The Effects of Ethanol on Zebrafish Development
来自 : 蚂蚁淘
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:ZebrafishembryoinepibolystageResults&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.
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