evolution of the dan gene family in vertebratesgene, which is also the most frequently mutated gene...

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RESEARCHARTICLE

Citeas:OpazoJC,HoffmannFG,ZavalaK,EdwardsSV(2020)EvolutionoftheDANgenefamilyinvertebrates.bioRxiv,794404,ver.2peer-reviewedandrecommendedbyPCIEvolutionaryBiology.doi:10.1101/794404 Posted:28June2020Recommender:KaterynaMakovaReviewers:2anonymousreviewersCorrespondence:[email protected]

EvolutionoftheDANgenefamilyinvertebratesJuanC.Opazo1,2,3,FedericoG.Hoffmann4,5,KattinaZavala1,ScottV.Edwards61InstitutodeCienciasAmbientalesyEvolutivas,FacultaddeCiencias,UniversidadAustraldeChile,Valdivia,Chile.2DavidRockefellerCenterforLatinAmericanStudies,HarvardUniversity,Cambridge,MA02138,USA.3MillenniumNucleusofIonChannels-AssociatedDiseases(MiNICAD).4DepartmentofBiochemistry,MolecularBiology,Entomology,andPlantPathology,MississippiStateUniversity,MississippiState,39762,USA.5InstituteforGenomics,Biocomputing,andBiotechnology,MississippiStateUniversity,MississippiState,39762,USA.6DepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,Cambridge,MA02138,USA.

Thisarticlehasbeenpeer-reviewedandrecommendedby

PeerCommunityinEvolutionaryBiologydoi:10.24072/pci.evolbiol.100104

ABSTRACTThe DAN gene family (DAN, Differential screening-selected gene Aberrant inNeuroblastoma) is a group of genes that is expressed during development and playsfundamental roles in limb bud formation and digitation, kidney formation andmorphogenesis and left-right axis specification. During adulthood the expression ofthese genes are associated with diseases, including cancer. Although most of theattention to this group of genes has been dedicated to understanding its role inphysiologyanddevelopment,itsevolutionaryhistoryremainspoorlyunderstood.Thus,thegoalofthisstudyistoinvestigatetheevolutionaryhistoryoftheDANgenefamilyinvertebrates,with the objective of complementing the already abundant physiologicalinformationwithanevolutionary context.Our results recovered themonophylyof allDAN gene familymembers and divide them into fivemain groups. In addition to thewell-knownDANgenes,ourphylogeneticresultsrevealedthepresenceoftwonewDANgene lineages; one is only retained in cephalochordates, whereas the other one(GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth.According to the phyletic distribution of the genes, the ancestor of gnathostomespossessed a repertoire of eight DAN genes, and during the radiation of the groupGREM1,GREM2,SOST,SOSTDC1,andNBL1wereretainedinallmajorgroups,whereas,GREM3,CER1,andDAND5weredifferentiallylost.Keywords:genefamilyevolution,Cerberus,differentialretention,evolutionarymedicine,evolutionaryslowdown,gremlin.

.CC-BY-NC 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted June 27, 2020. . https://doi.org/10.1101/794404doi: bioRxiv preprint

https://doi.org/10.1101/794404

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Introduction

Geneticvariationharbored innon-model species representsapowerful resource togain insights inour

understandingofthegeneticbasesofbiologicaldiversity(Opazoetal.2005;Storzetal.2007;Faulkesetal.,

2015).Thecomparativeapproachhasgainedpopularity since thediscoveryofnon-model species thatare

resistant to diseases that affect human health, and also because some non-model species develop

pathologies in a similarway as ourselves (Protas et al. 2006; Gorbunova et al., 2012; Castro-Fuentes and

Socas-Pérez,2013;Manovetal.,2013;Henningetal.,2014;Braidyetal.,2015).

TheDANgenefamily(DANstandsforDifferentialscreening-selectedgeneAberrantinNeuroblastoma)is

a groupof genes characterized by the presenceof aDANdomain (http://pfam.xfam.org/family/PF03045),

lowinter-paralogconservationandexpressionduringearlydevelopment.DANgenesplayfundamentalroles

in limb bud formation and digitation, kidney formation, and morphogenesis as well as left-right axis

specification(Nolan&Thompson,2014).Inadultorganisms,theexpressionofDANgenesisassociatedwith

pathologiessuchascancerandnephropathies(Kolietal.2006;Walshetal.2010;Guetal.2012;Droguettet

al. 2014; Liangetal. 2015).As currently recognized, theDANgene family comprises sevenparalogs:NBL1

(neuroblastoma 1); CER1 (cerberus 1); DAND5 (DAN domain BMP antagonist family member 5); SOST

(sclerostin);SOSTDC1(sclerostindomaincontaining1);GREM1(gremlin1);andGREM2(gremlin2) (Avsian-

Kretchmer et al. 2004;Walsh et al. 2010; Nolan & Thompson, 2014). Historically, DAN genes have been

associatedwiththe inhibitionofthebonemorphogeneticprotein(BMP)signalingpathway,althoughmore

recentstudieshaveshownthattheyalsoactasantagoniststonodal,wingless-typeMMTVintegrationsite

(Wnt)andvascularendothelialgrowthfactor(VEGF)signalingcascades(Piccoloetal.1999;Elliesetal.2006;

Chiodelli et al. 2011). Most of the work on DAN genes has focused on understanding their role in

development (Nolan & Thompson, 2014, and references therein), and less effort has focused on their

evolutionaryhistory(Avsian-Kretchmeretal.2004;Walshetal;2010;Nolan&Thompson,2014;LePetillon

etal.2013;Opazoetal.2017).Understandingtheevolutionaryhistoryofgenefamiliespermitsinsightsinto

thewaysinwhichgenesoriginateanddiversify,aswellasratesofevolution,therelativerolesofdifferent

mutationalprocessessuchaspointmutationsandduplicationsand,most importantly, relatingallof these

phenomenatothephysiologicalandlife-historyidiosyncrasiesofdifferenttaxonomicgroups.

