IntroductionInearlystagesofdevelopmentinamphibianembryos,veryfewcells"fatesarerigidlydetermined.However,previousexperimentsshowthatcellsinoneareaofthedevelopingembryoaredeterminedbeforegastrulation.Thisareaintheamphibianembryohassincebeentermedtheorganizerforitsroleincellfatedetermination.In1924HansSpemannandHildeMangoldshowedthatcellstransplantedfromthis"organizer"regionoftheblastoporelipofoneembryowillinducegastrulationinanykindoftissue.Theyusedthisinformationtoinducethegrowthofasecondneuraltubeonanalreadydevelopingembryo.ThisexperimentattemptstorepeatSpemannandMangold"sexperimentinXenopusembryos.

Detailedprotocol

1.Theoperationshouldbeperformedunderasepticconditions.AllphasesoftheoperationshouldbeperformedinSteinberg"ssolutiontopromotehealing.Afterhealing,however,embryosshouldbetransferredtospringwateror10%amphibianringerssolution.

2.Dipthetipoftheeyebrowknifein90%alcoholandthenpassedthroughasmallbeakercontainingtheoperatingmedium.Makesureallinstrumentsaresterile.3.TransfertheXenopusembryototheagardishcontainingoperatingmedium.Removejellycoatsandvitellinemembrane,handlethedenudedembryoCAREFULLY.

4.Withaballtippedglassneedleorwatchmakersforcepsmakeashallowdepressionintheagarinwhichtheembryocansitcomfortably,dorsalsideup.Usinganeyebrowknife,carefullyremovepartofthedorsallip.Withasinglemotion,bottomtotop,makeathintearintheepidermis.Followthefirstlongitudinalcutwithasecondlongitudinalcut.Thesubsequenttwocutsaretransversecuts,tocompletearectangularareaofdorsalliptissue.

5.Lifetheentirerectangularareaoftissueawayfromtheembryowiththetipoftheglassneedle.

6.Prepareasmallpocketintheventralsurfaceofthehostembryo.Donotattempttocutarectangularsection,allthatisneededisamereslit,orslighthole.Thepocketcanthenbeenlargedtoreceivethetransplantbycarefullyremovingsomeofthemesodermcells.

7.Transfertheexcisedpieceoftissuetotheslitinthehost.Gently,yetfirmly,pushthewholemassofcellsthroughthenarrowincision,itmaybenecessarytoenlargetheslittoaccommodatethetransplant,butatight-fittingtransplantisdesirable.

8.Oncethetransplanthasbeenpushedintotheslit,quicklycoveritwiththeflatsurfaceofathincoverslip.Theweightofthecoverslipshouldholdthetissueinplace.

9.Afterandhour,thepieceofcoverslipshouldbecarefullyremoved.Ifthetransplanthasnothealed,placeitagainunderpressure,withapieceofcoverslip,foranadditionalhalfhour.Afterhealingiscomplete,transfertheembryotospringwaterorringerssolution.

10.Observetoseeifthetransplantedtissueformsasecondneuralfold.

Results/DiscussionTheXenopusembryoswereveryfragileandeasilyinjuredduringthetransplantsurgery.Theywerealsoreluctanttohealwell.However,therewereafewsuccessfultransplants,withminimalinjury(figure1A).SeveraloftheembryosdevelopedvisIBLesecondaryaxis".Theoriginalaxisisdiscerniblebywhatwillformtheeyeandmouthandthesecondaryaxisischaracterizedbyalesswelldevelopedbud(figure1B,C).NoneoftheembryossurvivedtoformadultXenopus.