The Left-Right Axis in the Mouse

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Hidetaka ShiratoriandHiroshi Hamada* The left-rightaxisinthemouse:fromorigintomorphology Development 133,2095-2104(2006)doi:10.1242/dev.02384 *Author forcorrespondence (e-mail:[email protected]) Yamada-oka, Suita,Osaka565-0871,Japan. University, andCREST, JapanScienceandTechnology Corporation(JST),1-3 Developmental GeneticsGroup, GraduateSchoolforFrontier Biosciences,Osaka understood. of theasymmetricsignalintomorphology–isalsolittle lateral plate.Thefinalstepinleft-rightpatterning–translation asymmetric signalgeneratedinthenodeistransferredto it isnotyetknownhowthisflowfunctionsorthe be thesymmetry-breakingevent,atleastinmouseembryo, extra-embryonic fluidinthenodecavity(nodalflow)islikelyto this processremainunanswered.Althoughaleftwardflowof vertebrate embryos.However, manyimportantquestionsabout understanding ofhowleft-right(LR)asymmetryisgeneratedin The pastdecadeorsohasseenrapidprogressinour Fig. 2D)of~15to20 node cavity occursataspeed(visualizedwithfluorescent beads,see al., 1998).Thisleftward laminarflow ofextra-embryonic fluidinthe and Fig.2A).Thisfluidflow isreferredtoasnodalflow (Nonakaet anterior tipoftheprimitive streakinmouse embryos,seeFig.1B node isanembryonicstructurethatlocatedatthemidline, the by theunidirectionalflow ofextra-embryonic fluidinthenode(the mouse, thebreakingofLRsymmetryismostlikely tobe achieved asymmetry inothervertebrates (seeTabin, 2005),atleastinthe Although thereissomecontroversy concerningtheinitiationofLR Leftward fluidflowbreaks LRsymmetry vertebrates, seeLevin andTabin (Levin, 2005;Tabin, 2005).] differences inLRpatterningbetweenthemouseandother specifically mentioned.[For recentreviews ofthesimilaritiesand discuss findingfromstudiesinothervertebrates, except where particular, wefocusongeneticdatafromthemouse;donot mechanism ofleft-right(LR)patterningduringdevelopment. In thatdevelop leftside-specificmorphologies. as thelungandheart, receive Nodalsignalingcontribute tovarious visceralorgans, such Nodal results inthesecondstep:asymmetricexpression of thegene mesenchyme ofvarious visceralorgans.) Thetransferofthissignal somite-stage mouseembryoandlatercontributes tothe (LPM). (Thismesodermislocatedinthelateralregion of theearly- preferentially towards theleftsideoflateral platemesoderm an asymmetricsignal(s)thatisgeneratedinthenodetransferred embryonic day(E)7.5ofmousedevelopment (Fig.1).Inthisstep, symmetry-breaking event thattakes placeinthenodearound left-right (LR)patterninginthemouse.Thefirststepis There aretwo key stepsthatcontribute totheearlyestablishmentof Introduction in thesamedirection(clockwise, asviewed fromtheventral side)at node cavity (Suliketal., 1994)(Fig.2C).These200-300ciliarotate protrude fromcellslocatedontheventral sideofthenodeinto of microtubules but thatlackapairofcentral microtubles),which movement of9+0monocilia(theseareciliathat have ninedoublets In thisreview, wediscussourcurrentunderstandingofthe in theleftLPM(seeFig.1A,B).Cellsthat ␮ m/second andisgeneratedbytherotational cilia protrudevertically fromasurface. However, iftheciliaare rotational movement ofciliawould generateavortex only ifthe et al.,2005).Hydrodynamic principlesdictatethatasimple to lieintherotationangleof cilia(Okadaetal.,2005;Nonaka they generateaunidirectional flow? all thenodeciliarotateinsame clockwisedirection,how can into thegenerationofleftward flow inthenode?Furthermore,if is now known tobeequivalent to:how issuchinformationtranslated AP andDV informationtranslatedintoLR polarity?Thisquestion LR axismustderive fromAPandDV axisinformation.Buthow is conceptual F-moleculemodel(Brown etal.,1991),theoriginof and mediolateralaxes. AsBrown andWolpert proposedintheir existing positionalcues:theanteroposterior(AP),dorsoventral (DV) The mechanismofsymmetrybreakingmustmake useof thepre- The originofLRpolarity tested. Zucker etal.,2005),but theroleofflow hasnotbeendirectly patterning (Essneretal.,2005;Kawakami etal.,2005;Kramer- mutants thatlacktheciliainKufpper’s vesicle show impairedLR are motileandgenerateaunidirectionalflow inthevesicle. Zebrafish 2002; Essneretal.,2005;Kramer-Zucker