HighMagnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology

EstherK.Kieserman¹,ChanjaeLee,RyanS.Gray²,TaeJooPark¹,andJohnB.Wallingford³

HowardHughesMedicalInstituteandSectionofMolecularCellandDevelopmentalBIOLOGy,UniversityofTexas,Austin,TX78712,USA

¹Presentaddress:DepartmentofMolecularandCellBiology,UniversityofCalifornia,Berkeley,CA94720,USA.

²Presentaddress:CenterforCellDynamics,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD21205,USA.

³Correspondingauthor(wallingford@mail.utexas.edu).

INTRODUCTION

EmbryosofthefrogXenopuslaevisareanidealmodelsystemforinvivoimagingofdynamicbiologicalprocesses,fromtheinnerworkingsofindividualcellstothereshapingoftissuesduringembryogenesis.Theirexternallydevelopingembryosaremoreamenabletoinvivoanalysisthaninternallydevelopingmammalianembryos,andthelargesizeoftheembryosmakethemparticularlysuitablefortime-lapseanalysisoftissue-levelmorphogeneticevents.Inaddition,individualcellsinXenopusembryosarelargerthanthoseinothervertebratemodels,makingthemidealforimagingcellbehaviorandsubcellularprocesses(e.g.,followingthedynamicsoffluorescentfusionproteinsinlivingorfixedcellsandtissues).Xenopusembryosareamenabletosimplemanipulationsofgenefunction,includingknockdownandmisexpression,andthelargenumberofembryosavailableallowsevenaninexperiencedresearchertoperformhundredsofsuchmanipulationsperday.Transgenesisisquiteeffectiveaswell.Finally,becausethefatemapofXenopusembryosisstereotypical,simpletargetedmicroinjectionscanreliablydeliverreagentsintospecifictissuesandcelltypesforgenemanipulationorforimaging.Althoughyolkopacitycanhinderdeepimaginginintactembryos,almostanycellintheearlyembryocanbeplacedintoorganotypicculture,suchthatthecellsofinterestaredirectlyapposedtothecoverglass.FurThermore,liveimagingtechniquescanbecomplementedwithimmunostainingandinsituhybridizationapproachesinfixedtissues.Thisprotocoldescribesmethodsforlabelingandhigh-magnificationtime-lapseimagingofcellbiologicalanddevelopmentalprocessesinXenopusembryosbyconfocalmicroscopy.

RELATEDINFORMATION

ProtocolsforLow-MagnificationLiveImagingofXenopusEmbryosforCellandDevelopmentalBiology(Wallingford2010a)andPreparationofFixedXenopusEmbryosforConfocalImaging(Wallingford2010b)arealsoavailable,asaredetailsonperformingknockdownormisexpressionstudiesinXenopusembryos(Guille1999;Siveetal.2000).InformationisalsoavailableonEmbryoDissectionandMicromanipulationTools(Siveetal.2007).

ExamplesofconfocalimagingofliveXenopusembryoscanbeseeninFigures1and2.Themethodsdescribedherehavealsobeenusedtomonitortissue-levelmorphogeneticevents,suchasgastrulationandneuraltubeclosure(WallingfordandHarland2002;Haigoetal.2003;Ewaldetal.2004).Imagingcanalsobeperformedsimultaneouslywithmeasurementoftheforcesgeneratedbymovingtissuesduringdevelopment(Zhouetal.2009).

Viewlargerversion(89K):[inthiswindow][inanewwindow]

Figure1.ImagingofmorphogenesisinliveXenopusembryos.(A)Stillframesfromatime-lapsemovieofneuraltubeclosureinXenopustakenwithastereomicroscope.MountingforthisapplicationisdescribedinLow-MagnificationLiveImagingofXenopusEmbryosforCellandDevelopmentalBiology(Wallingford2010a).(B)TheimagesshowninthiscolumncorrespondtostagessimilartothoseshowninA,butathighermagnificationtoshowcellsoutlinedwithmembraneGFP(memGFP)intheregionindicatedbytheyellowboxinA;seealsoLeeetal.(2007).(C)Single3Dprojectionofmucus-secretingcellsontheepidermisofaXenopusembryo.GolgistructurescanbelocalizedwithGalT-RFP(Nicholsetal.2001)andapicalexocyticvesiclesarehighlightedusingmemGFP(Hayesetal.2007).(D)StillframesofamovieofadividingcellintheneuralepitheliumofXenopus,showingthemicrotubules(-GFP;KwanandKirschner2005)andthecellmembrane(memRFP). Viewlargerversion(26K):[inthiswindow][inanewwindow] Figure2.Invivotime-lapseimagingofXenopusembryoscanbeperformedacrossawidevarietyofsizeandtimescales.