethreeLiCltitrationsusedintheoriginalexperimentisincludedintheMaterialsandMethodssectionofthispage.

4.TransfereachpopulationofembryosfromtheembryomediumtotheLiCl-containingdishesusingaseveredpipetteandimmersetheminsolutionfor10minutes.

5.After10minutes,rinsetheembryosbyplacingeachgroupinaseparatedishcontaining10millilitersofembryomedium,andthentransferbacktotheoriginalembryo-medium-containingdishes.BesuretolabelthedisheswiththecorrespondingamountofLiCltowhichtheywereinduced.

6.Photographtheembryosafter24hourstoobserveanteriordevelopment,carefullynotingdeviationsinanteriorpatterningandformationfromthecontrolpopulation.

MaterialsandMethods

Theembryosusedinthisexperimentwereatthespherestageofdevelopment,whichisequivalenttoapproximatelyfourhoursofincubationpost-fertilization.Moreimportantly,thespherestageoccursafterthemidblastulartransition,whereupontheembryobeginstotranslateitsowngenes,cellmovementsoccur,andapatternofslow,asynchronouscleavagebegins(Gilbert,2003).Theexposureofeachofthevariantgroupstolithiumchlorideconsistedofaten-minuteincubationperiodforeachsothatthetimeofincubationwouldremainconcurrentwiththespherestageandnotextendbeyondthisperiodofdevelopment.Sphere-stageembryoswereisolatedaccordingtoStagesofEmbryonicDevelopmentoftheZebrafish(ZebrafishInformationNetwork,1995).IsolatedembryoswerecollectedinZebrafishEmbryoMedium,whichwaspreparedaccordingtothedevelopmentalBIOLOGylabwebsite(DBLab,2003).Duringtheinductionperion,approximately10-15BrachidaniorerioembryosweretreatedwiththefollowingtitrationsofLiCl:0.45M,0.30M,and0.15MmadeupinZebrafishEmbryoMedium.ThecalculationsfortheconcentrationsofLithiumChloridethatwereusedareasindicatedinTableI:

TableI:VariableMolarityofLithiumChlorideTitrations

ThefourthgroupdidnotcontainanyLiClandservedasacontrol,thusnocalculationswererequiredforthistitration.Subsequenttotheten-minuteincubationperiodofthethreevariablegroupsintheLiClsolutions,allembryoswererinsedwithZebrafishembryomediumandincubatedovernightat28°C.

Results

Inthecontrolgroupembryos,anterior-posteriorpatterningwasnormal,withfullformationoftheterminusstructures,theretina,andthelensoftheeye(Figure1A).Inembryostreatedwith0.15MLiCl,theeyesappeartohavedevelopednormally,buttheeyeshowninFigure1BisrelativelyclosertotheanteriorterminusthantheeyeinFigure1A.The0.15MLiCl-treatedembryosexibitedslightlystuntedgrowthattheiranteriorandposteriortermini;thetailswerealsoslightlybentandroundedatthetip(Figure2B.The0.30MLiCl-treatedembryosdidnotdevelopeyes,andexhibitedslightcurvatureofthedorsalregion(Figure1C).Theseembryosexhibitedevenlessdevelopmentoftheanteriorandposteriorterminithanthe0.15Membryos,withstubbytailsandlargeyolksacs(Figure2C).

AllembryosinFigure1exhibitdevelopmentofears,liver,andtheCNS.Theaveragesurvivalrateforembryosinthe0.00M,0.15M,and0.30Msolutionswere19/19,18/20,and15/21respectively.The0.45MLiCl-treatedembryoshadlowerviABIlity,withonlysevenoftheoriginalnineteenembryossurvivingthe24-hourperiodfromthetimeofincubationtothetimeofdatacollection.Theresultingembryoswerefragile,andsomedidnotsurvivethedechorionationprocessandtheircellsbecamedisaggregatedbythepulloftheforcepsandtheinfluxofZebrafishembryomediumintotheopenedchorion.Thisgroupdisplayedalackofanterior-posteriordevelopment,withnodefinableheadstructures,andstuntedtaildevelopment(Figure2D).

Figure1(A)Controlembryo,typicalanterior/posteriorpatterningwithfully-formedanteriorstructuresincludingtheeye,forebrain,CNS,earandliver(B)Embryoinducedwith0.15MLiClshowsliver,ear,eye,forebrain,andCNSdevelopment(C)0.30MLiCl-inducedembryoshowsnoeyeformation,thoughearandliverarepresent.

A0.00MLiCl	B0.15MLiCl

Figure2Photographstaken26hoursafterLiClinduction(A)Controlembryoshowsnormaltailformation(B)Embryoshowsnormaltaildevelopment(C)Tailappearsstuntedandcrooked,gutisshortened,headissmaller,andyolkisenlargedandmisshapen(D)Headandtailarestunted,anteriorstructuresaremissing,yolkisenlarged.