etal.,2005).Thesecilia the embryonicorganizer equivalent tothemousenode(Essneretal., zebrafish, forexample, ciliahave beendetectedinKupffer’s vesicle, mechanism mayoperateinothervertebrates (Okadaetal., 2005).In LR patterningevents. Recentevidence suggeststhatasimilar et al.,2002),demonstratingthattheflow persedirectssubsequent artificial flow leadstothereversal ofLRpatterninginmice(Nonaka Nonaka etal.,1998). and Anderson,2005;Huangfuetal.,2003;Murcia2000; Informatics) andKif3proteins(Garcia-Garciaetal.,2005;Huangfu Genome Informatics),wimple(Wim; Ift172–MouseGenome intraflagellar transportproteins,suchaspolaris(Ift88–Mouse barrier. Thebestexamples ofsuchmutantsarethosethatlack isomerism) becausethey alsohave functionaldefectsinthemidline phenotypes (typically, bilateralleftsidedness,known asleft However, mostmousemutantsthatlacknodeciliaexhibit complex possesses immotilecilia(Suppetal.,1999;Supp1997). exemplified bythe appears tobetherandomizationofLRorientation,asisbest al., 1999).Thedirectconsequenceoftheabsencenodalflow of nodeciliaarereportedtohave abnormalLRpatterning(Okadaet mutants thatlacknormalflow asaresultoftheabsence orimmotility be immotile(Okadaetal.,2005). the notochordalplate(Nonakaetal.,1998),but they arereportedto expression ontheleftsideofLPM.Monociliaarealsopresentin Nodal flow maythereforeoccurspecificallyto initiate LPM atthetwo-somite stageanddisappearsbythesix-somitestage. somite stage.Theasymmetricexpression of two-somite stage,persistsforseveral hoursandendsbythesix- only ashortperiodoftime.Itis,thus,firstapparentattheone-to a speedof600rpm(Nonakaetal.,1998).Nodalflow takes placefor The key totheanswer tothislastquestionwas recentlyshown Reversal ofthedirectionnodalflow bytheimpositionofan Nodal flow isessentialforLRdetermination.Various mouse iv/iv (inversus viscerum)mousemutant, which Nodal REVIEW begins inthe Nodal 2095

DEVELOPMENT (National Institute forBasicBiology, Japan). (arrow). Scalebar:10 visualize fluidflow. Thebeadsmove towards theleftsideofnode arrow indicatesthedirection ofnodalflow. Scalebar:10 Anteroposterior (AP)andleft-right (LR)orientationsare indicated.Red magnification. Scalebar:1 100 ( 2096 node cilia. Fig. 2.Leftward fluidflowgeneratedbyrotational movementof to thesurface, themovement offluidnearthesurface willbe al., 2005;Okadaet2005).Thus,whenaciliummoves closer generate aunidirectionalflow (Cartwrightetal.,2004;Nonaka tilted towards aspecificdirection,itispossible forthemto line). Itisalsopossiblethattheperinodal(crown) cellsrespond tothechemicaldeterminantandsendasignalviared r line). Alternatively, cellsinornearthenodemaysensemechanicalstress andsendanunknownsignal(s)totheleftLPMt is secreted intothenodecavityandtransportedtowards theleftside,where itssignalmaybetransducedbytheendodermand (pink line)orthrough thePAM (red line).According to thedeterminant-transportingmodel,anunknownmoleculeproduced inthe Asymmetric signal(s)generatedbytheleftward flowinthenode(blackwavylines)mightbetransferred totheleftLPM,either paraxial mesoderm(PAM), LPM,endodermandprospective floorplateare shown.Arrows indicatehowsignalsare transferred during expression inthenodeandleftLPM.( during development,from earlierembryonic(E7.5)stagestolaterones(E10-11.5).( ofthelateralplatemesoderm(LPM)andasymmetricorganogenesis.Theblackarrowin thenode,patterning onleftrepre inmouseembryo. Fig. 1.Mechanismsofleft-right(LR)patterning D Ventral viewofthenodecavity, withfluorescent beadsusedto ) A ␮ m. ( E10.5 E11.5 REVIEW E9.5 E8.5 E7.5 B ) Monociliainthenodeofamouse embryoshownathigh ( A ) Lateralviewofamouseembryoat8.0dpc.Scalebar: Symmetry breakingatthenode Situs-specific organogenesis by Nodal and Leftyproteins ␮ m. ImageinDcourtesyofShigenori Nonaka Patterning ofLPM ␮ m. ( C ) Ventral viewofthenodecavity. C ) Atransversesectiontakenatthelevelindicatedbyred linesinB.Thelocationofpitcells,crown cells, ␮ m. LR B LPM ( A ) Three stepsinthegenerationofLRasymmetry:asymmetry-breaking event Node may determine the positioningofciliaaccording toapicobasal ear (KleinandMlodzik,2005).Similarly, DV positionalinformation in responsible forthecoordinated localization andorientationofhairs