Developmental Biology of the Sea Urchin and Other Marine Invertebrates: Methods and Protocol PDF
Preview Developmental Biology of the Sea Urchin and Other Marine Invertebrates: Methods and Protocol
Methods in Molecular Biology 2219 David J. Carroll Stephen A. Stricker Editors Developmental Biology of the Sea Urchin and Other Marine Invertebrates Methods and Protocols Second Edition 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. Developmental Biology of the Sea Urchin and Other Marine Invertebrates Methods and Protocols Second Edition Edited by David J. Carroll Department of Biochemistry and Molecular Genetics, Midwestern University, Glendale, AZ, USA Stephen A. Stricker Department of Biology, University of New Mexico, Albuquerque, NM, USA Editors DavidJ.Carroll StephenA.Stricker DepartmentofBiochemistryand DepartmentofBiology MolecularGenetics UniversityofNewMexico MidwesternUniversity Albuquerque,NM,USA Glendale,AZ,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-0973-6 ISBN978-1-0716-0974-3 (eBook) https://doi.org/10.1007/978-1-0716-0974-3 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2014,2021 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. 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Preface Marine invertebrates have contributed greatly to our understanding of many biological processes—fromecologicalandenvironmentalissuestocell,developmental,andmolecular biologicalproblems.Therearemanyreasonsfor this,includingthefactthatmanyofthese organismsareeasilyaccessibleandhavelongstimulatedthecuriosityofhumankind.Youcan probablyrememberbacktoatimeinyourchildhoodwhenyouwerefascinatedwithsome interesting creature—a sea urchin at the beach or an earthworm in your backyard, for example. Even Charles Darwin was captivated by invertebrates, publishing a monograph on barnacles [1] before pondering the immense diversity of marine organisms he found during his voyage on the HMS Beagle, no doubt contributing to his ideas on natural selection [2]. In some cases, the relative simplicity of marine invertebrates has allowed breakthroughs that would be more difficult to obtain in research involving complicated higher vertebrates.Thediscoveryofcyclinisaprimeexampleofthis.Rosenthal,Hunt,and Ruderman noted the change in synthesis of proteins in the surf clam after fertilization, preciselybecausetheseeggsareeasilyaccessible,canbefertilizedinvitro,anddividerapidly [3]. The sea urchin provided an even better model for studying these early cleavage stages andledtothenamingoftheseproteinsasthecyclins[4].Alsocontributingtotheusefulness of marine invertebrates is the fact that a wide variety of natural products are produced by marine invertebrates and have been exploited for applications related to human health, including compounds with anticancer, antimicrobial, and other therapeutic properties [5]. In this volume, a variety of marine invertebrate model systems are described along with novel experimental protocols for taking advantage of their unique properties. The techni- ques range fromculturing the organisms to modifying their DNA.We have much to learn aboutthenaturalworld,andtheseorganismswillplayalargeroleinhelpingusdoso. In Chapter 1 on the “Laboratory Culture and Mutagenesis of Amphioxus (Branchios- tomafloridae),”HollandandLidescribetechniquesforcontinuousbreedingofthecepha- lochordate Branchiostoma floridae in the laboratory and methods for mutagenesis using TALENandTol2.Amphioxusoccupiesaspecialplaceinthehierarchyoforganisms,beinga sister group to vertebrates and tunicates. Studies on these organisms could provide novel insightsintohowvertebratesevolved. In Chapter 2 on the “Handling and Manipulation of Gametes and Embryos of the AnnelidanWormPseudopotamillaoccelata,”DeguchiandNakanoprovidedetailedmethods for utilizingPseudopotamillaoccelataasamodelsystemforoocytematuration,signalingat fertilization and early development. This polychaete worm is widely distributed in the northernPacificcoastmakingitlikelytobeuniversallyuseful.Theyhavedevelopedmethods forthecareandexperimentalmanipulationofgametesandembryos,includingmethodsfor