114 *Author ([email protected]) for correspondence Pennsylvania, Philadelphia,PA Medicine, University of 19104,USA. School of CellandDevelopmental Biology, of Program,Department Perelman epigenetic innatureandarethe subjectofintenseinvestigation, sure thatlineage-committedcells remaintruetotheirlineage,are maintain cellidentityovermultipleroundsofdivision,making (Bonasio etal.,2010a).Specifically, themolecularmechanismsthat give risetodifferent originatingfromthesamegenotype responsible forthemaintenanceofdiscretetranscriptionalstates that 1A,B).Inthelattercase,epigeneticinheritanceis mitosis (Fig. (Heard andMartienssen,2014),butitalsooccurscommonlyduring to thephenomenonoftransgenerationalepigeneticinheritance take placeduringmeiosisandaffect multiplegenerations,givingrise sequence (Bonasioetal.,2010a;Riggs1996). of phenotypictraitsthatoccurswithoutchangesinthe DNA for theremainderofthisreviewIwilltakeittomeaninheritance epigenetics hasevolvedand,withoutdwellingonthecontroversies, is called‘developmentalgenetics’.Inthemeantime,meaningof development (Waddington, 1942);thatdisciplineexiststodayandit that wouldstudythemechanisticactionofgenesincontext 2007). Waddington wishedtoestablishanewbiologicaldiscipline (Berger etal.,2009;Bird,2007;Bonasio2010a;Ptashne, and debatethatwouldstillsurroundthiswordalmost75yearslater (Waddington, 1942)hecouldnothavedreamedofthecontroversy When ConradH.Waddington coinedtheterm‘epigenetics’ KEY WORDS:Chromatin,Epigenetics,Genomics,Polyphenism becoming accessibletomolecularapproaches. systems, includingsocialinsects,fishandflatworms,are non-model organismsthathaveemergedaspotentialexperimental investigation. Here,Ireviewexamplesofepigeneticphenomenain and non-modelorganisms,openingnumerousnewavenuesfor and genomeeditingtoolshavebeguntoblurthelinesbetweenmodel molecular level;however, recentprogressonsequencingtechnology organisms andhavesofarbeenonlysporadicallyinvestigatedata examples ofepigeneticplasticitylieoutsidetherealmmodel including yeast,flies,plantsandhumans.Manymorefascinating history ofchasingdiscoveriesinavarietymodelorganisms, these reasonschromatinbiologyandepigeneticresearchhavearich conserved molecularpathwaysthatconvergeonchromatin.For phenomena canbetracedbacktoevolutionaryadaptationsoffew of theinherentflexibilityepigeneticregulation,avarietybiological stable adaptationtotheirphysicalandsocialenvironment.Because in allbranchesofthetreelifebyorganismsthatrequireplasticbut differentiation andtranscriptionalmemory, theyarealsowidelyused single .Althoughtheseprocessesareessentialtocellular Epigenetics studiestheemergenceofdifferent phenotypesfroma Roberto Bonasio* The expandingepigeneticlandscapeofnon-modelorganisms REVIEW © 2015.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2015)218,114-122 doi:10.1242/jeb.110809 Introduction ABSTRACT It isimportanttonoteattheoutsetthatepigeneticinheritancecan might belosingsightoftheforestfortrees. and thatbytryingtomakethemnarrowermoreaccuratewe testimony tothefactthatourdefinitionsareoflittleconsequence advantage oftheepigeneticmachinery, asreviewedbelow, isa could notexistwithoutthem. mechanisms, theyarefundamentaltobiologyandmulticellularlife Regardless ofwhatwechoosetocalltheunderlyingmolecular established aftercelldivisionand/ororganismal reproduction. often forlongperiodoftimes,and,onoccasion,mustbere- phenotypic statesmustpersistaftertheinitialstimulushassubsided, phenotypic responsestochangingenvironments.Someofthese previously (Nanney, 1958).Cellsandorganisms needtostabilize concept suchasepigeneticsmightbemisdirected,stated investigation, theenergy spentintheattemptstodefineanabstract term. which, Isuspect,isthecruxofcontroversysurroundingthis slippery slope,wherebyanyformofgeneregulationisepigenetics, broader usageof‘epigenetics’ (Bird,2007),althoughitcreatesa involved. Somemolecularbiologistspreferstoincludethisina following generation,butthesamemolecularmachinerymightbe 2012). Inthiscase,nobiologicalinformationistransmittedtoa mitotic cells(Fig. participate inthestabilizationoftranscriptionalpatternspost- mitotic andmeioticinheritanceofepigeneticstatesmightalso confusingly –thesamemolecularpathwaysthatareresponsiblefor 2009; DawsonandKouzarides,2012).Finally–most cancer treatmentandregenerativemedicine(AmabileMeissner, develop fromasinglefertilizedeggandholdgreatpromisefor because theyunderpintheabilityofmulticellularorganisms to form adiscoidstructure aroundwhich147 copies eachoffourshortpolypeptides knownashistones,which (Goll etal.,2005). by enzymesoftheDNMT1family, explainingitsepigenetic nature DNA methylationiscopiedontonewlysynthesizedstrandsofDNA properties ofaDNA sequence,toregulateitsfunction(Bird,2002). to cytosineresiduesineukaryotesandcanalterthebinding and Pugh,1975).Thisisachemicalmodificationthatrestricted to bediscoveredwasDNA methylation(Holliday, 1987;Holliday 2).The firstepigeneticmark information atthemolecularlevel(Fig. features ofchromatinhavethepotentialtoencodeepigenetic complex ofDNA,RNA andproteinscalled‘chromatin’.Several is comprehensibletoabroadrangeofbiologists. introduce relevantterminologysothattheremainderofthisreview et al.,2010a;Margueron and Reinberg, 2010).Here,Iwillsimply to themanyreviewsthathavebeenpublishedontopic(Bonasio information isencoded,storedandutilizedbythecellshould refer The readerinterestedinthemoleculardetailsofhowepigenetic Molecular epigenetics The varietyofbiologicalphenomenainwhichevolutionhastaken Although preciseterminologyisessentialtoscientific The basicunitofchromatin,the nucleosome,iscomposedoftwo The eukaryoticgenomeispackagedinsidethenucleusinto a