scenario issimilartotheplanar cellpolarity(PCP)pathway thatis may explain thetiltof theciliatoward theposteriorside.This membrane andtheposteriorlyshifted localizationofthebasalbody spherical. Assuch,theprotrusionofciliaperpendicularto cell posteriorly withinthecell(Fig.3).Nodepitcellsareroughly 1C andFig.3).Thebasalbodyofeachciliumisalsopositioned (cells ontheventral surface ofthenodethathave monocilia,seeFig. most ofthemprotrudefromtheposteriorregion ofnodepitcells node cilia(Nonakaetal.,2005;Okada2005)hasrevealed that cilium inaposteriorlytiltedmanner?Acarefulexamination ofthe proposed byBrown andWolpert (Brown etal.,1991). The nodeciliumthuscorrespondstothehypotheticalF-molecule axis isthus,indeed,dependentonAPandDV positionalinformation. axis isrepresentedbytheirventral protrusion.TheoriginoftheLR 3). TheAPaxisinfluencesthetiltofnodecilia,whereasDV AP andDV axes, andbytheunidirectionality ofciliaryrotation(Fig. (Nonaka etal.,2005;Okada2005). cilia areindeedtiltedposteriorly, atanaverage angleof30° High-speed microscopicobservations have revealed thatthenode generated ifrotatingciliaaretiltedtowards theposteriorside. Hydrodynamics thereforepredictthataleftward flow canbe and towards theleftwhenthey arefar fromthesurface (seeFig.3). be moving towards therightwhenthey comeclosetothesurface effectively. Ifciliaaretiltedtoward theposteriorside,they would surface, itinducesthemovement oftheneighboringfluidmore (Liron, 1996)].Conversely, whenaciliummoves away fromthe prevent thefluidthatoverlies itfrombeingdraggedbythecilia move; owing toitsviscousforce, thisstaticfluidlayerwould [according tothiseffect, fluidcontactingasolidwall willnot restricted asaresultoftheso-calledno-slipboundaryeffect How doesAPinformationdictatethepositioningofeachnode The directionofnodalflow, then,isdeterminedbythepre-existing Drosophila B PosteriorviewofamouseembryoatE8.0showing ) cuticles andofsensoryhairsin thevertebrate inner L C Endoderm LPM LPM Prospective floorplate oute. Crown cells horough thePAM (red PAM cells Development 133(11) through theendoderm Pit finally bytheLPM(pink sents atimecourse node/perinodalcells LRpatterning. Nodal R

DEVELOPMENT Modified, withpermission, from Nonakaet al.(Nonakaetal.,1998). generation andsensing ofmechanicalstress byleftward flowatcilia. and theunidirectionality ofciliaryrotation. combination ofdorsoventralinformation,anteroposterior information instead ofavortex.LRasymmetryisthusformeddenovothrough a posterior tiltoftheciliaryrotation axisgeneratesaleftwards flow rotates inaclockwisedirection whenviewedfrom theventralside.The the basalbody(green) isposteriorlyshiftedwithineachcell.Eachcilium cilium isalsoposteriorlytilted,mostprobably becausethepositionof cell (lightblue)towards theventralsideofmouseembryo.The embryo are illustrated.Eachcilium(red bar)protrudes from anodepit sources ofpositionalinformation. Fig. 3.Denovogenerationofleft-right(LR)asymmetryviathree Development 133(11) ( Fig. 4.Two modelsforthemechanismofaction ofnodalflow. Indeed, recentevidence (Tanaka etal.,2005)indicatesthatnodepit into thenodecavity, itwould bereadilytransportedbythefluidflow. (such aspitcellslocatedinthenodecavity orperinodalcrown cells) flow towards theleftside.Ifmoleculeissecretedbynodecells molecule thatactsastheLRdeterminantistransportedbynodal et al.(Nonakaal.,1998).Accordingtothismodel(Fig.4A),a This isanobvious possibilitythatwas initiallyproposedbyNonaka Does nodalflowtransportadeterminant? (Fig. 4). models have beenproposed,but there aretwo principalhypotheses which itachieves thiseffect hasbeensubjecttodebate.Several during development, atleastinthemouse,but themechanismby Nodal flow istheevent thatmostprobablybreaksLRsymmetry How doesfluidflowbreak LRsymmetry? in thenodepitcells. to PCPoperatescorrectlypositionthebasalbodyofeachcilium polarity oftheepithelium.Itappearslikely thatamechanismsimilar A A ) Thetransportationofaleft-right (LR)determinantor( Determinant molecule Determinant neirPosterior Anterior Left Generation ofleftside-specificcharacter signal,intra- orinter-cells Left-determinant Cilium Nodal flow molecule Determinant Left Ventral Dorsal Node pitcellsofE8.0mouse