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Effect of ethanol on development of Danio reriro
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Objective

Theobjectiveoftheexperimentistodeterminetheeffectsofethanolexposureontheembryonicdevelopmentofzebrafishthroughobservationofphysicaldefomities.

Introduction

Embryonicdevelopmentofzebrafish,Danioreriro,isaffectedbyethanolinamannersimilartohighervertebrates(BladerandSträhle,1998).Therefore,investigatingtheeffectsofethanolonzebrafishembryomanypromoteknowledgeandunderstandingoffetalalcoholsyndromeinhumans.BladerandStrahleconfirmedthatexposureofzebrafishembryostoethanolcausescyclopiaandcraniofacialabnormalitiesandaltersgeneexpressionintheventralaspectsoftheforeandmidbrain(1998).Exposureofzebrafishembryostoethanolalsoinducesdevelopmentalabnormalitiesofthenotochordandspinalcord,andmalformationofthebodytrunk(BladerandSträhle,1998).

Inordertounderstandtheoriginofthedeformities,itisnecessarytoknowhowembryoniccellsofzebrafishnormallymigrateduringgatrulation.Duringgastrulation,blastodermcellsepibolizeovertheyolk.Whenabouthalftheyolkcellbecomescoveredwithblastodermcells,themarginofepibolizingblastodermcellsformtheepIBLastandthehypoblast.Epiblastandhypoblastintercalatetoformtheorganizer,knownastheembryonicshield,atthedorsalsideoftheembryo(Gilbert,2003).Hypoblastcellsoftheembryonicshieldconverge,extendanteriorly,andnarrowalongthedorsalmidlineofthehypoblast(Gilbert,2003).Someofthesecellseventuallyformtheprechordalplateandthenotochord(Gilbert,2003).Prechodalplatecellsseemtobecrucialinexplainingtheoriginofethanolinduceddeformities.

Thoughlittleisknownabouttheunderlyingmechanismsbehindthedeformities,itisknownthattheectopicpositionoftheprechordalplatecellsischaracteristicofzebrafishexposedtoethanol(BladerandSträhle,1998).Itis,therefore,believedthatethanolisresponsiblefortheabnormalmigrationofprechordalplatecellsthatultimatelycausescyclopiaandotherdeformities.Theprechordalplatecellsexpressgeneslikegoosecoidandislet-1,whichcontrolcelldifferentiationintheanteriorregionoftheembryo(BladerandSträhle,1998).Hence,prechordal-specificgenesareexpressedectopicallytobringabouttheobserveddeformities.

Inadditiontothedeformities,ethanolappearstocauseabnormalcelldeath(Suliketal.,1988).Ethanolexposureatearlydevelopmentstagesresultsinsignificantdeathamongthecellsdestinedtogiverisetofacialstructures(Suliketal.,1988).Ethanolseemstoachieveapoptosisbyactivatingthecells’self-destructionmachineries(Suliketal.,1988).Therefore,inadditiontotheectopicprechordalplate-specificgeneexpression,ethanolinducedapoptosisappearstocontributetotheobserveddeformitiesinzebrafishembryos(Suliketal.,1988).

Thisexperimentseekstodeterminetheeffectsofexposureofethanolonzebrafishdevelopmentthroughobservationofphysicaldeformities.MostoftheprocedureswereadoptedfromBladerandSträhle’sexperiment(1998).30%epibolystage,alsocalledthelateblastulastage,isfollowedimmediatelybygastrulation(Gilbert,2003).Therefore,embryosat30%epibolystageatthelatestwillbeusedtoensurethatethanolwillaffectcellmigrationbeforeitoccurs.TheresultswillbecomparedtoBladerandStrahle’s.