1C), suchas,forexample,neurons(Bonasio,

bp ofDNA arewrapped

The Journal of Experimental Biology of noncodingRNAs (ncRNAs),someofwhich appeartofunction that hasbeenreportedinmost genomes givesrisetoawidevariety increasingly clearthatthelarge amountofnoncodingtranscription signals byhistonereaders(Ruthenburg etal.,2007b).Itisbecoming combination ofdifferent histonemarksareinterpretedas separate histone marksrecognizesits targets inthegenomeand howa understand howthecomplex machinerythatwritesanderases (Mersfelder andParthun,2006). function ofthisgroupmarksisnotaswellunderstood globular domainofhistones,but,withfewexceptions,thebiological downstream functionalconsequences.PTMsarealsofoundon the or histone‘readers’ (Ruthenburg etal.,2007a), thatmediatetheir are believedtoformbindingsurfacesforchromatin-bindingfactors, properties ofchromatin(Marmorstein,2001),butbyandlarge they acetylation, canfunctionbydirectlyfine-tuningthebiophysical advent ofgenome-widetechnologies.Histonemarks,especially (Allfrey etal.,1964),andevenmoresoinrecentyears,after the research hasbeenincreasinglyfocusedeversincetheirdiscovery These aretheso-called‘histonemarks’ onwhichmostchromatin methylation andothers(Kouzarides,2007;Margueron etal.,2005). modifications (PTMs),suchasacetylation,phosphorylation, and areavailabletoenzymesthatplacepost-translational have disorderedN-andC-terminaltailsthatprotrudefromthedisc (Luger etal.,1997).Inadditiontotheglobulardomain,histones Fig. REVIEW of interesttoepigeneticresearch. mechanisms, becauseofthesharedmolecularfeaturestheyarenonetheless Although theinheritance-baseddefinitionofepigeneticswouldexcludesuch transcriptional patternsinterminallydifferentiated, non-dividingcells. transmit informationthroughoutcelldivisionareusedtostabilize remains unknown.(C)Incertaincasesthesameepigeneticsignalsthat the natureofepigeneticsignalingermcells(yellowflag,middle) mouse. Inmanycases,althoughtheepigeneticstateisvisiblytransmitted, Several casesareknowninplantsandahandfulvertebratessuchas transmission ofinformationthatdoesnotresideintheDNA sequence. modifications. (B)Meioticepigeneticinheritancecontrolstransgenerational fashion includeDNA methylationand,tosomeextent,certainhistone division inMphase(rightcells).Epigeneticmarksthatcanbeinheritedthis throughout DNA replicationinSphase(middlecell)andthroughoutcell controls thereplicationofepigeneticmarks(yellowflagonchromosome) A centralgoalofcurrentresearchinchromatinbiologyisto C B A

.Types ofepigeneticmemory. 1. (A) Mitoticepigeneticinheritance and trxGfunction, respectively. H3 lysine27(H3K27me)and H3K4me3, whicharelinkedtoPcG intensely studiedhistonemarks, suchasthemethylationofhistone act onchromatinbyeitherplacing orrecognizingsomeofthemost complexes containingtheproducts ofthesegenesrevealedthatthey Paro, 2004).Thebiochemical characterizationoftheprotein maintenance ofcellidentityand embryonicpatterning(Ringroseand which wereshowndecadesagotomediatetheepigenetic belonging tothe established in link betweenepigeneticphenomenaandhistonemarkswas first product actedonchromatin(Schottaetal.,2003).Anotherstrong of variegationledtotheidentificationseveralgeneswhose underlying genotype.Geneticscreensforenhancersandsuppressors variegated manner, givingrisetomosaicism despiteahomogeneous of thechromosomeasaresulttranslocationisexpressed ina a genethatisrepositionednearrepressedheterochromaticregions double-helix ofdoublehelices(Songetal.,2014). a recentstudythatreportedthestructureof30 continued progressinthedescriptionofchromatinfiber, suchas conformation captureapproaches(Dekkeretal.,2002)and things arelikelytochangethanksthedevelopmentof challenging, becauseofalackquantitativeapproaches;however, possibly, epigeneticinheritance.Researchinthisfieldhasbeen expression (FraserandBickmore,2007;Misteli,2007)and, also beenlongspeculatedtoparticipateintheregulationofgene Cremer, 2001;Dixonetal.,2012;Phillips-Cremins2013),has topological domainsandchromosometerritories(Cremer looping (Kageyetal.,2010)andinthelongrange,suchas chromatin, bothintheshortrange,suchasenhancer-promoter press; CastelandMartienssen,2013;RinnChang,2012). modifiers toactontherelevantgenes(BonasioandShiekhattar, in as recruitmentorregulatorysignalsthatdirectthesechromatin repressed chromatin,respectively. (left) versusdense,compact(right)chromatincorrespondtoactiveand structures mightalsobevehiclesofepigeneticinformation.Inthiscaseloose methylation (me)orhistonePTMs(yellowflags).(B)Higher-orderchromatin information canbeencodedbychemicalchangestotheDNA,suchas into nucleosomes.(A)InadditiontotheDNA sequence,biological composed ofDNA (redline)packagedaroundhistoneoctamers(bluediscs) Fig. positional effect variegation in to bedueepigeneticeffects (Allisetal.,2007).Firstcame of observabletraitsmadeinvariousmodelorganisms allturnedout that disparateobservationsofunexpectednon-mendelianinheritance different modelorganisms asepigeneticshas.Thereason mightbe Few fieldsofmolecularbiologyhaveseensuchawidespreaduse Epigenetics inmodelorganisms B A Finally, thehigher-order, three-dimensionalorganization of