microinjectionandcalciumimaging. Continuing with the theme of providing methods for scientists to adopt these useful animals as novel model systems is Chapter 3 on “The Starfish Asterina pectinifera: Collec- tion and Maintenance of Adults and Rearing and Metamorphosis of Larvae” by Murabe et al. This chapter takes us from collecting these starfish during their breeding period, to maintaining gravid adults, to inducing larvae to undergo metamorphosis in the lab. Not onlywillthesemethodsallowforanalysisofdevelopmentallthewaythroughthestarfishlife v vi Preface cycle,itcouldalsoopenthepossibilityofusingthisanimalforstudiesofgenetics,whichhas beenlargelymissingfromthePhylumEchinodermata. InChapter4,“ExperimentalToolstoStudyRegenerationintheSeaAnemoneNema- tostellavectensis”byAmielandRo¨ttingeraddressesthephenomenonofanimalregeneration using the sea anemone Nematostella vectensis as a whole-body regeneration model. Amaz- ingly,regenerationinthisseaanemoneoccurswithin7days!Thechapterdescribesmethods ofinducingregenerationinthisorganism,alongwiththeaccompanyingmethodsoffixation andstainingtoallowdetailedmorphological,cellular,andmolecularstudiesoftheprocess. This chapter should be enjoyed along with Chapter 14, which details methods for tran- scriptomeanalysisusingthesameorganism. In Chapter 5, “Staining and Tracking Methods for Studying Sponge Cell Dynamics,” Borchiellini et al. present protocols for staining cells of three phylogenetically different sponge species to detect dynamic states of cell death, cell proliferation, and cell migration. Theyapplythesemethodstostudydifferentcellbehaviorsandfatesinthesespongesduring development.Sincespongesoccupysuchastrategicphylogeneticpositioninthetreeoflife, thiscomparativeworkcanprovidefundamentalknowledgeaboutcellbehaviorandphysiol- ogyduringthesedynamicprocesses. Chapter 6, “Microscopy Studies of Placozoans” by Smith et al., describes advanced techniquesforlightandelectronmicroscopicstudiesofTrichoplaxadhaerensandisagood companionforChapter18ongenomicsinPlacozoans.Thisorganismisafascinatinganimal withverysimple cellularorganizationand morphology. Thesemicroscopytechniques have increased our knowledge regarding the diversity and function of different cell types in placozoans. Understanding this phylogenetic group promises to provide insight into the evolution,development,differentiation,andphysiologyofcellsandtissues. In Chapter 7, “Identification of SH2 Domain-Mediated Protein Interactions That Operate at Fertilization in the Sea Star Patiria miniata,” Bates et al. present methods for the isolation of tyrosine phosphorylated proteins that interact with a specific signaling molecule in the sea star egg during fertilization. The signaling mechanisms controlling internal calcium release at fertilization in animals are still largely unknown. This chapter begins with methods for preparing the protein samples from the eggs and describes a method for producing fusion proteins for affinity interactions with these egg lysates. Methods for mutagenesis of the fusion protein and resolution of the bound proteins by gelelectrophoresisandwesternblottingarealsodescribed. Chapter8,“MarineNemerteanWormsforImmunoblottingStudiesofOocyteAging” by Stricker, outlines detailed methods for immunoblotting analysis of oocyte protein sam- plesusingphospho-specificantibodies.Thenemerteanwormisanestablishedmodelsystem for understanding oocyte maturation and fertilization. In this chapter, the use of this organismasamodelsystemforunderstandingtherolesplayedbyproteinkinasesasoocytes ageandlosetheirabilitytoundergonormalfertilizationisdescribed.Methodsforobtaining adult worms and for handling their gametes in experiments assessing oocyte aging arealso included. InChapter9,“RecoveryofSeaStarEggCellSurfaceProteinsReleasedatFertilization” by Wiseman et al., we return to the sea star Patiria miniata with a method for visualizing egg cell surface proteins that are released during fertilization. There is a lot of interest in understanding the proteins on the surface of gametes and how they may function during fertilization. However, only a few have