Reassessing the Role of Hox Genes during Vertebrate Development and Evolution

(1)

Review

Reassessing

the

Role

of

Hox

Genes

during

Vertebrate

Development

and

Evolution

Moisés

Mallo

1,

*

Since their discovery Hox genes have been at the core of the established modelsexplainingthedevelopmentandevolutionofthevertebratebodyplan aswellasitspairedappendages.Recentworkbroughtnewlighttotheirrolein thepatterningprocesses alongthemain bodyaxis.Thesestudies showthat Hoxgenesdonotcontrolthebasiclayoutofthevertebratebodyplanbutcarry out region-specific patterninginstructions loaded on the derivativesof axial progenitorsbyHox-independentprocesses.Furthermore,thefindingthatHox clusters are embedded in functional chromatin domains, which critically impacts their expression, has significantly altered our understanding of the mechanisms of Hox gene regulation. This new conceptual framework has broadened our understanding of both limb development and the evolution ofvertebratepairedappendages.

TheVertebrateBasic BodyPlan IsLaidOutIndependentlyofHoxGenes Vertebratesdisplayaremarkablevarietyofbodysizesandshapes,typicallyinvolvingfeatures alongtheirmainbodyaxisandtheirpairedappendages(seeGlossary).However,thebasic developmentalprinciplesgeneratingthevariousstructuresarelargelysharedamong verte-brates.Thevariousbodyattributesalongthemainbodyaxisareassembledsequentiallyina head-to-tailsequenceastheembryoextendsatitsposteriorend.Thisresultsfromtheactivity ofdedicatedaxial progenitorsproducingtherawmaterial thateventuallyformsthevarious embryonictissues[1,2].Althoughcontinuous,axialextensioncanbedividedintothreemajor stepseachregulatedbyadistinctgenenetwork[3–5].Duringtheﬁrststep,typicallyknownas headdevelopment,theembryogeneratesthebrainandheartprimordiatogetherwith mus-culoskeletalstructuresoftheheadandneck.Thisisfollowedbyformationofthetrunk,which holdsmostoftheinternalorgansinvolvedinvitalandreproductivefunctions.Theﬁnalstepof axialextensionisdevotedtotailformation,essentiallycomprisingvertebrae,muscles,anda variableamountofneuraltissue.Differencesintheoverallextentofbodyelongationduring development,aswellastheportionsofthisaxialgrowthdevotedtomakinghead,trunk,ortail structures,areamongthemostrelevantparametersgeneratinganatomicaldiversityin verte-brates.For thisreason both themechanisms controllingtheseprocessesand theirrole in vertebrateevolutionhaveattractedplentyofattentionoverthepastfewdecades.

SincetheirdiscoveryHoxgenes(Box1)havebeenconsideredthemajordriversof morpho-logicalevolutionintheanimalkingdom[6].Comparativeexpressionanalysesinembryosof vertebrateswithclearly distinctbodydistributionsrevealedaclose correlationbetweenthe expressionboundariesofparticular Hoxgenesandspeciﬁclandmarksintheaxialskeleton

[7,8],suggestingkeyrolesforthesegenesinsettingupthebasicvertebratebodyplan.Alarge varietyofgeneticstudies,mostlyinthemouse,conﬁrmedtherelevantcontributionsofHox genestopatterningprocessesalongthemainbodyaxis[9,10].Forinstance,Hox4genesare involvedinproperpatterningoftheneckvertebrae[11],Hox5,Hox6,andHox9genescontrol

Highlights

Thebasiclayoutofthevertebratebody

is outlined in the axial progenitors

mostly through Hox-independent

mechanisms.

Hoxgenesarecarriersofpatterning

informationloadedontothederivatives

ofaxial progenitors that guides the

productionofbodystructures

congru-entwiththeiraxiallevel.

Selectivetargetinactivationallowsthe

shutting down ofa subset of

Hox-dependent functions while keeping

othersactiveinthesamedomain.This

increasestheﬂexibilityofevolutionary

processes.

Theprocesses regulating Hoxgene

expressionintheproximalanddistal

regionsofthelimbbudsoccurintwo

alternative functional chromatin

domains.

Modiﬁcation of Hox regulatory

pro-cesses within chromatin domains

mighthaveplayedaroleinthe

evolu-tionofvertebratepairedappendages.

1

InstitutoGulbenkiandeCiencia,Rua

daQuintaGrande6,2780-156Oeiras,

Portugal *Correspondence: mallo@igc.gulbenkian.pt(M.Mallo). URL: http://www.igc.gulbenkian.pt/mmallo. 209

(2)

variousaspectsofribcagedevelopment[12,13],andHox10andHox11genesareessentialfor the formation of the lumbar and sacral areas of the axial skeleton, respectively [14,15]. However,these studies consistently failedto showsigniﬁcantchanges in the basichead/ trunk/taildistribution of the body,even incases where alterations in Hox geneactivity or expressionproducedextremephenotypesintheaxialskeleton.OnlyHox13geneshavebeen foundtocontributetothisprocess,byplayingaroleindeterminingtheﬁnallengthofthetail region [16,17].Overall thesedata indicated that the basic head/trunk/tail structure of the embryoismostlikelynotunderdirectHoxregulation,whichseemedtocontradicttheprevailing modelfortheevolutionofthevertebratebodyplanalongthemainbodyaxis.