(Top)Amovietakenat~370frames/secandspanningonly~35msecshowsthebeatingof20-mm-longmotileciliaonasinglemulticiliatedcellintheXenopusepidermis(Parketal.2008).(Bottom)Confocalstackscollectedevery5minandspanning~12hshowthedispersalofindividualfluorescentmyeloidcellsthroughoutaXenopusembryofromtheirorigininsurgicallytransplantedventralbloodislands.Eachindividualcellis~30µmacrossandtheentireembryoshownhereis~1mmlong.MountingforbothimagingapplicationswasasdescribedinSteps2.iv-2.vi(Fig.7B). MATERIALS Reagents Agarose(2%,preparedin1/3XMMR)(forimagingembryosthroughtheneurulastage) Agarose,low-melt(0.8%)(forimagingtailbud/tadpole-stageembryos) Marc’smodifiedRinger’s(MMR)(1X) Plasmidsencodinggreenfluorescentprotein(GFP)orredfluorescentprotein(RFP)fusionproteins ThereareavarietyoffluorescentproteinssuitableforimaginginXenopusembryos.EnhancedGFP(eGFP)andmonomericRFP(mRFP),inparticular,offerexcellentperformancewhenbalancingbrightnessversusphotobleaching.Generally,makingfusionstoXenopusproteinsispreferable,becausethesearemorereliable.However,fluorescentfusionstomammalianproteinsexpressedinXenopuscanalsobeused.ForexpressioninXenopus,vectorsoftheCS2family(CS2+,CS107,etc.)arerecommended.BecausemanyGFPfusionproteinsaregeneratedusingtheClontecheGFPvectors,wehavecreatedausefulCSfamilyvector(CS10R)designedforeasyshuttlingfromtheClontechvectors.Thisvectorisavailableuponrequest.ManyofourplasmidsaredepositedwiththeEuropeanXenopusResourceCentre(http://port.ac.uk/research/exrc/). Tricaine(0.15%)(optional;seeStep5) Xenopusembryosofthestageofinterest Equipment Comb Combscanbepreparedbycarefulmeltingofaplastichaircomb. Computer,equippedwithimageprocessingsoftware TheprotocoldescribedhereusesApplecomputersequippedwithAdobePhotoshopandQuickTimePro,althoughnumeroussoftwarepackagesareavailable,withImageJbeingperhapsthemostcommonlyused. Coverglass Careshouldbetakenwhenselectingcoverslipsforhigh-resolutionimagingexperiments.Differentmicroscopemanufacturerscalibratetheirobjectivelensesforslightlydifferentthicknessesofglass.Additionally,differentcoverslipmanufacturersuseslightlydifferentglasscompositionsandmaketheircoverslipsinavarietyofthicknesses.Consultyourmicroscopeservicerepresentativefortheidealcoverslipthicknessandglasscomposition.Somehigh-resolutionlensesalsoprovidecorrectioncollarstoenablefinescalematchingtothecoverslipthickness. Embryodissectionequipment(e.g.,forceps,hairloops,hairknives[Keller1991],razorblades) Foradditionalinformation,seeEmbryoDissectionandMicromanipulationTools(Siveetal.2007). EquipmentforinjectingXenopusembryos Microscope,inverted,equippedwithvideo-recordingcapABIlities(e.g.,ZeissLSM5PASCALandLSM5LIVEconfocalmicroscopes),equippedwithPlan-NeofluarandPlan-Apochromatobjectives(10X,numericalaperture[NA]=0.3;20X,NA=0.5;40Xoil,NA=1.3;63Xoil,NA=1.4). Dependingontheapplication,awidevarietyofconfigurationsmightbenecessary.Ingeneral,itisessentialtoinvestasignificantamountoftimeintrialanderrortooptimizeeachimagingapplication. Xenopusimagingchambers Commercialchambersareavailable,butreusableimagingchambersareeasytomanufactureandeasytouse.Forimaginginaqueoussolutions(e.g.,alltheliveimagingapplicationsdescribedhere),asimplePetridishviewingchamberwithacoverglassbottomisusedforimagingoninvertedmicroscopes.Theassemblyconsistsofathreadedplasticinsertandacounterthreadedmetalbase.Theselockacircularcoverglassintoplacewithano-ring.PhotographsofthecomponentsareshowninFigure3andaschematicofthechamberisshowninFigure4.Manymachineshopscanfabricatethesedishesbasedoncomputer-aideddesign(CAD)drawings(madeusingSolidWorks),availablefromtheauthoruponrequest. Viewlargerversion(115K):[inthiswindow][inanewwindow] Figure3.Componentsofthecustom-madechamberforimaginginaqueoussolutionswithaninvertedmicroscope.SeeFigure4forassembly. Viewlargerversion(29K):[inthiswindow][inanewwindow] Figure4.Schematicofacustom-madechamberforimagingembryosinaqueoussolutions.CADdrawingsofthesecomponentsareavailablefromtheauthoruponrequest. METHOD 