ThegoalofthisstudyistoinvestigatetheevolutionaryhistoryoftheDANgenefamilyinvertebrates,with

theaimofcomplementingthealreadyabundantphysiologicalinformationwithanevolutionaryperspective.

Therefore, we examined the diversity of DAN genes in representative species of all major groups of


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vertebrates, inferred homologous relationships by examining gene phylogenies and synteny conservation,

reconstructed ancestral gene repertoires, examined patterns of differential gene retention and quantified

rates ofmolecular evolution. Our results recovered themonophyly of all DAN gene familymembers and

groupedthemintofiveclades. Inadditiontothewell-knownDANgenes,ourphylogeneticresultsrevealed

the presence of two additional DAN gene lineages; one is only retained in cephalochordates (e.g.

amphioxus),whereasthesecondone(GREM3)hasonlybeenretainedbycartilaginousfish,holosteanfish,

and coelacanth. The phylogenetic distribution of genes suggests that the ancestor of gnathostomes

possesseda repertoireof eightDANgenes, and thatduring the radiationof vertebrates,GREM1,GREM2,

SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were

differentiallylost,creatingcomplexpatternsofretentionanddiversification.

Methods

DNAdataandphylogeneticanalyses

Tominimizepotentialerrorsintheannotationofthesequences,wemanuallyannotatedDANgenesin

representativespeciesofallmajorgroupsofchordates.Todoso,wefirstidentifiedgenomicfragments

containingDANgenesinEnsemblv86(Zerbinoetal.,2018)orNationalCenterforBiotechnologyInformation

(NCBI)databases(refseq_genomes,htgs,andwgs;Geeretal.,2010)basedonconservedsyntenys.Once

identified,genomicpieceswereextractedincludingthe5ʹand3ʹflankinggenes.Afterextraction,we

manuallycuratedtheexistingannotationorweannotatethenovobycomparingknownexonsequences

(querysequence)fromaspeciesthatshareacommonancestormostrecentlyintimetothespeciesofwhich

thegenomicpiece(subjectsequence)isbeinganalyzedusingtheprogramBlast2seqv2.5withdefault

parameters(Tatusova&Madden,1999).SequencesderivedfromshorterrecordsbasedongenomicDNAor

complementaryDNAwerealsoincludedtoattainabroadtaxonomiccoverage.Weincludedrepresentative

speciesfrommammals,birds,reptiles,amphibians,lobe-finnedfish,holosteanfish,teleostfish,cartilaginous

fish,cyclostomes,urochordatesandcephalochordates(SupplementaryTableS1).Aminoacidsequences

werealignedusingtheFFT-NS-istrategyfromMAFFTv.6(Katoh&Standley,2013).Ouralignmentcontained

424sequencesandalengthof797characters.Phylogeneticrelationshipswereestimatedusingmaximum

likelihoodandBayesianapproaches.Ourgoalwastoestimatethehistoryofagenefamily,notthephylogeny

oftheorganismallineagesthemselves,henceweusedtraditionalgene-treebasedphylogeneticmethods

(Edwards,2009;Yang&Rannala,2012).WeusedIQ-Tree(Kalyaanamoorthyetal.,2017)toselectthebest-


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fittingmodelofaminoacidsubstitution(JTT+I+G4)andtoobtainthemaximumlikelihoodtree

(Trifinopoulosetal.2016)andassessedsupportforthenodeswith1,000bootstrappseudoreplicatesusing

theultrafastroutine.BayesiansearcheswereconductedinMrBayesv.3.1.2(RonquistandHuelsenbeck,

2003),settingtwoindependentrunsofsixsimultaneouschainsfor5x106generations,samplingevery2,500

generations,andusingdefaultpriors.Oncetheanalysesweredone,weverifiedthattheestimatedsample

size(ESS)exceededtherecommendedvalueof200usingTracerver1.7.1(Rambautetal.2018).Therunwas

deemedconvergedoncethelikelihoodscoresreachedanasymptoticvalueandtheaveragestandard

deviationofsplitfrequenciesremained<0.01.Wediscardedalltreesthatweresampledbefore

convergence,andweevaluatedsupportforthenodesandparameterestimatesfromamajorityrule

consensusofthelast4,000trees.

AssessmentsofConservedSynteny

WeexaminedgenesfoundupstreamanddownstreamofeachmemberoftheDANgenefamilyonspecies

representativeofallthemajorgroupsofvertebrates.Syntenyanalyseswereperformedinhumans,chicken,

spottedgarandelephantshark.InitialorthologpredictionswerederivedfromtheEnsemblCompara

database(Vilellaetal.2009)andwerevisualizedusingtheprogramGenomicusv84.01(Muffatoetal.2010).

