site intheWolffian ductstotheurogenitalsinusepithelium,where undergo transposition, moving fromtheirprimaryinsertion to thebladder. mediate myogenicperistalsis,propellingurinefromtherenalpelvis differentiates intotheureters,which aremusculartubesthat mesenchyme. Theportionoftheuretericbud lying outsidethe morphogenesis inresponsetosignalsfromthemetanephric blastema andundergoes successive roundsofbranching caudal sproutfromtheWolffian ductthatinvades themetanephric renal collectingductsystemandextra-renal , formsasa bladder andurethra.Theuretericbud, whichwillgive risetothe differentiates intotheurogenitalsinus,primordiumof paraxial mesoderm.Wolffian ductsopenintothecloaca,which mesoderm, astripoftissuelyingbetweenthelateralplateand of themalegenitaltract,arelargely derived fromintermediate ureters andWolffian ducts,pairedepithelialtubesthatformmost the kidneys tothebladderforstorageandexcretion. Thekidneys, at mid-gestationtoformanoutflow tractthatconductsurinefrom (bladder andurethra)urinarytractformindependently, connecting The organs thatcomprisetheupper(kidney andureter)lower substances suchasfood,airandwaste intoandoutoftheembryo. independently formedorgans intocomplex systemsthatconduct A crucialfeatureinembryonicdevelopment istheassemblyof INTRODUCTION Accepted 27July2007 1 KEY WORDS:Bladder, Reflux,Trigone, Ureter, Urinarytractformation,Mouse,Human abnormal development. development occursdifferently thanpreviouslythought,providingnewinsightsintothemechanismsunderlyingnormaland formation dependsatleastinpartonintercalationofureteralandbladdermuscle.Thesestudiessuggestthaturinarytract trigone isformedmostlyfrombladdersmoothmusclewithamoreminorcontributiontheureter, andthattrigone not beentestedexperimentally. UsingtheCre-loxrecombinationsysteminlineagestudiesmice,wefind,unexpectedly, that however, thesemodelshave develops independentlyofthebladder, fromtheureters,Wolffian ductsoracombinationofboth; events underlyingtheseconnectionsandtrigoneformationarenotwellunderstood.Accordingtoestablishedmodels,the however, thedevelopmental between theuretersandtrigonearecrucialforproperfunctionofureteralvalvemechanism; Wolffian ductstothetrigone,amuscularstructurecomprisingbladderfloorjustaboveurethra.Preciseconnections compartments formindependently, connectingatmid-gestationwhentheuretersmovefromtheirprimaryinsertionsitein tothekidneythatcancauseseveredamageandinduceend-stagerenaldisease.Theupperlowerurinarytract bladder viaperistalsis.Onceinthebladder, theureteralvalve,amechanismthatisnotwellunderstood,preventsbackflowof The urinarytractisanoutflowsystemthatconductsurinefromthekidneystobladderviaureterspropel Renata Viana anti-reflux mechanism The developmentofthebladdertrigone,center (2007)doi:10.1242/dev.011270 Development 134,3763-3769 DEVELOPMENT ANDDISEASE 2049, 109ZinaPitcherDr, AnnArbor, MI481093,USA. *Author forcorrespondence (e-mail:[email protected]) USA. Genetics, UniversityofTexas M.D.Anderson Cancer Center, Houston,TX77030, 10032, USA. Richard R.Behringer York, NY, USA. Columbia University, DepartmentofUrology, 650West 168thStreet, NewYork, NY The upperandlower urinarytractcompartmentsjoinwhenthe 2 Department ofUrology, NewYork UniversitySchool ofMedicineNew 3 Department ofPathology, UniversityofMichigan,MSRB1,BSRB 1 , EkatherinaBatourina 4 , EllenShapiro 2 4 Department ofMolecular , Terry Hensle 1 , HongyingHuang 1 , SarahLambert 2 , Gregory R.Dressler and preservingrenal function. for formationoftheureteralvalve thatiscrucialforpreventing reflux mechanisms controllingurinary tract assemblythatarealsoimportant independently oftheureter. Thesestudieselucidateimportant that theureteralpathway is presentinthebladderwall andforms (Waldeyer, 1892)(reviewed byHutch,1972).We find, paradoxically, through thebladderisformed byasheathofureteralmuscle structure. Anumberofstudiesalsosuggestthattheureteralpathway muscle andthatureteralfibers areanimportantcontributor totrigone We find, unexpectedly, thatthetrigonederives largely frombladder