Abbie CasperandMarkVan Doren* The controlofsexualidentityinthe Development 133,2783-2791(2006)doi:10.1242/dev.02415 *Author forcorrespondence (e-mail:[email protected]) Charles Street, Baltimore, MD21218,USA. Department ofBiology, 302MuddHall,JohnsHopkinsUniversity, 3400North is establishedinthe other cellsoftheembryo.Here,weexplorehowsexualidentity that thisprocessisverydifferent ingermcellscomparedwith this decisionandhowisitexecuted?Onethingthatclear for theperpetuationofspecies.Howdoesagermcellmake cells, whichmustproduceeitherthespermoreggsnecessary development. Nowhereisthismoreimportantthaninthegerm Whether tobemaleorfemaleisacriticaldecisionin topics, whereappropriate. and referreaderstomore-comprehensive reviews onrelated entire review, wehave, bynecessity, focusedonspecificfindings represents several distinctsubjects,eachof whichisworthy ofan mammalian germcelldevelopment andhuman fertility. Asthis these events in determination. Finally, webrieflydiscusshow understanding development andhow thisaffects ourthinkingaboutgermlinesex then discussouremerging understanding ofgermcellsexual greatly toourknowledge ofhow this processiscontrolled.We discuss work ongermlinesex determinationthathascontributed sexual identityplaysintheseprocesses.Therefore,wefirst cell development requiresanunderstanding oftherolegermcell Similarly, understandingotherevents inmaleversus femalegerm developmental pathsofmaleandfemalegermcellsdiverge. determination dependsonourunderstandinghow the one withouttheother. Anunderstanding ofgermlinesex always beenstudiedtogether, itisclearthatwecannotunderstand germline sex determinationandsexual development have not cells andentryintospermatogenesisoroogenesis.Although events, suchastheformationofmaleorfemalegermlinestem female identity, but alsohow sexual identityinfluencesother determination, bywhichagermcellacquiresmaleversus discussion ofterms).Thisincludestheprocessgermlinesex the decisionsthatgermcellsface alongtheway (seeBox1fora context ofthedifferent stagesofgermlinesexual development and sexual reproductionandfertility. produce eitherspermoreggs, iscentraltoourunderstanding of germ cellisguidedalongamaleorfemalepath,toeventually initiate development ofanew individual. Thus,thestudyofhow a production ofdistinctmaleandfemalegametesthatmustuniteto However, onethingthatallsexual animalshave incommonisthe mechanisms oftendiffer drasticallybetweendifferent species. a mate,courtingmateandgettingthegametestogether. These conducting sexual reproduction,includingthoseusedfor attracting Animals have evolved afascinating arrayofmechanismsfor Introduction Drosophila aspects ofgermcelldevelopmentandwhatstudiesin In thisreview, wediscussgermlinesexual identitywithinthe can teachusaboutmammaliangermcells. Drosophila Drosophila can shedlightonsimilarevents in germline, howthisaffects other soma orthegermlinein chromosome isnotimportantforsex determinationineitherthe XY ismale(Fig.1)(reviewed byClineandMeyer, 1996).TheY autosomes), suchthat,innormaldiploidanimals,XXisfemaleand number ofXchromosomes(orratioto soma). Inthesoma,maleversus femaleidentityisregulated bythe occur differently fromintherestofbody(the somaticcellsor The establishmentofsexual identityinthegermlineisthoughtto Germline sexdeterminationin activate expression of are requiredforspermatogenesis.Inthesoma,XXembryos interrelated andcannotbe readily understoodinisolation. different aspectsof sex-specificgermcelldevelopmentare this mayseemabroad term, itemphasizesthethinkingthat stem cellsandtheultimateproduction ofspermoreggs.Although embryonic gonad,through the formationofmaleorfemalegermline include theearliestaspectsofgerm cellsexualdimorphisminthe of sex-specificterminaldifferentiation. Inthegermline,thiswould the timeacellfirstestablishesitssexualidentitythrough theprocess This termencompassesallaspectsofsex-specificdevelopment,from Sexual development oogenesis. differentiation torefer totheprocesses ofspermatogenesisor ‘undifferentiated’ state.Thus,wewillonlyusethe term sexual germline stemcellsare stillconsidered tobein arelatively female germlinestemcellsisaninteresting sexualdimorphism, for thegermlinebecause,eventhoughformationofmaleversus phenotype (sexualdimorphism).However, thistermcanbeconfusing How acellortissueusesitssexualidentitytoproduce asex-specific Sexual differentiation stages ofcommitment. types, sexdeterminationmaybeanongoingprocess withmultiple irreversibly establishesamaleversusfemaleidentity. Inothercell identity. Insomecelltypes,thismayinvolveasingleeventthat The mechanismbywhichacell,ororganism,acquires itssexual Sex determination development. between thesomaandgermlineinfluenceallaspectsofgermcell be particularlytrueinthegermline,where continuedinteractions additional sex-specificinputsthatinfluencesexualidentity. Thismay identity mayalsorequire maintenance,andacellmayreceive indicate thatsexualidentityhasbeeninitiated.However, sexual in acelltype,eventheexpression ofasinglesex-specificgene,can equivalent celltype,inmalesversusfemales.Anysexualdimorphism A difference inidentityordevelopmentalpotentialofanotherwise Sexual identity sex-specific development. ofgeneexpression,specific pattern aswellanyotheraspectsof equivalent celltype,inmalesversusfemales.Thisincludesasex- Any difference inthecharacteristics(phenotype)ofanotherwise Sexual dimorphism Box 1.Aglossaryofterms Drosophila Sex-lethal Drosophila ( Sxl germline , but doescontaingenesthat ), whichfunctionsthrough Drosophila REVIEW 2783

