Procedure

1)WashalbinoAxolotlamphibiangastrulaeinadishfilledwith100%HEPES-bufferedModifiedSteinberg"sSolution(HBSt).UsingalbinoAxolotl"swillmakeiteasiertotrackthedye.

2)Placeembryosin10%HBStandremovejellycoatusingusingfineforceps.

3)Transportembryosand10%HBSttoblackagarosedishforcontrast.

4)Applyanilebluesulfatedyechiptooneend(animalpoleorvegetalpole)ofthegastrulaeandaneutralreddyechiptotheotherendofthegastrulae(animalpoleorvegetalpole)(figure1).Replace10%Steinberg"ssolutionwithfresh10%HBSt.

5)Observegastrulaeinmicroscopeandtakeapictureondayoneofexperiment.Takeapictureeachdayforthenextfewdaystoobservethemovementofthedyedcellsofthegastrulae.(Note:Todelaygrowth,placegastrulaeat14oC).

ResultsandConclusions

Dyewasappliedtooppositeendsof6Axolotlembryos(figure2).Overathreedayperiod,theelongationofthestainedmaterialwasnoted.Theembryojustbelowthegreenstar(figure2)wasdyedatoppositeends(leftandright)oftheblastopore.Adye-markedembryosimilartothestar-markedembryo(figure2)wouldbeexpectedtogiverisetotheleftandrightsideoftheneuralectoderm(figure3).Lookingdownontheday2dye-markedembryo,theblueandreddyeappearaslongitudinalstripesonoppositesides.Thebluedyecanbeseenontherightsideoftheneuralectodermandreddyeontheleftsideoftheneuralectoderm(figure3a).Theday3neurula,whichislyingonitsleftside(whichwouldbeexpectedtocontainreddyeontheneuralectoderm),showsthebluedyeontherightsideoftheneuralectoderm(figure3b).

Itwasnotedthatwithtime,itbecamehardertosustainthemovementofthedye-markedpopulationsofcells,wherethedyebecamelighterandmuchmoredifficulttotrace.Also,someembryosbecamesickanddied.Inthefuture,itwouldbeagoodideatodyemoreembryosandpossIBLyeventakephotosmorefrequently(i.e.,twiceaday).

Figure2.DyemarkedAxolotlembryosondayone.Theembryojustbelowthestarwasdyedontheleftandrightsideoftheblastoporelip.Thatpopulationofcellswouldbeexpectedtofollowthepathwaythroughtheleftandrightneuralectodermshownbelow(figure3).

Figure3.(A)dye-markedembryoonday2showingabluelongitudealstipeontherightsideoftheembryoandaredlongitudalstripeontherightsideoftheembryo.Thedyedregionisontheleftandtherightsideoftheneuralectoderm.(B)day3dyemarkedneurulalyingonitsleftside.Thepopulationofcellscontainingthebluedyecanbeseenalongtherightneuralectodermoftheneurula.

Acknowledgements

IwouldliketothanktheFranklinandMarshallBIOLOGydepartmentforallowingmetoconducttheexperimentintheirlaboratoryfacilitiesandfortheavailABIlityoftheirlaboratorysupplies.AnotherspecialthankyoutomylabpartnersMatthewsBandaandJohnDeLongfortheirassistanceindyingtheembryos.IwouldespeciallyliketothankprofessorCebra-Thomas,mydevelopmentalbiologyprofessor,forherassistnaceinconductingtheexperimentandinterpretingtheresultsoftheexperiment.