trigone andtodeterminewhichlineagescontribute toitsformation. these questions,weusedmousemodelstostudythestructureof the translateintorefluxandobstruction?To begin toaddress mechanism established,andhow dopositionalabnormalitiesofthe remain: whatisthederivation ofthe trigone, how istheanti-reflux of thetrigonestructure(Meyer, 1946).Hence,anumberofquestions studies suggestthatthebladdermuscle(detrusor)mightalsobe part (Hutch, 1972;Tanagho, 1981;Weiss, 1988;Wesson, 1925).Other , differentiating fromthecommonnephricductandureter suggestion thatthetrigoneisstructurallydistinctfrombladderand controversial. Analysisofhumanandanimalspecimenshasledtothe and roleofthetrigoneinanti-refluxmechanismremains problems includingend-stagerenaldisease. obstruction, whichcandamagethekidney andcausesevere health of urinefromthebladdertoureters,amajorcauserefluxand crucial forfunctionofthevalve mechanismthatprevents backflow al., 2005).Preciseconnectionsbetweenuretersandthetrigoneare urogenital sinusepitheliumatthelevel ofthetrigone(Batourinaet Wolffian ducts,andfusion,inwhichtheureterorifice insertsintothe involves apoptosis,which enablestheureterstodisconnectfrom previous studiessuggestthatformationofthesefinal connections trigone, situatedbetweenthebladderandurethra(Fig.1).Our they make final connectionsinatriangularstructure,known asthe Despite itscentralimportanceinurinarytractfunction,theorigin 1 and CathyMendelsohn 3 , AkioKobayashi RESEARCH ARTICLE 1, * 4 , the 3763 the

DEVELOPMENT TGG ATATG-3 et al.,2005). 5 Laboratory andgenotypedusing:5 lacZ Rosa26 ACTCTCCTTCC-3 Laboratory andgenotypedbyPCR using:5 GCCTCTAGGGTG-3 CTG-3 3 CGTCCCTTCCTTTTCTCCTCA-3 were genotypedusingthefollowing threeprimers:Pax2F, 5 Genotyping was withPCRusingprimers:5 from DrFrankCostantini(ColumbiaUniversity, New York, NY). taken tobeE0.5. 16.00-17.00 h,andthemorningwhenvaginal plugwas visualizedwas For timedmatings,malesandfemaleswereplacedinacage togetherat Animals andgenotyping glycerol. 2-3 timeswithPBS,post-fixed with4%PFA andstoredat4°Cin80% mg/ml X-Galindimethylsulfate). After staining,sampleswerewashed mM potassiumferrocyanide, 2mMmagnesiumchlorideinPBSand1.2 X-Gal solutionfor2-5hoursat37°C(5mMpotassiumferricyanide, 5 cold 2%PFA inPBSfor5minutesat4°C,washed inPBS,andstained To reveal X-Gal histochemistry light microscopy counterstained withHematoxylin,dehydrated,mountedandobserved by containing 3,3 with avidin-biotinylated peroxidasecomplex anddeveloped inasolution goat anti-mousesecondaryantibodywas applied.Slideswerethentreated overnight incubationat4°Cwithanti-smoothmuscleactin,abiotinylated Carpinteria, CA)was usedtodetectthe humansmoothmuscleactin.After 10% normalgoatserum.Mousemonoclonalantibody(M0851,Dako, microwave treatment(900W)for20minutes,followed byblockingwith sections withantigenunmaskingsolution(Vector Labs#H-3300)and for 5minutes.Antigenretrieval was performedbyincubatingparaffin Endogenous peroxidaseactivity was blocked with3%hydrogenperoxide Representative tissue sectionsweredeparaffinized andrehydrated. Immunohistochemistry forsmoothmuscleactin fixed, paraffin-embedded andseriallysectionedat4 measurements ofcrown rump andfootlength.Specimenswereformalin- estimated fromdateoflastmenstrualperiodaswellsonographic was obtainedbytheconsultingobstetrician. Thegestationalageswere fetuses rangingingestationalagefrom19to22weeks.Informedconsent Research Associates,lower urinarytractswereremoved fromfourhuman With approval fromtheNew York University InstitutionalBoardof Human tissues overnight at4°Cthenwashed andmounted. incubated in(ASMA)FITC-orCy3-conjugatedantibodies(Sigma) hours atroomtemperature.Afterwashing andreblocking,thetissuewas washing, thesecondaryantibody(donkey anti-rabbitIgG)was appliedfor2 T. T. Sun(New York University, NY)was appliedovernight at4°C.After differentiation (Wu etal.,1994).UP3antibody(clone#744)was agiftofDr then primaryuroplakinantibody, amarker ofurothelialterminal were incubatedinblockingsolution(2%horseserumwashing