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 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: 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). A0.00MLiCl B0.15MLiCl C0.30MLiCl 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 C0.30MLiCl D0.45MLiCl 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.