1.Prepareembryoswiththedesiredlabel: Toexaminecellularmorphology Expressionofcell-membrane-targetedfluorescentproteins(e.g.,memGFP)providesanexcellentmeansforexaminingcellmorphologyinlivingtissues. i.Inject60-500pgofmRNAforvisualizationatgastrulastages. Toexaminesubcellularlocalizationoffusionproteins Becauseoverexpressioncanleadtoectopicorabnormalproteinlocalization,careshouldbetakentoinjectthelowestdoseofmRNAthatallowsvisualizationoftheconstruct.Acarefuldose-curveexperimentshouldbeperformedtodeterminetheproperexpressionlevelempiricallybeforeproceeding.Injectionsof15-75pgofmRNAaretypical,butconcentrationsaslowas5pgorashighas500pgmightbeneeded. ii.InjectthemRNAofinterest.Whenimaging(seeStep3),useahigh-NAobjective. Togeneratemosaicembryos iii.Inject4-cellembryoswithanmRNAencodingafusionproteinofinterest.Alternatively,mosaicexpressioncanalsobeachievedbyinjectionofplasmidDNA(Vizeetal.1991).BecauseplasmidDNAisnottranscribeduntilafterthemid-blastulatransition,thisapproachavoidsearlyexpressionoftheMarkergene,whichmightbedesirable.Moreover,expressionlevelswillvaryfromcelltocellusingthisapproach(Fig.5C). Viewlargerversion(32K):[inthiswindow][inanewwindow] Figure5.GenerationofmosaicXenopusembryosbytargetedmicroinjectionofplasmidDNAs(Vizeetal.1991).(A)mRNAencodingafluorescentmarkerisinjectedfirsttolabelthetissueuniformly.(B)AsecondinjectionofplasmidDNAismadesubsequently.(C)MosaicismisobservableinembryosinjectedwithmRNA(red)andplasmidDNAthatsegregatesandtranscribesheterogeneously(green). iv.Injectagainatalatercleavagestagetomanipulategenefunctioninonlyasubsetofcellsexpressingthereporter.Inthislaterinjection,includeboththemorpholino-oligonucleotideormRNAstomanipulategenefunctionandalsoacomplementarylineagetracertomarkthemanipulatedcells.Largeclonesofmanipulatedcellscanbemadewithsecondinjectionsattheeight-cellstage(Fig.6A).Smallerclonesofmanipulatedcellscanbemadewithsecondinjectionsatthe16-cellto32-cellstages(Fig.6B).PlasmidDNAcanalsobeinjectedfollowingmRNAinjection,allowingmosaicexpressionofonemarkerinauniformbackgroundofacomplementarymarker(Fig.5). Viewlargerversion(56K):[inthiswindow][inanewwindow] Figure6.GenerationofmosaicXenopusembryosbytargetedmicroinjection.(A)Tissue-levelmosaicscanbemadebysequentialinjectionsatthefour-andeight-cellstages(Kiesermanetal.2008).(B)Cell-levelmosaicscanbegeneratedbysequentialinjectionsatthefour-cellandthe16-or32-cellstages(Grayetal.2009). 2.Immobilizeembryosforconfocalimaging:Thesemethodsformountingandimagingarealsoeffectiveforfixedembryosfollowingimmunostainingorinsituhybridization,providedthatclearingisnotrequired.Forimagingclearedembryos,seePreparationofFixedXenopusEmbryosforConfocalImaging(Wallingford2010b). Forimagingblastula,gastrula,orneurulastages SeeFigure7Aforanillustrationofimmobilibzationatthesestages. Viewlargerversion(26K):[inthiswindow][inanewwindow] Figure7.Mountingofembryosforinvivotime-lapseimaging.(A)Mountingforblastula,gastrula,andneurulastages(seeSteps2.i-2.iii).