DEVELOPMENT cell types. Gap junctions(red) mayalsofacilitatecommunicationbetweenthetwo ligands, suchasUnpaired (Upd),whichpromotes maledevelopment. influence germlinesexdeterminationandactthrough extracellular development. Interactionsbetweengermcellsandsomaticalso identity, and male orasfemale.Inthegermcells,X:Aratioalsoinfluencessexual ( affecting the‘sex’ ofthegerm cells.For example, animalsthatare these genesisanotherway tochangethe‘sex’ ofthesomawithout Wieschaus, 1978;Schupbach,1982).Thus,themanipulationof genes producenormaleggs orsperminwild-typehosts(Marshand transformer 2 as 2784 form ofDSXisproducedbydefault. transcription factor. InXYanimals,thispathway isoff andamale of transformer ( chromosomes toautosomes(X:A)influencestheactivityof somatic gonadandgermline. Fig. 1.Asimplifiedviewofsexdeterminationpathwaysinthe that functionin 1977). Schupbach, 1982;Steinmann-Zwicky etal.,1989;Van Deusen, a testisdonotmake functionalsperm(MarshandWieschaus, 1978; cells inanovary donotmake functional eggs, andXXgermcellsin important conclusionfromthiswork isthat,in study thefate ofXYgermcellsinanXXsoma,andvice versa. An embryo toanother(IllmenseeandMahowald, 1974)allows oneto also Oliver, 2002).For example, transplantinggermcellsfromone the opposite‘sex’, suchasthroughtheuseofgeneticmosaics(see has beenanalyzedbyplacinggermcellsofone‘sex’ intoasomaof versus thegermcell-autonomouscontrolofthisprocess.Often, focused onstudyingtherelative contribution ofsomaticinfluence Much ofthework ongermlinesex determinationin Studying germlinesexdetermination germline. soma, andthesex ofthesomaalsoinfluencesthisdecision inthe sex inthegermcellsarevery different fromthoseactinginthe (Ryner etal.,1996).Aswediscussbelow, therulesfordetermining pathway, gonad, whileinthenervous system,asecondtarget forthis female development inmostsomaticcells,includingthe dsx Sxl Germ celltransplantationhasalso revealed thatmostofthegenes tra ), which, in turn, activates ), which,inturn, doublesex ), whichdetermineswhetherthesomaticgonadwilldevelopas and REVIEW fruitless dsx ovo ( , have norolein germcells;cellsmutantforthese ( tra tra2 tra2 , ( Somatic gonad Drosophila dsx ovarian tumor ) and ), controlsRNAsplicingof thealternative , regulates mostaspectsofsex-specific behavior X:A dsx ) RNA, producingafemaleformofthis Sxl tra transformer 2 transformer to determinethesex of the soma,such ( In somaticcells,theratioofX

otu Upd ) and ( tra2 ( ovo otu Sxl Sxl dsx tra Germ cell Germ toregulate thesplicing ) ). promote femalegermcell X:A regulates maleversus tra, Jak/Stat Drosophila along with Drosophila Sex-lethal doublesex , XYgerm has Wieschaus, 1978;Schupbach, 1982). proper germlinesex determination (Nöthigeretal.,1989;Marshand tra2 Overexpression of similar tothetransplantexperiments describedabove. germline, thisresultsinXXgermcellsdeveloping inamalesoma, a malesomaticgonad.However, as XX but mutantfor required inthesomaforpropersomatic controlofgermcellsex. cells themselves forproper germcellsex determination,itis determination pathway in many animals,including fliesandmice.Althoughthesomaticsex Somatic controlover germcellsex determinationisobserved in Somatic control overgerm cellsex signals andgermcell-autonomouscues. germ cellsex determinationrequiresacombinationofbothsomatic are notabletodevelop aseitherfullymale orfemale.Thus,proper opposite sex isvariable, oneconclusionisclear andconsistent:they compromised viability. stochastic decisionwhethertobemale,orfemale,andalsohave Thus, XXgermcellsinamalesomaappeartomake amore 1995; Nöthigeretal.,1989;Oliver etal.,1993; Staabetal.,1996). Hinson andNagoshi,1999;Horabinetal.,1995;Nagoshi degenerating (Andrews andOliver, 2002;Brown andKing,1961; rarely, femalecharacter, whilemany othercysts appeartobe remaining germcellsformcysts thatcanhave eithermaleor, more Nagoshi, 1999;Nöthigeretal.,1989;Sturtevant, 1945)].The cells [e.g. transformed toamaleidentity, many gonadscontainfew ornogerm animalswherethesomahasbeen al., 1989).Similarly, inXX transplantation (Steinmann-Zwicky, 1993;Steinmann-Zwicky et recovered atagreatlyreducedfrequency relative tothereciprocal in amalesoma.XXgermcellstransplantedintosomaare XX germcellsarelikely tofail toproliferateordiewhenpresent Female germcellsinamalesoma compromised viability. manner, thesegermcellsarenotfullymale,andmayalsohave maintaining aspectsofmaleidentityinacell-autonomous commonly observed, andislikely aresultofXYgermcells al., 1989).Thus,althoughtheovarian tumorphenotypeis in afemaleenvironment (Schüpbach,1985;Steinmann-Zwicky et et al.,2000).Furthermore,XYgermcellsdonotalways survive Nagoshi, 1999;JanzerandSteinmann-Zwicky, 2001;Waterbury have bothmaleandfemalecharacteristics(Hinson cells and analysesusingmolecularmarkers reveal thatthesegerm these experiments (Nagoshietal.,1995;Waterbury etal.,2000) However, characteristicsofoogenesiscanalsobeobserved in XY germcellscanretainamaleidentityinfemalesoma. spermatocytes (Steinmann-Zwicky etal.,1989),indicatingthat unambiguously malewithcharacteristicsofdifferentiating as inanormalovarian cyst. Attimes,thesegermcellsappear germ cellsinsteadof15interconnectednurseandanoocyte, cysts intheovary containtenstohundredsofsmallindividual review], theresultisoftenan‘ovarian tumor’,wheregermline methods andothers;seeOliver (Oliver, 2002),foranextensive When XYgermcellsdevelop inafemalesoma[usingtheabove Male germcellsinafemalesoma (McKeown etal.,1988),creatingtheoppositesituation. Although thephenotypeofgermcellsplacedinasoma and dsx tra are allrequiredinthesoma,and notthegermcells,for mutants (Brown andKing,1961;Hinson tra tra develop asmales(Sturtevant, 1945),including is sufficient tofeminizeanXYsoma Drosophila Sxl is notrequiredinthegerm tra , bycontrast,isrequired in is notrequiredinthe Development 133(15) tra ,