Materials

SiphonZebrafishEmbryoSolutionFinefrynetormeshfilterethanollarge-boreglasspasteurpipets60mmglassPetridishes(8)dissectingmicroscopezebrafishembryosatdome/30%epibolystage(40)Incubator,28°C

Procedure

A.IsolationofZebrafishEmbryos

1.Thenightbeforeembryocollection,vacuumthetankwellwithasiphontoremovedebrisseveralhoursafterthelastfeeding.2.Depositalayerofwashedmarblestocovertheentirebottomsurfaceofthetank.3.Donotdisturbforthefirsthourafterdawntoallowfishtomate.4.Collectembryosandfertilizedeggsfrombetweenthemarbleswithasiphonthenandcollectwithafinefrynetormeshfilter.5.Transferthenettoaglassdishfulloftankwaterandexaminetheembryoswithadissectingmicroscopeatlowmagnification.6.TransferhealthyembryostoacleanglassPetridishcontainingembryomediawithlarge-boreglassPasteurPipettes.Discardanythatarecloudyorruptured.7.Whennotbeingusedfortheexperiment,theembryosshouldbekeptat28°C(Zebrafishembryos’rapiddevelopmentmakesconstanttemperaturecrucialforobtainingareproduciblegroupofembryosforexperimentation).*protocoladoptedfromDBlabWebsite:IsolationofZebrafishEmbryos

B.EthanolTreatment

1.Select40healthyzebrafishembryosthatareat30%epibolystage(Figure1below).2.Preparefourseparatepetridishesthatcontaineachofthefollowingfour:normalzebrafishembryomedium,1%,2%,and3%ethanolzebrafishembryomediumsolutions.3.Takeembryosoutoftheincubator(setat28°C)andplace10zebrafishembryosintoeachpetridish.Placeallfourdishesintheincubator.Thenwaitfor3hoursfortheethanoltotakeeffect.4.Afterthe3hours,removethedishesfromtheincubatorandmovetheembryosintonewpetridishescontainingnormalzebrafishembryosolution.Differentbatchesofembryosmustgointodifferentdishes.Placethedishesintheincubatorandwaitfor24hours.5.Returnafter24hours.Observetheembryosforabnormalitiesandphotographthem.6.Return24hourslatertoobservetheembryosforabnormalities.Photographthem.*protocoladaptedfrom:TheEffectsofEthanolonZebrafishDevelopment:JeffMindel,F&M.

Figure1.Zebrafishembryoat30%epibolystage(43/2h).Topistheanimalpole.

Results

24hoursaftertheexposureofethanol,cyclopiawasobservedamongtheembryosculturedat3%ethanol(Figure1,Table1).Nocyclopiawasobservedinembryosat1%or2%ethanolsolutions.Forbothconcentrations,eyesdevelopednormally;thevesiclesweredarklypigmentedandtheeyeswerenormallyspaced(Figures1B,1C).At3%,1cyclopicembryowasobserved.Therestofthesurvivingembryosshowednosignsofcyclopia.Thecyclopicembryo’seyeswerefusedandweredeficientinpigmentation(Figure1D).

48hoursaftertheethanolexposure,nocyclopiawasobservedinembryosgrownat1%and2%ethanolsolution(Figure2,Table1).Forbothsolutions,eyedevelopmenttookplacenormally(Figures2B,2C).At3%,thecyclopsobservedatthe24hourstageadvancedwithitsabnormaleyedevelopmentasevidencedindarkopticalpigmentationnotseenpreviously(Figure2D).

Inadditiontocyclopia,embryosexposedto3%ethanolshowedvaryingdegreesofimpaireddevelopmentofposteriorstructures,morespecificallyofthetrunkandtail,24hoursaftertheirexposuretoethanol(Figure3).Again,nodeformitieswereobservedintheposteriorregionsofthoseat1%and2%ethanolsolutions(Figures3B,3C).2embryos,1ofwhomwastheaforementionedcyclops,culturedin3%ethanolsolutiondisplayedmalformationofthetrunkandtail(Figures3D,3E).Thenon-cyclopicembryo(eyeswerenotfusedbuthadshorteneddistancebetweenthem)withposteriordeformitieshadenlargedallantoiswithashortenedtail(Figure3D).Moreover,muchofthepigmentationsnormallyobservedalongthedorsalsideofuntreatedembryoswereabsent(Figure3D).Thecyclopicembryodisplayedsimilarsymptomswithincreasedseverity(Figure3E).