Gdf11andOct4AreUpstreamofHoxGenesintheBody-Patterning Hierarchy

Recentﬁndingsbroughtnewlighttothisissue,identifyingOct4andGdf11signalingasmajor playersintheestablishmentofthebasicbodyplan,actingupstreamofHoxgenes.Gdf11,a memberoftheTgfbfamilyexpressedintheposteriorembryonicareastartingatmid-gestation

[18,19],wasshowntobeakeyactivatorofthetrunk-to-tailtransition[20],aprocessinwhichit showspartialredundancywithGdf8 [21].Mice withimpairedGdf11 signaling havelonger trunksresultingfromdelayedactivationofthistransitionfromtheearlystagesofdevelopment, asreﬂectedinthesigniﬁcantlymoreposteriorpositionofthehindlimbsandcloaca,whichmark the posterior end of trunk-associated structures such as the lateral mesoderm and the endodermaltissuescontributingtotheinternalorgans[19,20].Conversely,premature activa-tionofGdf11signalingresultedinmoreanteriorinductionofthetrunk-to-tailtransition,which placedthehindlimbnextto theforelimbbudandthusledtoembryoswithoutatrunk[20]. Gdf11expressionindifferentvertebratesseems to givefurther supportfor therole ofthis signalingintheevolutionofvertebratetrunklength[22,23].

OtherstudiesrevealedthatthepluripotencyfactorOct4playsasomewhatcomplementaryrole inthelayoutofthebasicbodyplan,asitpromotesextensionthroughthetrunkregion.Such newroleforOct4,consistentwithitsexpressiondynamicsduringmousedevelopment[24,25], was suggested by genetic studies. In particular, conditional Oct4 inactivation in mouse embryosatearlystagesoftrunkformationresultedinembryoswithoutatrunkbutthatstill containedrecognizabletails[26].AdifferentsetofstudiesshowedthatOct4isalsosufﬁcientto extendthe vertebrate trunk [23].Prolonging theperiodof Oct4activity inmouse embryos resultedinlongertrunksattheexpenseofthetails.Inaddition,molecularanalysesinsnake embryosindicatethattheirremarkablylongtrunksmightbetheresultofanincreasedperiodof Oct4activityduringembryonicdevelopment[23].Altogether,variouslinesofevidenceplace the balance between Oct4 and Gdf11 activities at the top of the hierarchy of regulatory processescontrollingthebasicfeaturesofthevertebratebodyplanbyplayingfundamental rolesindeterminingthe relativecontributionsofthe differentbody sectionsto theanimal’s anatomy.

WhereDoHoxGenesFit inThisScheme?

ExpressionanalysesindicatedthatinmouseembryoswithmodiﬁedGdf11orOct4activity,50 Hoxgenesbecameactivatedataxiallevelscongruentwiththenewpositionofthehindlimb budand,thus,thetrunk-to-tailtransition[19,20,23,27,28].Interestingly,someHoxgenesof 30groupsshowedacomplementarybehavior,bestseeninembryoswithlongertrunks(i.e., Gdf11mutantsortransgenicswithsustainedOct4expression),wheretheirexpressionspread intomoreposteriorembryonicareas[19,20,23,28](Figure1).ThesameglobalpatternsofHox geneexpressionwereobservedinthenaturalsettingpresentedbythesnakeembryo[8,29]. ThesestudiesthusplaceHoxgeneexpressiondownstreamofOct4andGdf11signaling.A

Glossary

30and50Hoxgenes:aconvenient

wayofgroupingHoxgenesinthe

clusters(sometimesalsoreferredto

asanteriorandposteriorHoxgenes,

respectively).Althoughthiscanvary

slightlydependingonthespeciﬁc

sources,30Hoxgenesnormally

includethoseofgroups1–9and50

Hoxgenesthoseofgroups10–13.

Amniotes:acladeoflimbed

vertebrates,includingreptiles,birds,

andmammals.Embryosofamniote

speciesdevelopinaﬂuid-ﬁlledcavity

formedbyaseriesofextraembryonic

membranesincludingtheamnion,

whichgivesnametotheclade.

Pairedappendages:locomotor

structures(normallytwopairsper

animal)typicalofjawedvertebrates;

includethepectoralandpelvicﬁnsof

ﬁshesandthelimbsoftetrapods.

Teleosts:agroupofbonyﬁshes

containingamovablepremaxilla.The

largestpartoflivingspeciesofﬁsh

belongstothisgroup.

Topologicallyassociating

domains(TADs):3Dchromatin

structuresfoundininterphasenuclei;

identiﬁed,bymethodsallowing

high-throughputanalysisofmutual

genomicDNAcontacts,asregions

thatinteractwitheachothermore

frequentlythanwithotherpartsof

thegenome.Regulatoryinteractions

aretypicallycontainedinTADs.

Zeugopod,stylopod,and

autopod:thethreebasicsectionsof

thetetrapodlimb,fromproximalto

distal.Thezeugopodtypically

containsonebone(thehumerusin

theforelimbandthefemurinthe

hindlimb),thestylopodcontainstwo

bones(theulnaandradiusinthe

forelimbandthetibiaandﬁbulain

thehindlimb),andtheautopod

(3)

similarhierarchywasobservedatthecell/tissuelevel,asOct4andGdf11regulatepatterning directlyontheaxialprogenitors(andthusatthetopofthecellularhierarchycontrollingbody formation)whereasHoxgeneactivityismostimportantintissuesderivedfromthose progen-itors[20,23].Hence,theglobalpictureemergingfromthesestudies(Figure2)isthat,inafirst step,thebasiclayoutofthevertebratebodyisoutlinedintheaxialprogenitorsmostlythrough Hox-independentmechanisms.Asanintegralpartofthesebasicprocesses,thedifferentcell typesproducedbytheseprogenitorsbecomeloadedwithpatterninginformation correspond-ing to the axial level where they will differentiate. Hox genes thus belong to this second patterninglayerprovidingaxialidentitytomesodermal,neural,andmaybealsoendodermal derivativesoftheaxialprogenitors[9,30,31].Thus,theirroleintheevolutionofthevertebrate anatomy isexerted inthese tissueswhere specific combinations ofHox gene expression determine regional variations in the main body domains. Again, snakes provide a natural exampleto illustratethishypothesis.Inparticular,themorphologyofthetrunkvertebraeof differentsnakespeciesrevealedthatthisregionisnotuniformaswasclassicallyconsideredbut regionalizedalongtheanterior–posterioraxis followingspecies-specificpatterns,mostlikely resultingfromspecificvariationsinHoxgeneexpression,setindependentlyoftheirtrunklength

[32].

