hatchingsebao

DOI10.
1007/s00018-013-1288-2Cell.
Mol.
LifeSci.
(2013)70:3829–3845REVIEWExpandingrolesfortheevolutionarilyconservedDmrtsextranscriptionalregulatorsduringembryogenesisEricJ.
Bellefroid·LucasLeclère·AmandineSaulnier·MarcKeruzore·MariaSirakov·MichelVervoort·SarahDeClercqReceived:11October2012/Revised:18January2013/Accepted:31January2013/Publishedonline:5March2013SpringerBasel2013IntroductionTheDmrt(doublesexandmab3-related-transcriptionfactor)familyderivesitsnamefromtwofoundingmembers,dou-blesex(dsx)inDrosophilamelanogasterandmaleabnor-mal(mab-3)inCaenorhabditiselegans.
ItisdefinedbythepresenceoftheDMdomainDNAbindingmotif,anunusualcysteine-richzincDNAbindingmotifconstitutedoftwointertwinedzincfingers[1,2].
Dmrtgenesarewellknowntoplayaconservedroleinsexdetermination,sexualdimorphismorotheraspectsofsexualreproduction[3,4],butagrowingbodyofevidenceindicatesthattheyarealsoconservedregulatorsofotherdevelopmentalprocesses[5].
Inthisreview,weprovideanupdatedoverviewoftheevo-lution,structureandmechanismsofactionofDMgenes.
Wesummarizerecentfindingsontheirfunctioninsexualdevelopmentanddiscussmoreextensivelyrecentfunctionalstudiesdemonstratingtheirimportantfunctioninsomi-togenesisandneuraldevelopment.
Inparticular,wehigh-lighttheimportantroleofasubgroupofthemincludingDmrt3,Dmrt4andDmrt5,characterizedbythepresenceofanadditionalhighlyconserveddomaindesignatedDMA,inneurogenesisandpatterningofthedevelopingnervoussystem.
TaxonomicdistributionandarchitectureofDmrtproteinsAlthoughDMdomaingeneshavebeenextensivelystudiedinmodelorganisms,littleisknownabouttheevolutionofthisgenefamily.
DMdomaingeneshavebeendetectedinmanybilaterianspecies,includingintheamphioxus[6],theoysterCrassostreagigas[7],andintwocnidarianspecies,thecoralAcroporamillepora[8]andtheseaanemoneNematostellavectensis[9].
Nevertheless,theevolutionaryoriginofDmrtgenesinanimalshasnotbeenfurtherinvestigatedduetoaAbstractDmrtgenesencodealargefamilyoftranscrip-tionfactorscharacterizedbythepresenceofaDMdomain,anunusualzincfingerDNAbindingdomain.
WhileDmrtgenesarewellknownfortheirimportantroleinsexualdevelopmentinarthropodes,nematodesandvertebrates,severalnewfindingsindicateemergingfunctionsofthisgenefamilyinotherdevelopmentalprocesses.
Here,weprovideanoverviewoftheevolution,structureandmechanismsofactionofDmrtgenes.
Wesummarizerecentfindingsontheirfunctioninsexualregulationanddiscussmoreexten-sivelytheroleplayedbytheseproteinsinsomitogenesisandneuraldevelopment.
KeywordsDmrt·Sexualdifferentiation·Somitogenesis·Neurogenesis·Olfactoryplacode·TelencephalonElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.
1007/s00018-013-1288-2)containssupplementarymaterial,whichisavailabletoauthorizedusers.
E.
J.
Bellefroid(*)·A.
Saulnier·M.
Keruzore·M.
Sirakov·S.
DeClercqLaboratoiredeGénétiqueduDéveloppement,InstitutdeBiologieetdeMédecineMoléculaires(IBMM),UniversitéLibredeBruxelles,ruedesProfs.
JeeneretBrachet12,6041Gosselies,Belgiume-mail:ebellefr@ulb.
ac.
beL.
LeclèreSarsCentreforMarineMolecularBiology,UniversityofBergen,Thormhlensgt55,5008Bergen,NorwayM.
VervoortInstitutJacquesMonod,CNRS,UMR7592,UniversitéParisDiderot,SorbonneParisCité,75205Paris,France3830lackofdatafrombasalmetazoans.
Usingavailablewhole-genomesequencesfromseveralbasalmetazoans,weidenti-fiednovelDMgenesinthecnidarianAcroporadigitifera,intheplacozoanTrichoplaxadhaerens,inthectenophoreMnemiopsisleidyiandinthespongesSyconciliatumandOscarellacarmella(Fig.
1a;Suppl.
Table1).
However,DMdomaingeneswerenotdetectedinthegenomeofthespongeAmphimedonqueenslandica,andinseveralnon-metazoanholozoansincludingchoanoflagellates,theclosestrelativestometazoans.
Thus,theDMdomaingenefamilyprobablyaroseduringearlymetazoanevolutionafterthedivergencewithchoanoflagellates,andexpandedinthemetazoanlineage.
VertebrateshavemultipleDmrtgenes(e.
g.
,Dmrt1toDmrt8inmiceandhumans).
Dmrtproteinshavebeenclassifiedintoseveralsubfamilies,basedonthepres-enceofadditionalconservedproteindomainsandontheexon–intronstructureofthecorrespondinggenes.
Dmrt4(Dmrta1),Dmrt5(Dmrta2)andDmrt3(Dmrta3)constituteonesuchsubfamilycharacterizedbythepresenceofacon-servedDMAdomainattheirC-termini(Fig.
1b).
Eachgeneofthissubfamilyhastwocodingexons,withtheDMdomainencodedbythefirstcodingexonandtheDMAdomainencodedbythesecondone[10].
InN.
vectensis,aDMAdomainispresentinalleightDmrtgenes,suggest-ingthatitspresencemayrepresenttheancestralconditionforcnidariansandbilaterians[9].
WeobservedthepresenceofaDMAdomaininsomeoralltheDmrtgenesinmostmetazoanspecieswelookedat.
Thisincludesnon-bilate-rianssuchastheplacozoanTrichoplaxandthectenophoreMnemiopsis(Fig.
1a;Suppl.
Table1),indicatingthatDmrtgeneswithbothDMandDMAdomainswerealreadypre-sentduringearlymetazoanevolution.
Uncertaintiesaboutthephylogeneticrelationshipsofnon-bilateriananimals[11,12]andtherelativepaucityofgenomicdatafromnon-bilaterianspeciesdonotallowthedrawingoffirmconclu-sionsaboutwhetheraDM+DMAarchitecturemayrepre-senttheancestralstateofDmrtgenesinmetazoans.
FurtherstudiesarealsorequiredtodefinewhetherthelastcommonancestorofbilateriansandthatofbilateriansandcnidariansonlyownedDmrtgeneswithDMandDMAdomains,asisobservedinsomepresent-dayspecies(suchasNematostellaandSaccoglossus),orwhetherbothgeneswithandwithoutDMAdomaincoexistedintheseremoteancestors,asfoundinmanyextantspecies(suchasvertebrates).
PhylogenyoftheDmrtgenesWeperformedaphylogeneticanalysisoftheDmrtsequencestostudytheevolutionofindividualDmrtgenemembers.
ThephylogeneticrelationshipamongDmrtpro-teinsisnotobvious,asthereislittlesequencesimilarityoutsidetheshortDMandDMAdomains.
Here,welimitedourstudytodeuterostomesincludingsequencesfromvari-ouschordates,echinodermsandhemichordates.
Athor-oughphylogeneticanalysisisneededtodrawconclusionsabouttheoriginandtheevolutionoftheDmrtfamilyatthelevelofthemetazoans.
PhylogeneticanalysesofbothDMandDMAdomains(Fig.
1c,d)suggestthattheDmrt2,Dmrt3andDmrt4/5vertebratesubgroupismonophyleticandthatorthologsofeachofthesegeneswerealreadypre-sentintheancestorofdeuterostomesandcontainedbothDMandDMAdomains.