DEVELOPMENT and somatic signalsas 1995; Oliver etal.,1994).Bycontrast, (Andrews andOliver, 2002;Bielinskaet al.,2005;Nagoshiet germ cellsregardless of whetherthey areinamaleorfemalesoma Nagoshi etal.,1995; Waterbury etal.,2000). in afemalesomaticenvironment (HinsonandNagoshi,1999; expression isregulated bytheX:Aratioand appears toberesponsive togermcell-autonomous cues,as normally requiredinfemale,but notinmale,germ cells. these genesactispresentedinFig.1.Bothof are these genesisstillbeingresolved, onepossiblemodelforhow candidates foractingwith that, like differently andgermcellsalsorespondtosomaticsignals?Genes female germlineremainelusive. (King, 1979)]and regard togermcellsex determination,are development. Themostextensively studiedofthesegenes,with downstream target of germline (MarshandWieschaus, 1978),sothiscannotbethe Steinmann-Zwicky, 1993).Similarly, the germcells(Granadinoetal.,1993;Schüpbach,1985; and toactivate Sxl male-specific geneexpression isobserved inXXgermcellsthatlack tra both thesomaandgermline(below). Theeffects ofmutationsin Development 133(15) sisterless-b differently. Somaticcellsdependonfactors suchas depend ontheX:Aratio,they counttheirnumberofXchromosomes different inthegermlinethansoma.Althoughgermcells germline (Cline,1983;HagerandCline,1997;Oliver etal.,1993). might notactasa‘switch’betweenmaleandfemaleidentityinthe sufficient toactivate femalegermcelldevelopment and,therefore, development, asitdoesinthesoma,indicatingthat Sxl Nagoshi, 1999;Steinmann-Zwicky etal.,1989).However, gain of promote femalegermcelldevelopment inotherassays(Hinsonand indicating that when XYgermcellsarepresentinfemales(asdiscussedabove), Steinmann-Zwicky etal.,1989).Thisresembleswhat is observed 1988; Perrimonetal.,1986;Salz1987;Schüpbach,1985; Sxl cells require germ cells(Schüpbach,1985),asitisinthesoma,andfemale sex autonomously(Fig.1).ThisisdependentontheX:Aratioin In additiontosomaticinfluences,germcellsalso‘know’ theirown Germ cell-autonomouscontrol and activator oftranscription)pathway (Wawersik etal.,2005). mechanism actsthroughtheJak/Stat(Januskinase/signaltransducer germline sexual identity, ithasrecentlybeenshown thatonesuch Although littleisknown abouthow thesex ofthesomainfluences absence of germline assomefeminizingaffects of additional, and actsthrough same asthatcontrollingotheraspectsofsomaticsexual development controls somaticinfluenceover germcellsex determinationisthe How is In addition,thepathway bothupstreamanddownstream of , cannot make eggs but give risetoovarian tumors(Oliver etal., (Staab etal.,1996;Wei etal.,1994),and otu function inXYgermcellsdoesnotinterferewithmale tra2 is requiredbybothXXandXY germcellswhenthey are and Sxl Sxl dsx- and maternal dsx Sxl regulated inthegermlineifX:Aratiois‘readout’ dsx , give risetoovarian tumorswhenmutatedare Sxl independent mechanismforsomaticcontrolover the Sxl for properdevelopment. XXgermcellsmutantfor (Horabin etal.,1995;Waterbury etal.,2000). dsx on thegermlineindicatethatpathway that mutant XXgermcellsaremasculinized.Indeed, in females,but thesefactors arenotrequiredin ovo otu (Fig. 1).However, othershave argued foran Sxl expression isactivated byafemalesoma, (Oliver etal.,1987).Althoughtheroleof daughterless in thesecells.Thetarget(s) of Sxl to promotefemalegermcell to determinetheirX:Aratio tra tra otu are stillobserved inthe is notrequiredinthe appears torespond ovo ovarian tumor ovo is requiredinXX Sxl encodes several functions to Sxl Sxl scute/ is not in the Sxl [ ovo ovo otu is additional rolethatisindependentof genetically downstream of RNA regulation (Goodrichetal.,2004).Finally, The molecularfunctionofOTU isunknown, but itmayinvolve Andrews andOliver, 2002;Luetal.,1998;andOliver, 2001). 1995) thatdirectlyregulate Garfinkel etal.,1994;Mevel-Ninio etal.,1991;Mével-Ninio etal., related zinc-fingertranscriptionfactors (Garfinkel etal.,1992; thought toactinthisprocess,suchas 1987), unlike for germcellsurvival infemales(KingandRiley, 1982;Oliver etal., Hinson andNagoshi,1999),thatboth can alsobehave geneticallydownstream of Oliver andPauli, 1998;Pauli etal.,1993). form (Boppetal.,1993;Nagoshi1995;Oliver etal.,1993; required forthepropersplicingof (Salz, 1992),and sexual development inordertounderstandgerm cellsex essential tohave abetterunderstandingofallstagesgermcell continuous inputs,suchasthose fromthesomaticgonad.Thus,itis further alongthemaleorfemale developmental pathsbymore the onsetofgametogenesis,or if thegermcellsaresimplyguided know ifsex inthegermlineisever irreversibly determinedpriorto other aspectsofgermcelldevelopment. Moreover, wedonotyet or experimental manipulationaffects germline sexual identityor observed, andmakes itdifficult toknow