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Methods in Molecular Biology 2562 Ashley W. Seifert Joshua D. Currie Editors Salamanders Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Salamanders Methods and Protocols Edited by Ashley W. Seifert Department of Biology, University of Kentucky, Lexington, KY, USA Joshua D. Currie Department of Biology, Wake Forest University, Winston-Salem, NC, USA Editors AshleyW.Seifert JoshuaD.Currie DepartmentofBiology DepartmentofBiology UniversityofKentucky WakeForestUniversity Lexington,KY,USA Winston-Salem,NC,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2658-0 ISBN978-1-0716-2659-7 (eBook) https://doi.org/10.1007/978-1-0716-2659-7 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface For nearly threecenturies,salamanders haveremainedkeyresearch organisms fordevelop- mental,evolutionary,andecologicalstudies.Buildingonadvancesingeneticmodelsystems (e.g., Drosophila,C.elegans,and Mus),the last20 yearshasseen adizzyingacceleration of molecularandgenomictechniquesthathavemadetheir wayintoamphibianresearch.This breadthanddepthhavemadesalamanderandnewtmodelsidealresearchorganismstostudy processesaffectingbiologicalscalesfrommoleculestoecosystems. OurgoalineditingthisneweditionofMethodsinSalamanderResearchwastoassemble a definitive desk reference for researchers either new to using salamanders for research or expert labs that want to bring new techniques and approaches to bear on their scientific questions.Thechapterscontainedinthisvolumehighlightnewvoicesinthisfieldalongside the experience and perspective of veteran salamander scientists. We want to extend a heartfeltthankstoallthisvolume’sauthorsfor theireffortsinthefaceofpandemic-related disruptions. We hope this reference will be a catalyst for researchers using amphibians to dialoguewiththeauthorsandthebroadercommunityinordertoextendtheboundariesof salamander research. Lexington,KY,USA AshleyW.Seifert Winston-Salem,NC,USA JoshuaD.Currie v Contents Preface ..................................................................... v Contributors................................................................. xi 1 SalamandersasKeyModelsforDevelopmentand RegenerationResearch ..... ........ ....... ....... ........ ....... ........ 1 MalcolmMaden PART I LABORATORY COLONY HUSBANDRY 2 EstablishingaNewResearchAxolotlColony........ ........ ....... ........ 27 AnastasiaS.YandulskayaandJamesR.Monaghan 3 HealthMonitoringforLaboratorySalamanders ..... ...... .... ..... ........ 41 MarcusJ.CrimandMarciaL.Hart 4 Husbandry,CaptiveBreeding,andFieldSurveyofChinese GiantSalamander(Andriasdavidianus) .. ... ...... ....... .. .... ... ........ 75 WanshengJiang,HaifengTian,andLuZhang PART II TRADITIONAL MOLECULAR TECHNIQUES 5 Whole-MountInSituHybridization(WISH)forSalamander EmbryosandLarvae ....... ........ ....... ....... ........ ....... ........ 95 SruthiPurushothamanandAshleyW.Seifert 6 HybridizationChainReactionFluorescenceInSituHybridization (HCR-FISH)inAmbystomamexicanumTissue...... ........ ....... ........ 109 AlexM.Lovely,TimothyJ.Duerr,DavidF.Stein, EvanT.Mun,andJamesR.Monaghan 7 EthylCinnamate-BasedTissueClearingStrategies ......... .. ...... ......... 