ComparisonbetweenDMandDMAdomainphylogeniessuggestthatvertebrateDmrt2havelosttheirDMAdomainsincerelatedsequencesincephalochordatesandhemichordatescontainthisdomain.
VertebrateDmrt1,Dmrt6andDmrt7alsoappeartobemonophyletic.
Theycouldnotberelatedunambiguouslytonon-vertebratedeuterostomesequencesorotherDmrtvertebratesubgroupsduetoweakphylogeneticsignal.
Thequestionoftheiroriginremainsunsolved.
Dmrt8(Dmrtc1)showstrongsimilarityovertheirentiresequencetoDmrt7(Dmrtc2)butlacktheDMdomain[13].
Bothhavebeenfoundinmammalsbutnotinothervertebratespecies.
InDmrt8fromhuman,chimpandorangutan,theopenreadingframeisdisruptedbyastopcodon5′oftheDMFig.
1TaxonomicdistributionandphylogenyoftheDmrtgenesanddomainarchitectureoftheproteins.
aDistributionofDmrtgenesinrepresentativesofmetazoans.
Foreachspecies,thenumberofDmrtparalogs,withorwithoutaDMAdomain,isreportedinparentheses.
Accessionnumbersofthesequencesincludedinthisanalysisarepro-videdinSuppl.
TableS1.
Metazoaarehighlightedinred,Eumetazoainyellow,Bilateriainblue,CnidariaingreenandPoriferaingray.
Thephylogeneticrelationshipsofplacozoansandctenophoreswiththeothermetazoangroupsarestillcontroversialandarethereforerepresentedbydashedlines.
Insomephylogeneticstudies[12],cten-ophorestogetherwithcnidariansconstitutethesistergrouptobilate-rians,whileinotherstudies[11],ctenophoresarefoundasthesistergrouptoallothermetazoansincludingsponges.
Similarly,placozo-anshavebeenreportedeitherasbelongingtoeumetozoans[12],orasthesistergrouptoallothermetazoans[102].
bDomainarchitectureofthemouseDmrtparalogs.
ForeachDmrtprotein,theadditionalnamesareprovided.
Dmrtproteinssharehighlyconservedproteinmotifs,includingtheDMdomain,andaresubdividedintoafewsub-groupsbasedonsequencesimilarity.
Dmrt3,Dmrt4,andDmrt5arecategorizedasasubgroupbecausetheirproductsshareahighlycon-servedDMAdomainattheirC-termini.
Pro/Ser-,Ala-andGly-richdomainsarealsoindicated.
c,dPhylogeneticrelationshipsbetweendeuterostomianDmrtsinferredbyneighbor-joining(NJ)analysesoftheDM(c)andDMA(d)domains.
Sequencesincludedbelongtothemajorgroupsofdeuterostomes:hemichordates(sequencenamesinred),echinoderms(green),cephalochordates(blue),urochordates(orange)andvertebrates(black);Bf,Branchiostomafloridae;Dr,Daniorerio;Hs,Homosapiens;Mm,Musmusculus;Gg,Gallusgal-lus;Sk,Saccoglossuskowalevskii;Sp,Strongylocentrotuspurpuratus;Ci,Cionaintestinalis;Xt,Xenopustropicalis.
SeeTableS1forinfor-mationaboutsequences.
Foreachgenomes,allidentifiedsequenceshavebeenincludedexcepttheverydivergentB.
floridaeDmrtC,DmrtDandDmrtE.
Branchescrossedbyagreenorredbarindicatebootstrapvalue≥70and≥95%,respectively(1,000NJbootstrapreplicates)3831ABCD3832domain.
Therefore,primateDmrt8genesdonothavethepotentialtoencodeaDMdomain[14].
MechanismsofactionofDmrtproteinsDespitetheimportanceofDmrtproteins,howtheyregu-latetheexpressionofspecifictargetgenesremainselu-sive.
Dmrt3andDmrt5havebeenshowntoencodenuclearproteins[9,15].
Sequence-specificDNAbindingviatheDMdomainhasbeendeterminedinvitroformostverte-brateDmrtproteins[9,15,16].
TheyallbindverysimilarDNAsequencesresemblingthoseboundbyDSX[17,18]andMAB-3[19].
Theconsensusconsistsinapalindromicsequence(G/A)NNAC(A/T)A(A/T)GTNN(C/T)composedoftwohalf-sitesaroundacentral(A/T)basepair.
Aspre-dictedfromthesymmetricnatureofthissite,DmrtproteinsbindDNAashomodimersorheterodimerswithotherDMdomainproteins[16,17].
DMdomainproteinsareabletoactastranscriptionalactivatorsorrepressors.
ReportergenetranscriptionassaysanddeletionanalysishaveindicatedthattheirregulatorydomainsarelocatedattheirC-termini[9,20,21].
Todate,transcriptionalregulationbyDmrtproteinshasonlybeeninvestigatedatalargescaleinthecaseofDmrt1inmousesexualdifferentiation(seebelow).
Incellcultures,Dmrt1activatesorrepressestranscriptiondependingoncelltypeandpromoterstructure.
Invivo,inthedevelopingmousemaleandfemalegonads,Dmrt1actssimultaneouslyatmul-tiplesitesacrossthegenome,activatingsomegenesandrepressingothers.
Thus,Dmrt1functionsasabifunctionaltranscriptionalregulator.
ItalsobindsitsownpromoteraswellasoneoftheotherDmrtgenes,suggestingauto-andcross-regulationofthesegenes.
Chromatinimmunopre-cipitation(ChIP)analysisusingconditionalmutanttestesshowedthatDNAbindingandtranscriptionalregulationofindividualtargetgenescandifferbetweengermcellsandSertolicells,andthatsomegenesexhibitDmrt1bindingonlyinonecelltype.
DifferentialresponseofgenestolossofDmrt1appearstocorrespondtodifferencesinthebindingmotif,suggestingthatothertransactivatingfactorsmodulateitsactivity[22–25].
WhetherotherDMproteinsfunctionlikeDmrt1asbifunctionaltranscriptionalregulatorsisunclear.
MAB-3repressestranscriptionofyolkgene(vitellogenin)intheintestineandblockstranscriptionoftheantineuralbHLHgeneref-1,arepressoroftheproneuralproteinlin-32inmalerayprecursorcells[19,26].
dsxisalternativelysplicedintosex-specificisoformsthatencodeproteinsthatsharetheDMdomainbuthavedistinctC-termini.
DSXMinmalesblocks,whereasDSXFinfemalesactivates,transcrip-tionofgenessuchasthefemale-specificyolkproteingenesorthebricabrac1(bab1)andbab2geneswhichcontrolpigmentation[27–29].
Recently,novelDSXtargetsdur-inggenitaldevelopmenthavebeenidentified,somebeingactivatedandothersbeingrepressedbyDSX[30].
However,itisnotknownwhetherthesenoveldownstreamgenesaredirectorindirecttargets.
Thus,sofar,onlyDmrt1hasbeenshowntobebifunctional.
HowdoDmrtproteinsachievetranscriptionrepressionoractivationinacell-specificmannerIncontrasttootherzincfingerproteins,DMproteinsinteractwithDNAintheminorgrooveratherthanthemajorgroove[2,31].
ThisenablesthemtobindtoDNAonsitesoverlappingthoseofmajorgroovebindingproteinsandtophysicallyinterferewiththeirbindingortocooperatewiththem.
SuchamechanismhasbeenobservedinthecaseofDSXandMAB-3[26,28,29,32,33].
WhetherthismechanismisalsousedbyDmrtproteinsinvertebrateisnotknown.
Whethertheyrecruitcoactivatorsorcorepressorsandassociatewithchromatin-modifyingenzymesalsoremainstobeinvestigated.
DmrtsareconservedregulatorsofsexualdevelopmentDMdomain-containinggenesareconservedgeneticcom-ponentsinvolvedinsexdifferentiationinallanimalsthathavebeenstudied.
Inflies,dsxactdownstreaminthesex-determinationpathway.
dsxisexpressedinsomaticgonadalprimordiumanditsfunctioninthesomaticgonadisrequiredforsex-specificgermlinedevelopment.