SofarlittleisknownabouthowOct4andGdf11signalingcontrolsHoxgeneexpression.The two-waycomplementarityofHoxregulationbyOct4andGdf11(i.e.,that50Hoxgenesare repressedbyOct4andactivatedbyGdf11whereas30Hoxgeneexpressionexpands posteri-orlyfollowingextendedOct4activityoronGdf11inactivation)mightprovidecluesto under-standthisprocess.TheregionsoftheHoxclusterunderdifferentialregulationbyOct4 and Gdf11 roughly correspondto their distribution within the two topologically associating domains(TADs)coveringtheHoxAandHoxDclusters[33,34].TADsdemarcatechromatin territoriesfacilitatingregulatoryinteractions[35,36]andtheTADstructureassociatedwiththe

Box1.BasicConceptsofVertebrateHoxGeneOrganizationandExpression

Inmammals,whichprovideaparadigmtooutlinethebasicconceptsofHoxgeneorganization(FigureI),Hoxgenesare

distributedinfourclusters,namedHoxA,HoxB,HoxC,andHoxD,thoughttoresultfromtwosuccessiveduplicationsof

anancestralcluster.Hoxgenesaresubdividedin13groups(fromHox1toHox13)basedonsequencehomologyand

theirpositioninthecluster.Hoxgeneswithlowernumbersarelocatedatthe30sideoftheclusterandthosewithhigher

numberstowardthe50end.IngeneralHoxgeneactivationissequential,followingtheirorderintheclusterina30to-50

direction,apropertyknownascollinearity.BecauseHoxgeneactivationisconcurrentwithaxialextensionandlimb

growth,thedifferentareasofthebodyandlimbsexpressdistinctcombinationsofHoxgenes.Othervertebrateshave

differentnumbersofHoxclusters.Forinstance,thezebraﬁshhassevenclusters,knownasHoxAa,HoxAb,HoxBa,

HoxBb,HoxCa,HoxCb,andHoxDa,resultingfromanadditionalduplicationfollowedbythelossofonewholecluster.

Order of acƟvaƟon of gene expression

3ʹ 5ʹ HoxB HoxA a1 b1 d1 a2 b2 a3 b3 d3 a4 b4 c4 d4 a5 c5 b5 a6 b6 c6 a7 b7 c8 d8 b8 a9 b9 d9 c9 a10 c10 d10 a11 c12 c11 d11 d12 a13 b13 c13 d13 HoxC HoxD

(4)

HoxclustershasbeenshowntoberelevantfortheregulationofHoxgeneexpressionduring limbbuddevelopment[33,37].Inparticular,asmentionedbelow,asthelimbbudgrowsdistally theHoxregulatorylandscapeswitchesfromthe30tothe50TADcoincidentwiththeactivation ofHoxgenesatthe50endofthecluster(Figure2).Hoxgeneregulationinthemajorbodyaxis mightalsoﬁtinasimilargeneralschemeinvolvingaregulatoryswitchbetweenTADs orches-tratedbythebalancebetweenOct4andGdf11signalingactivities.InvolvementoftheTAD structureinHoxgeneregulationinthemainbodyaxisissupportedbyrecentﬁndingsshowing

Gdf11 Oct4 5ʹTAD 3ʹTAD

?

a1 b1 a2 a3 a4 a5 a6 a7 b7 b9 a9 b8 b6 c6 c8 c9 c11 a11 c10 a10 c12 c13 b13 a13 b5 c5 b4 c4 d4 d8 d9 d10 d11 d12 d13 b3 d3 b2 d1

Figure 1. Oct4 Promotes Trunk Elongation and Gdf11 Activates theTrunk-to-TailTransition(Marked bytheHindlimbPosition).Aspartof

theseactivities,theyregulateHoxgene

expression.50Hoxgenesarekept

inac-tiveattrunklevelsbyOct4andbecome

activated when Gdf11 signaling takes

over.Regulationofseveral30Hoxgenes

follows a complementary pattern. Hox

geneexpressionintheareasofthelimb

budgeneratingthearmandforearmis

underthecontrolofregulatory

interac-tionsinthe30 topologicallyassociating

domain(TAD) (in blue). The regulatory

landscapeswitchestothe50TADinthe

distallimb(purple)toproduce

hand-spe-ciﬁc Hox gene expression. Does TAD

organization also impact

Oct4/Gdf11-mediatedHoxregulationinthemainbody

axis? PaƩerning body structures Determine size of body secƟons Neur al/mesodermal deriv aƟ ve Axial pr og enit or s Trunk Head HL FL Tail Oct4, Gdf11 Ho x

