Ultraviolet irradiation impairs epiboly via microtubules in Zebrafish embryos相关交流-蚂蚁淘在线

IntroductionZebrafishhavetransparentembryosthatdevelopoutsidethemother.Theydeveloprapidly,sothatat24hoursafterfertilization,theembryohasformedmostofitstissueandorganprimordiaanddisplaysthecharacteristictadpole-likeform.Also,thecelldivisionoccuronlyintheanimalpoleblastodisc.Thesedivisionsarerapid,withaperiodicityofabout15minuteseach.Thefirst12divisionsoccursynchronously,formingamoundofcellsthatsitsattheanimalpoleofalargeyolkcell.Threekindofmovements(epiboly,involutionandconvergence)canbedistinguishedintheearlydevelopmentofzebrafishembryo.Involutionandconvergenceresultofmigrationofdeepcellsmovingpresumablyusingfibroblasts,butthemolecularnatureofthemechanismdrivingtheepibolyisnotknownyet.PreviousexperimentsshowedthatirrADIationofzebrafishzygoteswithultravioletlightselectivelyimpairsepibolyresultinginembryoswithopenblastoporesbutwell-formedanterioraxes.Theultravioletlighteffectisnotrestrictedtothezygotestageasirradiationoflaterembryonicstagesalsoimpairsepiboly.Theultraviolet-sensitivetargetmaythusbematernallyencodedcomponentsofthemachinerydrivingepiboly.

Reference:U.StrahleandS.Jesuthasan,Development119,909-919(1993)

ObjectiveTheobjectiveofthisexperimentwastoexposeseveralembryosatdifferentlevelofUVirradiationandobservethepotentialmalformations.

Selectionofembryos1)Embryoswerefertilizedonthemorning.

2)Afterfertilization,weselectedseveralnewlyfertilizedzygoteswhichconsistofalargeregionrichwithyolkvesiclesandasmalleryolk-freeblastodisc.Weusedtheembryosjustbeforethe2-cellstage(firstcleavage).

ProcedureforUVirradiation

1)Weput15embryos,randomlyorientedintheirchorionin3differentpetridishes.ToproducetheUVirradiationweuseda254nmCL-1000UVcrosslinker.TochangetheUVdoseswecanchangetheexposuretimes.Nevertheless,inthisexperimentweincreasedtheirradiationwithoutchangethetimeofexposure.

2.)ExposureI(1.8mJ/cm2)Weplaced15embryosinapetridishandthenweputthepetridishesintheCL-1000UVcrosslinkerasexplainedpreviously.

3)ExposureII(3.6mJ/cm2)Samethingbutthelevelofirradiationwasdoubled.

4)Wealsoput15embryosinapetridishanddidnotirradiatedthem.Theyservedasreferenceduringtheobservation(thenextday).

5)Weplacedthe3petridishestoincubateat28ºCinembryomedia.

ResultsIntheearlydevelopment,therapidcleavagedivisionsarenotaffectedbytheUV-light.Anomaliesstartwhenepibolystarts.Treatedembryosprogressslowerthanuntreatedembryo.So,thefundamentalcomponentsdrivingtheepibolyshouldstarttoworkinthelatezygote.Theexperimentsgaverisetothreelevelofimpair:

TypeA-->Slightlyaffected.Theblastodermhascoveredtheyolkcellupto80%andshowsawell-formedanterioraxisrudimentonthedorsalside.

TypeC-->Themoreaffected.TypeB-->Affected.RetardationofepibolybutdonotformTheblastodermroundedupformingavesicleonanaxisrudiment.topoftheyolkmass.

Allthecontrolgaverisetonormalembryowithnormalepibolymovement.Asshownonthetable,theimportanceofthe"mutation"areproportionalwiththelevelofUVexposure.

Theirradiatedembryosaredisorganizedandlessmicrotubuleswerefoundinthecytoplasmiclayeroftheyolksphere(Strahle).Thus,sinceepibolyistheonlyonemovementintheembryowhichisaffected,itshouldbedrivenbymotorsthatusemicrotubules.

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