Laterindevelopment,dsxiswidelyexpressedinasubsetofnon-gonadalcellswhereitactscellautonomouslyandnon-cellautonomously,viainteractionswithconservedHoxgenesandsignalingpathways,tointegratesex-specific,spatialandtemporalcuesandinducelocalizedsex-specificdifferentia-tion.
ThreeotherDMdomaingenesarepresentinflies,butonlydsxhasbeenshowntoregulatesexualdimorphism.
Caernorhabditiseleganshas11DMdomaingenes.
Amongthem,3areknowntoregulatesexualdevelop-ment:mab-3,mab-23andDMdomain3(dmd-3).
Theyaredispensabletogonaddevelopmentbutareinvolvedinthesexualdifferentiationofsomatictissuesincludingcopulatorystructuressuchassensoryrays,spiculesandmatingmuscles.
Asinflies,DMdomaingenesinnematodesintegratesexualandvariouspositionalandtemporalcuestoinitiateasex-specificdevelopmentalprogram(reviewedin[3,4]).
InDaphniamagna,afreshwaterbranchiopodcrusta-ceanwhichparthenogeneticallyproducesmalesinresponsetoenvironmentalcues,aDMdomaingene,DapmaDsx1,hasbeenidentified.
DapmaDsx1showshigherexpressioninmale-specificstructures.
KnockdownofDapmaDsx1inmaleembryosdirectstheproductionoffemaletraitswhereasectopicexpressionofDapmaDsx1infemaleresultsinthedevelopmentofmale-likephenotypes[34].
InAcroporamillepora,transcriptsoftheDMdomaingeneAmD1arepresentathigherlevelsduringsexualdif-ferentiation[8],suggestingthattheimplicationofDmrt3833genesinsexualregulationmayhavepredatedthedivergencebetweencnidariansandbilaterians.
Invertebrates,allDmrtfamilymembersareexpressedintheindifferentgonadandinmostcasestheyaresub-sequentlymaintainedathigherlevelsinmaleasopposedtofemalegonads(Table1).
AmongtheeightDMdomaingenesinvertebrates,threeofthemhavebeenestablishedtohaverolesingonadand/orgermlinedevelopment,namelyDmrt1,Dmrt4andDmrt7(Table2).
Inthefollowingsection,webrieflyhighlightthefunctionsoftwoofthem,Dmrt1andDmrt7,andshowthatDmrt1oracloseparaloghasrecentlymoveduptheregulatoryhierarchyfromdownstreamposi-tionsingonadandgermlinedevelopmenttothetoplevelofsexdeterminationduringevolutioninfish,birdsandfrogs.
TheroleofDmrt4ingonadalandnon-gonadaltissuesisdiscussedinthenextsection.
RecentexcellentreviewshavemoreextensivelydiscussedtheroleofDmrtsinthedevelop-mentandevolutionofsexdimorphism[3,4,35,36].
Dmrt1triggersmale-specificandrepressesfemale-specificdifferentiationInhumans,Dmrt1islocalizedinaregionontheshortarmofchromosome9(9p24.
3)includingthreeDmrtgenes(Dmrt1–3).
Deletionsofthisregionareassociatedwithtesticulardysgenesisand,insomecases,causesexrever-saloftheXYembryonicgonadintoovariantissue[37–40].
Amongthethreegenes,Dmrt1isthestrongestcandidateforXYgonadaldysgenesis.
Itisindeedexpressedinthehumanembryonicgenitalridgesofhumanmale,butnotfemaleembryos[41].
DeletionsormutationsofDmrt1cancauseXYgonadaldysgenesis[42–45].
Therefore,Dmrt1wasthoughttohaveanimportantfunctioninsexdetermination.
FeminizationassociatedwiththelossofDmrt1is,however,morelikelyduetoafailureofmalegonadaldifferentiationortothereprogrammingofSertolicellsintogranulosa-likecells,asrecentlydiscoveredinmice(seebelow).
Dmrt1isalsoassociatedwithtesticulargermcellcancer[46–48],whichisconsistentwitharoleofDmrt1asatumorsuppres-sorin129vmice[22].
Inmouseembryos,Dmrt1isexpressedinthegenitalridgeofbothsexesbeforeanyovertsignsofsexdiffer-entiation.
Later,Dmrt1expressiondeclinesintheovarybutismaintainedinthetestis,whereitisrestrictedtopre-meioticgermcellsandSertolicells[49].
Inmice,Dmrt1isnotrequiredforprimarysexdeterminationasXYDmrt1mutantsarebornasmales.
Itis,however,involvedinmul-tipleaspectsofthemalegonaddifferentiation.
Indeed,inthosemutants,Sertolicellsoverproliferate,loseexpressionofthemale-specificSox9proteinandacquireexpressionoffemalespecificForkheadboxL2(Foxl2)andothergranu-losamarkers.
Germcellsfailtoundergoradialmigration,toreactivatemitosis,toentermeiosisandtosurvivebeyondP10[50].
InordertoidentifythefunctionofDmrt1inSer-toliandgermcells,Kimetal.
haveusedconditionalgenetargeting.
ThisapproachrevealedthatDmrt1isrequiredinSertolicellsfortheirpostnataldifferentiation,forgermlinemaintenanceandformeioticprogression.
Ingermcells,Dmrt1isrequiredforradialmigration,formitoticreactiva-tionjustafterbirth,andforsurvivalbeyondthefirstpost-natalweek.
Thus,inmice,Dmrt1isrequiredautonomouslyinbothcelllineages.
Dmrt1activityinSertolicellsisalsorequirednon-cellautonomouslytomaintainthegermline[51].
ArecentstudyhasaddressedthefunctionofDmrt1inSertolicellsinthepostnataltestis.
Inmammals,sexisdeter-minedinthefetalgonadbythepresenceorabsenceoftheYchromosomegeneSry,whichcontrolswhetherbipoten-tialprecursorcellsdifferentiateintotesticularSertolicellsorovariangranulosacells.
Thispivotaldecisioninasinglegonadalcelltypeultimatelycontrolssexualdifferentiationthroughoutthebody.
SexdeterminationcanbeviewedasabattleforprimacyinthefetalgonadbetweenamalegeneticnetworkinwhichSryactivatesSox9andafemalenetworkinvolvingWntsignalingandFoxl2,afemale-specifictran-scriptionfactorexpressedingranulosaandthecacells.
LossofDmrt1,eveninadultSertolicells,activatesovary-specificgenessuchasFoxl2andcausesthelossofmale-promotinggenessuchasSox9,andreprogramsSertolicellsintogranu-losacells.
Inthisenvironment,thecacellsform,oestrogenisproduced,andgermcellsappearfeminized[24,25].
Con-versely,lossofFoxl2inadultgranulosacellscausesectopicexpressionofDmrt1andtheirreprogrammationintoSertolicells[52].
Thus,Dmrt1isrequiredtopreventfemalerepro-gramminginthepostnatalmammaliantestis,andthesexualfateofthesomaticgonadispostnatallycontrolledbytheopposedactivityofDmrt1andFoxl2.
AnotherfunctionofDmrt1inmalegermcellswasrevealedwhenDmrt1wasdeletedinpostnatalundifferenti-atedspermatogonia.
Inmammals,meiosisbeginsatpuberty,andspermisproducedthroughoutlife.
Spermatogenesisoccursinthreephases:amitoticproliferativephaseinvolvingspermatogoniastem/progenitorcells,tworeductivedivisionsofmeiosis,andthenapostmeioticphaseofspermiogenesis.
Theswitchfrommitosistomeiosisrequiresretinoicacid(RA),whichactivatesmeioticinducers,includingStra8[53,54].
Dmrt1isdetectedinallmitoticspermatogonia,anditsexpressiondecreaseswiththeonsetofspermatogonialdifferentiationanddisappearsattheinitiationofmeiosis.
LossofDmrt1inundifferentiatedspermatogoniacausesthemtoprecociouslyexitthespermatogonialprogramandentermeiosis.