Atthe48hourstage,therewerenoposteriordeformitiesobservedamongembryosculturedat1%and2%ethanolsolution(Figure4,Table1).Allsurvivingembryosgrownat1%and2%solutionthathatcheddisplayednormalswimmingmovements.At3%,thecyclopicembryoswithdeformedposteriorstructurescontinuedtodevelopwiththeaforementioneddeformitieswhich,atthe48hourstage,ultimatelyrenderedtheembryosimmobile.Thenon-cyclopicembryowithdefectivetrunkandtaildied.Thecyclopicembryofailedtohatch,andwhenartificiallypulledoutoftheshell,didnotshownormalswimmingmotions.Infact,theembryowascompletelyimmobileduetotheseverityofitstrunkandtaildeformites.

AnothernotableobservationmadewastheviABIlityofthezebrafishembryos(Table2).24hoursaftertheethanolexposure,itwasobservedthatincreasingofethanolconcentrationcorrelatedwithincreasingdeath.Therewasnodeathamongembryoscultureincontroland1%ethanolsolution.However,therewere2deathsin2%solutionand5in3%solution.Whenchecked48hoursaftertheethanolexposure,viabilityoftheembryosineachconcentrationwasasfollowing:90%incontrol,80%in1%,50%in2%,and30%in3%(Table2).

Clickonthepicturetoviewtheenlargedversion

Figure1.Ethanoltreatmentcausescyclopiaat3%.Zebrafishembryostreatedatthedome/30%epibolystagewith1%,2%,and3%ethanol-ZebrafishEmbryoMedium(ZEM)solutionfor3handculturedinZEMfor24hintheabsenceofethanol.Nocyclopiawasobservedin1%and2%.1cyclopicembryowasobservedoutof10at3%.(A)Untreatedcontrolembryo.(B)1%ethanoltreatedembryo.(C)2%ethanoltreatedembryo.(D)3%ethanolwithcyclopia.Eyevesiclesarefusedalongthemidlineofthehead.Arrowsindicateeyes.Clickonthepictureforenlargedversion.

Clickonthepicturetoviewtheenlargedversion

Figure.2.Ethanoltreatmentcausescyclopiaat3%.Embryostreatedatthedome/30%epibolystagewithincreasingconcentrationofethanolfor3handculturedfor48hinZebrafishEmbryoMediumintheabsenceofethanol.Cyclopiaobservedat3%only.(A)Untreatedcontrolembryo.(B)1%ethanoltreatedembryo.(C)2%ethanoltreatedembryo.(D)3%ethanolwithcyclopia.Eyevesiclesarefusedalongthemidlineofthehead.Embryosareshowninfrontalviews,dorsalup.Arrowsindicateeyes.Clickonthepictureforenlargedversion.

Clickonthepicturetoviewtheenlargedversion

FIG.3.Ethanoltreatmentimpairstrunkandtaildevelopmentat3%.Embryosweretreatedfromthedome/30%epibolystagewithincreasingconcentrationofethanolfor3handthenculturedfor24hoursintheZebrafishEmbryoMediumintheabsenceofethanol.(A)Untreatedcontrolembryo.(B)1%ethanoltreatedembryo.(C)2%ethanoltreatedembryo.(D)Non-cyclopicembryogeneratedby3%ethanoltreatmentshowingmilddeformitiesofthetrunkandtail.(E)Cyclopicembryogeneratedby3%ethanoltreatmentshowingseveretrunkandtaildeformities.Arrowsindicatedefectivetrunkandtail.