(B)Mountingfortailbudstages(seeSteps2.iv-2.vi). i.Pouralayerof2%agaroseintotheimagingchamber. ii.Usingasmallcomb,makeembryo-sizedwellsintheagarose. iii.Devitellinizeembryos.Placethemintothewellssuchthatthesurfaceofinterestcanbeimaged(e.g.,theneuralplateduringneuraltubeclosure[Leeetal.2007;Kiesermanetal.2008];theanimalcapofblastulastagesinwholemounts[Woolneretal.2008]).TakecaretopositiontheembryoascloseaspossIBLetothecoverglassbottomtoaccommodatetheworkingdistanceoftheobjective.Also,thelessagarosebetweensampleandobjective,thebetter.Thismethodiseffectiveforimagingupto6h. Forimagingtailbudandtadpolestages TheepidermisoftailbudandtadpolestageXenopusembryosisanexcellentinvivomodelforstudyingepithelialbiology(Hayesetal.2007;Kiesermanetal.2008;Parketal.2008).Imagingthistissuealsousesthespecializedchamber,butinvolvesadifferentmountingapproach(Fig.7B).Thisapproachisalsoeffectiveforlong-termtime-lapseanalysis(>12h). iv.Placetheembryosonthecoverglassofanimagingchamberinasmalldropof1/3XMMR. v.Workingquickly,removetheMMRentirely.Replacewithadropoflukewarm0.8%low-meltagarose.Useahairlooptoquicklypositiontheembryoasdesired. vi.Aftertheagarosehardens,fillthechamberwith1/3XMMRtopreventdesiccation.Thesampleisnowreadyforimaging. 3.Imageusingappropriateobjectivesandparameters:Whensettinguptheimagingparameters,acriticalconsiderationisthebalancebetweenphotobleachingandthebrightnessofthesample.Toerronthesideofcautionwithregardtophotobleaching,decreasethelaserpowerandthencompensatebyincreasingthedetectorgainorincreasethebrightnesspost-acquisition;caremustbetakenwithpost-acquisitionprocessing(seeStep6).Athighmagnification,onecanreliablyimagetoadepthofonlyaboutonecelldiameter.Higherlaserpowercanadddepth,butisclearlyphototoxic.Insomecases(usuallyatlowermagnifications),brightsignalsunderneathunlabeledcellscanbeimagedclearly.Forexample,thecrawlingmyeloidcellsinFigure2arelabeledandimagedthroughtwotothreelayersofunlabeledepidermalcells.Imagingdeepertissuescanalmostalwaysbeachievedbyimagingthecellsinorganotypicculture(seeDiscussion). Fortrue4Dimaging i.Collectstacksofslicesovertime(Fig.8). Viewlargerversion(32K):[inthiswindow][inanewwindow] Figure8.Schematicprotocolforcollectionof4DdatasetsfromXenopusembryos.(A)Protocolforimagingneuraltubeclosure.(B)Protocolforimagingtheepidermis. Thesizeofstacks,theamountofoverlapbetweenthesliceswithinstacks(z-resolution),andthecollectiontimes(time-resolution)mustbeoptimizedempiricallyforyourspecimen,fluorophore,andapplication(see,e.g.,Kiesermanetal.2008,SupplementalTable2). Fortime-lapseanalysisofindividualcells ii.Imagewith40X,63X,or100Xobjectives. Fortissuelevelimaging iii.Imagewith5X-20Xobjectives.Imagingat40Xisalsopossible,althoughthelowermagnificationsarepreferred. 4.Collectimagesasastackofindividualtifffilesforatime-lapsemovie. 5.Checkthemovieevery15-30minduringacquisition.Correctfocusdriftorembryomovementasneeded.Forlong-termimagingoftadpolestages,addasolutionof0.15%Tricainetothemediumtopreventtwitchingoftheembryo. 6.Withcarefulimaging,littleornopost-acquisitionprocessingisneeded.Ifnecessary,however,performimageprocessingtoimprovetheimageforthecomputerscreenandprintedpage:Manyjournalsnowrequestthatalloriginal,unprocessedimagesbedepositedwiththejournal.TheoriginalfilesMUSTthereforebesaved.ALWAYScreatearenamedcopyofimagesbeforeanyprocessingisperformed(e.g.,byadding"_PROCx"totheoriginalfile,wherexisanumber;differentiterationsoftheprocessingthusgetconsecutivelyhighernumbers)andperformenhancementsontherenamedcopy.Alwaysapplyfiltersandenhancementstotheentireimage.NEVERapplyenhancementsorfilterstoselectedregionsofanimage.Likeimaging,post-processingismosteffectiveiftrialanderrorisused. i.ContrastcanbeenhancedusingPhotoshop’s"Levels"function(ImageAdjustmentsLevels).ThisfunctionismoreusefulthantheBrightness/Contrastfunction. ii.ImageclaritycanbeimprovedusingPhotoshop’s"UnsharpMask"function(FilterSharpenUnsharpMask). iii.NoisecanbereducedusingtheMedian(FilterNoiseMedian)orDust&Scratches(FilterNoiseDust&Scratches)filter. 