buffer) For doublestainingwithuroplakinandsmoothmusclealphaactin,samples PBS/0.1% Triton X-100for30minutesthenprocessedimmunostaining. with 0.3%hydrogenperoxideincoldmethanolfor20minutes,washed in PBS andthenembeddedin3%agarose.Sectionswerepermeabilized sections (100-150 for 1-3hoursat4°CandembeddedinOCTcompound.For vibratome For cryosections(10 Immunostaining MATERIALS ANDMETHODS 3764 Ј Ј . -GCGAAGAGTTTGTCCTCAACC-3 Sm22-Cre Ј RESEARCH ARTICLE and 5 lacZ mice (Soriano,1999)werealsoobtained fromtheJackson mice (Holtwicketal.,2002)wereobtained fromtheJackson Ј -ACGATCCTGAGACTTCCACACT-3 Ј expression, vibratomeorcryostatsectionswerefixed in . Rarb2-Cre Ј Hoxb7-Gfp -diaminobenzidine (DAB). Allsectionswere ␮ Ј m), tissuewas fixed overnight in4%PFA, washed in ␮ and 5 Ј ) isewsfxdi %prfradhd (PFA) m), tissuewas fixed in4%paraformaldehyde ; andPGK,5 Ј -CGCATAACCAGTGAA ACAG CATTGC-3 mice weregenotypedasdescribed(Kobayashi mice (Srinivas etal.,1999)wereakindgift Ј -AAAGTCG CTCTGAGTTGTTAT-3 Ј Ј -AGACTGCCTTGGGAAAAGCGC- ; Pax2R, 5 Ј and 5 Ј -AGCGCGATCACATGGTC - Ј Ј Ј -CAGACACCGA AGCT - AGCT -CAGACACCGA -GAAAGGCCAGTGTG - -GGAGCGGGAGAAA - ␮ m. Ј . Pax2 mutant mice Ј -CCCAC - Ј Ј , . ob ato h rgn,includinganintra-uretericbar whichissaid to bepartofthe trigone, vibratome sectionsdidnotreveal additionalmusclegroupsreported analysis ofwhole-mounturogenital tracts,cryosectionsand and themuscleassociatedwith theintramuralureter. Extensive thebladdermuscle(detrusor) muscle typesarepresentinthetrigone: urothelium (Fig.2F,G). Thesefindings suggest thattwo major with thebladdermuscleandterminate inthesubmucosa,below the extend intotheintramuralregion, wherethey appeartointercalate onlyasmallsubsetoflongitudinalureteralfibers Baskin, 2004), asreportedbyothergroups(Yucel and muscle (Fig.2E).However, thick, containingatleastthreelayersofcircularandlongitudinal bladder (Fig.2C,D,G,H).Theureteralwall outsidethebladder is covered byathickmuscularissubmucosa,similar tothatinthe urethra. Itssurface was smoothandfreeoffoldslike theurethrawas trigone appearedatthisstagetobeahybridbetweenthebladder and revealed additionalsmoothmusclelining the intramuralureter. The detrusor andsubmucosa(Fig.2B,E,F).Analysisatadultstages fibers surroundingtheintramuralureterextending throughthe coat surroundingtheextra-vesicular ureterand afew longitudinal bladder) inthetrigonalregion (Fig.2A). lining theintramuralureter(theportionofwithin bladder), but therewas littleifany detectablesmoothmuscle in theextra-vesicular ureters(theportionofureteroutsidethe smooth muscledifferentiation (green)inthebladder, urethraand expression ofsmoothmusclealphaactinrevealed extensive developmental andpost-natalstages.AtE15,analysisfor analyzing itsformationinmouseurogenitaltractsatdifferent we first establishedwhichmusclesarepresentinthetrigoneby forming Bell’s muscle(Fig.1C).To begin toaddressthisquestion out laterallyforminganinter-ureteric ridgeandposteriorly that thetrigoneisformedinlarge partfromureteralfibers thatfan a different mannerthanpreviously thought.Otherstudiessuggest during uretertransposition,hencethetrigoneislikely toformin case becausethecommonnephricductundergoes apoptosis duct. However, ourprevious studiessuggestthatthisisnotthe common nephricductthatisthecaudal-mostsegment of Wolffian originates fromnon-urogenitalsinustissue,inparticularthe and physiologicalpropertieshave ledtothe ideathatthetrigone 1C). Theuniquefeaturesofthetrigoneincludingitsappearance by theureterorifices extending posteriorly to theurethra(Fig. consider thetrigonetobemusculartriangleboundedlaterally The trigonehasbeendefined inanumberofways; here,wewill Development ofthetrigone distinct fromthebladder. smooth, whichhasledtothesuggestionthatitsoriginmightbe the bladder, whichiscovered byfolds,thetrigoneisgenerally shaped ureterorifice openingintotheurothelium(Fig.1F).Unlike lateral edges(Fig.1E).Highermagnification reveals theeyelet- bladder muscleandsubmucosaopenintothetrigoneatits 1D, theurotheliumisred).Theintramuralureterspassthrough