123 WouterMasselinkandEllyM.Tanaka 8 InductionandCharacterizationofCellularSenescenceinSalamanders ........ 135 QinghaoYu,HannahE.Walters,andMaximinaH.Yun 9 MethodsforStudyingAppendicularSkeletalBiologyinAxolotls...... ........ 155 CamiloRiquelme-GuzmańandTatianaSandoval-Guzmań 10 FluorescenceInSituHybridizationofDNAProbeson MitoticChromosomesoftheMexicanAxolotl ...... ........ ....... ........ 165 MelissaKeinathandVladimirTimoshevskiy 11 TheUseofSmallMoleculestoDissectDevelopmentaland RegenerativeProcessesinAxolotls.......... ....... ........ ....... ..... ... 175 Ste´phaneRoy 12 COMETAssayforDetectionofDNADamageDuring AxolotlTailRegeneration........... ....... ....... ........ ....... ........ 183 BelfranCarbonell,JenniferA´lvarez,GloriaA.Santa-Gonza´lez, andJeanPaulDelgado vii viii Contents PART III EXPERIMENTAL MANIPULATIONS AND SURGERIES 13 InVivoandExVivoViewofNewtLensRegeneration....... ....... ........ 197 GeorgiosTsissios,AnthonySallese,WeihaoChen,AlyssaMiller, HuiWang,andKatiaDelRio-Tsonis 14 BeadImplantationandDeliveryofExogenousGrowthFactors....... ........ 209 RenaKashimoto,SayaFurukawa,SakiyaYamamoto, andAkiraSatoh 15 TheAccessoryLimbModelRegenerativeAssayandItsDerivatives ........... 217 MichaelRaymondandCatherineD.Mccusker 16 EmbryonicTissueandBlastemaTransplantations.... ........ ....... ........ 235 MarittaSchuez,ThomasKurth,JoshuaD.Currie, andTatianaSandoval-Guzmań 17 RetinoicAcid–InducedLimbDuplications.......... ........ ....... ........ 249 MalcolmMadenandTreyPolvadore 18 IsolationandCharacterizationofPeritonealMacrophages fromSalamanders......... ......... ....... ....... ........ ....... ........ 259 AnthonySallese,GeorgiosTsissios,J.Rau´lPe´rez-Estrada, ArielleMartinez,andKatiaDelRio-Tsonis PART IV BIOINFORMATICS AND GENOMICS 19 NavigationandUseofCustomTrackswithintheAxolotl GenomeBrowser .......... ........ ....... ....... ........ ....... ........ 273 SergejNowoshilowandEllyM.Tanaka 20 ChromosomeConformationCaptureforLargeGenomes .... .... ... ...... .. 291 AkaneKawaguchiandEllyM.Tanaka PART V TRANSGENICS AND LINEAGE-TRACING 21 AxolotlTransgenesisviaInjectionofI-SceIMeganucleaseor Tol2TransposonSystem.... ........ ....... ....... ........ ....... ........ 321 MarittaSchuezandTatianaSandoval-Guzmań 22 APracticalGuideforCRISPR-Cas9-InducedMutationsinAxolotls .......... 335 KonstantinosSousounis,KatharineCourtemanche, andJessicaL.Whited 23 ApplyingaKnock-InStrategytoCreateReporter-Tagged KnockoutAllelesinAxolotls ........ ....... ....... ........ ....... ........ 351 LiqunWang,Yan-YunZeng,YanmeiLiu,andJi-FengFei 24 BaculovirusProductionandInfectioninAxolotls.... ........ ....... ........ 369 PrayagMurawala,CatarinaR.Oliveira,HelenaOkulski, MaximinaH.Yun,andEllyM.Tanaka 25 CellDissociationTechniquesinSalamanders........ ........ ....... ........ 389 GabrielaJohnson,NadjibDastagir,ZacharyBeal, AndrewHart,andJamesGodwin Contents ix PART VI PHYSIOLOGICAL AND ORGANISMAL TECHNIQUES 26 AxolotlMetabolism:MeasuringMetabolicRate ..... ........ ....... ........ 407 MosheKhurgel 27 ArtificialInseminationinAxolotl .... ....... ....... ........ ....... ........ 