whetheraparticulargene contributes tothevariable natureofthegermcellphenotypes germ celldevelopment occurmuch earlier. Thisprobably gametogenesis inadults,even thoughmany ofthecrucialevents in phenotypes atrelatively latestages, usuallyaspectsof germline sex determination,discussedabove, hasinvolved analyzing germ cellsexual development. Consequently, muchofthework on Until recently, weknew relatively littleabouttheearlystagesof Germline sexualdevelopmentin chromosome dose. identity ofthegermline,independentany othereffects ofX that thenumberofXchromosomeshelpsdeterminesexual cells andnotjusttheirsurvival (asdiscussedabove). Thisindicates clearlyaffects themaleversus femalephenotypeofthegerm ratio chromosome numberaffects germcelldevelopment, astheX:A However, itisunlikely thatthisistheonlyway inwhichX while malegermcellscannottoleratea2Xchromosomedose. possible thatfemalegermcellsrequirea2Xchromosomedose, than duetospecificeffects ongermlinesex determination;itis may beduetogeneralproblemsresultingfromthisdifference, rather time. Someoftheeffects ofXchromosomenumberinthegermline would have different levels ofXchromosomegeneexpression atthis to resetthissystemforthenext generation.XXandXY germcells compensation, atleastsomepointintheirdevelopment, inorder compensation. However, germcellsmustturnoff dosage relative levels, aprocessknown asXchromosomedosage expression toensurethatfemales(XX)andmales(XY) have similar Embryos needtoadjusttheamountofXchromosomegene compensation The problem ofgermlineXchromosome dosage soma, remainsahighpriority. influences sex determination,incombinationwithsignals fromthe understanding ofhow theX:Aratioisinterpretedin thegermlineand expression (Sahut-BarnolaandPauli, 1999).Thus,achieving abetter Complications tothesimplifiedmodelinFig.1includethat Sxl (Schüpbach, 1985),suggestingthey have an stand still otu ovo ovo , whichappearstoregulate expression (Andrews etal.,2000; Sxl and Sxl sans fille RNA intothefemale(active) otu . Therearealsoothergenes ovo , andthesegenesare otu Drosophila , a and in someassays(e.g. Sxl REVIEW otu splicing factor are required Sxl 2785 acts ovo otu

DEVELOPMENT

2786 stage canbeunderstood. component parts sothatthemechanismsregulating eachdistinct now istobreak germline sexual development down intoits between thegermcellsand somaticgonad.Anessentialgoal Importantly, eachofthesestepsrequiresextensive interaction completion ofspermatogenesis versus oogenesis(Fig.2). versus femalegermline stemcells,andthecommitmentto it includesthemaintenanceofthisidentity, theformationofmale initiation ofmaleversus femaleidentityinthegermline.Inaddition, determination fully. Germcellsexual development includesthe

Adult

Stage 12 Stage 15 Stage 17 L1 L3 L3 L1 17 Stage 15 Stage 12 Stage cells stem Escort REVIEW SGPs Cap cells filament Terminal Germ cells progenitor stem cells Germline Cyst cells Hub cells msSGPs brown; folliclecells,yellow. spectrosomes andfusomes,limegreen; somatic(follicle)stemcells, purple; basalcells,lightblue;escortstemgreen; terminal filamentcells,orange;capred; stalkcellprecursors, testis sheath,yellow. Female:germcells,darkpink;GSCs,light fusomes, limegreen; cystprogenitor cells,lightgreen; cystcells,green; light blue;msSGPs,brown; embryonicandadulthubcells,orange; brown; germcells,yellow. Male:germcells,darkblue;putativeGSCs, (follicle) stemcells.Stage12:SGPs,green; male-specificSGPs(msSGPs), escort cellsare replaced byfolliclecellsproduced bythesomatic cells withbranchedfusomes.Laterinfemalecystdevelopment,the form differentiating oogenicorspermatogeniccystsofinterconnected the niche.GSCsandsomaticstemcellsproduce daughtercellsthat progenitor cellsinmalesandescortstemfemales)alsocontact cells infemalesandhubmales.Somaticstem(cyst germline stemcells(GSCs)contactthesomaticnicheformedbycap during stage17andspermatogenesisbeginsL1.Inadults, oogenesis beginsinearlypupae.Inthemale,embryonichubforms and Laski(GodtLaski,1995)]tomakeindividualovarioles, undergoes ovarymorphogenesisduringlateL3[modeledafterGodt gonad are already sexuallydimorphicatthistime.Thefemalegonad form theembryonicgonadbystage15.Bothgermlineandsomatic male. Thegermcellsandsomaticgonadalprecursors (SGPs)interactto the apicalendofasingleovarioleinfemaleandtestis 1985). L1,1stinstarlarvae;L3,3rd instarlarvae.Theadultstagedepicts stages are asdescribedpreviously (Campos-OrtegaandHartenstein, Fig. 2.Diagramofgermcellsexualdevelopment. Wawersik etal.,2005).Last,the femalegermlineismoresensitive female germcells donot(Kerkis, 1931; Steinmann-Zwicky, 1994; after gonadformationasmalegerm cellsbegin toproliferate,while in themale).Thedifference ingermcellnumberisthenamplified differences inthesomatic gonaditself(e.g.thepresenceofmsSGPs differences inhow thegermcellsrespondtosomaticgonador formation (Sonnenblick,1941; Suetal.,1998),thismaybedueto cells arearrestedinthecellcycle duringmigrationandgonad be differences ingermcellformationbetweenthesexes, andgerm (Poirie etal.,1995;Sonnenblick,1941).Astherearenotthought to are incorporatedintothemalegonadrelative tothefemalegonad embryonic gonadformation.Aslightlygreaternumberofgerm cells Sexual dimorphisminthegermlineisalsoevident atthetimeof Initial germcellsexualidentity germline canbesex specific. females, indicatingthatcommunicationbetweenthesomaand the ligand fortheJAK/STAT