InthecaseoftheelephantsharkthegenomicsegmentscontainingDANgeneswereannotated,and

predictedgeneswerethencomparedwiththenon-redundantproteindatabaseusingBasicLocalAlignment

SearchTool(BLAST)(Altschuletal.1990).

Resultsanddiscussion

Genephylogeniesandsyntenyanalysesdefinehomology

OurphylogeneticanalysesrecoveredthemonophylyofeachmembersofthegenesintheDANgenefamily

withstrongsupportwiththeexceptionofDAND5(Fig.1).Inaddition,ourtreessuggestanarrangementin

whichthesegenesaredividedintofivemaingroups(Fig.1):1)acladecontainingtheSOSTDC1andSOST

genes;2)acladethatcontainstheCER1andDAND5genelineages;3)acladecorrespondingto2cerberus-

likesequencesofcephalochordates;4)acladecorrespondingtotheNBL1gene;and5)acladecontainingthe

GREMgenelineages(Fig.1).

Inthefirstclade,themonophylyofSOSTDC1andSOSTiswellsupported,asisthesistergroup

relationshipbetweenthem(showninshadesofred;Fig.1),suggestingthattheyshareacommonancestor


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morerecentlythanwithanyothermemberofthefamily(SupplementaryFig.S1A).Thesistergroup

relationshipbetweenthesetwogenelineageshasalsobeenrecoveredinotherstudies(Avsian-Kretchmeret

al.2004;Walshetal.2010;Nolan&Thompson,2014).Syntenyanalysesprovidefurthersupportforthe

identityofthesetwogenelineagesbecausegenesfoundup-anddownstreamareconservedinmost

surveyedspecies(Fig.2).ThetreetopologyinwhichtheSOSTDC1/SOSTcladeisrecoveredsistertoallother

membersofthegenefamilyhasalsobeenrecoveredinotherstudies(Avsian-Kretchmeretal.2004;Walshet

al.2010;Nolan&Thompson,2014).

Inthesecondgroup,showninshadesoforange(Fig.1),themonophylyofCER1genesiswell

supported,whereasthemonophylyofDAND5genesisnot(Fig.1).ThelackofsupportfortheDAND5clade

couldbeduetothepresenceofacoelacanthDAND5sequenceforwhichthephylogeneticpositionisnot

wellresolved(SupplementaryFig.S1B).However,genesfoundupstream(GADD45GIP1andRAD23A)and

downstream(NFIX,LYL1)ofthecoelacanthDAND5sequenceconfirmthatthegeneisintheexpected

positionforaDAND5ortholog,providingindependentsupportforthetopologyobtainedinourphylogenetic

analyses(Fig.1).Thus,thedatasuggeststhatthecoelacanthDAND5geneisatrueorthologoftheDAND5

geneofvertebratesandthatthelackofresolutionislikelyduetohighsequencedivergenceinthe

coelacanthgene.Inadditiontothecoelacanthsequence,syntenyanalysesprovidefurthersupportforour

phylogeny,becausegenesfoundup-anddownstreamofCER1andDAND5areconservedacrossspecies(Fig.

2).AlthoughwerecoveredasistergrouprelationshipbetweenCER1andDAND5(Fig.1),therelevantnode

wasnotsupported(Fig.1).Intheliterature,thereisnoclearpatternregardingthesistergrouprelationship

betweentheseparalogs(Avsian-Kretchmeretal.2004;Walshetal.2010;Nolan&Thompson,2014).

AlthoughthestudyofWalshetal.(2010)supportsthesistergrouprelationshipbetweenCER1andDAND5,

thestudyofAvsian-Kretchmeretal.(2004)recoveredatopologyinwhichCER1isthesistergroupofaclade

containingDAND5,NBL1andGREMgenelineages,whereas,Nolan&Thompson(2014)recoveredDAND5

sistertoacladecontainingCER1andGREMgenelineages.Insupportofourtopology,anaminoacid

alignmentthatincludesallhumanDANfamilymembersshowsthatCER1andDAND5aremoresimilarto

eachotherthantoanyothermemberofthegenefamily;moreover,theEnsemblandgenecardsplatforms

suggestthattheonlyparalogofCER1isDAND5andviceversa.Finally,accordingtoouranalyses,theclade

thatincludesCER1andDAND5wasrecoveredsistertothecladethatincludestheNBL1andGREMgene

lineagesandathirdcladeofDANgenesthatincludestwocerberus-likesequencesfromtwolanceletspecies

(Fig.1).