transitional epitheliumthatprevents leakageanddamage(Fig. like thebladder, iscovered bytheurothelium,aspecialized urethra begins (Fig.1D,E).Thesurface oftheurethra andureters, the ureterslaterally, terminating atthebladderneckwhere urogenital tractsasasmoothtriangularshapedregion boundedby (Fig. 1A,C,D).Thetrigonecanbevisualizedindissected trigone atthebaseofbladderbetweenandurethra thick layerofmusclecalledthedetrusoranduretersenter In newborn mouseurogenitaltracts,thebladderis encircledbya RESULTS Analysis ofurogenitaltractsatP0revealed athicksmoothmuscle Development 134(20)

DEVELOPMENT (green) and smoothurothelial surface (red). Magnification: longitudinal fibers thatintercalate withthe bladder muscle(yellowarrows). ( longitudinal musclefibers(green). ( muscle layersbelow. ( trigone inanadultmouse.( intramural ureter crossing thebladdermuscleandsubmucosa.Notelongitudinal fiberssurrounding theintramuralure intramural ureter compared withtheextra-muralureter, whichalready has athicksmoothcoat.( (red) toreveal theurothelium, andsmoothmusclealphaactin(green) to reveal smoothmuscle.Notetheabsenceofmusclesurro revealed few, ifany, differences. Theureterisensheathedinathin the ureter(Fig.3A,C).Analysisofmousetrigoneatsimilarstages distinct fromthethinlongitudinalsmoothmusclefibers thatsurround of thebladdermuscle,whichisorganized inbundles, was seentobe bladder muscleandintothesubmucosa(Fig.3A).Themorphology smooth musclealphaactinrevealed theureterpassingthrough Sections throughthetrigoneofa22-weekhumanfetusstainedfor address thisquestion,wecomparedthetrigoneinhumanandmouse. differences inthestructureofmouseandhumantrigone.To differently thanpreviously thought,orthattherearesubstantial associated withthetrigonesuggeststhateitherisformed The failure toidentifystructuresinthemousethoughtbe The trigoneisevolutionarilyconserved trigone apex (Tanagho etal.,1968). which issaidtoextend caudallyfromtheureterorifices tothe to extend laterallybetweenthetwo ureterorifices, andBell’s muscle Development ofthebladdertrigone Fig. 2.Developmentofthetrigone. E ) Highermagnificationoftheureteral tunnelshowninB.( D h lde fanwonmuesoigtede od ofthelining,andmuscularismucosasmooth mouseshowing thedeepfolds ) Thebladderofanewborn G ) Highermagnificationoftheregion inCshowingthepositiontrigonewhere the ureter joins.Notethe ( A ) Brightfield/darkfieldcompositeshowingafrontal sectionthrough anE15embryostainedforuroplakin ϫ 50 inA-C; ϫ 100 inD,E,G,H; 1964). However, given thecomplexity ofthe trigonalregion itisnot trigone (Roshanietal.,1996;Tanagho etal.,1968;Woodburne, Ureteral muscleisthoughttomake amajorcontribution tothe muscle Lineage analysisreveals theoriginoftrigonal formed primarilyfromtheureterandbladdermuscle. develops inasimilarmannerbothspecies,andislikely tobe muscle arrangementtothatinhumansuggeststhetrigone observation thatthemousetrigonedisplayssimilarmorphologyand through themousetrigoneatacomparablelevel (Fig.3C,D).The are mostlikely ureteralsmoothmuscle,similartothatinthesection human trigoneissurroundedbyathinlayeroflongitudinalfibers that extensive similarityacrossspecies. Theintramuralureterinthe sections throughtheureterasitpassesbladderrevealed surrounded byanddistinctfromthebladdermuscle(Fig.3B).Cross- layer oflongitudinalsmoothmuscleoneortwo celllayersthick, H ) Theurethra mouseshowingthethick musclecoat inanewborn F ) High-magnificationimageofthe intramuralureter showingthe uroplakin). Magnification: shape atthepointitopensintourothelium (red, magnification oftheureter orifice,showingitseyelet (yellow) are locatedatthebaseoftrigone.( trigone inanadult reveal smoothmuscle.( urothelium, andforsmoothmusclealphaactin(green) to an adultmousestainedforuroplakin (red) toreveal the apex toformBell’s muscle.( form theinter-ureteric ridgeandextenddowntoward the bladder viaWaldeyer’s sheath,fanoutacross thebaseto is thoughttobecomposedofureteral fibersthatenterthe ureter. ( ( prevent back-flowofurinetotheureters andkidneys. intramural ureter segmentthatisnormallycompressed to base anditsconnectionswiththeureters showingthe mechanism. Fig. 1.Thetrigoneisthesiteofanti-reflux B ϫ ) Schematicshowingcompression oftheintramural 200 inF. C B ) Adetailedrepresentation ofthetrigone,which ) The trigone in a newborn mouseshowing the ) Thetrigoneinanewborn ( A ). Schematicofthetrigoneatbladder Hoxb7-Gfp E ) Openedbladdershowingthe RESEARCH ARTICLE ϫ D 100 inD,E; ) Avibratomesectionfrom mouse. Theureter orifices ϫ unding the ter. ( 200 inF. C ) The F ) High 3765