417 YukaTaniguchi-SugiuraandEllyM.Tanaka 28 ScreeningSalamandersforSymbionts ....... ....... ........ ....... ........ 425 ElliVickersandRyanKerney 29 AssessingLeukocyteProfilesofSalamandersandOtherAmphibians: AHerpetologists’Guide.... .... .... .... ... ....... ........ ....... ........ 443 AndrewK.DavisandJohnC.Maerz PART VII EPILOGUE 30 BestPracticestoPromoteDataUtilityandReusebythe Non-TraditionalModelOrganismCommunity...... ........ ....... ........ 461 GarrettS.DunlapandNicholasD.Leigh 31 NowthatWeGotThere,WhatNext? ....... ....... ........ ....... ........ 471 EllyM.Tanaka Index ...................................................................... 481 Contributors JENNIFERA´LVAREZ • GrupoGene´ticaRegeneraci(cid:1)onyCańcer,InstituteofBiology,University ofAntioquia,Medellin,Colombia ZACHARYBEAL • MountDesertIslandBiologicalLaboratory(MDIBL),KathrynW.Davis Center forRegenerativeBiologyandAging,BarHarbor,ME,USA BELFRANCARBONELL • GrupoGene´ticaRegeneraci(cid:1)onyCańcer,InstituteofBiology, UniversityofAntioquia,Medellin,Colombia WEIHAOCHEN • CellularMolecularandStructuralBiologyProgram,MiamiUniversity, Oxford,OH,USA;DepartmentofChemical,PaperandBiomedicalEngineering,Miami University,Oxford,OH,USA KATHARINECOURTEMANCHE • DepartmentofStemCellandRegenerativeBiology,Harvard University,Cambridge,MA,USA MARCUS J.CRIM • IDEXXBioAnalytics,Columbia,MO,USA JOSHUA D.CURRIE • DepartmentofBiology,WakeForestUniversity,Winston-Salem,NC, USA NADJIBDASTAGIR • MountDesertIslandBiologicalLaboratory(MDIBL),KathrynW.Davis Center forRegenerativeBiologyandAging,BarHarbor,ME,USA ANDREWK.DAVIS • OdumSchoolofEcology,TheUniversityofGeorgia,Athens,GA,USA;D. B.WarnellSchoolofForestryandNaturalResources,TheUniversityofGeorgia,Athens, GA,USA KATIADELRIO-TSONIS • DepartmentofBiology,MiamiUniversity,Oxford,OH,USA; Center forVisualSciencesatMiamiUniversity,Oxford,OH,USA;CellularMolecular andStructuralBiologyProgram,MiamiUniversity,Oxford,OH,USA JEANPAULDELGADO • GrupoGene´ticaRegeneraci(cid:1)onyCańcer,InstituteofBiology, UniversityofAntioquia,Medellin,Colombia TIMOTHYJ.DUERR • DepartmentofBiology,Northeastern University,Boston,MA,USA GARRETTS.DUNLAP • BiologicalandBiomedicalSciences,HarvardMedicalSchool, Cambridge,MA,USA JI-FENGFEI • KeyLaboratoryofBrain,CognitionandEducationSciences,Ministryof Education,InstituteforBrainResearchandRehabilitation,SouthChinaNormal University,Guangzhou,China;DepartmentofPathology,GuangdongProvincialPeople’s Hospital,GuangdongAcademyofMedicalSciences,Guangzhou,China SAYAFURUKAWA • DepartmentofBiologicalScienceFacultyofScience,OkayamaUniversity, Okayama,Japan JAMESGODWIN • MountDesertIslandBiologicalLaboratory(MDIBL),KathrynW.Davis Center forRegenerativeBiologyandAging,BarHarbor,ME,USA ANDREWHART • MountDesertIslandBiologicalLaboratory(MDIBL),KathrynW.Davis Center forRegenerativeBiologyandAging,BarHarbor,ME,USA MARCIAL.HART • IDEXXBioAnalytics,Columbia,MO,USA WANSHENG JIANG • JishouUniversity,Zhangjiajie,Hunan,China GABRIELAJOHNSON • MountDesertIslandBiologicalLaboratory(MDIBL),Kathryn W.DavisCenter forRegenerativeBiologyandAging,BarHarbor,ME,USA RENAKASHIMOTO • OkayamaUniversity,GraduateschoolofNaturalScienceand Technology,Okayama,Japan xi