B Mechanicalstress B ) the Left necessary toclarifytheprecise site ofFgf8function. Nodal signal,forexample. Thesite-specificablationof LPM tomaintainthecompetenceofrespond the a roleinLRpatterning.Fgf8maythusfunctionthenodeor the 2005), but itremainsuncertainexactly whenandwhereFgf8plays Fgf8 maybenecessaryforthesecretionofNVPs(Tanaka etal., left side.ExperimentswithanFgfinhibitor(SU5402)suggest that Superficially, Fgf8appearstoberequiredfordeterminationofthe LPM andexhibit rightisomerism(Meyers and Martin,1999). Mice conditionallydeficientin candidate fortheLRdeterminantthatistransportedbynodalflow. et al.,2005;Vermot andPourquie,2005). symmetry duringsomiteformation(Kawakami etal.,2005;Vermot at thenode.Instead,RAisrequiredformaintenanceofbilateral contained inNVPs(Tanaka etal.,2005),regulates theLRdecision et al.,2005).Itis,thus,unlikely thatRA,whichwas shown tobe plate; thatis,asymmetric expressed nearthenode,exhibit normalLRpatterninginthelateral mice thatlackRALDH2,theonlyRA-synthesizingenzyme detected intheperinodalregion (Vermot etal.,2005).However, revealed byexpression ofanRA-responsive transgene,hasalsobeen retinaldehyde dehydrogenase2(RALDH2).RAsignaling,as involved inLRdetermination. of afunctionalmidline,includingthenode,but thatitisnotdirectly suggest that,inthemouse,Hhsignalingisrequiredforformation in normalmouseembryos(Zhangetal.,2001).Theseobservations genes, suchas al., 2000).Moreover, anexamination oftheexpression ofHhtarget required forasymmetric be causedbytheassociatedlackof 2001). However, theLRdefectsoftheseanimalsaremostlikely to asymmetric ( Ihh, similartosmoothened (Ihh), isalsoimplicatedinLRpatterning.MicelackingbothShhand the midline).AnotherHedgehogfamily protein,Indianhedgehog midline abnormality(seebelow foradiscussionof thefunctionof develop bilateral midline, includingthenode,and other vertebrates, includingthemouse.Shhisexpressed inthe LR determinationinavian species(Levin etal.,1995)but notin should resultinthelackof determinant shouldbeproducedinornearthenode,anditsloss required criteria.Accordingtothesecriteria,acandidatefortheLR ( proposed fortheputative LRdeterminant,suchassonichedgehog determinant signalisunknown. Several candidateshave been Furthermore, theidentityofcellsonleftsidethatreceive the molecular determinantofLRpolarityremainstobeestablished, into thenodecavity. However, theidentityofsuchatransported that appeartocontainHedgehog(Hh)proteinandretinoicacid(RA) cells actively secretevesicles, callednodalvesicular parcels(NVPs), node cells,either pitcellsorcrown cells(Fig.4B)[crown cellsare determinant isthatitgenerates mechanicalstressthatissensedby An alternative tothehypothesisthatnodalflow transportsan LR Does nodalflowgeneratemechanical stress? is initiallynormalinthe isomerism asaresultofimpairedmidlinefunction.TheLRdecision (Meyers andMartin,1999).However, Shh Fibroblast growth factor 8(Fgf8)hasalsobeenconsideredasa RA issynthesizedinregions nearthenodeby enzyme Shh ) andRA(Tanaka etal.,2005),but noneseemstofulfillallthe is asymmetricallyexpressed andplaysanimportantrolein Nodal Ptch1, Nodal expression inthelateralplate(Zhangetal., failed toreveal any asymmetryinHhsignaling expression inthelateralplatebecauseof Shh Nodal Nodal Nodal -null embryos,but they subsequently Smo Fgf8 expression intheLPM(Rankinet expression ismaintained(Vermot expression inthelateralplate. Shh )-null mice,fail todevelop lack –/– Gdf1 Shh mice exhibit LRdefects Nodal –/– expression, whichis embryos show left expression inthe REVIEW Fgf8 will be 2097

DEVELOPMENT for ciliaryrotationisonly~5 force andviscousisvery low formicroscopicphenomena) mechanosensory model.First,asymmetricCa downstream ofnodalflow, whichpossiblyfavor the with fluorescentbeads,is~15to20 the fluid.Asmentionedabove, thespeedofnodalflow, asvisualized several factors, includingthespeedofflow andtheviscosityof magnitude ofmechanicalstressgeneratedbyaflow dependson which isthesensingofbloodflow byvascular endothelialcells.The stress (forareview, seeOrretal.,2006),perhapsthebestknown of are several examples ofcellsthatsenseflow-generated mechanical Cerl2 peri-nodal cellsofendodermoriginthatexpress 2098 Two