Introduction:

Theearlystagesofzebrafish,Danioreriodevelopmentarecharacterizedbymeroblasticdicoidalcleavage.Initiallycelldivisiononlyoccursintheblastodisc,whichisathinregionontheanimalcapoftheegg.Thisblasotdisccontainsalloftheegg"scytoplasmandorganelles,therestoftheeggisfilledwithyolk.Afterabout10celldivisionsthemidblastulatransitionbeginsandthecellsdifferentiateintothreegroups.Theyolksyncytiallayer(YSL)ismadeupofthecellsonthevegitalendoftheblastulawhichfusewiththeyolk.Thesecondpopulationofcellsistheenvelopinglayercells(EVL)whicharepositionedontheouteredgesoftheembryo.Thedeeplayercellsmakeupthethirdpopultaionandarethecellsthatactuallygiverisetotheembryoitself.DuringgastrulationtheYSLcellsmigratedowntheedgesoftheembryotowardsthevegitalpoleinaprocesscalledebibolythatenvelopstheyolk.Attheedgeofthismigrationthedeepcellsinvolute/ingressunderthemselvesandformthemesodermallayer.Ofthesenewmesodermcells,someoftheonesthatmigrateanteriorlybecometheprechordalplatemesoderm.Whenzebrafisharetreatedwithethanolduringearlygastrulastagesaconditionknownascyclopiacanbeinduced(BladerandSträhle,1998).Thisisaconditioninwhichthereisonenarroweyeslitandusuallytherearenoeyesatall.Itisbelievedthatexposuretoethanolcausestheprechordalplatemesodermtomigrateabnormally(BladerandSträhle,1998).Thisabnormalmigrationcausestheprechordalplatemesodermcellstoendupinanincorrectlocation,anditisthisthatisbelievedtocausecyclopia.Theprechordalcellsexpressthegenesgoosecoidandislet-1whichcodeforproteinsthathelpcontrolcelldifferentiationintheanteriorregionoftheembryo,andtheirreleaseintheincorrectlocationisthecauseofdeformations(BladerandSträhle,1998).Lackofsonichedgehogexpressionisalsoknowntocausecyclopiainmiceandchicks,andethanolmayacttodisruptitsexpressiontoo(Gilbert,2000).

Procedure:

1.Selectembryosthatareatthedome/30%epibolystagestage(Figure1).

2.Preparethreeseparatepetridishesthatcontainnormalzebrafishembryomedium,a2.5%ethanolzebrafishembryomediumsolution,anda1%ethanolembryosolution.

3.Placeatleast10oftheselectedembryosintoeachofthepetridishesandletthemsitfor3hours.

4.After3hourshaspassed,placeall3setsofembryosintopetridisheswithnormalzebrafishembryosolution.

5.24hourslaterobserveembryosforabnormalitiesandphotographthem.Figure1:Zebrafishembryoinepibolystage

Results&Conclusions:

Thezebrafishinthecontrolgroupdevelopednormallyaswasexpected.Atthestagethattheywereobserved,theyshowednormaleyedevelopment.(figure2).Theembryosinthe1%ethanolsolutionalsoshowednormaldevelopmentintheregionofthehead(figure3).Apparentythisconcentrationofethanolwasnotsufficienttocausecyclopia.The2.5%ethanolexperimentalgroupshowedalimitednumberofdeformedembryos.Outofthetenoriginalembryostwofailedtosurvive,thoughthiswasconsistentwiththesurvivalrateofthecontrolgroup.Outoftheeightthatsurvived,sixdevelopednormallyandtwowereslightlydeformed.Itappearsthatundertheseexperimentalconditionsahigherconcentrationofethanolwasneededtoproduceahigherfrequencyofteratogenicresults.ItwasdifficulttodeterminewhetherthetwodeformedemrbyoshadfullblowncyclopiabuttheydidnothavevisIBLeeyes(figure4).Figure2:Anembryofromthecontrolgroup,eyepigmentcanbeclearlyseen.Figure3:Embryofromthe1%ethanolgroupshowingnormaleyedevelopment.Figure4:Embryofrom2.5%ethanolgroupshowingunderdevelopedorabsenteyes.