.Molecularencodingofepigeneticinformation. 2.

me The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110809 Drosophila Polycomb and originatedfromthestudyofgenes group (PcG)andtrithorax Drosophila PTM (Muller, 1941),whereby Chromatin is

nm fiberasa group (trxG), 115

The Journal of Experimental Biology transmission ofepigeneticinformationin of silencingwasonethefirstformaldemonstrations segregation ofthishistonemarkandthecorresponding a hallmarkofconstitutiveheterochromatin,and,infact,meiotic Gasser, 2013).However, S.pombe maintain silencingatheterochromatinregions(Grunsteinand complex networkofhistoneacetylationanddeacetylationto histone markscommoninplantsandmetazoans,butstillutilizesa DNA methylationandtheenzymes involvedinseveralofthe 2007). subtelomeric sequencesandthematingtypelocus(Allisetal., regions thatmustbekeptsilentinmostcircumstances,notably seem tohavelittleneedforepigenetics,theyinfactcontaingenomic Although thesearemonocellularorganisms that,atfirstsight,would also contributedtoourunderstandingofepigeneticphenomena. prevalently inmammals. imprinting (LeeandBartolomei,2013),havebeenstudied phenomena, suchasXchromosomeinactivationandgenomic quickly becomethecellsystemsofchoice.Severalepigenetic (Takahashi etal.,2007;Takahashi andYamanaka, 2006)]have derived fromdifferentiated cellsbyepigenetic‘reprogramming’ humans, aswellinducedpluripotentstemcells[pluripotent this, embryonicstemcellsfrommiceand,morerecently understanding generegulationandcellidentityinmammals.For mechanism (GrewalandKlar, 1996). 116 REVIEW and possiblyother epigeneticsignalsappearto havededicatedroles development ofallotherorgans. However, chromatinmodifications epigenetic pathwaysofgene regulationthatcontributeto brain isacomplexorgan anditsontogenymustaccessthe same in suitablemodelorganisms. modeled incellcultureandarenot alwaysavailableforinvestigation regulation oforganism-level phenotypesthatcannotbeeasily of theseepigeneticpathwaysareincreasinglyimplicatedin the contributed somuchtoprogressinthefield.However, components investigated inthesamemodelorganisms thathavealready natural thatsomeofthesespecific,mechanisticquestionsshould be species, ncRNAsofvarioussorts(Bonasioetal.,2010a).Itseems include histonemarks,DNA methylationand,atleastinsome controversy, althoughfewwoulddoubtthatproximatesignals cases theidentityof‘true’ epigeneticsignalremainsamatterof organisms, manymorequestions remainunanswered.Inseveral Despite thesefundamentaldiscoveriesinepigeneticsmademodel Saccharomyces cerevisiae plants. chromatin changes,havebeendissectedinmuchmoredetail including transgenerationalinheritanceandRNA-mediated that areonlynowbeginningtobeunderstoodinmammals, adapt andsurvive.Thismightexplainwhyepigeneticphenomena relocation, areparticularlydependentonepigeneticmechanismsto detrimental changesintheirsurroundingenvironmentbyphysical In fact,itcanbeargued thatplants,lackingtheabilitytoescape hasbeencrossedoutforseveralgenerations(Chandler, 2007). present intheancestorsaremanifestprogeny, evenafterthe classic exampleofepigenetics,becausetheeffects ofacertainallele paramutation inmaize,firstdescribed1958(Brink,1958)isa particularly maizeand Open questionsinepigenetics Finally, epigeneticresearchhasmadegreatstridestoward One exampleisbrainfunctionand behavior(Bonasio,2012).The The mosttraditionalmodelorganism ofall,theyeasts Much insightintoepigeneticshascomefromthestudyofplants, S. cerevisiae has lostmostepigeneticmachinery, including Arabidopsis thaliana and Schizosaccharomyces pombe has retainedH3K9methylation, cis . Thephenomenonof by achromatin-based have their environment. variety oflifeformsthatuseepigenetic mechanismstobetterfitin genomic andgenetictoolsjustifiestakingasecondlookat the word for‘monster’ (Gustafsson,1979).Thegreatprogressin of thetoadflax Linnaeus’ horrifiedreactionwhenfirsttoldaboutepigeneticvariants disqualified organisms from beingselected,asevidencedby epigenetic effects onvisible phenotypeswouldhaveprobably fast generationtimes.Asamatteroffact,evidencemajor homogeneous andstablephenotypes,easeofcultureincaptivity and which wereselectedforspecificcharacteristics,including common innature,justnottheconventionalmodelorganisms, to fullyenvision. learning andbehavior, withramificationsthatarecurrentlydifficult a paradigmshiftinthewaywethinkaboutepigeneticinheritance, widespread anditsmolecularmechanismidentifieditwouldcause function andbehavior. Ifsucha phenomenonwerefoundtobe Ressler, 2014)hasrenewedinterestintheepigeneticsofbrain might betransmittedtosubsequentgenerationsofmice(Diasand specifically fear-conditioning inresponsetoanolfactorystimulus, and alsocontroversial–observationthatsomepaternalexperiences, and affect thephenotypeofawholeneworganism? The recent– content andlong-termstabilitytobetransmittedintothegermline features otherthantheDNA sequence hastherequiredinformation 2014). Howwidespreadisthisphenomenon?Whatmolecular organisms aretheexceptionorrule(HeardandMartienssen, unclear whetherthefewcasescharacterizedinothermodel Although examplesofthisphenomenoninplantsabound,itremains stop orevenreversetheclock(RandoandChang,2012). understanding therootcausesofthisdeteriorationmightallowusto clock isbyandlarge drivenbyepigeneticdeterioration,andthat germline isvirtuallyimmortal,ithasbeenproposedthattheaging in one ofthefewhistonemodifiersresponsibleforepigeneticsilencing longevity, theso-called‘sirtuins’,aredeacetylasesrelatedtoSir2, controversy (Guarente,2013)–onthemolecularregulationof the mammalianenzymesatcenterofmuchresearch–and pathways inregulatinglongevity. Itcanhardlybeacoincidencethat (Wang etal.,2013). the developmentoflong-lastingrelationshipsamongindividuals learning andmemory(Dayetal.,2013;Gupta2010),even histone acetylationandpossiblymethylationarerequiredfor DNA methylation.Increasingexperimentalevidencesuggeststhat for higherbrainfunction.Buttheconnectionsarenotrestrictedto with theneedforlong-termstabilityofneuronalstatesnecessary potential forlong-termmemoryoftranscriptionalstatesresonates dynamically tobrainactivity(Guoetal.,2011), andbecauseits connection mightbeDNA methylation,becauseitslevelsrespond functional –ratherthandevelopmentalrole.Theculpritforthis intheadultbrain(Luikenhuisetal.,2004),suggestinga MeCP2 itwasdemonstratedthatthegenecontributesto and atleastinthecaseofmethyl-cytosinebindingprotein cause neurologicalphenotypes(JakovcevskiandAkbarian,2012), in theadultbrain.Mutationsseveralknownepigeneticregulators known aspolyphenisms (West-Eberhard, 2003).Polyphenismis that allowdifferent phenotypestoarisefromasinglegenotype, Another wayofdefiningepigenetics istoreferthoseprocesses Developmental andphenotypicplasticity Cases ofepigeneticregulationwhole-organism phenotypesare A thirdandfinalexampleisthatof transgenerationalinheritance. Another exampleisoffered bythepotentialroleofepigenetic S. cerevisiae The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110809 Linaria vulgaris,whichhenamed (Houtkooper etal.,2012).Consideringthatthe Peloria, theGreek