linesofevidence indicatearoleforCa Role ofaCa mPa·s). TheReynold number( amniotic fluidis3to5mPa·s, whichissimilartothatofblood(14 present inthenodecavity isunknown, but theviscosityofbovine mm/second). Thepreciseviscosityoftheextra-embryonic fluid slower thanthatofbloodflow inperipheralarteries(~10 asymmetric expression of morphologically normalandmotileciliabut fail todevelop are consistentwithimpairedmechanosensation.They, thus,possess cells (Luoetal.,2003). been reported(Tanaka etal.,2005).Second,aputative Ca to searchfortheexpression ofTRPmembersinornear thenode. a smalllevel ofshearstress(Leeetal.,2006).Itmaybeinteresting ankyrin repeats,whichmayserve asamolecularspringtoamplify 2005). Interestingly, someoftheTRPfamily memberscontain systems, includinghearingandtouchsensitivity (Pedersenetal., channels hasbeenimplicatedinmechanotransductionsensory by signalingcascades. membrane ofnodecells,thesignalmaybeamplifiedwithincell cilia. Alternatively, ifthemechanicalstressissensedby by theslow nodalflow mightbeamplifiedbythebendingof et al.,2003).Ifthelatteriscase,asmallphysicalforcegenerated endothelial cells),aswellpotentiallybynonmotilecilia(McGrath stress canbedetectedbythecellmembrane(asinvascular which subcellularorganelle isresponsibleforitsdetection.Shear sensed bythecellsurface. the shearstressgeneratedbynodalflow mightbe toosmalltobe suggesting thattheassociatedinertialforceisnegligible. Therefore, asymmetric elevation ofCa detected attheleftmargin ofthenode(McGrathetal.,2003).The encodes aproteinthatfunctionsasCa for autosomalrecessive polycystic kidney diseaseinhumans, has beenimplicatedinLRdecisionmaking. 2002). They alsoexhibit alow level ofbilateral 2001) andisrequired toupregulate mediates Nodalsignaling(Hoodless etal.,2001;Yamamoto etal., Meno andH.H.,unpublished Nakamura, N.Mine,E.Nakaguchi, M.Yamamoto, K.Yahsiro, C. theoretical modelknown asthereaction-diffusion system(T. Foxh1 in theLPM,given thatasimilarphenotypeisapparentinconditional in thelevel ofthesignalthatisrequiredtoactivate (Shiratori etal.,2001)].However, thisisprobablyduetoadecrease expression ofwhichisinducedinleftLPMbyNodalsignaling the posteriorlateralplate(LP)[Pitx2isatranscriptionfactor, the Recently, afamily ofmechanically gatedchannelscalledTRP The mechanicalstressexperienced byacellmayalsodependon ( mutants (Yamamoto etal.,2003)andcanbe simulatedbya Dand5 REVIEW 2+ – MouseGenomeInformatics);seeFig.1B].There signal Pkd2 Nodal 2+ ). Foxh1 isatranscriptional factorthat mutant micedevelop LRdefectsthat Re and itslateralpropagationhave also ϫ , whichistheratiobetweeninertial 10 in theLPM(Pennekampetal., Nodal –4 2+ ␮ (Cartwright etal.,2004), m/second, whichismuch release channelincultured and PKD2 2+ 2+ in LRdetermination Lefty2 Pitx2 signaling hasbeen Nodal , acausative gene Nodal expression in expression in , expression 2+ Gdf1 channel and 2003). MechanicalstressalsostimulatesCa (MDCK) cellsabolishesflow sensing(PraetoriusandSpring, as removal oftheprimaryciliumfromMadin-Darbycaninekidney both nodeciliaandkidney cilia(Watanabe etal.,2003). homozygotes (Yokoyama etal.,1993).TheInv proteinlocalizesto to situsinversus, but it alsoresultsinpolycystic kidney in al., 2000).Asmentionedabove, asymmetric Ca genes thatcausepolycystic kidney, including monocilia, but, unlike thenodecilia,they areimmotile.Many ofthe Epithelial cellsofthekidney collectingductalsopossess 9+0 Hints from kidneycilia Lrd-negative cilia,whichpresumably actasmechanosensors. called Lrd(Suppetal.,1997),whichgeneratetheflow; andimmotile node ciliaexist inthenode:motileciliathatarepositive foradynein al., 2003;Tabin andVogan, 2003),whichproposesthattwo kindsof mechanosensory, asproposed bythetwo-cilia model(McGrathet localized tothesurface ofnodecilia,Pkd2mayserve asa spindle (Rundleetal.,2004)andcilia(Yoder etal.,2002).Ifitis plasma membrane,Golgiapparatus(Scheffers etal., 2002),mitotic detected inassociationwithavariety oforganelles, includingthe localization ofPkd2hasalsobeencontroversial, asithasbeen any sitebetween,andincluding,thenodeLPM.Thesubcellular ubiquitously, Pkd2maypotentially functionduringLRpatterningat determination remainstobeclarified.Given