Dmrt1appearstoactinspermatogoniabysuppressingRAviatranscriptionalrepressionofStra8,andbypromotingtheproductionofthespermatogonialdiffer-entiationbasichelix-loop-helixtranscriptionfactorSohlh1[55].
ThesedataindicatethatDmrt1isatranscriptional3834Table1AnoutlineofreportedDmrtfactorembryonicexpressioninvertebratesFactorSpeciesEmbryonicexpressioninnon-gonadaltissue(insitu)ExpressioningonadsReferencesDmrt1MousendGenitalridgepriortosexualdifferentiation.
ExpressionbecomesXYspecificduringgonadogenesis.
BothinSertoliandgermcells(mitoticspermatogonia).
[49,55,103]ChickennrGenitalridge,higherinmale.
Becomestestis-specificaftertheonsetofsexualdifferentiation.
[49,103]XenopusnrFirstdetectedinprimordialgonadinbothZWandZZtadpolesincellssurroundingthePGCs.
Expressionismaintainedandbecomeshigherintestisthanovaryaftermetamorphosis.
[20,71,104](dm-w)XenopusnrExpressionexclusivelyinprimordialgonadsofZWtadpolesincellssurroundingthePGCs.
ExpressionisnotmaintainedintheZWandZZgonads.
[71,72]PlatyfishndSpermatogoniaandSertolicellsinadulttestis.
[61,105]ZebrafishnrTestisandovary,higherintestisthanovary.
Germcells.
[106]MedakanrNoexpressioningonadsinembryosbeforestage20ofdevelopment.
Expressedinspermatogonium-supportingcellsaftertesticulardifferentiations(20daysafterhatching).
[62,67,93](Dmy)MedakanrExpressedduringdevelopmentfromneurulastageinsomaticcellssurroundinggermcellsexclusivelyinXYgonads.
Expressionmaintainedinadulttestes.
[62,63,67]Dmrt2MousePresomiticmesodermanddermomyotomeofsomites.
Barelydetectableexpression,higherintestisthanovaryduringembryonicdevelopment.
[80,83,85,86,107]ChickenTransientasymmetricexpressioninthechickHensen'snode,anteriorpresomiticmesodermanddorsalcompartmentofsomites.
nr[84]MedakaSomites.
Sertolicellsinadulttestis.
Earlystageoocytesinadultovaries.
[93]ZebrafishKupffer'svesiclein3-somitestageembryos;presomiticmesodermandnewlyformedsomitesinbudstageembryos(Dmrt2a).
Somitesandbranchial(Dmrt2b).
Adulttestis(Dmrt2b).
[80–83]PlatyfishSomites;branchialarches(Dmrt2a).
nd[105]Dmrt3MouseNasalplacode,telencephalon,spinalcordinterneurons.
Expressioninitiallysimilarintestisandovary;higherintestisafterE13.
5.
Interstitialcells.
[87,107]ChickenNasalplacode,telencephalon,dorsalspinalcordinterneurons,somites,müllerianducts(higherinfemalesthanmales).
nd[87]ZebrafishNasalplacode,anteriorneuraltube,dorsalspinalcordinterneurons.
Undifferentiatedgonadsfrom17dpf.
Adulttestis(spermatogoniaandspermatocytes)andovary(oocytes).
[15]MedakaDorsalspinalcordinterneurons.
Differentiatinggonads(12–20dph)andadulttestis.
[93]Dmrt4MouseUbiquitous,highexpressioninolfactorytissues.
Similarlevelsintestisandovary(fromE11.
5).
[89,107]XenopusNasalplacode,telencephalon,foregut,gallbladder.
Adulttestis.
[88]MedakaOlfactorysystem,telencephalon.
Differentiatinggonads,adulttestisandovary.
[93,108]PlatifishOlfactoryplacode,forebrain,branchialarches.
nd[105]3835gatekeeperthatcontrolsthemitosisversusthemeiosisdeci-sioninmalegermcells.
TheroleofDmrt1inthemammalianfetalovaryhasalsobeeninvestigated.
Infemales,meiosisbeginsinthefetus,andgermcellsremainarrestedinthediplotenestageofprophaseIuntilafterpuberty.
Asinmales,meioticactiva-tionoccursundertheinfluenceofRAandthedownstreammeiosisinducerStra8[54,56].
Dmrt1mutantgermcellswerefoundtohaveseverelyreducedStra8expressionandtoundergoabnormalmeioticprophase[23].
mRNAprofil-ingandChIPsuggestthattranscriptionalactivationofStra8isthemainfunctionofDmrt1inthefetalovary,andthatthisregulationislikelytobedirect.
Thus,Dmrt1controlsStra8sex-specifically,activatingitinthefetalovaryandrepress-ingitintheadulttestis.
StudiesonthefunctionofDmrt1insexualdevelopmenthavealsobeenconductedinnon-mammalianvertebrates.
Inspecieswithtemperature-dependentsexdeterminationsuchasturtles,lizardsandalligators,Dmrt1expressionwasfoundtobehigherindevelopingmalegonadsthaninfemaleones,suggestingthatitisinvolvedinthisprocess[57–59].
Inthemedaka,Oryziaslatipes,whichlikemammalsusestheXX/XYsexdeterminationsystem,Dmrt1hasundergoneduplication.
GeneduplicationgeneratingDmyrecentlyoccuredduringevolutionofthegenusOryziasasthegeneisabsentinotherfishes,includingotherOryziasspecies[60,61].
Oneofthenewlyderivedparalogs,calledDmdomainonY,alsoknownasDmrt1bYorDmy,islocatedonthesex-determiningregionoftheYchromosome.
DmyisexpressedexclusivelyinsomaticcellsofXYgonadsintheearlygonadalprimordiumbeforemorphologicalsexualdimorphismisobserved.
Dmyisamasterregulatorofsexdeterminationasitisbothrequiredandsufficientformaledevelopment[62–66].
Theotherparalog,Dmrt1,isexpressedinspermatogonium-supportingcells,whichisthesamelin-eageofcellsexpressingDmy,butaftertestisdifferentiation[67].
HightemperatureorsteroidtreatmentinducesDmrt1expressioninXXembryosandleadstoXXsex-reversedtestis[68,69].
Conversely,inaDmrt1mutantline,XYindi-vidualsdevelopedintonormalegg-layingfemales.
TheXYmutantgonadsfirstdevelopedintothenormaltestistype,butbyday10afterhatching,thegonadstransdifferentiateintotheovarytype[70].
ThesedatasuggestthatDmrt1inmedakaisessentialtomaintaintestisdifferentiationafterDmy-triggeredmaledifferentiationpathway.
XenopuslaevisusesthefemaleheterogameticZZ/ZW-typesexdeterminingsystem.
Similarlytomedaka,adupli-catedvariantofDmrt1residingonthefemale-specificWchromosomehasbeenisolated(termeddm-w).
WhileDmrt1isexpressedcontinuouslyinbothZZandZWdevel-opinggonads,dm-wisexpressedexclusivelyinfemaleZWprimordialgonadsatsexdetermination.
dm-wisanovary-determininggeneinX.
laevisasexogenousdm-wcausesdevelopingovotestesinZZtadpolesanddm-wknockdowninZWindividualsinducesmaledevelopment[71].
Dm-wappearstodirectfemalesexbyantagonizingtheautoso-malDmrt1genetodetermineatestisfate.
Dm-wencodesatruncatedDmrt1proteinwhichhasaDMdomainbutlacksmorecarboxy-terminalsequences.
Itisproposedthatdm-wblocksDmrt1bydimerizingandantagonizingDmrt1nrnotreported,ndnotdetectedFactorSpeciesEmbryonicexpressioninnon-gonadaltissue(insitu)ExpressioningonadsReferencesDmrt5MouseNasalplacode,dorsaltelencephalon,ventralforebrain/midbrainborderatE9.
5extendinglatertotheentireventralmidbrain,opticstalk,lateralheadectoderm,maxillaryandmandibularprocesses,eyes,hypophysis.
Higherlevelsinovaryversustestis(E12.
5toE15.
5).