The Journal of Experimental Biology context isthatmany that ismaintainedepigeneticallyforthelifeofindividual. developmental trajectory, whichresultsinadifferent adult phenotype signaling cascaderesultsintheactivationofanalternative predator onlyappearsbrieflyduringembryonicdevelopment, a developmental plasticitycontrolledbyexternalstimuli.Evenifthe exposure duringthefirstinstarsandisthereforeacaseof has notbeentested,itsdevelopmentcanbeinducedbytransient Dodson, 1999).Althoughthetransgenerationalinheritanceofthistrait protrusions intheneckregion,knownas‘neckteeth’ (Tollrian and induces thedevelopmentofastrengthenedexoskeletonandsmall changes arerecorded intheepigenomeandaffect futuregenerations. waters mightrevealnewmechanisms bywhichenvironmental comparisons ofthe although onlyonelocuswas considered.Future,broader transgenerational epigenomicdamage (Vandegehuchte etal.,2009), methylation uponexposuretopolluting agentsfoundnoevidenceof questions. A pioneeringstudythatanalyzedchangesinDNA constitute animportantmodelsystemtoaddresssomeof these and continuousexposuretotheaquaticenvironment, investigation invertebratebiology. With itsfastergenerationtimes the individual,butalsoitsdescendantsisasubjectofintensive environmental exposurealterstheepigenomeandaffects notonly in aquatictoxicology(Martinsetal.,2007).Thepossibility that epigenetic generegulation. molecular basesin other animalsandplants(Agrawaletal.,1999),studyingtheir given thatsimilarinduceddefensemechanismsarealsoobservedin of bonafidetransgenerationalepigeneticinheritance.Nonetheless, exposure oftheF1germlineduringembryonicdevelopmentandnot suggests thatthemodifiedphenotypemightbeaconsequenceof discussed byBurggren inthisspecialissue(Burggren, 2015)– effects –whichmaybeanexampleoftheepigenetic‘washout’ (Agrawal etal.,1999).However, thefactthatF2showedsmaller in subsequentgenerationsnotdirectlyexposedtothestimulus (Fig. development ofaprotectivecranialextensionknownas‘helmet’ presence ofchemicalsreleasedbypredatorsinthewatercauses exposure topredators(Tollrian andDodson,1999).Forexample,the exposure tocertainstimuliduringdevelopment. can nonethelessdisplaydiversephenotypesdependingontheir parthenogenetic individuals,whicharegeneticclonesofthemother, reproduce bothsexuallyandasexuallythefactthat biology thatmakeitvaluabletoepigeneticistsareitsability arena ofmolecularepigeneticresearch.Thekeyaspects (Colbourne etal.,2011), markingtheentryofthisorganism intothe 2012) butaDaphnia research hasbeenadvocatedbefore(Burggren, 2014;Harrisetal., fascinated biologistsformorethanacentury. Itsuseforepigenetic Daphnia fashion (typicalofepigeneticchanges)asitsenvironmentchanges. allowing anindividualtochangeitsphenotypeinametastable phenotypes intheadultindividuals(morphs),oritcanbeplastic, originate duringdevelopmentandgiverisetodistinct,stable arthropods and,tosomeextent,invertebrates.Polyphenismscan eusocial insects,asdiscussedbelow, butitisalsofoundinother particularly commonininsectsandbroughttoanextremeby REVIEW Epigenetic metamorphosesofthewaterflea A finalaspectof In otherDaphnia One specificcaseistheinductionofdefensestructureby 3A). InDaphniacucullata is agenusoffreshwatermicrocrustaceansthathas species, exposuretopredator-derived chemicals Daphnia Daphnia Daphnia Daphnia genome wassequencedonlyrecently epigenetics worthmentioninginthis might revealcommonparadigmsin species areimportanttestorganisms methylomes incleanandpolluted , thesestructuresarealsoobserved Daphnia Daphnia Daphnia might change (Fig. pseudomales, andinthiscase withoutrequiringthetemperature chromosome fromthe‘pseudofather’ alsodevelopinto ‘pseudomales’. Interestingly, ZW offspring thatreceive theZ induce juvenileZW femalestodevelopintofertile ZW based onZZandZW chromosomepairs.Changesintemperature reversal pathwayinadditionto its geneticsex-determinationsystem chromosomes, thisfishspeciesretainedanenvironmental sex- 2014). Possiblybecauseoftherelativelyrecentorigin sex ancestral autosomepairstarting~30 and revealedthatitsZW sexchromosomesevolvedfroman cultured inChina.Thegenomeofthisfishwasrecentlysequenced semilaevis, acommerciallyimportantflatfishthatisextensively example isthatofthehalf-smoothtonguesole insects, fishes,amphibiansandreptiles.Oneparticularlyinteresting common throughoutthetreeoflife,affecting severalspeciesof most egregiousisthecaseofsexreversal,whichsurprisingly Among themanytypesofadultphenotypicplasticity, oneofthe Fig. half-smooth tonguesole in latergenerationsdespitetheabsenceoforiginatingstimulus.(B)In defensive structurecalleda‘helmet’.Thepresenceofthehelmetcanpersist F1 (productsofparthenogenesis)presentdifferences intheformofa predator-derived chemicals(kairomones).Inthiscase,geneticallyidentical alternative phenotypescanemergeasaconsequenceofexposureto Epigenetic sexreversalinthetonguesole sex conversion. if thetemperatureremainslow, suggestinganepigenetictransmissionofthe ZW progenyofthesepseudomaleswilldevelopintomoreeven phase induceconversionofZWfemalesto‘pseudomales’.Interestingly, the W chromosome(female).However, highertemperaturesduringthejuvenile determined bythepresenceoftwoZchromosomes(male)oroneand B