that be acomponentofmechanosensor, thepreciseroleofPkd2inLR so (Naulietal.,2003;NauliandZhou,2004).AlthoughPkd2may embryos fail torespondmechanicalflow, whilewild-typeonesdo observations supportthenotionthatPkd2actsasa Ca the leftLPM(Saijohetal.,2000;Yamamoto etal.,2003).Recent The Role ofInvs by bilateral results inpolycystic kidney, aswellinLRdefectscharacterized protein thatcontributes tointraflagellartransport,anditsdeficiency between theciliaofkidney andthoseofthenode. Polarisisa in LRpatterning,indicatingthattheremaybeasimilarityfunction 1998) andispreferentially localizedto9+0cilia, includingthoseof contains ankyrin repeats (Mochizukietal.,1998;Morgan etal., node ciliaareabnormallytilted. The report (Okadaetal.,2005)showed thatasmallproportionofthe in theirstructureormovement (Watanabe etal.,2003),but arecent node ciliaof that theendogenousflow isabnormal.However, examination ofthe embryos (Nonakaetal.,1998;Watanabe etal.,2003),suggesting leftward artificialflow isabletocorrectsitusinversus insuchmutant turbulent, but itsdirectionisstillleftwards (Okadaetal.,1999).A (Yokoyama etal.,1993).Nodalflow in instead ofLRrandomization,invirtuallyallhomozygotes which theLRasymmetryofvisceraiscompletelyreversed), 2003). such cellsdeficientinPkd2fail torespondflow (Naulietal., in theintracellularCa either withamicropipetteorbyartificialflow inducesanincrease epithelial cells.MechanicalbendingoftheciliumMDCKcells intracellular Ca 2001). Kidney epithelialcellsinprimaryculturealso manifest is gatedbymechanicalstress.First, al., 2003).Second,kidney epithelialcellsderived from Recent evidence suggeststhatrenalciliafunctionasflow sensors, inv mouse mutationinducessitusinversus (amalformationin Nodal inv/inv 2+ 2+ signaling intheperinodalendoderm(McGrathet signaling inresponsetoartificialflow. However, expression inLPMandleftisomerism(Murciaet embryos hasfailed toreveal obvious anomalies 2+ concentration (PraetoriusandSpring, inv Pkd2 is araremutationthatgives rise Invs inv –/– gene encodesaproteinthat embryos donotmanifest mutant miceisslow and Development 133(11) 2+ Pkd2 signaling inkidney Pkd2 , alsoplayarole 2+ is expressed channel that Pkd2 –/–

DEVELOPMENT Lefty1 signal from thenode.Theexpression domainsof nodal flow;pinkoval,asmallregion ofLPMthatinitiallyreceives the signal (Lefty1)negativelyregulates from thenodetoLPM is unknown.Inthefourthstep,amidline second andthird steps,but theidentityofsignalthatistransferred from themidlinetoLPM. TheNodalsignalistransferred inthe within theLPM;(3)from the leftandrightLPMtothemidline;(4) (1)fromtake placeduringLRpatterning: thenodetoleftLPM; (2) view ofanE8.0mouseembryo.Atleastfourstepssignaltransfer Nodal-Lefty loops.( generate asymmetricsignalinthenode.Foxh1isacomponentof LPM. Thebroken linerepresents themidline.Lrd andInvare required to mesoderm (LPM).Nodalactivityinduces initiates components are shown.Anasymmetricsignalgeneratedinthenode 2002). TheprecisefunctionofInvs remainsunknown. a componentoftheanaphase-promotingcomplex (Morgan etal., al., 2005), calmodulin (Yasuhiko etal.,2001),dishevelled 1 (Dvl1)(Simonset protein interactswithavariety ofotherproteins, including and nucleus.Biochemicalassayshave shown thattheInvs (inversin) however, and theencodedproteinispresentinbothcytoplasm the node(Watanabe etal.,2003). Development 133(11) (LR) patterning. Fig. 5.Geneticpathwayfor, andsignaltransferduring,left-right al., 2000;HuangfuandAnderson,2005).However, suchadifference exhibit bilateral cilia (suchasthosedeficientinKif3a,Kif3b,polarisandwimple) randomized iv those lackingciliafavors themechanosensorymodel.Thus,whereas the phenotypicdifference betweenmutantswithimmotileciliaand completely exclude thetransportmodel.Ithasalsobeenargued that which cellsreceive adeterminantmolecule,thesedatadonot physiological flows (Nonakaetal.,2002).Therefore, dependingon speed inthenodecavity (20 imposed onembryosinthestudybyNonakaetal.,effective flow this conclusionmightbeincorrect.Althoughafast flow was be expected todisperseasolubledeterminantmolecule.However, inconsistent withthetransportmodelbecausesuchfast flows would has beenthoughtthatdataobtainedwithartificialflows