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OurphylogeneticanalysesrecoveredathirdcladeofDANgenesthatincludestwocerberus-like

sequencesfromtwolanceletspecies(BranchiostomafloridaeandB.belcheri;purplelineage;Fig.1).These

sequenceswererecoveredsistertotheGREM/NBL1clade(Fig.1).Thisphylogeneticarrangementwouldbe

compatiblewithtwopossibleevolutionaryscenarios.ThefirstsuggeststhattheDANgenerepertoireof

lanceletsrepresentsthegenecomplementoftheancestorofOlfactores,fromlatinolfactus,thegroupthat

includesvertebratesandurochordates,andduringtheradiationofthevertebratestheancestralgene

lineagegaverisetotheextantGREMandNBL1genes.Thesecondscenarioimpliesthatthelanceletgene

lineagecouldbeanancientmemberoftheDANgenefamilythathasonlybeenretainedin

cephalochordates.Insupportofthesecondscenario,wefoundcephalochordatesequencesintheNBL1and

GREMclades(SupplementaryFig.S1C),suggestingthatthegenomesofchordatespossessanalready-

differentiatedmemberoftheGREMandNBL1clades.

ThefourthcladecorrespondstotheNBL1genewhichwasrecoveredwithstrongsupport(pinkclade;

Fig.1)(SupplementaryFig.S1C).Theconservationofthegenesfoundup-anddownstreamprovidesfurther

supportfortheidentityoftheNBL1genelineage(Fig.2).ThephylogeneticpositionofNBL1isstillamatter

ofdebatebecausedifferentphylogenetichypotheseshavebeenproposedinearlierstudies.Nolanetal.

(2014)recoveredNBL1assistertothecladecontainingGREM1,GREM2,CER,andDAND5genelineages;

whereasinWalshetal.(2010)NBL1sequenceswererecoveredinatrichotomywiththeGREM1/GREM2and

CER1/DAND5clades.However,insupportofourstudyAvsian-Kretchmeretal.(2004)recoveredNBL1as

sistertotheGREMgenelineages.

Thefifthcladecorrespondstoawell-supportedgroupcontainingGREMgenelineages(shownin

shadesofgreen;Fig.1).Withinthisclade,themonophylyoftheGREM1andGREM2genesiswellsupported

(Fig.1).Asinothercases,syntenyanalysessupporttheidentityofbothgenelineages(Fig.3).Weidentifieda

cladethatcontainsasinglecopygeneinlanceletsandtunicates,whichinturnwasrecoveredassistertothe

GREM2clade(SupplementaryFig.S1D).Thepresenceofasinglecopygeneincephalochordatesand

urochordatesisexpectedgiventhatapreviousstudyreportedthatthevertebrateGREMgenesdiversifiedas

aproductofthetworoundsofwhole-genomeduplicationsearlyinvertebrateevolution(Singhetal.2015;

Sacerdotetal.2018;Simakovetal.2020),andcephalochordatesandurochordatesdivergedfrom

vertebratesbeforethesewhole-genomeduplications.Unexpectedly,ourphylogeneticanalysesidentifiedthe

presenceofathirdGREMgenelineage(GREM3)amonggnathostomevertebratesthathasnotbeen

describedbefore(Fig.1).Thisnewgenelineagewasrecoveredwithstrongsupport,andwasidentifiedin

threedistantlyrelatedspecies:elephantshark(Callorhinchusmilii),spottedgar(Lepisosteusoculatus)and


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coelacanth(Latimeriachalumnae).Syntenyanalysesprovidefurthersupportfortheidentityofthisnew

lineageasgenesfoundup-(RASGRP4,FAM98CandSPRED3)anddownstream(RYR1,MAP4K1,andEIF3K)

areconserved(Fig.3).Moreover,wefoundthathumanorthologsofthegenessyntenictoGREM3mapped

tochromosome19,suggestingthatthischromosomeastheputativegenomiclocationoftheGREM3genein

humans(Fig.3).Asimilardifferentialretentionofanancestralgeneinasmallgroupofdistantlyrelated

specieshasalsobeenobservedinothergenefamilies(Wichmannetal.2016).

Itisnecessarytohighlightthatalthoughwereportdisagreementintreetopologiesbetween

publishedstudiesandourresults,phylogenetictreesarenotalwaysdirectlycomparableastheyhave

differencesinthetaxonomicand/ormembershipsampling,twofactorsknowntoaffectphylogenetic

inference.

Definitionofancestralgenerepertoires

Theinterpretationofthephylogeneticdistributionofgenesincombinationwiththeevolutionary

relationshipsamongthem(i.e.genetree),inthelightoftheorganismalphylogeny(i.e.speciestree),

representsanappropriatewaytounderstandtheevolutionoftheDANgenefamily.Accordingtoourresults,

GREM1,GREM2,SOST,SOSTDC1,andNBL1arepresentinthemajorgroupsofgnathostomes(Fig.4;

SupplementaryFig.S1E),andwithineachparalogcladethespeciesarrangementdoesnotsignificantly

deviatefromtheorganismalphylogeny.Thisindicatesthatallthesegeneswerepresentinthecommon

ancestorofthegroup,andagreeswiththeexpectationsofthesimplestmodelofmultigenefamily

diversification,thedivergentevolutionmodel(Neietal.1997).InthecaseofGREM3,CER1,andDAND5,

althoughtheyarenotfoundinallmajorgroupsofgnathostomes,theirphyleticdistributionincombination

withthecorrespondingtreetopology(Fig.4)indicatethatthesegeneswerealsopresentinthecommon

ancestorofgnathostomes.Thus,ourresultssuggestthatthelastcommonancestorofgnathostomes,dated

between615to476mya(Hedgesetal.2015),possessedarepertoireofeightDANgenes:GREM1,GREM2,

SOST,SOSTDC1,NBL1,GREM3,CER1,andDAND5.Wenotethattheancestralconditionofeightgenesisonly

presenttodayincoelacanths(Fig.4)..Thisreconstructionholdsunderanyofthealternativerootings

previouslyproposedintheliterature.