pathway, differently inmales versus express secretedfactors suchas Inaddition,theSGPs the somaandgermline(Tazuke etal.,2002). al., 2003),andgapjunctionsmayfacilitate communicationbetween the embryonicgonad,asSGPsensheatheachgermcell(Jenkinset ample opportunityforsomatoinfluencegermcelldevelopment in gene expression inmalesversus females(LeBras,2006).Thereis also have asex-specific identityandexhibit adifferent patternof undergo apoptosisinfemales(DeFalco etal.,2003).AnteriorSGPs (msSGPs) becausethey contribute toonly the malegonadand The mostposteriorSGPsareknown as‘male-specific’SGPs sexually dimorphicatthistime(seeFig.2andlaterin thereview). by Van Doren,2006).Interestingly, thesomaticgonadisalready the embryonicgonadatabout12hoursafterfertilization(reviewed Germ cellscoalescewithsomaticgonadalprecursors(SGPs)toform Gonad formation unpaired (Wawersik etal.,2005),a Development 133(15) Embryonic

DEVELOPMENT cells canexpress regulated differently fromitsmaintenance. Inaddition,XXgerm cells, andissufficient to induceexpression ofsome male-specific necessary forthemaintenance of in theembryonicgonad,amale-specific behavior. It isalso pathway isnecessaryandsufficient toinducegermcellproliferation either sex aslongthey contactamalesomaticgonad.TheJak/Stat females, andtheJak/Statpathway canbeactivated ingermcells of is expressed intheembryonic somaticgonadinmales,but notin (Wawersik etal.,2005).Aligandforthispathway, germline? OnemechanismactsthroughtheJak/Statpathway Steinmann-Zwicky, 1998). mgm1 male somaticgonad(Wawersik etal.,2005)andrepressionof and thereisevidence forbothinductionof initiation of female soma(JanzerandSteinmann-Zwicky, 2001).Thus,the the soma,asXYgermcellscannotmaintain Steinmann-Zwicky, 2001). genotype (HellerandSteinmann-Zwicky, 1998;Janzerand environment, indicatingthatitisregulated bythegermcell XY germcellsinitiate regulated bybothsomaticsignalsandgermcell-autonomous cues. versus femaleidentities. indicates thatthismaybewhengermcellsfirstacquiredistinctmale patterns arefirstobserved inthenewly formedembryonicgonad The fact thatsex-specific germcellphenotypesandgeneexpression 2002), alongwithothermale-specificgenes(Wawersik etal.,2005). reflects expression ofthetranscriptionfactor Development 133(15) [ cells exhibit male-specificexpression of germ cellssoonaftergonadformation(stage15).Atthistime, already sexually dimorphicatthistime. occurs duringembryonicstages,thisindicatesthatthegermlineis dysgenesis) thanisthemalegermline(Wei etal.,1991).Asthis to deathcausedbytheactivation ofPelementtransposons(hybrid male niche(hub)usesboththeJak/StatandTgf cap cells)usestheTgf identity, whiletheotherdaughterisdisplacedfrom thenicheandentersgametogenesis.Signalingfrom thefemaleniche(termi to thenicheviaDE-cadherin-richcell-cellcontacts.GSCsusuallydividesothatonedaughterremains associatedwiththenich Fig. 3.Enlargedviewofthemaleandfemalegermlinestemcell(GSC)niches. cells, red; escortcells,lightgreen; epithelialsheath,blue. orange; cystprogenitor cells,lightgreen; testissheath,yellow. Female:germcells,darkpink;GSCs,lightterminalfil mgm1 How doesthesomaticgonadregulate initial sexual identityinthe Sex-specific geneexpression inembryonicgermcellsis A sex-specific patternofgeneexpression canalsobeobserved in i.3, Sabe l,19),a , Fig.3C,D(Staabetal.,1996)], expression bythefemalesomaticgonad(Hellerand mgm1 mgm1 ␤ expression inthemalegermlinemaybe signaling pathwaytomaintainGSCsandtheJak/Statescortcellpopulation.Signalingfrom the mgm1 when inamalesoma(Staabetal.,1996), mgm1 expression even inafemalesomatic mgm1 expression isalsoregulated by lacZ expression inmalegerm male germline marker 1 mgm1 ␤ mgm1 escargot reporter thatprobably pathways tomaintaintheGSCs.Male:germcells,darkblue;putativeGSCs,lighthub expression bythe unpaired expression ina (Streit etal., ( upd ), Male versusfemalegermlinestemcells established shouldfocusonthisearlytimepoint. Future work tounderstandhow initialgerm cell sexual identityis autonomous cuesareimportantforgermlinesex determination. evidence thatbothsignalsfromthesomaticgonadandgermcell- first establishedinthegermcells.Even atthisearlystage, thereis this mayrepresentthepointatwhichmaleversus female identityis after they have joinedwithSGPstoformtheembryonicgonad,and sexual identityatthisearlystage. germ cell-autonomousfashion, contribute tospecifyinggermcell genes, suchas subset ofgermcells (LeBras,2006).Thisislikely torepresentthe of embryogenesis(stage17),which interactsspecificallywitha Bras, 2006).Thesecellsform a tightclusterduringthelaststage markers characteristicof theadulthub(Gönczyetal.,1992;Le SGPs inthemaleembryonic gonad express several molecular In addition,recentwork demonstrates thatagroupofanterior similar tothehubispresentin earlylarval stages(Aboïm,1945). (Aboïm, 1945;Hardyetal.,1979).Astructuremorphologically and iscreatedbyatightclusterofsomaticcellsknown asthe‘hub’ males, asingleGSCnicheispresentattheapicalendofeachtestis during thelaststageofembryogenesis(Fig.2,Fig.4E,F).Inadult However, inmalesthereisevidence thatthestemcellnicheforms become GSCsarestillunansweredquestionsinbothsexes. created byspecificsomaticcells.Whenandhow germcells become germlinestemcells(GSCs)andpopulateacellniche Drosophila produce acontinuoussupplyofdifferentiating gametes.In Germ cellsoftenformastemcellpopulationsothatthey can Germline stemcellformation other somaticsignals,orgenessuchas commitment tomalegermcellidentity. Itiscurrentlyunknown if sex determination,anddoesnotnecessarilyleadtoan irreversible Thus, thispathway isonlyoneofthefactors thatregulates germline germ cells,atleastearlystages,doesnotblockoogenesisinadults. al., 2005).However, activation oftheJak/Statpathway infemale Minichromosome maintenance5 In summary, germcellshave asex-specific identityatatimesoon In boththemaleandfemaleGSCniches,GSCsare attached , asubsetofgermcellsinbothmalesandfemales mgm1 , disc proliferation abnormal , infemalegermcells(Wawersik et Sxl ament cells,orange;cap , ovo ovo e, andretains GSC nal filamentand and REVIEW otu acting ina 2787 and