Figure 2. Patterning in the Main BodyAxisOccursinTwo Consecu-tiveStages.Intheﬁrststage,theﬁnal

sizeofthehead/trunk/tailregionis

deter-mined in the axial progenitors mostly

throughHox-independent mechanisms,

includingOct4andGdf11activities.The

transitionbetweentheseregionscanbe

identiﬁedinthedevelopingembryobythe

positionsoftheforelimb(FL)andhindlimb

(HL). Thesecond stageoccurs inthe

derivatives of the axial progenitors by

loadingthemwithaxiallevel-speciﬁc

pat-terninginformation(includingHoxgenes)

thatguidestheir differentiationinto the

(5)

thatWnt3/Wnt3asignalingandCdxproteinssequentiallyactivate30HoxAgenesintheepiblast throughspecificregulatory interactionsoccurringin the30TAD [38,39].Additional findings suggestthatOct4activitymightalsofitwithinthisregulatoryscheme.Inparticular,ithasbeen shownthatinembryonicstemcellsOct4primesHoxa1andHoxb1foractivationonexposure to retinoicacid[40],aphysiological activatorof Hoxgeneexpression. These observations indicatethatOct4mightindeedbeinvolvedinactivationof30Hoxgenes.Inaddition,Oct4has beenreportedtobindtheHoxAclusterattheintersectionoftheTADs,andthisbindingmight beimportantforproperchromatinstructureandHoxgeneexpressionincelllines[41].Even lessisknownaboutHoxregulationbyGdf11signaling.Currently,theonlyconnectionbetween Gdf11signalingandHoxgeneexpressionwassuggestedbythestudyofanenhancerinthe Hoxd11 30 UTR required for proper activation of Hoxd11 [42]. This enhancer contains a phylogenetically conservedSmad binding site essentialfor itsactivity intransgenic assays

[43].WhetherthisenhancerplaysamoregeneralroleintheregulationoftheHoxDcluster remainstobedetermined.

SelectiveInactivationofSpeciﬁcHoxTargets AddsEvolutionaryFlexibility AlthoughalargepartofHox-dependentmorphologicalvariationsintheaxialskeletonmight resultfromchangesinHoxgeneexpression,modulationofspeciﬁcdownstreamaspectsof Hoxactivitycanalsohavearelevantimpactonthisprocess.Aninterestingexampleistheorigin ofthelargeribnumbersofPaenungulata(includingelephantsandmanatees),extendingfurther posteriorlythaninothermammalstocovermostoftheirpresacralskeleton[44].Thisfeature lookssurprisinglysimilartotheskeletalphenotypeofmutantmicetotallylackingHox10activity

[14].SinceHox10genesplayotheressentialrolesinmammaliandevelopment(e.g.[45])itis unlikelythatPaenungulatajusthappenedtolosethesegenes.However,thegenomeofanimals belongingto thiscladecontain anSNPinanenhancermediatingHox10activity duringrib development[13]thatprecludesbindingofHox10proteins[46],thussimulatingafunctional Hox10inactivationrestrictedtothedevelopingaxialskeleton.Interestingly,thesame polymor-phismwasfoundinsnakes,whereitisalsoverylikelytointerferewithHox10rib-repressing activity.Inparticular,expressionofthesnakeHoxa10gene,whichblocksribformationwhen testedinmouse embryos [46], extendswellinto rib-formingsomites ofthe snake embryo withoutapparentnegativeeffects onrib development[8].Thisexampleillustrateswellhow regulationof Hox gene activity by selective interference withspeciﬁc downstream targets generatessubstantialevolutionaryﬂexibility,asitcanaffectasubsetoftheprotein’sfunctions whilekeepingotherrelevantactivitiesinthesamedomainorevenallowingtheacquisitionof additionalfunctionsinthatarea.

TheChromatinStructureofHoxClustersImpactsLimb Morphogenesis... Hoxgenesalsoplayessentialrolesinthemorphogenesisandevolutionofvertebratepaired appendages.Theirexpressioninthetetrapodlimbbudoccursintwosequentialphases,the ﬁrstassociatedwithproximallimbsegments(stylopodandzeugopod)andthesecondwith thedistaldomain,theautopod [47].Recentdataindicatethatthe regulationofthesetwo phasesofHoxgeneexpressioniscloselyconnectedtothe3Dchromatintopologycoveringthe Hoxclusters[33,34,37].InteractionanalysesperformedontheHoxAandHoxDclusters,the majorHoxplayersinlimbdevelopment,revealedthatduringtheﬁrstexpressionphaseHox genesinthe30TAD(uptoHox11)areunderthecontrolofregulatoryregionsinthisTAD[33,48]. This regulation is maintained in the proximal limb domain at later developmental stages. However,in thedeveloping autopodthe Hox regulatorylandscape switchesdrastically.In thisregionHoxgenesinthe50TAD(thoseoftheHox9toHox13groups),changetheirfunctional interactionstobecomeregulatedbyenhancersinthistopologydomain[33](Figure2).These latter interactions are required for the autopod-type Hox gene expression, including the

(6)

‘reversecollinearity’[49](i.e.,thestrengthandextensionofHoxgeneexpressiondecreasesina 50-to-30direction)and,importantly,theinactivationofHoxa11intheautopod[50],which,as discussedbelow, isdirectlylinkedtoHox13geneactivity.Thisregulatory switchcreatesa ‘Hox-free’areabetweentheproximalanddistallimbdomainsthoughttoberequiredforwrist/ anklejointdevelopment[33,47].

Althoughthemechanismsinvolvedinthisregulatoryswitchremainincompletelyunderstood,it isclearthatHox13genesareakeycomponentoftheprocess.Consistentwiththis,Hox13 mutantsareunabletoelicittheswitchandasaconsequencecharacteristicfeatures ofthe proximallimbbudextendintotheprospectiveautopodregion,hinderingproper morphogene-sisinthisarea[51,52].Hox13genesplayseveralrolesinthisprocess.Besidesdisconnecting regulatoryactivities thatinvolve the 30 TAD[51],Hox13 proteinsalsointeract withspeciﬁc enhancersinthe50TADtopromotethelatephaseofHoxgeneexpressioninthelimbs[51,52]. Finally,Hox13 proteinscontrolHoxa11 expressionas well,by activatingan enhancerthat promotesthetranscriptionofanantisenseHoxa11transcript(Hoxa11as)thatsilencesin-cis Hoxa11geneexpression[50].