7.UsePhotoshoptoresize(ImageImageSize)andcompress(FileSaveAs)individualtifffiles.ThiscanbedoneforalltheimagesusingtheFileAutomateBatchfunction.Alternatively,themoviecanberesizedafterassembly(seeStep9).Tinymoviesareusefulfore-mailingtocollaborators,butthelargest,highestqualitymoviesthatarepracticalshouldbesubmittedwithapaperforreview. 8.Afteradjustmentsaremade,assemblethemodified,individualtifffilesintoamovieusingQuickTimePro(FileOpenImageSequence). 9.(Optional)Resizeand/orcompressthemoviesafterassembly: i.Tochangethesize/qualityofamovie,usetheFileExportOptionsfunctioninQuickTimePro. ii.UsetheSettingsandSizefunctionsintheOptionswindow. DISCUSSION Xenopusembryosprovideanexcellentplatformforliveimagingacrossawiderangeofsizescales.Forexample,wholeembryoscanbefilmedwithastereomicroscopetoaskquestionsabouttissuemorphogenesis(seeLow-MagnificationLiveImagingofXenopusEmbryosforCellandDevelopmentalBiology[Wallingford2010a]).Theverysameembryocanthenberemovedfromthestereoscopestageandmountedintactonaconfocalmicroscopeforthree-dimensionaltime-lapseimagingofmigratorycellmovements(Fig.2),cytoskeletalorganization,orthedynamicsofGolgistructure(Fig.1C,D),tonamejustafewapplications.ItisimportantalsotonotethatXenopusembryoscanbeeffectivelyimagedacrossawidevarietyoftimescales.Intactembryoshavebeenusedtomake4Dconfocalmoviesofmyeloidcellmigrationthatspan15h,andthebeatingofciliahasbeenimagedat>370frames/sec(Fig.2). Variouscell-membrane-targetedGFPfusionsexistforexaminingcellmorphologyinlivingtissues,includingGFPstargetedtothemembranebyadditionoftransmembranedomainsorbyadditionoffarnesylationsequences.Evenathighlevelsofexpression,littleGFPisdetectedinthecytoplasm.Itshouldbenoted,however,thatintracellularvesiclesarelabeledwithhigherdosesofmRNA.Thesereagentsprovideverybrightlabelingofcellmembranes,allowingvisualizationnotonlyofthecellbody,butalsomembranefeaturessuchasexocyticpitsandfilopodia/lamellipodia(Wallingfordetal.2000;Hayesetal.2007;Davidsonetal.2008). Similarly,imagingofproteinlocalizationwithfluorescentfusionproteinsishighlyeffectiveinXenopus.Wehaveimagedawidevarietyofproteins,makingeffectiveuseofGFPandRFPfusionproteinstovisualizecytoskeletalstructures,suchasmicrotubules(Kiesermanetal.2008)andsubcellularcompartments,suchastheGolgi(Fig.1C,D).Finally,avarietyoffluorescentsensorscanbeappliedinlivingXenopusembryos,includingGFP-basedsensorsofGTPaseactivity(BeninkandBement2005)andfluorescentcalciumindicators(Wallingfordetal.2001).Becauseoverexpressioncanleadtoectopicorabnormalproteinlocalization,careshouldbetakentoinjectthelowestdoseofmRNAthatallowsvisualizationoftheconstruct.ThelargesizeofthecellsinXenopusembryosallowsresolutiontobesacrificedforphotons. Thegenerationofmosaicembryos,inwhichmanipulatedandunmanipulatedcellscanbevisualizedwithinasingletissueofasingleembryo,isanextremelypowerfulexperimentalapproach.Xenopusembryosareverywell-suitedformosaicanalysesusingsimpletargetedmicroinjectionapproaches(Fig.6).Forexample,reagentsformanipulatinggenefunction(e.g.,antisensemorpholino-oligonucleotides,mRNAs)caneasilybedeliveredspecificallytothedesiredtissuesbytargetedinjection.Targetingiseasilyachievedbecauseofthewell-knownpigmentationpatternsofearlyembryosandthestereotypicfatemapsforthefour-,eight-,16-,or32-cellstagesofXenopus(DaleandSlack1987;Moody1987;MoodyandKline1990).Basictargetedmicroinjectionisperformedbyconsultingfatemapstodeterminetheblastomereoriginofthetissueofinterestandinjectingthedesiredreagents(mRNAsormorpholino-oligonucleotides)accordingly.NotethattheanimalpigmentationofXenopusembryosisgenerallyasymmetric,andtwomore-darklypigmentedblastomerescanusuallybeidentifiedinfour-cellembryos.Thesetwodarkerblastomereswillformtheventraltissues,andthelighteroneswillformthedorsaltissues(Fig.6).Thus,injectionsintothelighterblastomereswilltargettissuessuchastheneuralplateornotochord,andinjectingthedarkerblastomereswilltargettheepidermis.Injectionsatearlystages(fourtoeightcells)canbeusedtotargetlargertissuedomains(germlayers),whereasinjectionsatlaterstagescanbeusedtotargetmorespecifictissues,suchasthegutorkidney(Wallingfordetal.1998;Lietal.2008).Alwaysrememberthatfatemapsarepredictive,notaguarantee.Injectedembryosshouldbescreenedatgastrulaorneurulastagestoensureexpressioninonlytheintendedtissues(WallingfordandHarland2002;KiesermanandWallingford2009). Liveimagingofintactembryosisanexcellentapproach,butislimitedtosurfacetissuesbytheopacityofamphibianembryos.Tocircumventthisproblem,cellsandsubcellularstructureswithintheembryocanbevisualizedquiteeasilythroughmicrosurgeryandsubsequentcultureoftissueexplants.Withpractice,nearlyanytissuecanbemicrosurgicallyisolatedfromanyregionoftheembryouptothelatetadpolestages(Keller1991).Briefly,explantedtissuesareheldinplacegentlyunderafragmentofcoverglasssecuredinplacewithmodelingclayorhigh-vacuumsiliconegrease.Whenculturedinanappropriatemedium(e.g.,DFAorSteinberg’s,whicharespeciallyformulatedtomatchthelow-chloride,high-pHconditionsoftheinterstitialfluidoftheearlyXenopusembryo),cellswithinexplantscontinueinvivoprogramsofcellmotilityandcellrearrangement(Kelleretal.1985).Ingeneral,cellswithinphysicallyisolatedexplantsautonomouslyrecapitulatemovementswithintheembryo.Forexample,imagingofdorsalmarginalzoneexplantsisusedcommonlytoexamineconvergentextension.Asisthecaseforwholeembryos,Xenopusexplantshavebeenusedtoimagecellprotrusiveactivity,proteinlocalization,extracellularmatrixorganization,andevencalciumdynamics(Wallingfordetal.2000,2001;DavidsonandWallingford2005;Davidsonetal.2008).CulturedXenopusanimalcaps,oftenusedforgeneexpressionstudies,arealsoveryamenableforimaging,havingbeenusedtoexamineproteinlocalization(Axelrodetal.1998;Parketal.2005),nucleocytoplasmicshuttling(Batutetal.2007),andmorphogendiffusion(Williamsetal.2004). Withthecombinationofexternaldevelopment,largecellsize,andawell-definedfate-mapfortargetedmicroinjectionandgenerationofmosaics,Xenopusembryosprovideanunparalleledsystemfortheinvivostudyofthecellbiologyofvertebratedevelopment.Examinationofcellsincultureisanimmenselypowerfulapproach,butmanybiologicalprocessesthathavebeenwell-studiedinculturehavesubsequentlybeenfoundtobesubjecttosubstantialdevelopmentalcontrolinembryosorintacttissues.Thatis,cellularprocessessuchasmigrationanddivisionhavefrequentlybeenfoundtodifferdramaticallyevenindifferentcelltypeswithinthesameembryo.Thus,extensiveinvivostudywillbeessentialbeforeacomprehensiveunderstandingwillemerge.Moreover,becausebasiccellbiologicalprocessesunderlieallofdevelopmentalbiology,fromsignalingtomorphogenesis,acomprehensiveunderstandingofdevelopmentwillrequirethatcellbiologicalapproachesbeappliedmoreandmoretodevelopmentalproblems.ThecellbiologystudiesthatcanbemosteffectivelycarriedoutinXenopusembryoswillcomplementthegeneticstudiesofmouseandzebrafishaswemoveforward. ACKNOWLEDGMENTS ThemethodsdescribedherehavebeendevelopedandoptimizedbyallthemembersoftheWallingfordlaboratory.WethankHye-JiChafortheGolgiimage.ThankstoAndrewEwaldfortheoriginalconstructionoftheXenopusimagingchambers.WealsothankAndrewEwaldfordiscussions.WorkintheWallingfordlaboratoryhasbeenfundedbytheNationalInstitutesofHealth/NationalInstituteofGeneralMedicalSciences(NIH/NIGMS),theMarchofDimes,TheBurroughsWellcomeFund,andtheSandlerProgramforAsthmaResearch. REFERENCES AxelrodJD,MillerJR,ShulmanJM,MoonRT,PerrimonN.1998.DifferentialrecruitmentofDishevelledprovidessignalingspecificityintheplanarcellpolarityandWinglesssignalingpathways.Genes&Dev12:2610–2622.