.Examplesofepigeneticplasticityin development. 3. A 22ºC The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110809 3B). Inotherwords, anenvironmentalstimulus causes ZW female ZZ male 22ºC Cynoglossus semilaevis

ZZ male Predator No predator

million yearsago(Chenetal., 1F2 F1 , sexisgenetically ZW pseudomale ZW pseudomale No predator ZZ male 28ºC (A) InDaphnia Cynoglossus 117 ,

The Journal of Experimental Biology 118 REVIEW Astatotilapia (Haplochromis) burtoni behavior (polyethism)isseenintheAfricancichlid fish dramatic caseofpolyphenismsgivingrisetoalternativesocial serve asversatilemodelstostudytheepigeneticsofbehavior. One With theirwidevarietyofbiologyandethology, fishmightalso processes inotherspecies. regulation ofdosagecompensationmight,ineffect, prohibitsuch chromosomes, becausethedifficulties ofrevertingtheepigenetic sex reversalismorecommoninspecieswith‘young’ sex be interestingtoanalyzewhethertheoccurrenceofenvironmental a completecompensatorymechanism(Shaoetal.,2014).Itwould suggesting thatthese‘young’ sexchromosomeshaveyettoevolve between malesandpseudomales,exceptforanarrow2 incomplete betweenmalesandfemalesalmostnon-existent the tonguesole.Initialstudiesshowedthatdosagecompensationis regulated duringaphenotypicsexreversalsuchastheonefoundin two sexes,raisingthequestionofhowdosagecompensationcanbe machinery, ascouldbeexpected,aredeployeddifferentially inthe (Augui etal.,2011). Thecomponentsofthedosage-compensation such as,forexample,Xchromosomeinactivationinmammals in geneticdosageiscompensatedbycomplexepigeneticprocesses, in birds)butonlyonecopytheheterogameticsex.Thisdifference the homogameticsex(e.g.XXfemalesinmammalsandZZmales the commonsexchromosome(ZorX)arepresentintwocopies compensation. Inspecieswithdifferent sexchromosomes,geneson epigenetic conundrum,whichishowtodealwithdosage reversal mightprovidedeeperinsightinthisprocess. juveniles beforeandafterthetemperatureshiftthatcausessex comparison ofgeneexpressionandDNA methylationprofilesin developed organisms wereanalyzed(Shaoetal.,2014).A obvious epigeneticculpritsforthesexreversal,butonlyfully full surveyofthetonguesole’s DNA methylomefailedtoreveal causal roleinthisenvironmentalsexreversal(Chenetal.,2014).A Zarkower, 2012;Smithetal.,2009),correlatewellwithapossible roles insexdeterminationchickenandotheranimals(Matson transcription andDNA methylationatthe astounding phenotypicplasticityremainundefined,changesin et al.,2014). development ofphenotypicmalesdespiteafemalekaryotype(Chen in anepigeneticmannertothenextgenerationandthatresult changes intheZchromosomeofZW individualsthataretransmitted Social interactionsandbrainepigeneticsincichlidfish fully reversible: removalofthesuper-dominant malewillcausethe 2 changes inbehaviorandphysiology, whichculminate, about as earlyafewminutesafterexposure. Theseareaccompaniedby dominant phenotype,withvisible changesincolorationappearing ‘super-dominant’ fish,formerlydominant malesswitchtothenon- sequences withinthegenome.In fact,whenpresentedwithalarger, categories, thedistinctphenotypesarenotencodedindifferent DNA 2012). compared withthedullcolorationofnon-dominants(Fernald, phenotypic traits,suchasthebrightcolorationofdominantmales physiology, suchassmallertestesinnon-dominantmalesandvisible differences inreproductive behavior areaccompaniedbydistinct population) andanon-dominantmalethatdoesnotreproduce. The and isreproductivelycompetent(about10–20%ofthe male interchangeable, phenotypes:adominantmalethatownsterritory

weeks later, inthelossofreproductivecompetence.Thisswitchis In thecaseoftonguesole,sexreversalposesyetanother Although themolecularmechanismsresponsibleforthis Despite thedramaticdifferences betweenthesetwosocial . MaleA.burtoni dmrt1 locus, whichhas exist intwo,