... andtheEvolutionofPairedAppendages

ProgressinunderstandingthecontrolofHoxactivityduringlimbdevelopmentalsoprovided newinsightsintohowthetetrapodlimbmighthaveevolvedfrompairedfinsoffishes.Whilethe earlystagesoffinandlimbdevelopmentaresimilar,theyclearlydifferduringtheformationof theirdistaldomains.Thedistal-mostlimbdomain,theautopod,isdominatedbymesenchymal tissuethateventuallyprovidesthesubstratefordigitformation,whereasthedistalfindomain comprisesanepithelialstructure,thefinfold,thateventuallyholdsthefinradials.Theoriginsof thedifferencesbetweendistalfinandlimbdevelopmentremainnottotallyunderstood, but recentdatasuggestthattheymightinvolvetheacquisitionbyamniotesofnovelregulatory regionsinvolvedinthesecondphaseofHoxgeneexpressioninthelimbs.3Dstructureand interactionanalysesindicatethatHoxclustersofteleostsandamnioteslargelyshareboththeir TADstructureandthepreferredcontactsbetweenHox13genesandgenomicregionsinthe50 TAD[37,53].Despitethis,functionalanalysesrevealeddifferentregulatorypotentialsforthe50 TADsequencesofteleostsandmammals.Inparticular,whenpufferfishBACclonescontaining theHoxAa,HoxAb,orHoxDaclustertogetherwiththeadjacent50regionwereintroducedinto mice,theyactivatedfishHoxgeneexpressioninproximallimbdomains,failingtoextendinto the autopod [37]. Consistent with this, the zebrafish 50 TADs lack several key regulatory elements required for autopod-type Hox gene expression, including regions controlling Hox13activity[54,55] andtheHox13-responsiveenhancerpromotingHoxa11astranscript expression[50].Typical features of finHox geneexpression, likeoverlapping Hoxa11 and Hoxa13expressiondomains[56],non-detectableHoxa11astranscript[50],andexpressionof 50Hox geneswithnosignofreversecollinearity[57],areconsistentwiththelackofthose enhancerelements.

Interestingly,atleast someofthe50regulatory elementsabsent fromteleostscan activate reporter expression in the zebrafish fin bud with appropriate spatial distribution [50,54], suggestingthatteleostpairedappendagescontainthemolecularmachineryrequiredtocontrol thoseenhancers.Thisobservation,togetherwiththesimilarchromosomalarchitecture and interactionprofilesoffishandamnioteHoxclusters,indicatethattheteleostfincouldeasily acquireanautopod-typeregulatorylandscapeonincorporationoftherelevantenhancersinto their 50 TADs. This scenario has been suggested to have contributed to the fin-to-limb evolutionary transition[37]. Interestingly, the garHoxA regionseems to have a regulatory structure somewhere between teleosts and amniotes, as it contains at least one of the

(7)

enhancersabsentfromzebraﬁshthatisabletoreproduceautopodalHoxa13geneexpression whentestedinmouseembryos[54].

ACentralRole forHox13Genesin DistalDevelopmentofPaired Appendages

A varietyoffunctionalexperiments suggestthat thespecificexpressionfeatures ofHox13 genes,includingtheirexpressionlevels, arerelevantforthedifferentialdevelopmental char-acteristicsofthedistal limbandfin.Gradualreduction oftheHox13dosage inthemouse correlated with the progressive acquisition of several fin-like features, including the distal extensionof Hoxa11expressionthatinvades theHox13 domain,digitshortening,andthe absenceofthesmallskeletalelementscharacteristicofthezeugopod/autopodjoint[58,59]. Conversely,increasingHoxa13levelsinzebrafishfinsexpandedthemesodermalcoreatthe expenseoftheectodermalfoldandpromotedtheexpressionofsometypicaldistallimbbud markers[55].

Hox13genesare alsorequiredfordistal findevelopment.Cell-tracingstudiesindicatethat distalradialsderivefromHox13-positivemesenchymeenteringthefinfoldandgenetic experi-mentsrevealedthatthosestructuresfailtoformintheabsenceofHox13activity[60].These observationsreopentheolddiscussionaboutthehomologybetweendigitsanddistalradials. Suchhomologycouldactuallyhelptoexplainthelargernumberofradialsversusdigitsonthe basisofquantitativeandqualitativeaspectsofHoxgeneexpression.ThecharacteristicHoxa11 andHox13expressionoverlapinfinbuds[56]couldbepartofthemechanism,asexperimental Hoxa11activationintheHox13expressiondomainresultedinpolydactyllimbs[50].Inaddition, reducedHox13activityinaGli3-nullcontext(thuswithreducedhedgehogactivity)leadstoa significantincreaseindigitnumber[59].ItwassuggestedthatundertheseconditionsHox13 activitywoulddeterminethewavelengthoftheTuring-typemechanismcontrollingdigitnumber

[59,61],withthisnumberincreasingwiththereductionofHox13activity.Itisthuspossiblethat aTuringsystemactingintherelativelylowHox13contextoftheﬁncouldcontributetothelarge radialﬁnnumbers,althoughtheprecisecomponentsofthissystemmightnotbethesameasin mammals.

CanSnakesBlameHoxGenesforHavingLostTheirLimbs?