Inferencesabouttherepertoireofthecommonancestorsofvertebrates,olfactoresandchordates

aresomewhatspeculativegiventhelimitedavailabilityofgenomesandthequalityofcurrentassemblies.

However,thephylogeneticpositionofkeytaxonomicgroups,suchascyclostomes,urochordates,and

cephalochordates,providessignificantinformationtoadvancehypothesesregardingthecomplementof


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genesinthesedifferentancestors.WeidentifiedGREM2andSOSTDC1sequencesincyclostomes

(SupplementaryFigs.S1AandS1D),indicatingthatatleastthesetwogeneswerelikelypresentinthe

commonancestorofvertebrates.However,giventhatcyclostomesequencesarenestedwithintheGREM2

andSOSTDC1cladesandnotsistertoacladewithmorethanoneDANgene,itispossiblethattheabsenceof

theotherparalogsincyclostomegenomesisduetogeneloss.

Inferencesaboutthecommonancestorofolfactoresarerestrictedgiventhelimitednumberof

urochordategenomesandthequalityoftheirassemblies.Tofurthercomplicatethissituation,urochordates

haveexperiencedanextraordinarynumberofgenelosses(Dehaletal.2002;Cañestroetal.2003;Cañestro

etal.2013;Albalat&Cañestro,2016),soitisdifficulttotellwhethertheabsenceofageneisduetothe

qualityofthedataorarealloss.Takingalltheseuncertaintiesintoaccount,wewereabletoidentifyone

urochordatesequence.ThissequencewasrecoveredassistertothreecephalochordateGREMgenes,which

inturnwasrecoveredassistertoGREM2(SupplementaryFig.S1D).GiventhatGREMgenesdiversifiedasa

productofthetworoundsofwholegenomeduplicationsinthelastcommonancestorofvertebrates(Singh

etal.2015;Sacerdotetal.,2018;Simakovetal.2020),asingleannotatedgenecopyinurochordatesis

expected.Thus,themostprobablescenarioisthatthecommonancestorofolfactorespossessedoneGREM

gene.Itisnotpossibletodrawinferencesfortheothermembersofthegenefamilyatthistime.However,

giventhattheancestorofolfactoresexistedbeforethetwovertebrate-specificwholegenomeduplications,

weexpectitsrepertoiretohavefewergenescomparedtotheancestorsofvertebratesorgnathostomes.

Finally,wefoundeightcephalochordatesequencesthathelpusdefinetherepertoireofgenesinthe

ancestorofchordates.OnesequencewasrecoveredsistertoalampreySOSTDC1gene,acladethatinturn

wasplacedsistertoallotherSOSTDC1sequences(SupplementaryFig.S1A).Agroupoftwosequenceswas

recoveredsistertothecladethatincludesNBL1andGREMgenes,andwethinkthiscladerepresentsan

ancientmemberofthegenefamilythatwasonlyretainedinthisgroup.Twoadditionalsequenceswere

recoveredassistertotheNBL1lineage;whereasthelastthreewereplacedsistertoaurochordateGREM

sequence,andthislatterclade,inturn,wasrecoveredsistertoGREM2(SupplementaryFig.S1C).Ourresults

agreewithLePetillonetal.(2013),whoalsofoundcopiesofNBL1andGREMorthologsincephalochordates.

However,incontrasttoLePetillonetal.(2013),wedidnotfindacephalochordateDAND5genelineagebut

insteadidentifiedacephalochordate-specificgenelineage(Fig.1,purpleclade).Bringingtheseresults

together,weproposethatthechordateancestorhadatleastfourDANgenes(SOSTDC1,NBL1,GREM,

DAND5andthecephalochordate-specificgenelineage).LePetillonetal.(2013)alsoidentifiedsequencesin

ambulacraria,thegroupthatincludesechinodermsandhemichordates,suggestingthatthedeuterostome


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ancestor,whichexistedbetween797and684mya(Hedgesetal.2015),hadatleastthreeDANsequences,

NBL1,GREM,andDAND5.

DifferentialretentionofDANgenesduringtheevolutionaryhistoryofgnathostomes

OurresultsshowthatcopiesofGREM1,GREM2,SOST,SOSTDC1,andNBL1havebeenmaintainedinthe

genomeofallmajorgroupsofgnathostomesduringthelast615mya(Hedgesetal.2015;Fig.4),whichcould

suggesttheyplaycriticalrolesduringdevelopment.Inlinewiththissuggestion,theinactivationordeletion

ofsomeofthesegenesproduceembryonicallylethaleffects(Khokhaetal.2003;Gazzeroetal.2006);in

othercases,thelossofageneisassociatedwithpathologicalconditions(Lietal.2008),althoughthereare

otherexampleswhereknockoutsdevelopminormorphologicalanomalieswithnosignificantconsequences

(Davisetal.2015;Vogueletal.2015).Althoughthesegenesarepresentinallmajorgroupsofgnathostomes,

someofthemcouldstillbeabsentinaparticulargroupwithinthesemoreinclusivetaxonomiccategories.