[90,91,107]XenopusNasalplacode,dorsaltelencephalon,ventraldiencephalon.
nr[9]ZebrafishNasalplacode,dorsalandventraltelencephalon,ventraldiencephalon.
Germcellsinadulttestisandovary.
[95,109]PlatifishForebrain,olfactoryplacode,midbrain,lens.
nd[105]Dmrt6Mousenrnd[107]Dmrt7MousendHigherlevelinovaryversustestisinembryonicandadultgonads.
Postnatally,expressionismalespecificandrestrictedtospermatocytesandsperm.
[75,76,107]Dmrt8MousenrHigherlevelintestisthaninovaryinembryonicgonads.
Testis-specificexpressionrestrictedtoSertolicellsintheadult.
[14]Table1continued3836Table2AnoverviewofthephenotypesassociatedwithlossorgainoffunctionofchordateDmrtgenesFactorSpeciesLoss-of-function/gain-of-functionphenotypesReferencesDmrt1MouseKODefectsingermradialmigration,reactivationofmitosisandsurvival.
FailureofSertolicelldifferentiation.
Teratomaformationinfetalgermcells.
Failuretocontrolthemitosisversusmeiosisdecisioninmalegermcells.
Femalereprogramminginthepostnataltestis.
Reductionofprimordialfolliclesinthejuvenileovary.
[22–24,50,51,56]MedakaMMale-to-femalesexreversalaftersexdetermination.
[70](Dmy)MedakaMFemaledevelopmentingeneticmales.
[62,65]GOFMaledevelopmentingeneticfemales.
Inhibitionofprimordialgermcellproliferation.
[66,110]KDLossofproliferationinhibitioninprimordialgermcells.
[110](dm-w)XenopusGOFOvariancavitiesingonadsofZZtadpolesoverexpressingdm-w.
[71,72]KDMaledevelopmentinZWindividuals.
[72]ChickenKDFeminizationofgonadsofembryonicmales.
[74]Dmrt2MouseKOEmbryonicsomitepatterningandmyogenicdefects.
[83,85,111]GOFIncreasedmyogenesis.
[86](Dmrt2a/Terra)ZebrafishGOFIncreasedapoptosis.
[80]KDRandomizationofleft-sidesspecificgenesanddesynchronizationofthesegmentationclock.
[84]Dmrt2bZebrafishKDDefectsinsomitogenesisandhedgehogsignaling.
Neuraltubepatterningdefects.
Randomizationofleft-sidespecificgenes.
Impairmentofslowmuscledevelopment.
[81]Dmrt3MouseKOMalesexualdevelopmentabnormalities.
Dentalmalocclusions.
[112]KODefectsinspinalcircuitsinvolvedinlocomotion[21]HorseMDefectsinpatternlocomotioninhorses[21]XenopusGOFmDmrt3promotesneurogenesisincaps.
[9]Dmrt4MouseKOFemaleswithpolyovularfolliclesandmalesexualdevelopmentabnormalities.
[89]XenopusKDImpairedneurogenesisintheolfactoryplacode.
[88]GOFPromotesneurogenesisincaps.
[88]Dmrt5MouseKOReduceddevelopmentofthecaudomedialcerebralcortex.
[91,92]GOFPromotesdopamineneuronsinEScells.
[90]KDInhibitionofEScellsdifferentiationtowardsaventralmedialmesencephaliccellfate.
[90]XenopusKDImpairedneurogenesisintheolfactoryplacode.
[9]GOFPromotesneurogenesisincaps.
[9]ZebrafishM,KDDefectsintelencephalicneurogenesis.
[95](Dmrt1)CionaMDefectsinanteriorneuralplatederivatives.
[100]KDImpairedSix1/2,Six3/6,MeisandZicLinthedevelopingbrainandFoxCinpalps.
[99]Dmrt7MouseKOInfertilitywithspermatogenicarrestinpachytenestageandsexchromatindefects.
[75,76]GOFgainoffunction,KDknockdown,KOknockout,Mmutation3837transcriptionalactivity[72].
Asinmedaka,thissex-deter-miningroleisarecentinnovation.
Indeed,acloselyrelatedspecies,Siluranatropicalis,lacksthedm-wgene[73].
BirdsalsouseafemaleheterogameticsexZZ/ZWsystem.
SexissupposedtobedeterminedbythehigherZchromo-somedosageinmales,bythepresenceofaWchromosomeinfemales,orpossiblybyacombinationofboth[35].
Inallbirds,Dmrt1islocatedontheZchromosome.
Inembryos,itisexpressedintheearlybipotentialgonadandshowshigherexpressionlevelsinZZversusZWembryos[49].
IfDmrt1activityisexperimentallyreduced,thegonadsofgeneti-callymale(ZZ)embryosarefeminized[74],demonstratingthatDmrt1isrequiredfortesticulardifferentiation.
Thus,inbirds,Dmrt1playsanimportantroleinsexdetermination;thehigherexpressionlevelinmale(ZZ)embryostriggersthetestisspecificationpathwaywhereasonlyalowerdose,asfoundinfemales,iscompatiblewithovariandevelop-ment.
WhetherelevatedDmrt1expressionissufficientinZWgenitalridgetoinducemaledevelopmentremainshow-evertobetested.
Dmrt7isrequiredformalemeiosisDmrt7ispresentinplacentalmammalsandmarsupialsbutnoorthologhasbeenreportedinnon-mammalianvertebrates.
Inmouse,Dmrt7isexpressedinbothmaleandfemalefetalgonads.
Intheovary,Dmrt7expressionisindependentofthegermline,as,inXXc-kitmutantswhichlackgermcells,thelevelofitsexpressionremainssimilartothatobservedinwild-types.
Inadults,Dmrt7expressionismalespecific.
Itispredominantlydetectedinmid-tolate-pachytenesper-matocytesandtheproteinpreferentiallylocalizestotheXYbody,adenselystainedchromatindomainharboringsexchromosomes,essentialformalemeioticprogression.
Con-sistentwiththisexpressionpattern,micedeficientinDmrt7areinfertileandmostmutantcellsshowspermatogenicarrestinpachytenestageandabnormalcellularorganizationofSer-tolicells[75,76].
ThegermcelldefectsofDmrt7mutantsarenotcausedbyaberrantSertolicellorganizationsinceanimalswithdeletionofDmrt7justinSertolicellshavenormaltestisandspermatogenesis.
Dmrt7mutantcellsestablishanormalXYbodyinmid-pachynema,butthenhavemultipleepige-neticdefectsinthesexchromatintransitionfrompachynematodiplonema.
ThissuggeststhatDmrt7playsaroleinthecontrolofthetransitionfrommeioticsexchromosomeinacti-vationtopostmeioticsexchromatininmales[76].
Dmrtsareimportantduringembryogenesisinnon-gonadaltissuesFollowingtheirinitialexpressioninthedevelopinggonads,asubsetoftheDmrtfamilymembers,includingDmrt2,Dmrt3,Dmrt4andDmrt5,showdifferentialexpressioninalimitednumberofnon-gonadaltissuesandorgans.
Inmostspecies,Dmrtgeneshavebeendetectedintissuessuchasthecentralnervoussystem,nasalplacodeorsomites.
Theirexpressionpatternisoftenconservedacrossspecies,buttherearealsodifferences.
Forexample,whileDmrt3isexpressedintheneuraltubeandinthepresomiticmesoderminchick,itisonlydetectedinthenervoussysteminfishandmousesuggestingitsfunctionhasshiftedduringevo-lution(Table1).
ThosefourDMdomaingeneshavebeenrecentlythesubjectoffunctionalanalysisinmouse,fishorXenopus.
Table2summarizessomeofthesegain-andloss-of-functionstudiesandtheresultingphenotypes.
Here,wediscusstheserecentstudieswhichshowthatDmrts,likeDSX,directavarietyofcelldifferentiationeventsandoftenfunctioninthespecificationofprogenitorcells.
TheyalsoindicatethatsomeoftheseDmrtfactors,asDSXinflies[33,77–79],actbymodulatingsignalingpathways,suggestingthatthismaybeacommonthemeforDMdomainproteinsacrossspecies.