been described in any model system. This method describes the collection and analysis of proteins (or peptides) that are either naturally released by the egg or cleaved by sperm proteases. These released proteins can be Preface vii concentrated by an affinity interaction and subsequently analyzed by western blotting and massspectrometrytodeterminetheidentityofeggmembraneproteins. Chapter 10, “Quantifying Cell Proliferation During Regeneration of Aquatic Worms” byZattaraandO¨zpolat,presentsmethodsforidentifyingandquantifyingcellproliferation by several methods, including very clever “click” chemistry. These methods are used to address therole ofcellproliferation inanimal regeneration,particularlyfor visualizationin thewholeworm.Whiletheyhavedevelopedthesemethodsforapplicationinwholemounts ofsmallmarineandfreshwater worms,theycouldalsobeappliedtolargersamples. Chapter 11, “In situ Hybridization Techniques in the Homoscleromorph Sponge Oscarella lobularis” by Fierro-Constain et al., addresses methods for understanding gene expression over space and time at different stages of development in the sponge Oscarella lobularis. The methods are applied to both whole mount individuals and paraffin sections. The Porifera are widely considered as the sister group to all other metazoans. Therefore, studies on this phylum are useful for understanding the early evolution of important morphologicalororganismalfeatures. InChapter12,“MethodologyforWholeMountandFluorescentRNAInSituHybri- dization in Echinoderms: Single, Double, and Beyond,” Perillo et al. detail methods for localization of RNA by in situ hybridization in echinoderms. Unique to this chapter is the useofco-labelingtechniquestoidentifymultipleRNAsinoneexperiment.Theauthorsalso includeanextensive“troubleshooting”sectionthatshouldproveinvaluabletoinvestigators adoptingthesetechniquesintotheirlabs. In Chapter 13, “Gene Editing in the Ascidian Phallusia mammillata and Tail Nerve CordFormation”byMcDougalletal.,thepowerfulCRISPR-Cas9systemisappliedtothe ascidianPhallusiamammillataforthestudyofnervecorddevelopment.TheCRISPR-Cas9 systemisemployedtoknock-outspecificgenesofinterestandisappliedalongwithimaging techniques for visualizing the nerve cord. Ascidians are a valuable model system for com- bining cell and molecular biology methods for understanding cell specification and mor- phogenesisduringdevelopmentbecauseoftheir relativesimplicityandrapiddevelopment. Chapter 14, “Transcriptomic Analysis in the Sea Anemone Nematostella vectensis” by Warner and Ro¨ttinger, is a companion chapter for Chapter 4. In this chapter, recent procedures for generating high-quality RNA for next generation sequencing are presented alongwithmethodsforquantificationofthisdata.TheseaanemoneNematostellavectensisis beingdeveloped as amodelsystemfor studyingembryonicdevelopment andtissue regen- eration at the multiple experimental levels. This organism shares similarities with mamma- liangenomesandhaslongbeenusefulforevolutionarydevelopmentalstudies. InChapter15,“RNAInterferenceonRegeneratingHolothurianGutTissues,”Alicea- Delgadoetal.describeausefultransfectionprotocolfortheintroductionofDicer-substrate interferenceRNAsintotissueexplantsforthestudyofgutregeneration.Thisaddsamuch- needed protocol to the toolkit of echinoderm biologists and expands the use of this technologypastembryosandintoadultanimals. Chapter16,“ATAC-SeqforAssayingChromatinAccessibilityProtocolUsingEchino- dermEmbryos”byMagrietal.,describesanovelmethodtodeterminechromatinaccessi- bilitycalledAssayforTransposase-AccessibleChromatinwithhigh-throughputsequencing (ATAC-seq). This allows for identification of putative cis-regulatory elements for under- standingtheregulationofgeneexpression.Themethodhasbeenspecificallyadaptedforuse inechinodermsandwillbeextremelyusefulforunderstandingtheregulationofgenesthat havecomplicatedtemporalandmorphologicalexpressionpatternsduringdevelopment. viii Preface In Chapter 17, “Usage of the Sea Urchin Hemicentrotus pulcherrimus Database, HpBase” by Kinjo et al., an overview of the very useful HpBase (http://cell-innovation. nig.ac.jp/Hpul/) is