Forexample,ithasbeensuggestedthattheGREM2genewaslostinthecommonancestorofruminants,

hippopotamuses,andcetaceansbetween56.3and63.5millionyearsagoasaproductofachromosomal

rearrangement(Opazoetal.2017).

OurresultsalsorevealedthatGREM3,CER1andDAND5weredifferentiallyretainedduringthe

evolutionaryhistoryofgnathostomes.Thedifferentialretentionofgenescouldbeastochasticprocess,in

whichtheresultingdifferencesingenecomplementdonottranslateintofunctionalconsequences.This

phenomenoncouldbefacilitatedbyadegreeofredundancythatcouldfunctionasabackupwith

functionallyoverlappingparalogues,incasethatoneofthegenesislostorinactivated(Gitelman,2007;

Cañestroetal.,2009;Félix&Barkoulas,2015;Albalat&Cañestro,2016).However,itisalsopossiblethat

possessingmultiplecopiescouldhelpdirectthetrajectoryofphysiologicalevolutionbyproviding

opportunitiesfortheemergenceofbiologicalnovelty(Ohnoetal.,1968;Ohno,1970;Forceetal.,1999;

Hughes,1994;Zhang,2003).Forexample,thedifferentialretentionandduplicationoftheγ-globingenes

mighthaveplayedanimportantroleintheevolutionoflifehistoryinanthropoidprimatesbyfacilitatingan

extendedfetaldevelopment(Goodmanetal.1987;Opazoetal.2008).Similarly,thedifferentialretentionof

functionalcopiesoftheINSL4genemayhavebeenassociatedwithuniquereproductivecharacteristicsin

catarrhineprimates(Arroyoetal.2012a,b).

Fromanotherperspective,theabsenceofgenesinnaturalknockoutsrepresentsanopportunityto

understandtheirphysiologicalrole(Albertsoneta.2009).Thissuggestionisbasedontheorthology-function

conjecture:theexpectationthatorthologousgenesaremostlikelytohaveequivalentfunctionsindifferent


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organisms(Altenhoffetal.2012).Thisapproachhasbeenusefulforunderstandinghumandiseases,

especiallyforthoseexhibitingsimpleMendelianinherence,suchascysticfibrosis,albinismandother

diseases(Albertsonetal.2009).Forexample,ithasbeenshownthatintheblindcavefish(Astyanax

mexicanus),lossofpigmentationisproducedbymutationsinactivatingtheproteinencodedbytheOCA2

gene,whichisalsothemostfrequentlymutatedgeneincasesofhumanalbinism(Protasetal.2006).Thus,

bothretentionandgenelosscouldbeseenasevolutionaryeventsthathelpustounderstandthegenetic

basesofbiologicaldiversity.

OurresultspointtotheGREM3geneasthemostextremecaseofdifferentialretentionamongDAN

genes.Ithasbeenretainedincartilaginousfish,holosteanfishandcoelacanths(Fig.4),suggestingthatitwas

presentinthecommonancestorofgnathostomes,between615and473mya(Hedgesetal.2015),andwas

subsequentlylostindependentlyinthecommonancestorsoftetrapodsandteleostfish(Fig.5).Asimilar

caseofdifferentialretentionofanewlydiscoveredgenelineagewasshowninatumorsuppressorgene

family(Wichmannetal.2016).Inthiscase,inadditiontotheelephantshark,spottedgarandcoelacanth,the

newlydiscoveredgenelineagewasalsoretainedbyteleostfish(Wichmannetal.2016).Theabsenceof

GREM3inasignificantfractionofgnathostomespeciessuggeststhatGREM3couldbedispensable.However,

giventhatGREM3representsanewgenelineageofunknownbiologicalfunction,itisprematuretoevaluate

thephysiologicalandevolutionaryimpactsofretainingorlosingthisgene.Experimentalevidenceforthe

othermembersoftheclade(GREM1andGREM2)providesconflictingresults(Khokhaetal.2003;Davisetal.

2015;Vogueletal.2015),makingitdifficulttoanticipatethedegreeofdispensabilityoftherelatedGREM3

gene.

CER1wasalsopresentinthecommonancestorofgnathostomesandwasdifferentiallyretained

duringtheradiationofthegroup(Fig.4),beinglostinthecommonancestorofray-finnedfish

(Actinopterygii)between425and375mya(Betancur-Retal.2013)(Fig.4).Syntenyanalysesprovidefurther

supportforourscenariobecausethegenomiccontextofCER1isconservedinfish(Fig.5).CER1isaprotein

possessingnineconservedcysteinesandacysteineknotregionexpressedduringearlygastrulationinthe

primitiveendodermandsubsequentlyinsomitesandanteriorpresomiticmesoderm(Shawlotetal.1998;

Pearceetal.1999;Piccoloetal.1999;Beloetal.2000).CER1hasdevelopmentalrolesassociatedprimarily

withanterior-posterior,left-right,anddorso-ventralasymmetries(Beloetal.2009),accomplishingthemby

bindingtoBMPs,NodalandWntligands,whichinturnblocktheligand-receptorinteractionandactivation

