DEVELOPMENTAL EFFECTS OF LITHIUM CHLORIDE ON XENOPUS EMBRYOS

PURPOSE:Theobjectiveofthisexperimentistostudytheeffectoflithiumchloridetreatmentonearlyblastulaxenopusembryos.

INTRODUCTION:Whenthedevelopingembryosofmanyorganisms,suchasthelytechinusvariegatus(seaurchin)andxenopus(frog),aretreatedwithlithiumchloride,theyexperiencealterationsorperturbationsindevelopment.LithiumisbelievedtobethecausetheseperturbationsduetoitsABIlitytoinhibitglycogensynthasekinase-ß(beta)whichinitiatesdevelopmentalpathways.

TheearlieststagesofXenopusdevelopmentafterfertilizationoftheeggischaracterizedbytherapidcleavageofcells,whichisholoblastic(uneven)duetothelargeconcentrationofyolkatthevegetalpole.Aftertheblastulahasformedandtheblastocoel,thehollowcavityinsidetheblastula,ispresent,thengastrulationbegins.Gastrulationinitiatesonthefuturedorsalsideoftheorganismwhichismarkedbythegraycrescent.Thegraycrescentistheregionwhichisoppositefromthelocationofspermentry.Thisregionhasrotated,thecortical(outer)cytoplasmrotates30û,sothatitslocationisbetweentheanimalandvegetalpole.Thecellswhicharemigratingintotheembryotravelinthroughthedorsalblastoporelip.Thedorsalblastoporelipisnotonlythepointofentryofcells,butalsosetsthedorsal-ventralaxisandmarkstheposterioroftheembryo.

Whenearlyembryosaretreatedwithlithiumchloridebeforethe64cellstage,ithasbeenshownthattheembryosundergodorsalization(KaoandElison1988,1989;Slack1988).Dorsalizationiswhenthedorsalblastoporelipformsaroundtheentireembryoatthesametime.Theresultisanembryothathasacontinousdoralside.ThemostextremeresultsofthistreatmentareembryoswithrADIalsymmetricheads,acylindricalheart,andbandsofeyepigment.(Siveetal.2000)ThiseventoccursatthesametimeindevelopmentthatthedorsalblastoporelipwouldformiftherehadbeennolithiumchloridetreatmentadmiNISTered.Asmentioned,lithiumhastheabilitytoinhibitglycogensynthasekinase-ß(beta),whichisasubstancewhichinititesthewntpathwayinxenopusembryosthatleadstodorsalaxisformation(KleinandMelton1996).

PROTOCOL:

1.Dejellyearlyblastulaembryosbeforethe32cellstage.

• 1)Selectandseparateembryosbelowthe32cellstage

• 2)Removejellymembraneusingsharpenedforceps.

• 4)Placedejelliedembryosin10%Steinberg"ssolution.

• 5)Selectembryosthatremainbeloworatthe32cellstage

2.Rinsehealthyembryosandtransfer3/4totheexperimentalpetridishcontaining0.3Mlithiumchloridein0.1xMBS.Transfertheremainingembryostoacontrolpetridishwith10%Steinberg"ssolution.

3.Gentlymixsolution.

4.Incubateembryosat18-20ûCfor10minutes.

5.Rinseincubatedembryosandtransfertopetridishwith10%Steinberg"ssolution.

6.Incubateembryosat18-20ûCandscoreperturbationsatearlygastrula.

*ProtocoladaptedfromEarlyDevelopmentofXenopusLaevis.

RESULTS:

After1day,thecontrolemryosweredevelopingatanormalpaceandwerecompletewithgastrulation.AsFigure1and2show,theembryoswerefaralongindevelopmentandnearlyatthetadpolestage.Lithiumexposedembryosweremuchclowerindevelomentattheonedaystage.Attheonedaymark,thedorsalblastoporeliphadformedbutwasirregularandelongated;ithadtheappearanceofan"eye"ascanbeseeninFigure3and4.ThisfollowsKaoandElison"sworkwhichsaysthatlithiumwillcauseapertubation,calleddorsalization,ofthedorsalblastoporelip.

ControlandLithiumExposedEmbryos-1Day
Figure1:control	Figure2:control
Figure3:lithiumexposed	Figure4:lithiumexposed

RESULTS(continued):

After2days,thecontrolgrouphasdevelopedintonormal,functioningtadpoles(Figure11-12).Thelithiumexposedgroupisundergoinganontraditionaldevelopmentalpattern.AscanbeseeninFigure5-8,theembryoshavenodefinedanteriorexceptfortheblackdotwhichisthemouth.Theorganismsasawholehaveminimalanterior-posteriordefinitionandappeartobedevelopingradially,alloftissuesareforminginmultiplenumbersasaresultofthedorsalizationoftheblastoporelip.InFigure9-10,theembryosappeartohavedevelopedtounhealthytadpoles;thisisunlikleytohavebeencausedbythelithium.Noneofthelithiumexposedembryossurvivedpastthe2daystage;theydisintegrated.

LithiumExposedEmbryos-2Days
Figure5	Figure6
Figure7	Figure8
Figure9	Figure10

ControlEmbryos-2Days
Figure11	Figure12

CONCLUSIONS:

Thisexperimentsuccessfulexhibitedtheabilityoflithiumtoalterthedevelopmentofthefrogembryos.Thecontrolorganismdevelopednormally,whilethelithiuminducedembryosunderwentdorsalization,altereddevelopment,anddeathaspredictedbypreviousresearch.Thealtereddevelopmentoftheembryoswasmarkedbyaminimalanterior-posterioranddorsal-ventralaxis.Iftheembryossurvivedlonger,radiallydevelopmentwouldmostllikleyhavebeenexhibited.Lithiumhasbeenshowntohavetheabilitytoinhibitglycogensynthasekinase-ß(beta),whichinititesthewntpathwayinxenopusembryosthatleadstodorsalaxisformation(KleinandMelton1996).