Dmrt2isinvolvedinestablishingleft–rightasymmetryandsomitogenesisThefirstDmrtgenesuggestedtohavearoleunrelatedtosexualdevelopmentwasDmrt2.
Itwasfirstidentifiedinzebrafishthroughasystematicsearchforgeneswithtissue-specificexpressionandwasselectedbecauseofitssomiteandpresomiticmesoderm-specificexpressionpattern.
Theidentifiedgene,originallycalledterra,wasfoundtoplayaroleinsomitogenesisbasedontheobservationthatitsover-expressioninducesrapidapoptosisinthemesoderm[80].
AduplicatedcopyoftheDmrt2genewaslaterdescribedinthezebrafishgenome,andDmrt2a/terraandDmrt2bhavebeendesignatedtodistinguishthem.
Bothgenesareexpressedduringsomitogenesis,butthefish-specificDmrt2bisalsoexpressedinbranchialarches[81,82].
Inadditiontothedevelopingsomites,Dmrt2aistransientlyasymmetricallyexpressedinthezebrafishKupffer'svesicleandintheequivalentstructureinthechick,theHensen'snode,whichsuggestaleft–rightpatterningfunctionduringdevelopment[83,84].
Indeed,inzebrafish,usingamor-pholino-basedapproach,Dmrt2awasfoundtoberequiredforleft–rightsynchronizationofthesegmentationclock.
Itisalsorequiredforleft–rightpatterninginthelateralplatemesodermandthusthecorrectpositioningoftheinternalorgansoneachsideofthemidline.
Assuch,Dmrt2aisakeyfactorlinkingleft–rightpatterningwithbilateralsyn-chronizationofthesegmentationclockinthemesoderm[84].
Dmrt2bmorphantsalsodisplaydefectsinheartandvisceralorganasymmetry,andsomelateralplatemesodermmarkersexpressedintheleftsidearerandomized.
Dmrt2bknockdownalsoleadstonotabledefectsinsomitogenesis3838andreducestargetgeneexpressionofHedgehogsignal-ing,whichresultsinsignificantimpairmentinslowmus-cledevelopment.
Dmrt2acannotcompensateforthelossofDmrt2bandviceversa.
Thesedataindicatethatfunctionaldivergencehasoccurredbetweenthetwoduplicatedgenes,withDmrt2bmaintainingthecommonfunctionforleft–rightestablishmentandcontributingtoadivergentfunctioninsomitogenesisthroughHedgehogsignaling[81].
Inthemouse,Dmrt2isexpressedinthepresomiticmesodermandisthenconfinedtothedermomyotome,thedorsalepithelialdomainofthedevelopingsomitescontain-ingmusclestemcells,butisabsentfromthenode[83].
Asinzebrafish,itstargeteddisruptionleadstoseveresomitepatterningdefects,firstvisibleatdayE10.
5.
Boththedermomyotomeandmyotomefailtoadoptanormalepi-thelialmorphology.
Accompanyingthesemorphologicaldefects,alterationsintheexpressionofdermomyotomalandmyotomaltranscriptionfactorssuchasPax3,Paraxis,Myf5,Myogenin,Mrf4andMyoDwereobserved[85].
Inagreementwiththeabsenceofitsexpressionfromthemousenode,Dmrt2homozygousmutantsdonotshowleft–rightdesynchronizationofsomiteformationordefectsinleft–rightasymmetricorganpositioning.
Thus,theroleofDmrt2insymmetricsomiteformationandintheregulationofthelateralitypathwayisnotconservedduringzebrafishandmouseembryonicdevelopment[83].
WhetherthislossofDmrt2functioninleft–rightpatterninginthemousearisefrommutationsoccurringintheenhancerresponsibleforthenodeexpressionorfromthelossofaprotein(s)nec-essarytoactivatespecificallythenodeenhancerremainsunknown.
Inarecentreport,Dmrt2hasbeenidentifiedasatargetofPax3,acriticalregulatorofskeletalmusclestemcells.
Furthermore,Dmrt2wasfoundtodirectlyregulateearlyactivationofthemyogenicdeterminationgeneMyf5,requiredfortheformationofthefirstskeletalmuscleinthesomites.
ConditionaloverexpressionofDmrt2inPax3-expressingcellsinthesomiteconfirmstheroleofthisfactorintheactivationofMyf5[86].
Thus,ageneticnetworkcom-prisingPax3/Dmrt2/Myf5operatesinthemusclestemcellsofthedermomyotomeinthemouseembryotoorchestratetheonsetofmyogenesis.
Dmrt3,Dmrt4andDmrt5playkeyrolesinneurogenesisSeveralstudieshaveshownthatmembersoftheDmrt3–5subfamilyofDMgenesareexpressedinarestrictedman-nerinthedevelopingnervoussystem.
Allthreegenesareexpressedinthedevelopingtelencephalonandolfactoryplacodeinthemouse(Fig.
2),andthisexpressionpatterninthedevelopingCNSisconservedinmostvertebrateembryosstudied,includingchick,mouseandzebrafishembryos(Fig.
3a–d)[87–92].
Inaddition,Dmrt3isalsostronglyexpressedinthespinalcordindorsalinterneurons[15,87,93]andDmrt5intheventralmedialmesencephalon(Fig.
3a)[90,91].
Thefunctionalrelevanceoftheseexpres-sionpatternshasrecentlybeeninvestigatedforDmrt4andDmrt5inXenopusolfactoryplacodeandzebrafishtelen-cephalonneurogenesisandforDmrt3inmousespinalcordneuronalspecification(Fig.
4).
InXenopus,Dmrt4andDmrt5arecoexpressedearlyintheanteriorneuralridge(ANR)andthenbecomeprogres-sivelyrestrictedtothedorsaltelencephalonandtheolfac-toryepithelium.
Bothgenesarepositivelyregulatedbyneuralinducersandnegativelybyproneuralfactors.
Theyarealsoactivatedbythecombinedactionofthetranscrip-tionfactorOtx2,broadlytranscribedintheheadectoderm,andofNotchsignaling,activatedintheANR.
KnockdownofDmrt4orDmrt5impairsneurogenesisintheembryonicolfactorysystemandinneuralizedanimalcaps.
Conversely,theiroverexpressionpromotesneuronaldifferentiationinanimalcapsasvisualizedusingmarkerssuchasthebHLHtranscriptionfactorsNgnr-1,Ebf2orAth5,apropertythatrequirestheC-terminalDMAdomainanddownstreamDmrt3Dmrt4E12.
5Dmrt5E11.
5E11.
5teloeteloeteloetelteltelACBACBFig.
2InsituanalysisofDmrt3,Dmrt4andDmrt5expressionintheheadofmouseembryos.
Coronalsectionsattheindicatedstagesinthenasalregion(a–c)andattheleveloftheanteriortelencephalon(a′–c′)areshown.
Allthreegenesareexpressedinagradedmannerinthedevelopingtelencephalonandintheolfactoryepithelium.
Oeolfactoryepithelium,teltelencephalon3839sequences.
Inanimalcaps,theNogginmediatedinduc-tionofNgnr-1andEbf2thatisaffectedbythedepletionofDmrt5canberescuedbyDmrt4overexpression.
Con-versely,theinhibitionofNgnr-1andEbf2inthecontextofDmrt4depletedexplantscanberescuedbyDmrt5overex-pression[9,88].
Together,thesedataindicatethatDmrt4andDmrt5haveoverlappingfunctionsupstreamofproneuralfactorsduringolfactoryplacodeneurogenesis.
Inthemouse,Dmrt3,Dmrt4andDmrt5arestronglyexpressedinthedevelopingolfactoryepitheliumbutitisnotknownwhethertheyplayaroleinitsformation[87,89,91].
Dmrt4-deficientmicehavebeengenerated.
Thosemiceareviableandfertilebuthavepolyovularfollicles,suggestingaroleinfolliculogenesis.
Interestingly,25%ofthemutantmalesexhibitedcopulatorybehaviortowardsothermales.