DEVELOPMENT hub (F).Imagescourtesy ofStephanieLeBras. germ cellsadoptaspecificrosette distributionaround theembryonic embryonic hubformsinmales,but notinfemales(E),andanterior (anti-Vasa, red) andembryonic hubcells(anti-Fasciclin3,green). The 2005). ( reproduced, withpermission, from Wawersik etal.(Wawersik etal., in maleembryos(D)andisnotexpressed infemales(C).Images in stage16gonads(outlined). and male-specific somaticgonadalprecursors (msSGPs)(anti-Sox100b,red) embryonic gonadlabeledtoreveal thegermcells(anti-Vasa, blue), to beGSCsatamuchearlierstage(AsaokaandLin,2004).Thus,an suggesting thatsomefemalegermcellsarealreadypredetermined female embryonicgonadaremorelikely tobecomeGSCs, work hasshown thatgermcellspopulatingtheanteriorregion ofthe for GSCformationatthistime(ZhuandXie,2003).However, recent between thenicheandGSCsinadultovary, isalreadyimportant suggests thattheTgf King, 1970;Sahut-Barnolaetal.,1995;Spradling,1993).Evidence which begins inthelate(thirdinstar)larvae (GodtandLaski,1995; cap cells.Thisstructureisformedonlyduringovary morphogenesis, contains astemcellniche,whichiscreatedlargely bythesomatic stage. functional GSCsinthelateembryoorearly(firstinstar)larval it ispossiblethatthegermcellsinteractingwithhubarealready spermatogenesis begins duringearlylarval stages(Aboïm,1945), formation oftheGSCnichethatpersistsinadulttestis.As 2788 courtesy ofStephanieLeBras.( germ cells,respectively), eventhoughbothare inthe and mgm1enhancertrapsare expressed differently (inmaleSGPsand Fig. 4.Sexualdimorphismintheembryonicgonad. identity astheyexpress esg in malegonadsatthisstage,andanteriorSGPshaveasex-specific In thefemale,eachof16orsoovarioles inanadultovary esg E G66B REVIEW , F ) Stage17embryonicgonadlabeled toreveal thegermcells enhancer trap(anti- ␤ G66B pathway, thecrucialsignalingpathway mgm1 C in malesbutnotfemales.Theesg ␤ , D -gal, green). msSGPsare foundonly ) mgm1 expression isspecifictogerm cells expression (X-galstaining) esg ( A locus. Images , B ) Stage15 G66B development. to prevent prematuregermcelldifferentiation earlierin same signalsthatregulate stemcellmaintenanceintheadultmayact undifferentiated stateuntilthestemcellnichehasbeenformed.The both sexes itislikely tobeimportant holdgermcellsinan premature differentiation (GilboaandLehmann,2004b).Thus,in morphogenesis, andtheTgf potential toentergametogenesisatearlystages,priorovary GSCs. Similarly, ithasbeenshown thatfemalegermcellshave the those thatcontactthenicheretainsignalandcontinuetoactas the nichelosesignalanddirectlyenterspermatogenesis,while remain undifferentiated. Thosegermcellsthatdonotinteractwith contributes totheirmaleidentity, andallows themtoproliferateand all germcellsinamaleembryoinitiallyreceive thissignal,which there isnorealdifference betweenthesetwo identities. Itmaybethat germ cellidentity, maleGSCidentityorboth?Onepossibilityisthat in theadulttestis.Thus,doesJak/Statpathway promotemale they enterthemaleembryonicgonad,but isthen active onlyinGSCs differential responsestothenicheenvironment. sexual identityofthegermcellsiscrucialfordetermining their signals andmaintaindistinctidentities.Itislikely thattheprior environments, maleandfemaleGSCsresponddifferently tothese 1996). Thus,despitebeingexposed tosimilar signaling male germcellsandbecomesrestrictedtoGSCs(Staabetal., including in expression inmaleversus femaleGSCs(Gönczyetal.,1992), and Spradling,2005).Inaddition,therearedifferences ingene (escort) stemcells;femaleGSCsdonotrequirethissignal(Decotto pathway alsooccursinthefemaleniche,itactsonsomatic and Ingham,2003).AlthoughsignalingthroughtheJak/Stat Tgf both theJak/Stat(Kigeretal.,2001;Tulina andMatunis,2001) (Xie andSpradling,1998),whileinthemale,signalsactthrough the niche,but inthefemalethissignalactsthroughTgf female GSCs.Inbothsexes, GSCsaremaintainedbysignalingfrom similarities, therearealsosomecleardifferences between maleand al.,2005;Yamashita etal.,2005).However, despitethese et 2005; GilboaandLehmann,2004a;Spradlingetal.,2001;Wong somatic stemcellsintheniche(Fig.3)(DecottoandSpradling, active intheniche,andcloseinteractionbetweenGSCs how they interactwiththeniche,signalingpathways thatare surprising similaritiesbetweenmaleandfemaleGSCs,including extensive studyoftheadulttestisandovary. Therearesome Most ofourknowledge ofGSCsin Adult GSCsandniches specifying GSCidentityinasubsetofgermcells. male andfemaleembryonicgonads,mayberesponsiblefor interesting possibilityisthattheanterior-most SGPs,inboththe Zwicky et al.,1989),indicatingthat GSCmaintenanceis germline isalsolostover timeinthesegonads(Steinmann- produce germlinecysts (Schüpbach,1985).However, the This suggeststhatGSCsare stillpresentandcontinuously produced inan‘assemblyline’ fashion asinnormalovarioles. ovarian tumor phenotype,individual germlinecysts arestill When malegermcellsarein afemalesomaandexhibit the versa? Littleiscurrentlyknown aboutthese interestingquestions. behavior? CanamalegermcellbecomefemaleGSC orvice How doesgermlinesexual identityaffect GSCformationand Sex andGSCs Interestingly, theJak/Statpathway isactivated inallgermcellsas ␤ pathways (Kawase etal.,2004;SchulzShivdasani mgm1 expression, whichisinitiallyexpressed inall ␤ pathway isimportanttoprevent this Drosophila Development 133(15) comes from ␤ pathway