DEVELOPMENT ureter. Magnification: point tothelongitudinalmusclefibersassociatedwithintramural urothelium andsmoothmusclealphaactin(green). Theyellowarrows the intramuralureter, stainedforuroplakin (red) toreveal the ( path oftheureter anditssurrounding thinlayeroffibers(blackarrows). ureter. ( the longitudinalureteral musclefibersthatencircle theintramural the urothelium stainedforuroplakin (red). The yellowarrows pointto showing thetrigonestainedforsmoothmusclealphaactin(green) and the intramuralmusclefibers.( stained forsmoothmusclealphaactin(brown). Blackarrows pointto section through thehumantrigoneatlevelofintramuralureter bladder muscle andsubmucosa(Fig.4D,E). Despitethelarge longitudinal fibers surrounding theureterthatextended intothe the trigonalregion, careful analysisrevealed but notinthebladderorurethra(Fig.4C).In cells observed atE14, which weremostlikely descendents ofthelabeledmesenchymal extra-vesicular ureter coatinbothcircularandlongitudinalfibers, At birth, smooth muscleprogenitorsinthebladderandtrigone(Fig.4A,B). expression inmesenchymalcellsaroundtheureters,but notin contribution ofureteralmuscletothetrigone. Cre activated incellsexpressing boththe ( with mesenchyme (Kobayashi etal.,2005), duct, inmesenchymalcelltypeswithinthekidney andinureteral recombinase inmesonephricmesenchymesurroundingthenephric Cre whether theirdescendentspopulatethetrigone.We crossed mesenchymal cellsatlatestagesofdevelopment todetermine recombination system.We thenfollowed thefate ofureteral labeling smoothmuscleprogenitorsintheureterusingCre-lox To addressthisquestion,weperformedlineagestudiespermanently possible todeterminewhetherthisisthecasebyvisualinspection. Fig. 3.Comparisonofthetrigoneinhumansandmice. 3766 D R26RlacZ ). Sectionthrough themousetrigoneatbirthshowingpathof Analysis of transgene andintheirdescendents,enablingustodeterminethe mice (Kobayashi etal.,2005),whichexpress theCre C RESEARCH ARTICLE ) Sectionthrough ahumantrigoneshowingtheintramural lacZ ) mice(Soriano,1999). Rarb2-Cre;R26RlacZ expression persistedin smooth musclecellsinthe ϫ 20. B ) Asectionthroughmouse anewborn lacZ Rosa26 embryos atE14revealed expression ispermanently reporter andthe Rosa26 lacZ lacZ activity inthe ( A ) A reporter Rarb2- Rarb2- lacZ trigone. wd;Wolffian duct.Magnification: E, showingtheureteral muscleembeddedinbladderthe smooth muscle.( of asectionserialtoD,showingthatthe surrounding theintramuralureter. ( ureter. ( expressing smoothmusclecellsliningtheextra-muralandintramural of anewborn and urethra. ( through a Fig. 4.Ureteral fiberscontributetothetrigone. ureteral fibers. To assessthe contribution ofbladder muscletothe the detrusor muscleofthebladder andlongitudinal trigonal region: Histological studiessuggestthat two musclegroupsresideinthe bladder muscle The trigoneisformedpredominantly from ureteral fibers thatismuch morelimitedthanpreviously thought. predominantly frombladdermuscle,withacontribution from differences. Thesefindings suggestthatthetrigoneisformed extensive contribution fromureteralfibers isnotduetointerspecies differences (Fig.4E,F),suggestingthatthefailure toidentifyamore in themouseandhumantrigoneatthisstagerevealed few, ifany, Mercier’s bar(Fig.4C,D).Comparisonofthedistribution ofmuscle extending toward theurethra,whichhave beenpostulatedtoform generate theinter-ureteric bar, norintotheposteriortrigone which have beenpostulatedto extending furtherintothetrigone, amount ofmuscleinthisregion, wedidnotobserve ureteralfibers panels). Notetheabsenceof mesenchymal cellssurrounding theureter (yellowarrowheads inall D ) Asectionthrough thetrigoneshowing Rarb2-Cre;R26RlacZ B Rarb2-Cre;R26RlacZ ) Highermagnificationofaregion ofA.( F ). Sectionthrough ahumanfetusatthesamelevelas lacZ embryo atE14showing -expressing cellsinthebladder, trigone urogenital tractshowing E ) Smoothmuscleuroplakin staining lacZ ϫ 100 inA; activity inDcorresponds to Development 134(20) lacZ ( A -expressing cells ϫ ) Sagittalsection lacZ C 200 inB-F. ) Whole-mount -expressing lacZ -