[Abstract/FreeFullText] BatutJ,HowellM,HillCS.2007.Kinesin-mediatedtransportofSmad2isrequiredforsignalinginresponsetoTGF-βligands.DevCell12:261–274.[Medline] BeninkHA,BementWM.2005.ConcentriczonesofactiveRhoAandCdc42aroundsinglecellwounds.JCellBiol168:429–439.[Abstract/FreeFullText] DaleL,SlackJM.1987.Fatemapforthe32-cellstageofXenopuslaevis.Development99:527–551.[Abstract/FreeFullText] DavidsonLA,WallingfordJB.2005.Visualizingcellbiologyandtissuemovementsduringmorphogenesisinthefrogembryo.InImaginginneuroscienceanddevelopment(eds.RYusteandAKonnerth),pp.125–136.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY. DavidsonLA,DzambaBD,KellerR,DeSimoneDW.2008.Liveimagingofcellprotrusiveactivity,andextracellularmatrixassemblyandremodelingduringmorphogenesisinthefrog,Xenopuslaevis.DevDyn237:2684–2692.[Medline] EwaldAJ,PeyrotSM,TyszkaJM,FraserSE,WallingfordJB.2004.RegionalrequirementsforDishevelledsignalingduringXenopusgastrulation:Separableeffectsonblastoporeclosure,mesendoderminternalizationandarchenteronformation.Development131:6195–6209.[Abstract/FreeFullText] GrayRS,AbituaPB,WlodarczykBJ,Szabo-RogersHL,BlanchardO,LeeI,WeissGS,LiuKJ,MarcotteEM,WallingfordJB,etal.2009.TheplanarcellpolarityeffectorFuzisessentialfortargetedmembranetrafficking,ciliogenesisandmouseembryonicdevelopment.NatCellBiol11:1225–1232.[Medline] GuilleM.1999.MicroinjectionintoXenopusoocytesandembryos.InMolecularmethodsindevelopmentalbiology:Xenopusandzebrafish(ed.MGuille),pp.111–124.Humana,Totawa,NJ. HaigoSL,HildebrandJD,HarlandRM,WallingfordJB.2003.Shroominducesapicalconstrictionandisrequiredforhingepointformationduringneuraltubeclosure.CurrBiol13:2125–2137.[Medline] HayesJM,KimSK,AbituaPB,ParkTJ,HerringtonER,KitayamaA,GrowMW,UenoN,WallingfordJB.2007.Identificationofnovelciliogenesisfactorsusinganewinvivomodelformucociliaryepithelialdevelopment.DevBiol312:115–130.[Medline] KellerR.1991.EarlyembryonicdevelopmentofXenopuslaevis.MethodsCellBiol36:61–113.[Medline] KellerRE,DanilchikM,GimlichR,ShihJ.1985.ThefunctionandmechanismofconvergentextensionduringgastrulationofXenopuslaevis.JEmbryolExpMorpholSuppl89:185–209.[Medline] KiesermanEK,WallingfordJB.2009.InvivoimagingrevealsaroleforCdc42inspindlepositioningandplanarorientationofcelldivisionsduringvertebrateneuraltubeclosure.JCellSci122:2481–2490.[Abstract/FreeFullText] KiesermanEK,GlotzerM,WallingfordJB.2008.Developmentalregulationofcentralspindleassemblyandcytokinesisduringvertebrateembryogenesis.CurrBiol18:116–123.[Medline] KwanKM,KirschnerMW.2005.Amicrotubule-bindingRho-GEFcontrolscellmorphologyduringconvergentextensionofXenopuslaevis.Development132:4599–4610.[Abstract/FreeFullText] LeeC,ScherrHM,WallingfordJB.2007.Shroomfamilyproteinsregulate-tubulindistributionandmicrotubulearchitectureduringepithelialcellshapechange.Development134:1431–1441.[Abstract/FreeFullText] LiY,RankinSA,SinnerD,KennyAP,KriegPA,ZornAM.2008.Sfrp5coordinatesforegutspecificationandmorphogenesisbyantagonizingbothcanonicalandnoncanonicalWnt11signaling.Genes&Dev22:3050–3063.[Abstract/FreeFullText] MoodySA.1987.Fatesoftheblastomeresofthe32-cell-stageXenopusembryo.DevBiol122:300–319.[Medline] MoodySA,KlineMJ.1990.Segregationoffateduringcleavageoffrog(Xenopuslaevis)blastomeres.AnatEmbryol(Berl)182:347–362.[Medline] NicholsBJ,KenworthyAK,PolishchukRS,LodgeR,RobertsTH,HirschbergK,PhairRD,Lippincott-SchwartzJ.2001.RapidcyclingoflipidraftmarkersbetweenthecellsurfaceandGolgicomplex.JCellBiol153:529–541.[Abstract/FreeFullText] ParkTJ,GrayRS,SatoA,HabasR,WallingfordJB.2005.SubcellularlocalizationandsignalingpropertiesofDishevelledindevelopingvertebrateembryos.CurrBiol15:1039–1044.