DEVELOPMENT which represents animportantareaforfuture investigation. First, discussion ofgermlinesexual identityin There aretwo mainideas onwhichwehave focusedour Conclusions that occurbetweengermcellsand thesurroundingsoma. how eachisaffected bygermcellsexual identityandtheinteractions the different stagesofgermcellsexual development individually, determination. Thisproblemhighlightstheneedtobetterunderstand gametogenesis thatareseparatefromtheevents ofgermlinesex phenotypes couldsometimesresultfromrelatively latedefectsin germline sex determination.However, itisalsopossiblethatthese between thesomaandgermlinethatisduetoearlierproblems in defects atthisstagecouldindeedresultfromsexual incompatibility extensive soma/germ-cellcommunicationduringgametogenesis, the work ongermlinesex determinationdescribedabove. Asthereis phenotypes, suchasovarian tumors,thathave beenusedformuchof spermatogenesis versus oogenesis,andothertypesoflatecyst Decotto andSpradling,2005).Itistheselatedifferences in the earlycysts dieandarereplacedbyfolliclecells(Spradling, 1993, development because,inthefemale,escortcellsinteractingwith Robinson etal.,1994).Therearealsodifferences insomaticcell time (Himeetal.,1996;HinsonandNagoshi,1999;Lin1994; differences inringcanalandfusomecharacterbecomeevident atthis that areeasilyrecognizablebytheirpolyploidnuclei.Inaddition, commits tomeiosisinfemales,whiletheother15becomenursecells create 64spermatids.Bycontrast,onlyonegermcell(theoocyte) cells ofthecyst grow significantlyinsizeandallcompletemeiosisto distinguish clearlybetweenspermatogenesisandoogenesis. 1993). Thus,upthroughtheearly16-cellcyst stage,itis difficult to by somaticcellsinbothsexes (DecottoandSpradling,2005;Fuller, and Ohlstein,1995).Finally, theearlygermcellcysts areensheathed protein asthey entergametogenesis(Gönczyetal.,1997; McKearin male andfemalegermcellsalsoexpress theBagofMarbles (BAM) a specializedorganelle, thefusome,extends betweenthesecells.Both The connectionsbetweengermcellsareformedbyringcanals,and cytokinesis toproducea16-cellcyst ofinterconnectedgermcells. that leaves thenicheundergoes fourmitoticdivisions withincomplete surprisingly similar. Inbothmales andfemales,theGSCdaughter processes, withdramaticallydifferent endproducts,they areinitially spermatogenesis andoogenesisareobviously very different fertilization, reviewed bySpradling(Spradling,1993)].Although not begin infemalesuntilmuchlater[earlypupae,5+daysafter days afterfertilization,reviewed byFuller(Fuller, 1993)]but does Gametogenesis begins inmalesduringtheearlylarval stages[1-2 Gametogenesis influenced bygermcellsexual identity. male versus femaleGSCsareestablishedandhow thisprocessis during development, willberequiredinbothsexes todeterminehow the GSCphenotypesinadults,andananalysisofformation whether any behave asGSCs.Clearly, amoredirectedanalysisof When suchgonadsdocontaingermcells,littleisknown about germ cellsdonotsurvive orcannotpopulatethemaleGSCniche. adult testeshave few ornogermcells(above), indicatingthatthese a femaleormixed identity. cells donotappeartohave maleGSCidentity, andprobablyhave and Steinmann-Zwicky, 2001;Waterbury etal.,2000).Thus,these markers, suchas defective. Theseputative GSCslackexpression ofmaleGSC Development 133(15) Subsequently, thedifferences becomeobvious. Inmales,thegerm When femalegermcellsdevelop inamalesoma,many resulting mgm1 , andexpress female-specificSxl(Janzer Drosophila , eachof identity iscontrolledateachstep. sexual development andforinvestigating how germlinesexual have thetoolsnecessaryforstudyingdifferent stagesofgermline morphogenesis, andofstemcellnicheformation.Thus,wenow to understandingtheearlierprocessesoftestisandovary stages. Thisalsoincludesapplyingourknowledge oftheadultgonad of thegermlineandsomaticgonadduringlateembryoniclarval embryonic gonadtounderstandthesexually dimorphic development stages. Asdescribedabove, thisincludesmoving forward fromthe ‘both ends’have madedramaticprogressinour abilitytostudythese somatic gonadoccurs.Recently, though,researchersworking from during whichmostofthesexual development ofthegermlineand has beendifficult tobridgethegapbetweenembryoandadult, about theadultovary andtestis.However, fortechnicalreasons,it about theearlydevelopment ofthegonadinembryo,andalso these ideasinthefuture.Considerableknowledge hasbeengained germ cellsexual development thatwillallow ustobetteraddress of thesestages. or oogenesis.Extensive germline-somainteractiongoverns each male orfemalegermlinestemcellsandthroughspermatogenesis embryonic gonad,andmustprogressthroughtheformationof determined. Thegermlineisalreadysexually dimorphicinthe yet know whenthesex