(Bouwmeesteretal.1996;Piccoloetal.1999;Beloetal.2000;Silvaetal.2003;Avsian2004).The

physiologicalsignificanceofthisgeneseemstovarywiththetaxonomicgroup:forexample,CER1produces


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strongerphenotypiceffectsinamphibiansthaninmammals(Simpsonetal.1999;Piccoloetal.1999;Beloet

al.2000).Infish,CER1developmentalfunctionsmaybeperformedbyitsparalogDAND5,whichis

functionallysimilar(Hashimotoetal.2004;Marquesetal.2004;Beloetal.2009;Lopesetal.2010;

Schweickertetal.2010;Hamadaetal.2012;Araujoetal.2014).Thus,fromanevolutionaryperspective,the

possessionofbothparalogs,CER1andDAND5,ingnathostomesotherthanfishwouldrepresentacaseof

functionalredundancy.Underthisscenario,thelossofoneofthesegenescouldbecompensatedbythe

retentionoftheother.

InthecaseofDAND5,wefoundthisgeneinallmainlineagesofgnathostomesotherthansauropsids

andcartilaginousfish(Fig.4).Inthecaseoftheelephantshark,webelievethattheabsenceofthegene

couldbeduetoaprobleminthecurrentgenomeassembly;accordingly,ourproposedscenariomainly

assumestheabsenceofthisgeneinsauropsids.OurresultsindicatethatDAND5waslostinthecommon

ancestorofsauropsids,thegroupincludingbirdsandnon-avianreptiles,between312and280mya(Hedges

etal.2015;Fig.5),eventhoughthecorrespondinggenomicregioniswellconserved(Fig.6).Ourproposed

lossofDAND5inbirdshasalsobeennoticedbyotherauthors(LePetillonetal.2013).DAND5hasbeen

describedasageneplayingfundamentalrolesdrivingasymmetriesintheearlystagesofdevelopmentby

inhibitingNodalactivity(Marquesetal.2004).Althoughinamphibians,fish,andmammalstheexpression

patternseemstobedifferent,themolecularmechanism,restrictingnodalsignalingintheleftsideofthe

embryoisconserved(Beloetal.2009).Inadditiontotherolesinearlydevelopment,duringadulthood

DAND5alsohelpspromotecancermetastasis,aswellasreactivationofmetastaticcellsinlungsbyreversing

theabilityofBMPtoinhibitcancerstemcellfunction(Guetal.2012).Experimentsinotherorgans,suchas

boneandbrain,showthatcancer-relatedfunctionsofDAND5arespecifictothelung(Guetal.2012).

Consequently,ithasbeenshownthatpatientsexpressinghighlevelsofDAND5haveoverallreducedsurvival

rates(Guetal.2012).Thelackofthisgeneinsauropsidsisdifficulttointerpret;wespeculatethat,here,the

lossofDAND5iscompensatedbyCER1,removinganyphysiologicalconsequencesandconsistentwiththe

ideathatgenefamiliespossesssomedegreeofredundancy(Gitelman,2007;Cañestroetal.,2009;Félix&

Barkoulas,2015;Albalat&Cañestro,2016).Thus,CER1andDAND5apparentlyperformsimilarphysiological

rolesduringdevelopment(Hashimotoetal.2004;Beloetal.2009).

Ratesofmolecularevolution:themammalianslowdown


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Thephylogenetictreesindicatethattheratesofmolecularevolution,asmeasuredbybranchlengths,vary

withineachparalogclade(Fig.1).Themostnotoriouscaseistheobservedslowdowninbranchesleadingto

mammalsrelativetosauropsids(SupplementaryFig.S1AtoS1D).Thisisinterestingbecausesauropsidssuch

asbirds,crocodilians,lizards,snakesandturtlestendtohavegenome-wideratesofevolutionmuchlower

thaninmammals(Greenetal.2014).UsingtherelativeratetestinMEGA7(Kumaretal.2016)we

confirmedthatratesinaminoacidchangearesignificantlyslowerinmammalsrelativetosauropsids

(SupplementaryTableS2).Inmammals,thedivergencevaluesrangedfrom0.0112(SOSTDC1)to0.5143

(DAND5)substitutionspersite,whereasinsauropsidstheyrangedfrom0.0326(GREM2)to0.9248(DAND5).

Inallcases,exceptforGREM2,thesaurospiddivergenceswerehigherthaninmammals,andtheratioof

ratesinsauropsidsvs.mammalsvariedfrom1.79(DAND5)to14.66(GREM1).Weexpectgenesexpressedin

earlystagesoflifetobesubjecttomorestringentpurifyingselectionthanthoseexpressedinlater

development,andthereforetohavelowerevolutionaryrates(Goodman,1961,1963;Roux&Robinson-

Rechavi,2008).Thus,theslowerrateofevolutioninmammalscouldreflectanincreasedleveloffunctional

orstructuralconstraintsonthesegenes.Aslowdowninaspecificsetofgenesshouldbedistinguishedfroma

lineage-specificslowdown.Inthefirstcase,thereducedevolutionaryrateisprobablyduetodifferencesin

thestrengthofpurifyingselectionbetweenthedifferentlineages,whereaslineage-specificslowdownsare

moreplausiblybeexplainedbychangesinthesubstitutionrate,whichcouldbereducedbyloweringofthe

mutationratebyimprovedsystemsofDNArepairmechanisms,longergenerationtimes,orotherfactors

(Goodman,1985).