Inadditiontotheﬁn-to-limbevolutionarytransition,Hoxgeneshavebeensuggestedtoplaya roleinthelossofpairedappendagesbysnakes[62],althoughtheprecisemechanismsoreven whetherthisisindeedtrueawaitsdirectexperimentalproof.IthasbeenreportedthatTbx5,a keyregulatorofforelimbbudinduction[63],isactivatedinthelateralmesodermbyHox4and Hox5genes[64],suggestingapossiblemechanismbywhichchangesinHoxgeneexpression mighthavecontributedtothelossofforelimbscharacteristicofsnakes.However,regardlessof whether theseHox genes are essential for forelimb induction [11,12], expression studies showedthatmembersofthoseHoxgroupsaswellasTbx5itselfareexpressedinthelateral mesodermofsnakeembryos[8,29],indicatingthatthelackofforelimbsinsnakesismostlikely torequirealternativeexplanations.

Still,modiﬁcationofHoxgeneactivitymightaccountfortheinabilityofthehindlimbbudsof ancientsnakestodevelopintofull-grownlimbs.IthaslongbeenshownthatexpressionofShh, akeyplayerinlimbbudgrowthandpatterning[65],isdefectiveinthesnakelimbbuds[62].Shh limbexpressionreliesonadistalenhancerthatisfunctionallycompromisedinsnakes[66,67]. Interestingly,themodiﬁcationsfoundinthepythonShhenhancerrenderitunabletorespondto Hoxproteins[67].ConsideringthatHoxgeneactivityisrequiredtoinitiateand/ormaintainShh expressioninthemouselimbbuds[68],itislikelythatthelackofresponseoftheShhenhancer

(8)

toHoxproteinshasplayedafundamentalroleinthedevelopmentalarrestofthesnakehindlimb bud.

ConcludingRemarksandFuturePerspectives

OverthepastyearsaconsiderableamountofworkhaschangedtheplaceheldbyHoxgenesin theoverallhierarchyofthepatterningcascaderegulatingthevertebratebodystructure and their variations among taxa. The resulting new model not only describes how patterning information can traverse the various hierarchical levels controllingaxial patterning and the productionoffunctionalbodystructures,butalsoshedslightonsomeunexplainedaspectsof Hoxmutantphenotypes.Sofarit isunclear howOct4 andGdf11 controlHoxgenes (see OutstandingQuestions).TheeffectofthesetwofactorsonHoxgeneexpressionsuggestsa mechanismrelyingonthemodulationofregulatory activitiesorganizedinTADs,akintothe modelrecentlydescribedinthelimbbuds.However,directexperimentalevaluationisneeded toelucidatewhetherthisisindeedthe caseorwhetherthesefactorsoperateaccordingto entirelydifferent principles. It might also be interesting to determine whether Gdf11/Gdf8 signalingparticipatesinHoxgeneregulationinthelimbbuds,asbothGdf11andGdf8are expressedintheseappendages[18,69]andtheirsimultaneousinactivationledtostronglimb malformations[21].AnotherinterestingquestionistheextenttowhichotheraspectsofHox regulationidentiﬁedinthelimbbuds,likethoseinvolvingHox13genes,alsooperateinthemain bodyaxis.ThesubsequentﬁndingswillshowtheextenttowhichbasicmechanismsofHox generegulationareconservedthroughoutdevelopmentalterritories.Finally,itwillbeimportant tounderstandhowthe mechanismsofHox generegulationcoordinatewithotherrelevant featuresassociatedwithHoxgeneactivation,liketheprogressiveopeningoftheHoxclusters

[70],aswellastheirrelationshipwithotherfactorsknowntoregulatepatterningprocessesin Hox-expressingembryonicareas,likethemainbodyaxisorpairedappendages.

Acknowledgments

TheauthorthanksAnaCasaca,RitaAires,andAndreDiasforinsightfulcommentsonthemanuscript.WorkinM.M.’s

laboratoryissupportedbygrantsPTDC/BEX-BID/0899/2014FundaçãoparaaCiênciaeaTecnologia(FCT,Portugal)and

SCML-MC-60-2014(fromSantaCasadaMisericordiadeLisboa,Portugal).

References

1. Wilson,V.etal.(2009)Stemcells,signalsandvertebratebody axisextension.Development136,1591–1604

2. Henrique,D.etal.(2015)Neuromesodermalprogenitorsandthe makingofthespinalcord.Development142,2864–2875

3. Tam,P.P.L.andBehringer,R.R.(1997)Mousegastrulation:the formationofthemammalianbodyplan.Mech.Dev.68,3–25

4. Steventon,B.andMartinezArias,A.(2017)Evo-engineeringand thecellularandmolecularoriginsofthevertebratespinalcord. Dev.Biol.432,3–13

5. Stern,C.D.etal.(2006)Head–tailpatterningofthevertebrate embryo:one,twoormanyunresolvedproblems?Int.J.Dev.Biol. 50,3–15

6. Pearson,J.C.etal.(2005)ModulatingHoxgenefunctionsduring animalbodypatterning.Nat.Rev.Genet.6,893–904

7. Burke,A.C.etal.(1995)Hoxgenesandtheevolutionof verte-brateaxialmorphology.Development121,333–346

8. Di-Poï,N.et al.(2010)Changesin Hoxgenes’structureandfunction during the evolution of the squamate body plan. Nature 464, 99–103

9. Mallo,M.etal.(2010)Hoxgenesandregionalpatterningofthe vertebratebodyplan.Dev.Biol.344,7–15

10.Wellik,D.M.(2007)Hoxpatterningofthevertebrateaxial skele-ton.Dev.Dyn.236,2454–2463

11.Horan,G.S.B.etal.(1995)Compoundmutantsforthe paralo-goushoxa-4,hoxb-4,andhoxd-4genesshowmorecomplete

homeotictransformationsandadose-dependentincreaseinthe numberofvertebraetransformed.GenesDev.9,1667–1677