Discussion

Theseresultsdemonstratethatlithiumchloridetreatmentaffectedtheanterior-posteriormorphogenesisofBrachydaniorerioembryossuchthathigherconcentrationsofthissubstanceleadtoincreasinglygreaterinhibitionofanteriorandposteriordevelopment.The0.15MLiCl-treatedembryoweremissingtheiranteriormoststructures.Theembryostreatedwith0.30MLiClexhibitedgreaterteratogeniceffectsonthedevelopmentofanteriorandposteriorterminusstructures.HigherconcentrationsofLiClalsoledtomoremorphologicaldefectsinboththeanteriorandposteriorterminalregions,furtherconfirmingthehypothesisthattheconcentrationsofLiClteratogenisdirectlyrelatedtothephenotypiceffectonZebrafish.

Lithium-inducedteratogenesishasbeenshowntoperturbthedevelopmentofventralstructuresinpremidblastularZebrafish,whentheblastulaisstillformingmorphogeneticgradientsofproteinsintheWntsignalingpathway(suchasb-catenin)andcontinuingtotranscribematernalmRNAs.Intheseearlyembryos,lithiumteratogenesisleadstotheexpressionofbustledandradializedphenotypes,whichexhibithyperdorsalization(Stachelet.al.,1993).Inthesphere-stageembryos,morphogeneticgradientshavealreadybeenestablished,sotheeffectoflithiuminthiscaseprobablyoccursdownstreamoftheseevents.

StudiesconductedbyStewartandGerhart(1990)showedthatlithiuminhibitsgastrulationmovementsinXenopus,resultinginincompleteinvolutionandthusincompleteinductionofanteriorstructures(StewartandGerhart,1990).Thiscouldpossiblyindicatethatlithiumteratogenesisperturbsdevelopmentinlaterembryosbypreventinggastrulation.However,thephenotypeoflithium-treatedZebrafishembryosdoesnotappeartobeequivalenttothatofXenopusembryos.Themoststrikingeffectisonthenormalformationofstructuresanteriortotheeye.Stachelandcoworkers(1993)havepresentedevidenceofthenormaldevelopmentofcentralnervoussystemstructuresanteriortothepresumptiveeyeregionwhenlensandretinadevelopabnormally.ThissuggeststhattheincompletegastrulationmodeloflithiumteratogenesisinXenopusdoesnotfullyexplainthephenotypiceffectsoflithiuminBrachydaniorerioembryos.ThisevidencealsosuggeststhattheremaybedifferentialsignalingpatternsinvolvedinXenopusascomparedtoZebrafisheyeformation.

Lithiumdorsalizesseaurchinembryosbyincreasingthelevelsofb-cateninthroughoutthecytoplasm,whichleadstotheradializationofgoosecoidexpressionandsubsequentaugmentationoforganizedmesodermthroughoutthemarginalzoneandintothepresumptiveventralregionsoftheembryo(Gilbert,2003).Theembryosinthisexperimentdidnotshowagreatdegreeofdorsalization,exceptpossiblythe0.45MLiClembryo,butfurthertestswouldneedtobeconductedbeforedeterminingwhetherornotlithiuminducesdorsalspecificationoftissues.

Fortunately,suchstudieshavebeenconductedbyStachelandcolleagues(1993).Theyusedafluorescentmarketgeneintercalatedinthegenomeadjacenttothegoosecoidtranscript,sothattheMarkerappearsinthewherevergoosecoidproteinispresent.TheresultsoftheirexperimentonlithiuminductioninZebrafishshowsthatthedistributionofgoosecoidtranscriptsincreasessubstantiallytenhoursafterdevelopment.Duringthisperiod,rostralcrescentswellingandmedialstrippatternsusuallyexhibitinggoosecoidtranscriptsappearnormal.However,thelateralwings,areasofgoosecoidexpressionfoundinnormalembryos,aremissing,whichsuggeststhatlate-lithiumexposureeitherreducestranscriptionofgoosecoidinthelateralwingregionorpreventscellmovementsdirectingtheanterolateralaccumulationofgoosecoid(Stachelet.al.,1993).Thisreductionintheexpressionofgoosecoidintheanterolateralregionsuggeststhatlithiumispartiallyresponsiblefortheinhibitionofnormalanteriordevelopment.

Acknowledgements

Iwouldliketorecognizeseveralindividualsformakingthisexperimentpossible.Thankyoufirstofalltomylabgroupmembers,KristiLaSalle,BrandtRakowski,KatyLewis,andErinBettersforisolatingthesphere-stageZebrafishembryosusedinthisexperiment.IwouldliketorecognizePeterBurgessofFranklinandMarshallCollegewhoconductedasimilarexperimentonMedakaembryosthatinspiredmetoconductthisexperiment.Thankstothelabpersonnelresponsibleforfeedingthefish,andtoProfessorJudyCebra-ThomasandFraserTanforpreparingtheLiClconcentrationsusedinthisexperiment.ThankstoScottStachelandhiscolleaguesforprovidingusefulinformationonpreviousexperimentsinlithiuminductionofZebrafishandtoScottGilbertforprovidingawonderfulresourceforbeingabletointerpretthedatacollectedinthisexperiment.Additionally,IwouldliketothankWilliamGreshforadvisingmeinthewritingofthislabreport.