Asolfactionandsexualbehaviorarestronglylinkedinmice,thissuggestpossibleolfactoryfunctiondefects[94].
Nevertheless,Dmrt4-deficientmicehaveahistologicallynormalolfactoryepitheliumandgeneralolfaction[89].
Incontrast,theolfactoryepitheliumisreducedinDmrt3andDmrt5knockout(KO)miceandisalmostcompletelyabsentinDmrt3:Dmrt5doubleKO(Saulnieretal.
,unpublisheddata).
ThelackofphenotypeinDmrt4mutantsisthuslikelytobeduetothepresenceofDmrt3andDmrt5.
Together,theseobservationsindicatethatDmrt3-5mayhaveoverlap-pingfunctioninvertebrateolfactoryplacodedevelopment,whichremainstobefurtherinvestigated.
Inzebrafish,aDmrt5mutantwasisolatedthatshowsdefectsintelencephalicneurogenesis.
ExpressionofNeurog1andothertelencephalicmarkergenessuchasFoxg1andEmx3weredownregulated,whileincontrast,Her6,aHes-relatedgenethatencodesanegativeregulatorofNeurog1,wasexpanded.
KnockdownofHer6rescuesNeurog1expres-sionintheDmrt5/telencephalon,suggestingthatDmrt5regulatesNeurog1expressionbyrepressingHer6[95].
SuchamechanismhasbeenpreviouslyreportedforMAB-3inthespecificationofsex-specificneuronsinC.
elegans[26].
So,Dmrt5regulatesneurogenesisinthezebrafishposterior-dorsaltelencephalon.
WhethertheotherDmrtgenesexpressedinthedevelopingtelencephalonalsoplayaroleinneurogen-esis,andwhetherthisfunctioninneurogenesisisconservedamongvertebrates,remainstobeinvestigated.
ADmrt4/5-relatedgenehasbeenrecentlyidentifiedintheseaanemoneN.
vectensis(designatedNvDmrtB),amodelMouseX.
LaevisChicktelnplediteldienpltelnpldiABCCionaEFZebrafishNematostelladinpltelDmesGanpsvFig.
3Whole-mountinsituanalysisofDmrt5expressioninthemouse,chick,frogandfishembryosandofrelatedgenesinascid-ianandcnidarian.
a–dInmouse(stageE10.
5),chick(stage18),Xenopuslaevis(stage28)andzebrafish(24hpf)embryos,Dmrt5isstronglyexpressedinthenasalplacodeanddorsaltelencephalon.
Itisalsohighlytranscribedintheventraldiencephaloninchick,frogandzebrafishembryosandinthemouseintheventraldien-cephalonandmesencephalon.
e,fInCiona64-cellstageembryos(e),Dmrt1thatisrelatedtoDmrt4/5,isexpressedinprogenitorsoftheanteriorneuralplateandlaterattailbudstage(f)isrestrictedtotheanteriorbrain.
(ImagesfromGhostdatabase,http://ghost.
zool.
kyoto-u.
ac.
jp/SearchGenomekh.
html).
gInNematostellavectensis,NvDmrtBthatisrelatedtoDmrt4/5isexpressedinscatteredcellsinboththeectodermalandendodermallayersinlateplanulaembryos.
Thestrongeststainingismostprominentatthatstageintheendo-derm,correlatingwiththeonsetofneurogenesis.
In(a–d),lateralviewsoftheheadoftheembryosareshownwithanteriortotheleft.
In(e),theanteriorsideoftheembryosistothetop.
In(f),lat-eralviewofatailbudstageembryoisshown.
In(g),theblastoporeistotheright.
Anpanteriorneuralplateprogenitors,Didiencephalon,eeye,mesmesencephalon,nplnasalplacode,svsensoryvesicle,teltelencephalon3840systemfromthesistergroupofbilaterians,thecnidarians.
InNematostella,NvDmrtBisexpressedinscatteredneuralcellsinboththeectodermalandendodermallayers(Fig.
3g).
InXenopus,itsoverexpressioninducesneurogenesisinani-malcapexplants.
Conversely,knockdownofNvDmrtBinNematostellareducesthenumberofElav-1positiveneu-rons[9].
TheseresultssuggestthatDmrt'sabilitytocon-trolneurogenesisderivesfromanancestralfunctionalreadypresentinthelastcommonancestorofcnidariansandbila-terians.
FurtheranalysesofDmrtgenesinbasallybranchinganimals,includingsponges,willberequiredtodefinetheancestralfunctionsofDmrtgenes.
AveryrecentstudyhasshownthataDmrt3nonsensemutationhasamajoreffectonthepatternoflocomotioninhorses.
ThephenotypeindicatesthatDmrt3+spinalcordneuronshaveacriticalroleforleft–rightcoordinationandforcoordinatingthemovementofthefore-andhind-legs.
Dmrt3isexpressedincellsoriginatingfromdI6progenitorsthatdevelopintoinhibitoryinterneuronsprojectingipsi-andcontralaterally.
Examinationofwild-typeandDmrt3nullmicedemonstratedthatittakespartinneuronalspecifica-tionwithinthissubdivision,andthatitiscriticalforspinalcircuitfunction.
ThemutationleadstoatruncatedDmrt3proteinretainingtheDMandDMAdomainsbutlackingthedownstreamsequences,whichhighlighttheirimportanceforitsactivity[21].
Dmrt5isrequiredforanteriorneuraltissuedevelopmentInthemouse,threerecentstudies,twousingKOmiceandthethirdusingembryonicstem(ES)cells,haveprovidedinsightsintothefunctionofDmrt5inthedevelopmentofthetelencephalonandmesencephalon,respectively(Fig.
4).
Regionalizationofthetelencephalonisinitiatedbymor-phogenssecretedfromlocalizedinductivecenters.
Thetwomajorpatterningcentersthatarethemostdirectlyimpli-catedintelencephalonpatterningaretheANRlocatedattherostralpoleofthetelencephalonandtheroofplateandcorticalhem(CH)regionatthedorsalcaudalmidlineandimmediateadjacentterritories.
TheANRsecretesFGFsandtheCHproducesavarietyofWntandBmpligandscriti-calforhippocampusdevelopment.
Thesesignalsestablishgradedexpressionofgenesencodingtranscriptionfactorsthatarecrucialfortheregionalizationofthetelencephalonandsubsequentarealizationofthecerebralcortexincorti-calprogenitors.
AmongthemareEmx2,promotingacau-domedialfate,andPax6,promotingarostrolateralidentity[96,97].
Inthemouse,similarlytoEmx2,Dmrt5isdetectedincorticalprogenitorsinahighcaudomedialtolowrostro-lateralgradientandisdependentonWntsignaling[91,92,98].
Withrespecttotheothertranscriptionfactorsplayingacrucialroleincorticaldevelopmentandpatterning,ithasbeenshownthatDmrt5isdependentontheearlyzincfingertranscriptionfactorGli3.
Incontrast,Emx2isnotrequiredforDmrt5expression.
Pax6,expressedinacomplemen-tarymannertoDmrt5,mayantagonizeitsexpression.
InDmrt5nullmutants,thecaudomedialcortex,includingthehippocampus,isstronglyreduced.
WntandBmpsignal-ingintheembryonicdorsomedialtelencephalonandthedownstream-dependenttranscriptionfactorssuchasEmx2arereduced.
Pax6,whichisinhibitedbymidlinesignals,isupregulated[91,92].
Incontrast,conditionalablationofGli3Dmrt5,Dmrt3Pax6Emx2,Lhx2,Cux2MidlineWntsMouseZebrafishDmrt5Her6Neurog1TelencephalonMesodi-encephalonDmrt5Nkx2.
2,Lhx1,Isl1Foxa2,Msx1,Lmx1mDAneurons(ventral-lateral)(ventral-medial)CaudomedialfateRostrolateralfateNeuronaldifferentiationBCDAscidianDevelopingbrainDmrt1FoxCZicLFgf(palpplacode)(anteriorneuraltissue)Dmrt1Six3/6Six1/2MeisFAnteriorneuralplateXenopusNogginXebf2Ngnr-1XDmrt5XDmrt4NeuronaldifferentiationOlfactoryplacodesEOtx2NotchDmrt3CommissuralinterneuronsWt1(dI6interneurons)SpinalcordAFig.