Mb region, individuals and because, atleastinmostspecies, castesarenotpre- Genoud, 1997). a fewmonths(Jemielityetal., 2005;Keller, 1998;Kellerand queen antis28.5 example, queenslivelonger;the longestdocumentedlifespanofa queens andworkersarenotlimited toreproductivebiology. For (HölldoblerandWilson, 1990). The differences between hymenoptera androyalcouples(kingsqueens)inthecase of few well-protectedindividuals:queensinthecaseofhaplodiploid construction, cleaning),withreproductiverightsrestrictedtoone or themselves withcolonymaintenance(foraging,defense, Typically, mostindividualsarefunctionallysterile andoccupy within thecolonies,whichrequiredistinctsetsofbehaviors. and physiologiesand,mostimportantly, carryingoutdifferent tasks categories ofadultindividuals(castes)withdifferent morphologies and Wilson, 1990).Theseinsectcolonies are composedofdifferent as theyarefoundedontheprincipleofdivisionlabor(Hölldobler pronounced polyphenismsamongtheirmembers(Bonasio,2014), (, termites,somebees)dependheavilyontheexistenceof animals belongtothisgroup.Indeed,coloniesofeusocialinsects insect worldmightexplainwhythelarge majorityofeusocial reviewed elsewhereinthisissue(Ernstetal.,2015). constructed, andtheoutstandingregenerativecapacityofplanarians. and polyethismsuponwhichthesocietiesofantsbeesare regulation ofthegenome:specifically, thepronouncedpolyphenisms two specialcasesthatstandoutasextremeusesofepigenetic for epigeneticresearchinnon-modelorganisms. Next,Iwillpresent The examplesdiscussedherearemeanttowhetthereader’s appetite social environmentinshapinggenomicoutput. anticipate thatitwillprovidemuchneededinsightontheroleof repertoires, andwithitsgenomenowavailable(Fernald,2012),we pathways instabilizingalternativebrainstatesandbehavioral burtoni place insociety. Thus,themetastable switchinsocialstatus who justlost(orwon)aconfrontationdoesnotlosesightofitsnew more stablereorganization ofthetranscriptomesothatanindividual different (Maruskaetal.,2013a). males wereforcedtolosetheirstatus,butthegenesinvolved Interestingly, thesamebrain network wasactivatedwhendominant involved insocialbehaviorvertebrates(Maruskaetal.,2013b). called ‘socialbehaviornetwork’ composedofbrainnucleithatare genes inthebrain(Burmeisteretal.,2005),specificallyso- to dominanceisaccompaniedbytheactivationofimmediate-early the cellularandmolecularlevel,transitionfromnon- converted maletoreturnitsdominantstatus(Fernald,2012).At locusts suchasLocustamigratoria polyphenism andpolyethismarethephasetransitionsdisplayedby economically important–examplesofnon-eusocialinsect 2011). Iwillpointoutthatamongthemostimpressive–and reviews onthetopic(EvansandWheeler, 2001;Simpsonetal., many tolisthereandIreferthereaderseveralexcellent information. Thetypesandspecificcasesofpolyphenismaretoo within asinglespeciesand,therefore,fromsetofgenomic are alsospecialistsinexhibitingmultiplealternativephenotypes As oneofthemostdiversegroupsanimalsonplanet,insects Extreme epigenetics Extreme Polyphenism andpolyethismineusocialinsects Because ofthesepronounced differences betweenrelated The availabilityofsuchavarietypolyphenicblueprintsinthe These rapidchangesingeneexpressionmustbeaccompaniedby is anidealcandidatetounderstandtheroleofepigenetic The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110809

years, comparedwiththeworker’s lifespanofjust and Schistocerca gregaria , as A.

The Journal of Experimental Biology of successfultransgenesis in genomes, withmanymoreinthe pipeline,aswellthefirstreport this epigeneticpuzzle.Theavailability ofseveralantandbee eusocial insects,whichwillprobably provideadditionalpiecesof 2013). Moreover, westilllackacompleteannotationofncRNAsin castes ofthecarpenterant caste-specific patternsofH3K27 acetylationindifferent worker unveiling apotentialrolefortheacetyl-transferaseCBP inshaping chromatin from post-translational modifications:onereportingtheirpresence in been laggingbehind,withonlytwostudiespublishedonhistone thought. regulatory mechanismsmightbemorewidespreadthanpreviously reported inhumancells(Shuklaetal.,2011), suggestingthatthese similar casesofsplicingregulationbyDNA methylationhavebeen candidate (Foretetal.,2012;Li-Byarlay2013).Interestingly, alternative splicingbydifferential DNA methylationisapromising remain inlarge partmysterious, althoughtheregulationof (Kucharski etal.,2008).Thedownstreammechanismsforthiseffect development ofqueensfromotherwiseworker-destined larvae determination inthatinhibitionofDNMTs resultedinspontaneous DNMT activitywasreportedtobecausallylinkedcaste al., 2012;Lyko etal.,2010).At leastinthecaseofhoneybees, ants andcorrelatedwithcaste-specificgeneexpression(Bonasioet DNA methylationbetweencastes have beenobservedinbeesand et al.,2010b)andtermites(Terrapon etal.,2014).Differences in 2006), whichwasfollowedbysimilarobservationsinants(Bonasio methyltransferases (HoneybeeGenomeSequencingConsortium, unlike thatofDrosophila observation thatthegenomeofeusocialhoneybee (Bonasio, 2012).A boosttothisinterest wasprovidedbytheinitial social insectshasattractedtheattentionofepigeneticsresearch 4A),theworldof assigned toembryosviageneticmechanisms(Fig. Fig. REVIEW all organsoftheadultanimal,includingnervoussystem(4). create aregeneratingstructurecalledblastema(3),whicheventuallyrestores adult stemcellsknownasneoblasts(reddots)migratetothewoundsite(2), regenerate allbodytissuesandstructuresafteramputation(1).Pluripotent morphology, butalsoinphysiologyandbehavior. (B)Adultplanarianscan and non-reproductives(workers).Thesediffer notonlyinsizeand give risetoentirelydifferent adults(right);specifically, reproductives(queens) embryos (left)andlarvae(middle)aregeneticallyindistinguishable,butthey different developmentaldestiniesinthesamegenome.Inmostspecies Research onepigeneticpathwaysotherthanDNA methylationhas mroLarva Embryo B A