provided along with a description of some of the useful analysis tools included. HpBase provides both genome and transcriptome resources for the sea urchin Hemicentrotuspulcherrimus.Inadditiontotheseresources,HpBasealsopresentsprotocols for biological experiments that have been assembled using H. pulcherrimus. This resource shouldbevaluableforinvestigatorsinawiderangeoffields. Chapter 18, “Functional Genomic Studies of Trichoplax adhaerens Neuronal Activity” by Gauberg et al., details methods for studying cellular and molecular activities in the placozoan Trichoplax adhaerens. Trichoplax has been generating a lot of interest recently becauseofitspositionasabasalmemberoftheanimalkingdomanditsarrayofinteresting behavioral responses to environmental stimuli. Great progress has been made on the developmentofcellularandmolecular methodsforstudyingthisbehavior. InChapter19,“ABioinformaticsTutorialforComparativeDevelopmentGenomicsin DiverseMeiofauna,”Sevignyetal.provideadetailedbioinformaticstutorial,withaspecific focusongenomeassembly,genomecomparison,andthecharacterizationofHoxclustersin meiofaunalspecies.Thesemethodswillbebroadlyapplicabletomanydifferentminiaturized benthicinvertebrateswhicharenotyetwellrepresentedinthetypicaldatabases,butwhich willbeveryusefulforaddressinguniquebiologicalandevolutionaryquestions. Glendale,AZ DavidJ.Carroll Albuquerque,NM StephenA.Stricker References 1. DarwinC(1852–1854)Amonographonthesub-classCirripedia,withfiguresofallthespecies,vol 1,2.TheRaySociety,London 2. DarwinC(1859)Ontheoriginofspeciesbymeansofnaturalselection,orpreservationoffavoured racesinthestruggleforlife.JohnMurray,London 3. RosenthalET,HuntT,RudermanJV(1980)SelectivetranslationofmRNAcontrolsthepatternof proteinsynthesisduringearlydevelopmentofthesurfclam,Spisulasolidissima.Cell20:487–494 4. EvansT,RosenthalET,YoungblomJ,DistelD,HuntT(1983)Cyclin:aproteinspecifiedbymaternal mRNAinseaurchineggsthatisdestroyedateachcleavagedivision.Cell33:389–396. 5. Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR (2012) Marine natural products. Nat ProdRep29:144–222 Contents Preface ..................................................................... v Contributors................................................................. xi 1 LaboratoryCultureandMutagenesisofAmphioxus (Branchiostomafloridae) ................................................. 1 LindaZ.HollandandGuangLi 2 HandlingandManipulationofGametesandEmbryosof theAnnelidanWormPseudopotamillaoccelata.............................. 31 RyusakuDeguchiandTakeshiNakano 3 TheStarfishAsterinapectinifera:CollectionandMaintenance ofAdultsandRearingandMetamorphosisofLarvae........................ 49 NaoyukiMurabe,Ei-ichiOkumura,KazuyoshiChiba EnakoHosoda,SusumuIkegami,andTakeoKishimoto 4 ExperimentalToolstoStudyRegenerationintheSea AnemoneNematostellavectensis........................................... 69 AldineR.AmielandEricR¨ottinger 5 StainingandTrackingMethodsforStudyingSponge CellDynamics.......................................................... 81 CaroleBorchiellini,SandieM.Degnan,EmilieLeGoff CarolineRocher,Ame´lieVernale,StephenBaghdiguian NinaSe´journe´,FlorentMarschal,Andre´LeBivic,NellyGodefroy BernardM.Degnan,andEmmanuelleRenard 6 MicroscopyStudiesofPlacozoans......................................... 99 CarolynL.Smith,TatianaD.Mayorova,ChristineA.Winters ThomasS.Reese,SallyP.Leys,andAndreasHeyland 7 IdentificationofSH2Domain-MediatedProteinInteractions thatOperateatFertilizationintheSeaStarPatiriaminiata ................. 119 LaurenBates,EmilyWiseman,JamieKitson, andDavidJ.Carroll 8 MarineNemerteanWormsforImmunoblottingStudies ofOocyteAging........................................................ 137 StephenA.Stricker 9 RecoveryofSeaStarEggCellSurfaceProteinsReleased atFertilization.......................................................... 151 EmilyWiseman,LaurenBates,andDavidJ.Carroll 10 QuantifyingCellProliferationDuringRegenerationof AquaticWorms......................................................... 163 EduardoE.ZattaraandB.DuyguO¨zpolat ix
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