Clickonthepicturetoviewtheenlargedversion

FIG.4.Ethanoltreatmentimpairstrunkandtaildevelopmentat3%.Embryosweretreatedfromthedome/30%epibolystagewithincreasingconcentrationofethanolfor3handthenculturedfor48hoursintheZebrafishEmbryoMediumintheabsenceofethanol.(A)Untreatedcontrolembryo.(B)1%ethanoltreatedembryo.(C)2%ethanoltreatedembryo.(D)Cyclopicembryogeneratedby3%ethanoltreatmentshowingseveretrunkandtaildeformities.Arrowsindicatetrunkandtaildeformities.

Table1.Developmentalabnormalitiesinducedbyethanolexposure.Embryosweretreatedfromthedome/30%epibolystagewith1%,2%,and3%ethanol-ZebrafishEmbryoMedium(ZEM)solutionfor3handculturedintheZEMforatotalof48hintheabsenceofethanol.10embryosweresubjectedtoeachconcentration.Cultureswereobservedtwice:24hafterthetreatmentand48hafterthetreatment.Deadembryoswithdeformitieswerenotcounted.

*Oneofthe2alsohadcyclopia,thesameembryodenotedby1inthe24cyclopiacolumn.

Table2.Viabilityofzebrafishembryosexposedtodifferentconcentrationofethanolconcentrationsattwo24hintervals.Embryosweretreatedfromthedome/30%epibolystagewithwith1%,2%,and3%ethanol-ZebrafishEmbryoMedium(ZEM)solutionfor3handculturedintheZEMforatotalof48hintheabsenceofethanol.10embryoswereexposedtoeachconcentration.Cultureswerecheckedtwice:24hafterthetreatmentand48hafterthetreatment.

Discussion

Physicaldeformitieswereobservedonlyamongthezebrafishembryosculturedin3%ethanol-ZEMsolutionandnotin1%and2%,suggestingthatthecriticalethanolconcentrationmaybeanywherefrom2%to3%,including3%(Table1).ThatrangeisoverlapssignificantlywithBladerandStrahle’scriticalethanolconcentrationof2.4%(1998).However,thefrequencyofthedeformitiesobtainedintheexperimentwasdifferentfromBladerandStrahle’s74.1%(1998).48hoursaftertheethanoltreatment,at3%ethanolsolution,approximately20%displayedanyphysicaldeformities.Suchdiscrepancymaybeduetomistakesmadewhenstagingtheembryosfortheexperiment.AccordingtoBladerandStrahle,onlyethanoltreatmentduringanarrowtimewindowcomprisinglateblastulaandearlygastrulastages(dome/30%epiboly)causescyclopia(1998).Moreover,deformitiesofthetrunkandthetailalsorepresentdome/30%epibolystagetreatmenteffects.Thus,itcanbesuggestedthattheembryosusedintheexperimentweregenerallypasttheir30%epibolystages.However,thelowpercentageofdeformedembryosisstatisticallyinsignificantduetothesmallsamplesizeusedintheexperiment;only10embryoswereusedforeachethanolconcentrationasopposedtoBladerandStrahle’s700(1998).

Thoughinlownumbers,cyclopiawasobservedinembryostreatedwith3%ethanol,supportingthefactthatethanoldoesinducecyclopiainzebrafishembryos(BladerandSträhle,1998).Cyclopiaisinducedinamphibiansandchickembryoswhentheprechordalplateisremoved,suggestingthattheprechordalmesodermisrequiredtoseparatethemonolithiceyefieldintotwolateraldomains(BladerandSträhle,1998).Therefore,itcanbesuggestedthattheembryowithseparate,butmorecloselyspacedeyeshaditsprechordalplatemildlyaffectedbyethanol.Theroleoftheprechordalplateisfurtherevidencedinthecylopicembryo;itwasclearlyobservedthatwhatlookedlikeasinglemedialeyewasactuallytwoeyevesiclesfusedatthecenteroftheheadwhenviewedunderthemicroscope(Figures1D,2D).However,itisnotclearastowhyembryostreatedwithethanoldisplayedsignificantlackofpigmentationwhenobserved24hoursaftertheethanoltreatmentbutnotafter48hours.Onepossibleexplanationcouldbethatethanolsloweddownthezebrafish’sdevelopment.Inthepresenceofethanol,eyedevelopmentmayproceedabnormallyslowly,resultinginretardingoftheproliferationofthepigmentcellsoftheeyes.Anotherpossibleexplanationisthatethanolmighthavecausedapoptosisoftheeyepigmentcellsat24handneighboringcellscompensatedforthelossbydifferentiatingintoeyepigmentcellsby48h.