12.McIntyre,D.C.etal.(2007)Hoxpatterningofthevertebraterib cage.Development134,2981–2989

13.Vinagre,T.etal.(2010)EvidenceforamyotomalHox/Myf cas-cadegoverningnonautonomouscontrolofribspeciﬁcationwithin globalvertebraldomains.Dev.Cell18,655–661

14.Wellik,D.M.andCapecchi,M.R.(2003)Hox10andHox11genes arerequiredtogloballypatternthemammalianskeleton.Science 301,363–367

15.Carapuço,M.etal.(2005)Hoxgenesspecifyvertebraltypesin thepresomiticmesoderm.GenesDev.19,2116–2121

16.Economides,K.D.etal.(2003)Hoxb13mutationscause over-growthofcaudalspinalcordandtailvertebrae.Dev.Biol.256, 317–330

17.Young,T.etal.(2009)CdxandHoxgenesdifferentiallyregulate posterioraxialgrowthinmammalianembryos.Dev.Cell17,516– 526

18.Nakashima,M.etal.(1999)Expressionofgrowth/differentiation factor11,anewmemberoftheBMP/TGFbsuperfamilyduring mouseembryogenesis.Mech.Dev.80,185–189

19.McPherron,A.C.etal.(1999)Regulationofanterior/posterior patterningoftheaxialskeletonbygrowth/differentiationfactor 11.Nat.Genet.22,260–264

OutstandingQuestions

How do Oct4 and Gdf11 signaling

controlHoxgeneexpression?

DoesHoxgeneregulationinthemain

bodyaxisalsoinvolvedynamic

regula-tory processes associated with the

TADstructureoftheHoxclusters?

DoesHoxgeneregulationfollowthe

samebasicprinciplesinthedifferent

embryonic areas or does it obey

region-speciﬁc rules?Ifthose

princi-ples aretosome extentconserved,

howextensiveisthisconservation?

What are the mechanistic links

between Hox regulatory processes

and the progressiveopening of the

Hoxclusterchromatin?

Itisknownthatgrowthandpatterning

processesalongthemainbodyaxis

andinthepairedappendagesarealso

underthecontrolofavarietyoffactors

otherthanHoxgenes,includingmany

signalingpathways.Whatisthe

func-tionalrelationshipbetweenthese

(9)

20.Jurberg,A.D.etal.(2013)Switchingaxialprogenitorsfrom pro-ducingtrunktotailtissuesinvertebrateembryos.Dev.Cell25, 451–462

21.McPherron,A.C.etal.(2009)Redundancyofmyostatinand growth/differentiationfactor11function.BMCDev.Biol.9,24

22.Matsubara,Y.etal.(2017)Anatomicalintegrationofthesacral– hindlimb unit coordinated by GDF11 underlies variation in hindlimb positioning in tetrapods. Nat. Ecol. Evol. 1, 1392–1399

23.Aires,R.etal.(2016)Oct4isakeyregulatorofvertebratetrunk lengthdiversity.Dev.Cell38,262–274

24.Downs,K.M.(2008)SystematiclocalizationofOct-3/4tothe gastrulatingmouseconceptussuggestsmanifoldrolesin mam-maliandevelopment.Dev.Dyn.237,464–475

25.Osorno,R.etal.(2012)Thedevelopmentaldismantlingof pluri-potencyisreversedbyectopicOct4expression.Development 139,2288–2298

26.DeVeale,B.etal.(2013)Oct4isrequiredE7.5forproliferationin theprimitivestreak.PLoSGenet.9,e1003957

27.Szumska,D.etal.(2008)VACTERL/caudalregression/Currarino syndrome-likemalformationsinmicewithmutationinthe pro-proteinconvertasePcsk5.GenesDev.22,1465–1477

28.Liu,J.-P.(2006)Thefunctionofgrowth/differentiationfactor11 (Gdf11)inrostrocaudalpatterningofthedevelopingspinalcord. Development133,2865–2874

29.Woltering,J.M.etal.(2009)Axialpatterninginsnakesand cae-cilians:evidenceforanalternativeinterpretationoftheHoxcode. Dev.Biol.332,82–89

30.Delpretti,S.etal.(2013)MultipleenhancersregulateHoxdgenes andtheHotdoglncRNAduringcecumbudding.CellRep.5,137– 150

31.Philippidou,P.andDasen,J.S.(2013)Hoxgenes: choreogra-phersinneuraldevelopment,architectsofcircuitorganization. Neuron80,12–34

32.Head,J.J.andPolly,P.D.(2015)Evolutionofthesnakebodyform revealshomoplasyinamnioteHoxgenefunction.Nature520, 86–89

33.Andrey,G.etal.(2013)Aswitchbetweentopologicaldomains underliesHoxDgenescollinearityinmouselimbs.Science340, 1234167

34.Berlivet,S.etal.(2013)Clusteringoftissue-speciﬁcsub-TADs accompaniestheregulationofHoxAgenesindevelopinglimbs. PLoSBiol.9,e1004018

35.Nora,E.P.etal.(2012)Spatialpartitioningoftheregulatory landscapeoftheX-inactivationcentre.Nature485,381–385

36.Dixon,J.R.etal.(2012)Topologicaldomainsinmammalian genomesidentiﬁedbyanalysisofchromatininteractions.Nature 485,376–380

37.Woltering,J.M.etal.(2014)Conservationanddivergenceof regulatorystrategiesatHoxLociandtheoriginoftetrapoddigits. PLoSBiol.12,e1001773

38.Neijts,R.etal.(2016)PolarizedregulatorylandscapeandWnt responsivenessunderlieHoxactivationinembryos.GenesDev. 30,1937–1942