ofthegermlinebecomesirreversibly of thedifferent stagesofgermlinesexual development. We donot germline sexual identityneedstobeunderstoodwithinthecontext interact tocontrolgermlinesexual identity. Thesecondisthat uncovered, muchremainstobelearnedabouthow thesefactors and somaticeffects ongermlinesex determinationhave been important playersthatmediateboththegermcell-autonomous signals andgermcell-autonomouscues.Althoughsomeofthe germ cellsex determinationrequiresacombinationofsomatic depends onthenumberofXchromosomes (asin proper germcellsexual development. Interestingly, thislargely gametes. Thus,germcell-autonomouscuesarealsoimportant for normally inasomaoftheoppositesex, andgive risetofewer orno also similartoflies,mousegermcellsdonotgoondevelop genotype ofthegermcells(reviewed by McLaren,2003).However, female atthistimedependsonthesex ofthesomaticgonad,not male germcellsdonot.Whetherbehave asmaleor gonad (genitalridge),whenfemalegermcellsentermeiosiswhile the mouseoccursoonaftergermcellsassociatewithsomatic the somaandgermline. each ofwhichrequiresextensive, sex-specific interactionbetween sexual identity, theformationofGSCsandentryintogametogenesis, several discretestages,includingtheestablishmentofgermcell cells. Inaddition,mousegermcellsexual development alsoinvolves and isdependentonthenumberofXchromosomesingerm germ cell-autonomouscues,isregulated differently frominthesoma germline sex determinationalsorequiresbothsomatic signalsand development in Interestingly, themainconclusionsaboutgermlinesexual the fly Mammalian germcellsexualdevelopment:aviewfrom survive andmake itfurther intospermatogenesisthan doXXor In addition,wheninamalesomatic environment, XOgermcells or XOgermcellsinasimilargonad environment (McLaren,1981). cells aremoreinclinedtofemale characteristicsthanareeitherXY determines sex inthemammalian soma).For example, XXgerm than onthepresenceorabsence ofaYchromosome(which Fortunately, anew window isnow openinginourabilitytostudy As inflies,thefirstsignsofsex-specific germcelldevelopment in Drosophila are alsotrueforthemouse.Mouse Drosophila REVIEW ) rather 2789

DEVELOPMENT Burgoyne, P. S. Brown, E.H.andKing,R.C. Bopp, D.,Horabin,J.I.,Lersch,R.A., Cline,T. W. and Schedl,P. Bielinska, B.,Lu,J.,Sturgill,D.andOliver, B. Asaoka, M.andLin,H. elucidating thesemechanismsandthegenesthatcontrolthem. mammals indicatesthat similarity betweengermcelldevelopment in needed tounderstandthedefectsthatoccurinsuchindividuals. The germ cellsexual identityandofgermcellsexual development is unknown origin.Abetterknowledge ofthemechanismsthatcontrol (sertoli cellonlysyndromeandprematureovarian failure) thatareof other patientsareseenwithsimilarsevere germcelllossphenotypes cells arepresentinamalesomathemouseorfly. Inaddition,many leading togermcelllosssimilarthatobserved whenfemale germ cells have two Xchromosomesandmightthereforebefemale, male becauseofthepresenceaYchromosome,whilegerm individuals withKlinefelter’s syndrome,forexample, thesomais between thesex ofthesomaandsex ofthegermline.In chromosome constitutionswould leadtoan‘incompatibility’ chromosome numberinfluencesgermcellsexual identity, these Lanfranco etal.,2004).AccordingtothehypothesisthatX in germcelldevelopment andgermcellloss(Abiretal.,2001; Turner’s (XOfemale)syndromesleadtosterilitywithsevere defects fertility. Humandisorderssuch asKlinefelter’s (XXYmales)and also crucialforunderstandinghumangermcelldevelopment and autonomous cuestocontrolpropergermcellsexual development is in thesespeciesmaybehighlyconserved. intriguing possibilitythattheprocessofgermcellsex determination dependent onthenumberofXchromosomes.Thisraises signals combinedwithgermcell-autonomouscuesthatare both fliesandmice,germcellsex determinationdependsonsomatic compensation discussedabove. However, thedatasuggestthat,in including theproblemofgermlineXchromosomedose explanations fortheroleofXchromosomenumberingermcells, Andrews, J.,Garcia-Estefania, D.,Delon,I.,Lu,J.,Mevel-Ninio,M.,Spierer, Aboïm, A.N. spermatogenesis inbothmouseand chromosome (therearealsoYgenesnecessaryfor Y chromosometobeinitially‘male-like’, but musthave onlyoneX (Burgoyne, 1987;Huntetal.,1998).Thus,germcellsdonotneeda XXY germcells,whichbehave initiallyasmalebut thendie 2790 Andrews, J.andOliver, B. Abir, R.,Fisch,B.,Nahum,Orvieto,Nitke,S.andBenRafael,Z. References by NIHgrantsGM63023andHD46619thePewCharitableTrust. Work from theVan Doren laboratorycitedinthisreview hasbeensupported Stephanie LeBrasandMattWawersik forproviding imagesusedinFig.4. anonymous reviewers forcriticalevaluationofthismanuscript.We alsothank We thankBrianOliver, membersoftheVan Doren Laboratoryandthe spermatogenesis. gene ofDrosophila melanogaster. Drosophila oogenesis. Expression ofthe Genetics contribute toovo-Bregulation intheDrosophila melanogastergermline. Development descend from polecellsinthe anteriorregion oftheembryonicgonad. 881-892. function antagonisticallyintheDrosophila femalegermline. A., Payre, F., Pauli,D.andOliver, B. transcription inDrosophila melanogaster germcells. Hum. Reprod.Update Turner’s syndrome andfertility:current statusandpossibleputativeprospects. 52 gonades normalesetagamétiquesdeDrosophila melanogaster. Understanding how somaticsignalscombinewith germcell- , 53-154. REVIEW 169 (1945). 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