are Additional observations mayberelevant totheroleofnodalflow. It Chemosensory versusmechanosensorymodels A R mutant mice,whichpossessimmotilenodecilia,show and Lrd Node Nodal-Lefty1 , Inv Lefty2 Midline Lefty1 ␤ -cadherin andN-cadherin(Nurnberger etal.,2002),and Nodal Foxh1 Nodal ( (blue) are indicated. A Nodal Pitx2 B ) A genetic pathway for LR patterning. Onlymajor ) AgeneticpathwayforLRpatterning. ) Signal transfer during LR patterning. Aventral ) SignaltransferduringLRpatterning. L expression intheLPM,embryoswithoutnode - Lefty2 LPM Foxh1 Foxh1 expression (Nonakaetal.,1998;Murcia Lefty2 regulatory loopsintheleftlateralplate ␮ m/second) was withintherangeof Nodal B Invs Pitx2 in theLPM.Pinkarrow, the R is expressed ubiquitously, expression intheleft Cardiogenic plate Midline Nodal 4 Node Lefty1 Nodal (red), and 4 3 1 LPM 2 2 L Lefty2 Nodal the nodehasbeenestablishedby geneticmeanstobeessentialfor LPM. that Nodalmayplayaroleinsignal transferfromthenodetoleft expression of Nodal antagonistproducedin the noderegulates theasymmetric of 1999). MicethatlackCerl2show bilateralorright-sidedexpression being substantiallyhigherthanthatontheleftside(Pearceet al., asymmetric manner, withthelevel ofexpression ontherightside the leftLPM(Marquesetal.,2004). expressed intheperinodalregion before et al.,2002). been shown toprevent The specificablationof expression intheleftLPM(Brennanetal.,2002;Saijoh2003). activate route isthesignaltransferredtoLPM?How doesthesignal remain unansweredaboutthisprocess.How and throughwhich expression of transferred tothelateralplate,whereitinducesasymmetric whether itismechanicalstressoramoleculardeterminant,mustbe The asymmetricsignal(orsignals)generatedinornearthenode, Signal transferfrom thenodetolateralplate transferred duringdevelopment. problem, whichremainsafascinating example of how asignalis evidence obtainedtodateprovides aconclusive answertothis over themechanosensorymodel,whereasothersdoopposite.No mutant doesnotsupportonemodelover theother. our view, thedifference inphenotypebetweenthesetwo typesof whereas thelattermutantsalldo(they alllack of midlinedefects.The in that LPM (seeFig.1C).Furthermore,geneticevidence hasestablished (in perinodalcrown cells)beforeitsexpression begins intheleft transfer oftheLRsignal. LR patterningofthelateralplate,andthey mayplaya roleinthe Signaling moleculesexpressed inthenodeareessential forcorrect How istheLRsignaltransferredfromnodetolateralplate? transferred? Is thesignalfrom thenode totheLPMrelayed ordirectly located fromthenode. crown cellsintheperinodalregion; orendodermcellsdistantly two-cilia model(McGrathetal.,2003;Tabin andVogan, 2003); rotating cilia;nodecellswithimmotilecilia,assuggestedbythe (Fig. 1C).Thisroutemayinclude:thenodepitcellswiththeir for thetransferofasignalgeneratedinornearnodetoLPM determinant ofitstransfer. Several potentialroutescanbeenvisioned mechanical stress,thetopologyofmouseembryoisanimportant Whether thecilia-derived asymmetricsignalisamoleculeor Route ofsignaltransfer there? Asymmetricelevation ofCa signal thatreachestheleftLPMandactivates address separateissues,but ratherareinterrelated. asymmetric between thenodeandLPM,but how isitrelatedtothe Nodal Nodal iscurrentlytheonlysignaling moleculewhosefunctionin In summary, someobservations favor thechemosensorymodel Nodal Nodal expression intheLPM(Brennanetal., 2002;Saijohetal., Nodal expression mightsimplybeduetothepresenceorabsence in theLPM(Marquesetal.,2004),suggestingthatthis expression inthenodeisessentialforsubsequent Nodal Cerl2 Nodal Nodal expression intheleftLPM?Whatisnatureof , whichencodesanantagonistofNodal,isalso expression inLPM?Thesequestionsdonot in theLPM.Theseobservations thusindicate (Fig. 1,Fig.5).Several importantquestions Nodal Nodal iv Nodal mutant doesnothave amidlinedefect, expression intheperinodalregion has expression intheleftLPM(Brennan is bilaterallyexpressed inthenode 2+ may beanintermediateevent Nodal Cerl2 Lefty1 expression begins in is expressed inan Nodal REVIEW expression). In expression Nodal 2099