DEVELOPMENT muscle progenitorsbycrossing trigone, wepermanentlylabeledbladderandurethralmesenchymal Development ofthebladdertrigone and urethra,but therewerefew ifany birth, expression was throughoutthemuscleinbladder, trigone in theuretersorWolffian ducts(Fig.5Aanddatanotshown). By thetrigoneandurethra,but not mesenchymal cellsinthebladder, Cre;R26RlacZ (Kuhbandner etal.,2000)(Fig.5).Beginning atE12, urogenital sinusmesenchymebut notinureteralmesenchyme Sm22-Cre region (Fig.5B,C).Thedistribution of cells intheureter, includingtheintramuralureterintrigonal agenesis resultsinanabnormallyshapedipsolateralhemitrigone. important forformationofthetrigoneisobservation thatureter One pieceofevidence supportingtheideathatureteralmuscleis ureteral fibersintercalate withbladdermuscle Ureters enterthetrigone through atunneland formed mainlyfrombladdermuscle. Hence, ureteralfibers make acontribution tothetrigone,whichis portion ofthetrigonein embedded inthebladderwall, correspondingtotheunlabeled ureteralmusclewas clearlypresent, smooth muscleconfiguration: sections fromhumantrigonerevealed remarkablesimilarityinthe correspond toureteralmuscle(Fig.5D,E).Comparisonwith the ureteraljunction,andthattheseunlabeledcellsarelikely to there isindeedmusclepresentinthislateralportionofthetrigoneat smooth musclealphaactininwild-typeembryos.Thisrevealed that region of Sm22-Cre;R26RlacZ mouse lineinwhichtheCrerecombinaseisexpressed in embryos displayedextensive Sm22Cre;R26RlacZ mice was comparedwiththatof R26RlacZ lacZ lacZ -labeled smoothmuscle activity inthetrigonal reporter micewitha mouse (Fig.5D-F). lacZ activity in Sm22- ureter in question, weanalyzedtrigoneformationintheabsenceof urogenital sinus-derived fibers. (Fig.6A).To further address this ureter throughthebladderandintercalationofureteral the trigonalstructuremightbeformedfromthispathway ofthe muscle exclusively atitslateraledges. Thesefindings suggestthat terminate inthetrigoneandintersectwithureteralbladder bladder wall inparallelwithbloodvessels. Ureteralmusclefibers E18 embryosrevealed thattheureterpassesthroughatunnelin Analysis ofmuscledifferentiation insagittalsectionsofwild-type musculature (Waldeyer, 1892)(reviewed byHutch,1972). the trigoneisencasedinasheaththatformedfromureteral itself and,accordingtotheliterature,ureteralpassageway to Ureteral muscleisthoughttocontribute extensively tothetrigone consistent withtherequirementforuretertomaintainraised bladder muscleoccursonlyatthelateralsidesoftrigone is bladder/trigone. Theobservation thatintercalationofureteraland indicates thatitisalmostcertainlyderived fromthe The presenceoftheureteraltunnelinabsenceureters contained onlybloodvessels owing tothe absenceoftheureter. urothelium. In blood vessels thatpassthroughthemuscleandsubmucosa intothe wild-type mice,thetunnelcontainedintramuralureterand bladder wall, whichprobablycorrespondstotheureteraltunnel.In mutants andinwild-typelittermates,agapwas presentinthe completely encirclethebladderneck.Interestingly, bothin mutant shown (Fig.6B)containsbladdermusclethatappearedto to agenesisofthecaudalWolffian duct.Thetrigoneinthe urogenital sinusdifferentiation but lackuretersandkidneys owing section through anE17 ϫ both wildtype(A)andmutant(B). det,detrusor. Magnification: muscle, andthetunnelthrough thebladder(red arrow) present in absence oftheureter. Note the abundantbladderandurethral shown inA,showingthestructure ofthetrigoneregion inthe arrows). ( the ureteral longitudinalfibers jointhebladderdetrusor(yellow intercalation ofureteral andbladdermuscle. Fig. 6.Thestructure ofthetrigoneislikelytodependon 100. Magnification: ureter through thebladdermuscleoftrigone. level ofahumanembryoshowingthepathintramural the actin toreveal muscleoftheintramuralureter, unlabeledby the samesampleasinD,stainedforsmoothmusclealpha the ureter inan transgene. ( muscle thatisextensivelylabeledbythe lacZ R26RlacZ urethra muscle.