.Extremeepigenetics. 4. 1 Apis mellifera 2 , encodedorthologsforallclassesofDNA (A) Eusocialinsects,suchasants,encode Camponotus floridanus Apis (Dickman etal.,2013)andone (Schulte etal.,2014), hasopened 3 (Simola etal., Apis mellifera, 4 Queen Worke r vertebrate adultstemcells. context ofregenerationmight helpusunderstandtheirrolein Sánchez Alvarado,2014)andcharacterizingtheirfunctionsin the their depositionarestronglyconservedinplanarians(Robb and be expected,histonemarksandthemachinerythatisresponsible for regulation ofneoblastpluripotencyanddifferentiation. Asitwould methylation. is mediatedviatheNuRDcomplexandindependentof DNA therefore, itispossiblethattheeffect ofMBD2/3onregeneration in thesameNuRDcomplexvertebrates(Zhangetal.,1999); Zeng etal.,2013).TheorthologsofMBD2/3andCHD4arefound that theyexpress(Jaber-Hijazi etal.,2013;Regev etal.,1998; their genomesorevenontheclassesofDNA methyltransferases the factthatthereisnoconsensusonwhetherplanariansmethylate Hijazi etal.,2013).Thelatterobservationisinterestinginlightof binding proteinMBD2/3isalsorequiredforregeneration(Jaber- measurable amountsofmethylatedcytosines,themethyl-CpG al., 2013).Despitethefactthatplanariansdonotseemtocontain the H3K9methylationreaderheterochromatinprotein1(Zenget 2010), varioushistonemethyltransferase(Hubertetal.,2013)and requirements forthechromatinremodelerCHD4(Scimoneetal., to regenerate.Indeed,pioneeringstudieshavereported cells and,assuch,epigeneticpathwaysmustcontroltheirability adult animal(Wagner etal.,2011), theyarepluripotentadultstem Iglesias etal.,2008;PetersenandReddien,2007). heads inplaceoftailsattheirposteriorend(Gurleyetal.,2008; patterning duringregenerationandcausesdissectedanimalstogrow gene encoding al., 2005).Forexample,knockdownoftheplanarianversion genes requiredforneoblastfunctionandregeneration(Reddienet allowed forforwardgeneticscreensthathaveidentifiedseveral knockdown byRNA interference (RNAi)intheseorganisms the regenerationofmorecomplexstructures.Theeasegene and SánchezAlvarado,2004),whichisanecessaryprerequisitefor after dissectionandformastructureknownasblastema(Reddien neoblasts, apopulationofadultstemcellsthatpopulatethewound mechanisms ofregeneration(SánchezAlvarado,2006). propelled themtotheforefrontofresearchonmolecular ability toregenerate,planariansoffer severaladvantagesthathave study (Morgan, 1898).Althoughseveralothermetazoanshavethe interest toThomasHuntMorgan, whopioneeredtheirsystematic regenerative capacitiesinthe18thcenturyandwereofparticular scrutiny ofbiologistssincetheoriginalobservationtheir from minusculefragments.Planarianshavebeenunderthe stage, includingmissingorgans andcomplexstructures,starting the capacitytoregeneratelarge regionsoftheirbodiesattheadult 2004) (Fig. ability offreshwaterplanarians(ReddienandSánchezAlvarado, Another caseofextremeepigeneticsistheastoundingregenerating insects. fascinations: thesophisticated,organized societiesofeusocial the doorstomolecularresearchononeofbiology’s longstanding few researchers wouldhaveundertakenit.To seriouslyconsideran for molecularresearchwouldhave beendauntingtothepointthat A few yearsagotheendeavorofestablishinganewmodelorganism Regeneration inflatworms Emerging technologies tostudynon-model organisms Emerging These areonlythefirststepsindissectingepigenetic Given thatsingleneoblastscanregenerateallthestructurein Planarian regenerationreliesonthefunctionofproliferating The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110809 4B). Theseflatworms(phylumPlatyhelminthes)have β -catenin resultsindisruptedanterior–posterior 119