4SchematicrepresentationoftheinvolvementofDmrtsinneu-raldevelopment.
aInthemousespinalcord,lossofDmrt3increasesthenumberofWt1+neuronsandresultsinfewercommissuralinterneurons.
bInmouseEScells,Dmrt5inducesventral-medialmidbrainprogenitormarkersandinhibitsventral-lateralones,sug-gestingthatinvivo,intheventral-medialmesencephalicneuroepi-thelium,itenhancestheacquisitionofamDAneuronalfate.
cInthemousetelencephalon,Dmrt5andDmrt3aretargetsforWntsignalingandaredependentonGli3.
DirectorindirectactionofGli3onWntsandonDmrt5andDmrt3,throughregulationoftheWntsignalingpathway,isindicatedbydashedlines.
Dmrt5inturnisrequiredforWntandBmpexpressioninthedorsalmidlinesignalingcenterandtheexpressionoftheirdownstreamtargets,thatspecifyacaudome-dialfate.
Pax6isupregulatedinDmrt5mutants,presumablythroughitsnegativeregulationbyWntsandEmx2.
dInzebrafish,Dmrt5isrequiredforneurogenesisinthetelencephalon,possiblybyrepress-ingHer6.
eInthefrog,Dmrt5andDmrt4actdownstreamofneuralinduction,Otx2andNotchandupstreamofNeurogenintocontrololfactoryplacodeneurogenesis.
fInCiona,Fgfsignalsplayacru-cialroleinnervoussystemdevelopment,activatingseveralneuralgenesincludingDmrt1.
Italsocontrolscellfatechoicebetweenthepalpplacodesandanteriorcentralnervoussystem,whichexpressFoxCandZicL,respectively.
FoxCandZicLexpressingcellsderivefromcommonprogenitorsthatexpressDmrt1andbothgenesrequireDmrt1fortheiractivation.
Dmrt1playsalsoaroleinthepromotionofSix1/2,Six3/6andMeisinthedevelopingbrainandnegativelyreg-ulatesitsexpression3841Dmrt5atneurogenicstagesusingtheNestin-Cretransgeniclineonlycausesaslightreductionintelencephalonsize[92].
Thus,Dmrt5isanovelWnt-dependent"earlymaster"regulatorofinitialregionalpatterningofthecerebralcortex.
Itislikelytofunction,atleastinpart,bypromotingdorsalmidlinesignalingcenterformationandtherebyhelpingtoestablishthegradedexpressionoftheothertranscriptionregulatorsofcorticalidentity.
WhetherDmrt3andDmrt4functionsoverlapwiththatofDmrt5incorticalpatterning,andwhethertheydirectlyorindirectlyregulateWntandEmxgeneexpression,aretwoimportantquestionsstilltobeaddressed.
Ascidianbelongstotheurochordates,whichrepresenttheclosestlivingrelativesofthevertebrates.
AgenerelatedtoDmrt4/5withaDMAdomainhasbeenidentifiedintheascidianCionasavigny(designatedDmrt1).
Inascidians,theCNSdevelopsvianeurulationofcellsoftheneuralplate,formingasimplebraincalledthesensoryvesicleandacaudalnervecord.
Theanteriorneuralplateproducesplacodalderivatives,suchastheadhesivepalpsandstomo-deum,andtheanteriorportionofthebrain,calledthesen-soryvesicle.
Dmrt1isexpressedfromthe64-cellstageinprogenitorsoftheanteriorneuralplateandislaterrestrictedtotheanteriorpartofthesensoryvesicle(Fig.
3e,f).
AnullmutationintheDmrt1geneaswellasknockdownexperi-mentshaveshownthatDmrt1isrequiredforthedevel-opmentofthepalpsandoralsiphon(mouth)thatderivefromthestomodeum.
Italsoleadstoextensivedisruptionofsensorystructures,suchaslight-sensitiveocellus,inthesensoryvesicle[99–101].
Furthermore,knockdownexperi-mentshaveshownthatDmrt1isactivatedbyFGFsignalsandisrequiredfortheexpressionofFoxCandZicLthatmarksthepalpplacodesandanteriorneuraltissue,respec-tively.
TheyalsoshowthatDmrt1promotesSix1/2,Six3/6andmeisinthedevelopingbrainandnegativelyregulatesitsownexpression[99].
InCiona,asinvertebrates,Dmrtgenesthusmarkanteriorneuralregions,includingplacodesandanteriorneuraltissue,andarerequiredfortheirdevel-opment(Fig.
4).
Theventral-medialcaudaldiencephalonandmesen-cephaloncontaindopaminergicneuronsthatareessentialforthecontrolofvoluntarymovementsandtheregulationofemotion,andareseverelyaffectedinneurodegenerativediseasessuchasParkinson'sdisease.
Inresponsetolocalinductivesignals(Shh,Fgf8,Wnt1),transcriptionfactorsareexpressedatspecificdorsalandventralpositionsinthemesodiencephaloninducingdistinctcellfates,includingintheventral-mostprogenitorsamidbraindopaminergiccellfate.
Asinthedevelopingtelencephalon,expressionofDmrt5intheventral-medialmesencephalonisrestrictedtoprogenitorsthatgiverisetodopamineneurons.
Inembryonicstem(ES)cells,overexpressionofDmrt5inducesaventral-medialprogenitorphenotypeandinhibitsventral-lateralmesencephalicmarkers.
Conversely,knockdowncompro-misesEScelldifferentiationtowardaventral-medialcellfate[90].
Thus,Dmrt5promotesmidbraindopaminergic(mDA)identityinEScellsbyenforcingaventralprogenitorfate.
WhetherDmrt5controlsinvivoventral-medialmesen-cephaliccellfateremainstobedemonstrated.
ConclusionBesidestheirroleinsexualdevelopment,Dmrtfactorshaveclearlyemergedasimportantregulatorsofvertebratedevelopment.
RecentfindingsindicatethatmembersofthegroupAfunctionascriticalregulatorsofthedevelopmentofthenervoussystem,functioningbothinneurogenesisandpatterningofthedevelopingneuraltissue,andthatthisabilitytocontrolneuraldevelopmentisanancestralfunction.
AbetterunderstandingofwhenandwheretheseDmrtfactorsbindstothegenomeisneededtoprovideinsightintothemolecularmechanismsemployedbythesefactors.
ElucidatingtheroleoftheconservedDMAdomainwillalsobecentraltounderstandhowDmrtfactorscoordi-natedevelopmentalprocesses,functioningasactivatorsorrepressorsdependingonthecellularcontextorlocus.
Futurestudiesshouldalsodeterminewhethertheyplayanycell-autonomousrolesinsexualdifferentiationofnon-gonadaltissuesandwhethertheirderegulationisassociatedwithsomehumanneuraldiseases.
AcknowledgmentsWearegratefultoDr.
YutakaKikuchi(Hiro-shima,Japan)fortheimageofDmrt5expressioninzebrafish,toDr.
FabianRentzsch(Bergen,Norway)fortheimageofNvDmrtBexpressioninN.
vectensisandtoMajaAdamska(Bergen,Norway)forallowingtheuseofunpublishedgenomicsequencesfromthespongeS.
ciliatum.
WealsothankDr.
YasuoHitoyoshi(Villefranche-sur-mer,France)forhelpfuldiscussions.
ThisworkwassupportedbygrantsfromtheBelgianFondsdelaRechercheScientifique(FRFC2.
4544.
12)andtheBelgianQueenElisabethMedicalFoundation(toE.
B.
)andtheInstitutUniversitairedeFrance(toM.
V.
).
S.
D.
isapost-doctoralfellowfromtheBelgianFondsdelaRechercheScientifique(FNRS).
M.
K.
isadoctoralfellowfromtheBelgianFondspourlafor-mationàlaRecherchedansl'Industrieetdansl'Agriculture(FRIA).
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