( mesenchyme thathavedifferentiated inthebladderand mouse showingdescendentsoftheurogenital sinus the bladderandurethra ofanadult but notintheureter orWolffian duct.( are visibleinthebladder, urethra andtrigone(whitearrow), R26RlacZ bladder muscle. Fig. 5.Thetrigoneisformedpredominantly from Pax2 B ) Asagittalsectionthrough a Sm22-Cre -expressing cells,anditspaththrough thebladder Pax2 mutants, whichdisplayapparentlynormal mouse showingtheureter, whichhasfewifany embryo atE14. D mutants, thetunnelwas alsopresent,but ) Asectionthrough theintramuralportionof transgene. ( ϫ Sm22-Cre-R26RlacZ Pax2 C 100 inA-C; ( ) Sectionthrough anadult A ) Asagittalsectionthrough a +/+ embryo showingthepointatwhich lacZ F RESEARCH ARTICLE ) Sectionthrough acomparable ϫ -expressing mesenchymalcells 200 inD-F. Pax2 Sm22-Cre-R26RlacZ adult. ( –/– B Sm22-Cre littermate ofthat ) Sectionthrough ( A E ) Asectionfrom Sm22-Cre- ) Asagittal Sm22-Cre- 3767 Pax2 Pax2

DEVELOPMENT Haraguchi etal., 2007).However, themorphologyofthese tissues the tailbud orcloacal mesoderm(Brenner-Anantharam etal.,2007; muscle progenitorsliningtheureter andurogenitalsinusderive from Recent studiesindicatethatmost, ifnotall,ofthemesenchymal tract alongtheurinaryoutflow Distinct patterning of theureter(Liangetal.,2005). indistinguishable fromthatofthebladder, but isdistinctfromthat demonstrating thattheurothelialcovering of thetrigoneis bladder (theurogenitalsinus)isconsistentwithstudies trigone isformedfromthesameprimordialtissueasrestof the as suggestedbyothers(Woodburne, 1964).Theobservation that the areinfact derived fromthebladder (Fig.7), formed fromtheureter, whichhave beenconsideredtobe Mercier’s barandBell’s muscle, mechanism. Thesestudiesalsosuggestthatmusclessuch as trigone formationandformaintainingtheureteralanti-reflux intercalation ofureteralandbladdermuscleislikely tobecrucial for is muchmorelimitedthanpreviously thought(Fig.7B).The contribution fromureterallongitudinalfibers atthelateraledgesthat trigone isformedpredominantlyfrombladdermuscle,witha fate ofureteralandbladdermuscleprogenitorsfind thatthe et al.,2005).Here,usingCre-loxrecombination,wefollowed the instead undergoes apoptosisduringuretertransposition(Batourina not contribute toany partofthebladder, trigoneorurethra,but formation. We have establishedthatthecommonnephricductdoes current lineagestudysuggestanalternatemodelofurinarytract other musculartissuesintheembryo.Ourprevious findings andthe trigonal muscleislikely tobederived frommesenchyme,asare epithelial tube,anextension oftheWolffian duct,whereas the the trigonethistissuewould form,asthecommonnephricductisan orifices inthebladderneck.However, itisunclearwhichportionof and expands duringuretertransposition,repositioning theureter segment, isthoughttocontribute tothetrigone asitdifferentiates common nephricduct,whichisthemostcaudalWolffian duct ureter (Brooks,2002;Tanagho etal.,1968;Wesson, 1925).The bladder viaatunnel(Waldeyer’s sheathorspace)derived fromthe form Bell’s muscle(Fig.7).Theureters aresaidtopenetratethe base toformtheureteralridge,andalsotoward thetrigonebaseto derived predominantlyfromureteralmusclethatstretchesacrossthe According totheliterature,structureoftrigoneiscomplex, Rethinking urogenital tractformation DISCUSSION deformation ofthetrigone. explaining whytheabsenceofipsolateralureterresultsin triangular structurenormallyassociatedwiththetrigone, 3768 RESEARCH ARTICLE formation and, bydefault, ureteralvalve function, dependon muscular backing.Theobservations