Developmental Biology of the Sea Urchin and Other Marine Invertebrates: Methods and Protocol

316 Pages·2020·9.582 MB·English

by  David J. Carroll, Stephen A. Stricker

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
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JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
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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)
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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