[Medline] ParkTJ,MitchellBJ,AbituaPB,KintnerC,WallingfordJB.2008.Dishevelledcontrolsapicaldockingandplanarpolarizationofbasalbodiesinciliatedepithelialcells.NatGenet40:871–879.[Medline] SiveHL,GraingerRM,HarlandRM.2000.EarlydevelopmentofXenopuslaevis:Alaboratorymanual.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY. SiveHL,GraingerRM,HarlandRM.2007.Embryodissectionandmicromanipulationtools.ColdSpringHarbProtocdoi:10.1101/pdb.top7.[Abstract/FreeFullText] VizePD,MeltonDA,Hemmati-BrivanlouA,HarlandRM.1991.AssaysforgenefunctionindevelopingXenopusembryos.MethodsCellBiol36:367–387.[Medline] WallingfordJB.2010a.Low-magnificationliveimagingofXenopusembryosforcellanddevelopmentalbiology.ColdSpringHarbProtoc(thisissue).doi:10.1101/pdb.prot5425.[Abstract/FreeFullText] WallingfordJB.2010b.PreparationoffixedXenopusembryosforconfocalimaging.ColdSpringHarbProtoc(thisissue).doi:10.1101/pdb.prot5426.[Abstract/FreeFullText] WallingfordJB,HarlandRM.2002.NeuraltubeclosurerequiresDishevelled-dependentconvergentextensionofthemidline.Development129:5815–5825.[Medline] WallingfordJB,CarrollTJ,VizePD.1998.PrecociousexpressionoftheWilms’tumorgenexWT1inhibitsembryonickidneydevelopmentinXenopuslaevis.DevBiol202:103–112.[Medline] WallingfordJB,RowningBA,VogeliKM,RothbächerU,FraserSE,HarlandRM.2000.DishevelledcontrolscellpolarityduringXenopusgastrulation.Nature405:81–85.[Medline] WallingfordJB,EwaldAJ,HarlandRM,FraserSE.2001.CalciumsignalingduringconvergentextensioninXenopus.CurrBiol11:652–661.[Medline] WilliamsPH,HagemannA,González-GaitánM,SmithJC.2004.Visualizinglong-rangemovementofthemorphogenXnr2intheXenopusembryo.CurrBiol14:1916–1923.[Medline] WoolnerS,O’BrienLL,WieseC,BementWM.2008.Myosin-10andactinfilamentsareessentialformitoticspindlefunction.JCellBiol182:77–88.[Abstract/FreeFullText] ZhouJ,KimHY,DavidsonLA.2009.Actomyosinstiffensthevertebrateembryoduringcrucialstagesofelongationandneuraltubeclosure.Development136:677–688.[Abstract/FreeFullText] 您的称呼： 单位名称： 您的邮箱： 您的电话： 品牌与型号： 其他要求： 新闻动态 行业前沿 技术文章 最新产品 Gendepot公司 GenDEPOT细胞afp免疫荧光 2017-12-04 Gendepot抗体品牌 GenDEPOT生物检测试剂盒 2020-10-05 代理 报价 2018-09-04 普瑞麦迪(北京)实验室技术有限公司_Premedical Laboratories Co... 2021-03-24 GenDEPOT代理创亚化工(上海)有限公司-创亚化工(上海)有限公司 2021-03-24 全部产品分类_普瑞麦迪(北京)实验室技术有限公司_丁香通 2021-03-25 普瑞麦迪-首页 2021-03-24 普瑞麦迪-首页 2021-03-24 [原创]石家庄凯达生物工程有限公司商业计划书doc下载_爱问共享资料 2020-03-27 三日疟原虫荧光PCR检测试剂盒优惠促销品牌:百奥莱博北京盖德... 2019-09-04 GenDEPOT中国特约代理商(2021年度) 2020-06-29 暗黑破坏神2_暗黑破坏神2下载_修改器_存档_补丁_攻略_专题_游侠... 2017-05-01 GenDEPOT 品牌介绍及代理商/经销商名单品牌专区 2019-03-09 厂家直供10ml玻璃瓶 透明棕色 留样瓶 试剂瓶 样品瓶 10ml ... 2020-09-24 feldan_产品目录 2021-04-03 GenDEPOT 品牌介绍及代理商/经销商名单品牌专区 2019-03-17 ...different methods for generation of singlestranded DNA... 2021-09-11 敌克松杀菌剂价格_敌克松杀菌剂价格商家/厂家/公司/ 2021-08-13 TissueHarvestProtocol资讯 2021-08-11 ...different methods for generation of singlestranded DNA... 2021-09-13 【进口代理清关】进口代理清关价格_进口代理清关报价 2021-08-08 Tissue Harvest Protocol General Surgery and … 2021-08-24 Tissue Harvest Protocol General Surgery and … 2021-08-17 gendepot 2021-08-31 Gendepot/ABTS显色剂（高对比度），A3510/A3510-050-500ml 2026-07-13 Gendepot/Celecoxib，>99%，BC016/300 mg/BC016-300 2026-07-13 Gendepot/His-Tag Mouse Monoclonal Antibody, 1mg/ml, Ab1005/1mg/Ab1005-101 2026-07-13 Gendepot/Distilled Water, W0325/500 ml/W0325-050 2026-07-13 Gendepot/X-Gal，5-溴-4-氯-3-吲哚基β-D-半乳吡喃糖苷，X0320/X0320-101-10 g 2026-07-13 Gendepot/牛血清白蛋白，细胞培养试验，CA068/100 g/CA068-010 2026-07-13 Gendepot/HRV 3C蛋白酶，T9800/1000单位/T9800-010 2026-07-13 Gendepot/离心管，50 ml，PP，无菌，塞式密封盖，自立式，袋装，LC8006/500/CS/LC8006-500 2026-07-13 Copyright@苏州蚂蚁淘生物科技有限公司 2003-2021  188进口试剂采购网版权所有     苏ICP备17049038号-9 友情链接：  其他 188进口试剂采购网 www.188bio.cn -中国试剂网,试剂网,化学试剂网,国药试剂,抗体公司,试剂公司,试剂盒公司,苏州试剂公司,北京化学试剂公司,天津化学试剂,试剂商城,试剂代理,流式抗体 细胞库查询 sitemap