fromourstudiesthattrigone length oftheintramuralsegment isincreasedandhasimproved insertion siteandreinsertedin thetrigoneinsuchaway thatthe reflux, wherebytherefluxingureter isdetachedfromitsoriginal al., 2002). transport orperistalsis(Airiketal.,2006;Chang2004;Yu et differentiation, canalsoresultinrefluxowing tofaulty urine Intrinsic ureteralabnormalities,suchasafailure in muscle normally separatesfromtheWolffian duct(Batourinaetal.,2005). abnormalities inuretertransposition,atthetimewhen 2000; Luetal.,2007;MiyazakiYu etal.,2004),orby 2005; Batourinaetal.,Grieshammer2004;Kume etal., Stephens, 1983)asseeninseveral mouse models(Bassonetal., than normal(MackieandStephens,1975;Popeetal.,1999; bud fromtheWolffian ductfromalocationmorecranial orcaudal the trigoneabnormally, canbecausedbysproutingoftheureteric orifice (reviewed byRadmayr, 2005). al., 1996)andinnervation thatregulates openingoftheureteral stereotypical positioninthetrigone(Kingetal.,1974;Stephens through thebladderandinsertionofureterorifice ata including sufficient lengthoftheintramuralsegment, itspathway terminal ureteralsegment isthoughttodependonseveral factors, smooth musclebladderwall. Theabilitytoeffectively compressthis ureters andkidney bycompressingtheintramuralureter againstthe mechanism, whichnormallyprevents back-flow ofurinetothe isthoughttobeimportantforfunctionoftheanti-reflux The pathway taken bytheureterthroughbladdermuscleand Application ofthisnewmodeltohumandisease also leadtotrigoneabnormalities. in normaltrigonedevelopment andwhethermutationsinthesegenes al., 2002).Futurestudieswilldeterminetheroleofthesepathways al., 2007;Raatikainen-Ahokaset2000;Scott2005;Yu et (Airik etal.,2006;Brenner-Anantharam etal.,2007;Haraguchi and includingHoxgenes, expressed signalingmolecules, embryological origin,ashasbeensuggested,but onspatially edges. Itscellularmorphologyislikely todependnotonits interaction betweenbladderandureteralmusclefibers atitslateral trigone issmoothandhasadistinctive shapeprobablygeneratedby composed ofdeepfoldsthatenablecontractionandexpansion. The layer ofsmoothmuscle,amuscularismucosaandsurface mediate myogenicperistalsis.Thebladderissurroundedbyathick is diverse. Uretersareensheathedby3-4layersofmusclethat The trigoneisthesiteatwhich surgery isperformedtocorrect A shorteningoftheintramuralsegment, orureterorifices joining Shh , thatarecrucialforpatterningotherurinarytracttissues around theureter thatfunctionsasatunnel. the structure derivedfrom bladdermuscleandthespace contribution from ureteral fibers(green) andthebulkof ( been considered toformindependentlyofthebladder. ureteric ridgeandBell’s muscle.Notethatthetrigonehas fibers thatfanoutacross thesurfacegeneratinginter- with theureters (green), formedinlargepartfrom ureteral trigone formation,showingthetobecontinuous i.7 oesoftrigoneformation. Fig. 7.Models B ) Current modeloftrigoneformation,showingasmall Development 134(20) ( A ) Oldmodelof Bmp4 , Tbx18

DEVELOPMENT Airik, R.,Bussen,M.,Singh,M.K.,Petry, M.andKispert,A. References to C.M.andHD30284R.R.B. uroplakin antibody. Thisworkwassupportedbygrantsfrom NIH:DK061459 University) forartwork;andDrT. T. Sun(NYUniversity)forthekindgiftof We thankChristopherChoifortechnicalassistance;NancyHeim(Columbia is paramountforrenalfunction. This willfurtherourunderstandingoftheanti-refluxmechanismthat better connectionswithunderlyingbladdermuscleandthetrigone. reimplantation ofuretersmightalsoinadvertently helpestablish addition toincreasingthelengthofintramuralureter, intercalation ofureteralfibers withbladdermuscle,suggestthatin Development ofthebladdertrigone Batourina, E.,Tsai, S.,Lambert,Sprenkle, P., R.,Dutta,S.,Hensle, Viana, Basson, M.A.,Akbulut,S.,Watson-Johnson, J.,Simon,R.,Carroll, T. J., Kuhbandner, S.,Brummer, S.,Metzger, D.,Chambon,P., Hofmann,F. and Kong, X.T., Deng,F. M.,Hu,P., Liang,F. X.,Zhou, G., Auerbach,A.B., Kobayashi, A.,Kwan,K.M.,Carroll, T. J.,McMahon,A.P., Mendelsohn,C.L. 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