39.Neijts,R.etal.(2017)Cdxiscrucialforthetimingmechanism drivingcolinearHoxactivationanddeﬁnesatrunksegmentinthe Hoxclustertopology.Dev.Biol.422,146–154

40.Simandi,Z.etal.(2016)OCT4actsasanintegratorof pluripo-tencyandsignal-induceddifferentiation.Mol.Cell63,647–661

41.Kim,Y.J.etal.(2011)Conserved,developmentallyregulated mechanismcoupleschromosomalloopingandheterochromatin barrieractivityatthehomeoboxgeneAlocus.Proc.Natl.Acad. Sci.U.S.A.108,7391–7396

42.Zákány,J.etal.(1997)DeletionofaHoxDenhancerinduces transcriptional heterochrony leading to transposition of the sacrum.EMBOJ.16,4393–4402

43.Gaunt,S.J.etal.(2013)DirectactivationofamouseHoxd11axial expressionenhancerbyGdf11/Smadsignalling.Dev.Biol.383, 52–60

44.Narita,Y.andKuratani, S.(2005)Evolutionofthevertebral formulaeinmammals:aperspectiveondevelopmental con-straints.J.Exp.Zool.BMol.Dev.Evol.304,91–106

45.Satokata,I.etal.(1995)Sexuallydimorphicsterilityphenotypesin Hoxa10-deﬁcientmice.Nature374,460–463

46.Guerreiro,I.etal.(2013)Roleofapolymorphismina Hox/Pax-responsiveenhancerintheevolutionofthevertebratespine. Proc.Natl.Acad.Sci.U.S.A.110,10682–10686

47.Woltering,J.M.andDuboule,D.(2010)The originof digits: expressionpatternsversusregulatorymechanisms.Dev.Cell 18,526–532

48.Montavon,T.etal.(2011)AregulatoryarchipelagocontrolsHox genestranscriptionindigits.Cell147,1132–1145

49.Montavon,T.etal.(2008)ModelingHoxgeneregulationindigits: reversecollinearityandthemolecularoriginofthumbness.Genes Dev.22,346–359

50.Kherdjemil,Y.etal.(2016)EvolutionofHoxa11regulationin vertebratesislinkedtothepentadactylstate.Nature539,89–92

51.Beccari,L.etal.(2016)AroleforHOX13proteinsintheregulatory switchbetweenTADsattheHoxDlocus.GenesDev.30,1172– 1186

52.Sheth,R.etal.(2016)Distallimbpatterningrequiresmodulation ofcis-regulatoryactivitiesbyHOX13.CellRep.17,2913–2926

53.Acemel,R.D.etal.(2016)Asinglethree-dimensionalchromatin compartmentinamphioxusindicatesastepwiseevolutionof vertebrateHoxbimodalregulation.Nat.Genet.48,336–341

54.Gehrke,A.R.etal.(2015)Deepconservationofwristanddigit enhancersinﬁsh.Proc.Natl.Acad.Sci.U.S.A.112,803–808

55.Freitas,R.etal.(2012)Hoxd13contributiontotheevolutionof vertebrateappendages.Dev.Cell23,1219–1229

56.Sordino,P.etal.(1996)ZebraﬁshHoxaandEvx-2genes:cloning, developmentalexpressionandimplicationsforthefunctional evolutionofposteriorHoxgenes.Mech.Dev.59,165–175

57.Ahn,D.andHo,R.K.(2008)Tri-phasicexpressionofposterior Hoxgenesduringdevelopmentofpectoralﬁnsinzebraﬁsh: implicationsfortheevolutionofvertebratepairedappendages. Dev.Biol.322,220–233

58.Fromental-Ramain,C.etal.(1996)Hoxa-13andHoxd-13playa crucialroleinthepatterningofthelimbautopod.Development 122,2997–3011

59.Sheth,R.etal.(2012)Hoxgenesregulatedigitpatterningby controllingthewavelengthofaTuring-typemechanism.Science 338,1476–1480

60.Nakamura,T.etal.(2016)Digitsandﬁnrayssharecommon developmentalhistories.Nature537,225–228

61.Raspopovic,J.etal.(2014)Modelingdigits.Digitpatterningis controlledbyaBmp–Sox9–WntTuringnetworkmodulatedby morphogengradients.Science345,566–570

62.Cohn,M.J.andTickle,C.(1999)Developmentalbasisof limb-lessnessandaxialpatterninginsnakes.Nature399,474–479

63.Rallis,C.etal.(2003)Tbx5isrequiredforforelimbbudformation andcontinuedoutgrowth.Development130,2741–2751

64.Minguillon,C.etal.(2012)HoxgenesregulatetheonsetofTbx5 expressionintheforelimb.Development139,3180–3188

65.Tickle,C.(2015)Howtheembryomakesalimb:determination, polarityandidentity.J.Anat.227,418–430

66.Kvon,E.Z.etal.(2016)Progressivelossoffunctioninalimb enhancerduringsnakeevolution.Cell167,633–642

67.Leal,F.andCohn,M.J.(2016)Lossandre-emergenceoflegsin snakesbymodularevolutionofSonichedgehogandHOXD enhancers.Curr.Biol.26, 2966–2873

68.Kmita,M.etal.(2005)Earlydevelopmentalarrestofmammalian limbslackingHoxA/HoxDgenefunction.Nature435,1113–1116

69.Amthor,H.etal.(2002)Theregulationandactionofmyostatinas anegativeregulatorofmuscledevelopmentduringavian embryo-genesis.Dev.Biol.251,241–257

70.Soshnikova,N.andDuboule,D.(2009)Epigenetictemporal controlofmouseHoxgenesinvivo.Science324,1320–1323