The Journal of Experimental Biology double-stranded breaksareuniversallyresolved eitherbynon- genome ofinterest(Congetal., 2013;Jineketal.,2012).Because genomic DNA,wasadaptedto introducethesecutsanywhereina which usesanucleasethatproduces double-strandedbreaksin grasped bymultiplegroupsand aparticularflavorofCRISPR, 2012). Theutilityofsuchapathway formolecularbiologywas sequences, whicharethencut anddegraded(Wiedenheft etal., guide RNAsrecruitnucleasestocomplementaryRNA orDNA evolved toprotectprokaryotesfromforeigngeneticmaterial.Small The CRISPR/Cassystemisamoleculardefensepathway that palindromic repeat)-mediatedgenomeediting(Congetal.,2013). by theadventofCRISPR(clusteredregularlyinterspaced short embryonic stemcells).Theseconsiderationshavebeenswept away happens atanunusuallyfrequentrate(e.g.yeastandmouse model organisms orcelltypes inwhichhomologousrecombination genetics, namelygenetargeting, wasonlyavailableinahandfulof necessarily large number of individualsinvolvedandreverse impossible bylonggenerationtimesordifficulty inhandlingthe in mostcasesmutagenesisandforwardgeneticsare made been therealobstacleinestablishingnewmodelsystems,because organism shouldbeamenabletogeneticmanipulation.Thishas desirable forroutineexperimentation. small-interfering RNAsbyindividualinjection,whichisclearlynot non-model organisms itrequiresdeliveryofdouble-strandedor However, inmostorganisms RNAiisonlytransientandinmany to manipulategenelevelsincellcultureandintactorganisms. known asRNA interference(RNAi), hasbecomeaninvaluabletool complementary sequence(Hannon,2002).Thisphenomenon, inhibiting thetranslationofmRNAswithstretches have theabilitytointerferewithgenefunctionbydegradingor have knownthatsmallRNA molecules (endogenousorartificial) second issue,interferingwithgenefunction.Sincethelate1990swe sequence) distance. annotated genomesofspeciesthatarecloserinevolutionary(and annotation, becomeincreasinglyfast,becausetheycanleanonthe limiting stepsof 2013). Infact,asmoreandgenomesarepublished,therate- groups, includinghelmints,birdsandinsects(i5KConsortium, tens, hundreds,eventhousandsofgenomesfromvariousorganism about $1000(Hayden,2014).Projectsareonthewaytosequence 120 REVIEW sequence ahumangenome(to30timescoverage)in3 Venter etal.,2001).Asof2014,thenewestinstrumentscan a roughdraftofthehumangenomein2000(Landeretal.,2001; and yearsofdedicatedworkbyhundredsresearcherstogenerate could nothavebeenimaginedjustadecadeago.Itcost$3billion revolution intheeaseandspeedofnucleicacidsequencingthat known asnext-generationsequencing)hasbroughtabouta decades. Times havechanged.Massiveparallelsequencing(also and onlythroughthetirelesslaborofcountlessscientistsover indefinitely. artificial crossesandpropagategeneticallymodifiedlines It mustbepossibletoraisetheorganism inthelaboratory, perform or transgenesisatleastbymeansoftransientmanipulations.(3) be awaytointerferewithgenefunction,ideallybytargeting cloned, characterizedandtheiractivityquantified.(2)Theremust and wellannotated,sothatcodingnoncodinggenescanbe Ideally theentiregenome,atleastindraftform,shouldbeavailable must bemet.(1)Itsgenesandregulatoryregionsshouldknown. organism asamodelformolecularbiologyfewbasicrequirements To overcomethelimitationsofRNAi,atrueexperimental Great stridesinRNA researchhave providedsolutionstothe Until recently, onlyafewspecies metthesethreerequirements de novo genome sequencing,assemblyand

days for time thesephenomenaatthemolecular level. efficient genomeeditingtoolswillallowustoprobeforthe first environment. Theavailabilityof cheapsequencingtechnologiesand phenotypes andbehaviorsthat makethembettersuitedfortheir epigenetic pathwaysinunique waystodevelopalternative date. been greatlysuccessfulinexplaininginheritanceandevolution to razor, Icontinuetosupportthemodern synthesis,whichhas absence ofsuchmolecularevidenceandindeferencetoOckham’s over evolutionarytimesforthousandsofgenerations.In the epigenetic signalscouldcauseanacquiredphenotypetobe fixed 2010a). Itisdifficult toenvision molecularmechanismsbywhich epigenetic statesisthattheyare al., 2014;Rechaviet2011). However, acharacteristicof prime candidates(HeardandMartienssen,2014;Rechavi et Darwin’s via acommunicationroutebetweenthesomaandgermline– epigenetic effects mightevenconferinheritanceofacquiredtraits occasion overseveralgenerations.Insomecasestransgenerational ,resultingintheirepigeneticinheritance,on mechanisms. Somephenotypescanindeedbemaintainedthrough activated inresponsetoastimulusandstabilizedbyepigenetic be encodedinthesamegenome,withappropriatephenotypes above arecompatiblewiththenotionthatmultiplephenotypescan examples ofpolyphenismsandepigeneticplasticitydescribed for example,http://creation.com/epigenetics-and-darwin).The 2012) anditisdangerouslypronetocreationistdistortions(see, understanding ofmolecularepigenetics(DickinsandRahman, I donotbelievethisisanaccuraterepresentationofour natural selection(Danchinetal.,2011; JablonkaandLamb,2008). evolutionary processesthatdonotrelyonrandomvariationand transgenerational inheritancehavebeeninterpretedassupporting times. toward attemptsatextendingitsrealmofactiontoevolutionary sophistication oflifeonEarth.Nonetheless,Ioffer awordofcaution important contributionsofepigeneticstothediversityand after development.Asanepigeneticsresearcher, Ibelieveinthe phenotypes atthecellularandorganismal level,before,duringand organisms. Itisbeyonddoubtthatepigeneticforcesshape epigenetic controlandlieoutsidetherealmofconventionalmodel incredible varietyofbiologicalphenomenathatcouldbeunder means constituteacompletelistandareonlymeanttoillustratethe on whichmolecularinvestigationhasalreadybegun.Theybyno In thisreview, Idescribeafew examplesofnon-modelorganisms laboratory. approaches invirtuallyanyorganism thatcanberearedinthe new organisms asgeneticmodelsandoffers thepowerofgenetic suitable stage,itdramaticallylowersthebarriertoadoptionof microinjection ofguideRNAsandnucleasesintoembryosata choice. high-efficiency introductionofanytypemutationinagenome homologous recombination,theCRISPR/Cassystemallowsfor function mutations)or, inthepresenceofasuitabledonor, by homologous end-joining(whichcausessmalldeletionsandloss-of- The authordeclares nocompetingfinancialinterests. Closing remarks Competing interests In conclusion,manynon-modelorganisms appeartohaveadapted At times,theexistenceofphenotypicplasticityand Although CRISPR-mediatedgenomeeditingstillrequires gemmulae The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110809 (Darwin, 1868)–forwhichncRNAsare meta-stable (Bonasioetal.,

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