Ethanoldidcausedefectivedevelopmentintheposteriorstructuresinzebrafishembryos.Althoughthemechanismsbehindtheobserveddeformitiesofthetrunkandthetailarenotknown,itcanbeconjecturedthattheimpairmentofepibolymayberesponsible.BladerandStrahlenotedthatepibolywasmarginallyimpairedbythetreatment,withtheadvanceoftheblastodermmarginlaggingbehinduntreatedcontrolsby10to15%towardtheendofgastrulation(1998).Epibolyestablishestheposteriorareaoftheembryoincludingthetrunkregionandthetailbudand,therefore,itseemsreasonablethatimpairingthelast10-15%ofepibolymaycausedefectivetrunkandtail(Gilbert,2003).Inadditiontoadefectivetrunkandtail,itwasalsoobservedthattherelackofpigmentationinthetruckregionsinembryosexposedto3%ethanol(Figures2D,2E,and4D).Onepossibleexplanationforthepigmentationdeficiencyisthatethanolmighthavecauseddeformitiesinthespinalcordfromwhichneuralcrestcellsdestinedtobecomemelanocytesarise(Gilbert,2003).However,itisnotconclusiveastowhetherlackofpigmentationcanbeattributedtoethanolexposure.Largelyduetosmallsamplesize,furtherexperimentationisneededtoconfirmethanol’sroleinpigmentationdefect.

Anothernoticeableeffectofethanolwasthemortalityrate(Table2).ThoughBladerandStrahlespecificallynotedthatethanoldoesnotseemtocausedeathinembryos,theexperimentsuggeststhatincreasingethanolconcentrationincreasedthenumberofdeaths(1998).Sixoutof10embryosincubatedinthe3%ethanolsolutiondiedasopposedto1outof10ofthecontrolsdyingseemsignificant.Moreover,itisdifficulttosimplyattributethelinearrelationshipdisplayedinthedatatochance.Itwasconjecturedthatabnormalapoptosis,anaccompanyingsymptomcausedbyethanolexposure,mayhavecausedthedeathsofembryos.ItisthoughtthattheenzymesthatcellsusetodegradeandclearethanoloutofthesystemproducefreerADIcalswhichchemicallyreactwithimportantcomponentsofthecell,suchasproteins,DNA,andlipids(Suliketal.,1988).Therefore,itdoesnotseemfarfetchedtosuggestthatethanolinducedapoptosismayultimatelycausedeathbydamagingtheessentialcellularstructuresoftheembryos.

Thisexperimentdemonstratedthatethanolinducesphysicaldeformities,morespecificallycyclopiaandtrunkandtaildeformities,inzebrafishembryos.Itisknownthat,inhumans,severefetalalcoholsyndromecausesmildformsofholoprosencephalies,agroupofdisordersthatincludecyclopia(Suliketal.,1988).Therefore,thefindingsofthisexperimentseemtobearatleastsomerelevancetoexperimentsinvestigatingfetalalcoholsyndromeinhumans.Thisexperiment,however,didlittletoinvestigatethepresenceofethanolinducedabnormalapoptosisanditsroleonphysicaldefectsanddeaths.Inanotherexperiment,embryosmaybeexposedtovaryingconcentrationsofethanol,stainedforapoptosisusingvitalstainssuchastheAcridineOrange,andobservewhethertherearecorrelationbetweendeformitiesandcelldeath.

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