ConclusionsWeperformedanevolutionarystudyofthedifferentialscreening-selectedgeneaberrantinneuroblastoma

(DAN)genefamilyinvertebrates.Accordingtoourresults,thisgenefamilyhasevolvedbyacombinationof

modelsincludingdivergentevolution,birth-and-death,anddifferentialretention.Werecoveredthe

monophylyofallrecognizedgenefamilymembers,whichinturnwerearrangedintofivemainclades.

Importantly,inthisworkwedescribedthepresenceoftwonewDANgenelineages;onethatisonlypresent

incephalochordates(e.g.amphioxus),andother(GREM3)thatwasonlyidentifiedincartilaginousfish,

holosteanfishandcoelacanths.Theancestorofgnathostomevertebratespossessedarepertoireofeight

DANgenes.Duringtheradiationofthegroup,someofthem(GREM1,GREM2,SOST,SOSTDC1andNBL1)

wereretainedinthegenomeofallmajorgroups,whileothers(GREM3,CER1andDAND5)weredifferentially


https://doi.org/10.1101/794404



retainedduringtheevolutionaryhistoryofthegroup.Therateofmolecularevolutionofmammalsisin

generallow,suggestinganincreasedevolutionaryconstraintregime.Finally,weexpectthatwithallthese

newinformationresearchersinthebiomedicalfieldcouldputtheirresultsinanevolutionaryperspective.

Dataaccessibility

Dataandsupplementarymaterialareavailableonline:https://zenodo.org/deposit/3911345

Acknowledgements

ThisworkwassupportedbytheFondoNacionaldeDesarrolloCientíficoyTecnológicofromChile(FONDECYT1160627) and Millennium Nucleus of Ion Channels Associated Diseases (MiNICAD), Iniciativa CientíficaMilenio,Ministry of Economy, Development and Tourism from Chile to JCO, National Science Foundation(EPS-0903787, DBI-1262901 and DEB-1354147) to F.G.H. S.V.E. acknowledges NSF grant DEB 1355343.Version 2 of this preprint has been peer-reviewed and recommended by Peer Community In Evol Biol(https://doi.org/10.24072/pci.evolbiol.100104)

Conflictofinterestdisclosure

Theauthorsofthispreprintdeclarethattheyhavenofinancialconflictof interestwiththecontentofthis

article.JuanC.OpazoisrecommenderforPCIEvolutionaryBiologyandPCIGenomics.

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SOST

SOSTDC1

CER1

DAND5

NBL1

GREM3

GREM2GREM1

0.7

87/0.91

88/0.83

95/0.9396/0.81

100/0.99

96/1

100/0.98

100/1

83/0.79

100/1

99/0.99100/1

83/0.7087/0.99


https://doi.org/10.1101/794404


Elepha

nt

shark

*Spo

tted G

ar

Chicke

n

MEOX1

SOST

DUSP3

C17orf105

MPP3

ETV4

DHX8

(*) (*)

ISPD

SOSTDC1

LRRC72

ANKMY2

BZW2

MEOX2

AGMO

(*)

Human

NFIB

ZDHHC21

CER1

FREM1

TTC39B

SNAPC3

PSIP1

MPDZ

RAD23A

GADD45GIP1

DAND5

NFIX

LYL1

CALR(*)

PQLC2

CAPZB

MINOS1

NBL1

HTR6

TMCO4

RNF186

AKR7A2

(*)(*)

A)

B)

C)


https://doi.org/10.1101/794404


ARHGAP11A

SCG5

GREM1

FMN1

AVEN

CHRM5

RYR3

CHRM3

FMN2

GREM2

RGS7

FH

KMO

RYR2

OPN3

MTR

(*) (*)Human

Elephantshark

Spo�edGar

Chicken

(*) (*) (*)Human

Elephantshark

Spo�edGar

Chicken

(*)

Human(Chr19)

Coelacanth

ElephantShark

Spo�edGar

RASGRP4

GREM3

RYR1

MAP4K1

EIF3K

FAM98C

SPRED3


https://doi.org/10.1101/794404


Mammals

Birds

Crocodiles

Turtles

Squamates

Amphibians

Coelacanths

Holosteanfish

Car�laginousfish

Teleostfish

Grem1Gre

m2

Grem3

2X 2X

Cer1

DAND5

1X

SOST

1X

SOSTD

C1

2X

NBL1

1X


https://doi.org/10.1101/794404


Human

Fugu

Tetraodon

S�ckleback

Amazonm

olly

Platyfish

Medaka

Tilapia

NFIB

ZDHHC21

CER1

SH3GL2

ADAMTSL1

HAUS6

PLIN2

PLAA


https://doi.org/10.1101/794404


SYCE2

FARSA

DAND5

GCDH

LYL1

RAD23A

GADD45GIP1

CALR

Anolelizard

Chineseturtle

Human

NFIX

Americanalligator

Burmesepython

(*)Chicken

(*)


https://doi.org/10.1101/794404


evolution of the dan gene family in vertebratesgene, which is also the most frequently mutated gene...

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