DEVELOPMENT lack GDF1donotmanifestasymmetric Xenopus Like Nodal,GDF1,whichsharessequencesimilaritywithVg1in that isexpressed inthenodeandthatplaysaroleLR patterning. to theleftLPMremainsbedirectlydemonstrated. (Saijoh etal.,2005).However, thetransportofNodalfromnode Foxh1 thatareessentialforenhanceractivity andNodalresponsive enhancers possessbindingsequencesforthetranscriptionfactor Norris andRobertson,1999;Saijohetal.,2000).Importantly, both asymmetric geneexpression intheleftLPM(Adachiet al.,1999; identified two enhancers,bothofwhichareabletoconfer Hamada etal.,2002). Geneticevidence hasrevealed thatNodalacts and Lefty2playacentralrolein thisprocess(Capdevila etal.,2000; understood stepoftheestablishment oftheLRaxis.Nodal,Lefty1 The asymmetricpatterningof the lateralplateisperhapsbest and negativesignalingloops bypositive Lateral plateasymmetric patterning node. understand thepreciseroleofNodal(andGDF1)producedin the to thelateralplate.Clarificationofthisissuewillrequireus to may playaroleinLRpatterningindependentlyofNodal. cells (Chengetal.,2003;Wall etal.,2000),suggestingthatGDF1 responsive reporterwhenoverexpressed infrogembryosorcultured signaling byNodalcomponents,aswella respond totheNodalsignal.Second,GDF1alonecanactivate somite stages,indicatingthatGDF1renderstheLPMcompetent to not onlyintheperinodalregion but alsointheLPMatearly also playadifferent roleinLRpatterning.First, interacting withNodal.However, evidence suggeststhatGDF1may role intransferringalateralitysignalfromthenodetoLPMby their respective mutantphenotypesindicatethatGDF1mayplaya al., 2000).Similaritiesin LPM andexhibit rightisomerismofthevisceralorgans (Rankinet regulatory sequencesthatcontrolasymmetric (Yamamoto etal.,2003).Second,asearchfortranscriptional vector intherightLPMinducesexpression ofendogenous Nodal itself.Thus,theectopicintroductionofaexpression possibility. First, the leftLPM.Several linesofcircumstantialevidence supportthis LPM. and Smad5(Changetal.,2000), mouse embryo,given that,intheabsenceofBMPeffectors Smad1 signal maynegatively regulate identified inthemouseorzebrafishgenomes.Nevertheless, aBMP vertebrates. ABMPantagonistcorrespondingtoCarontehasnotbeen known whetherasimilarsignalingmechanism operates inother in turn,induces encodes aninhibitorofbonemorphogeneticprotein(BMP).Caronte, activates theexpression of Esteban etal.,1999;Yokouchi etal.,1999),Shhproducedinthenode and activates cells) andinduceinthemasecondarysignalthattravels totheLPM from thenode,paraxialmesodermcellsorintermediate the LPM(suchasperinodalcells,endodermalcellsdistantlylocated in thenodemaythusactoncellsthatarelocatedbetweenand node? TheNodalsignalmightberelayedtotheLPM.produced consideration. But,whatisthepreciseroleofNodalproducedin 2003). Any modelforLRpatterningmustthereforetake thisfact into 2100 GDF1 isatransforminggrowth factor It thusremainsunknown how theLR signal travels fromthenode An alternative isthatNodalitselftransportedfromthenodeto REVIEW , isbilaterallyexpressed intheperinodalregion. Micethat Nodal Nodal Nodal expression. Inthechickembryo(Rodriguez expression intheleftLPM.However, itisnot expression intheLPMcanbeinducedby Gdf1 Caronte Nodal and Nodal in theparaxialmesoderm,which Nodal expression intheLPMof is expressed bilaterallyinthe ␤ Nodal expression domainsand (TGF- Nodal Gdf1 ␤ expression inthe )-related protein expression has is expressed Nodal produced inleftLPMinduces expands withintheleftLPMalongAPaxis(Fig.6).Nodal in asmallregion ontheleftatlevel ofthenodeandsubsequently transient. Inthemouseembryo, Nodal induces (Fig. 5).Nodalisalsothoughttotravel to the midline,whereit Meno etal.,2001). side, resultinginbilateral begins normally, but theNodalsignalsubsequentlyleakstoright of Lefty1orLefty2,asymmetric induced byNodalsignaling(Yamamoto etal.,2003).Intheabsence the midlineandleftLPM,respectively (Fig.5).Theirexpression is indicates). Figure courtesyofChikaraMeno (KyushuUniversity, Japan). expression), and( LPM alongtheAPaxis(red arrows indicatetheexpansionofNodal three-somite (s)stageatE8.0. ( a smallregion atthelevelof thenode(purplearrow) atthetwo-to views ofmouseembryos.( (four-somite stage)inthelateral platemesoderm(LPM).(C-E)Lateral Nodal signalingandthedurationof lungs, adoptrightisomerism. Nodal signaling,bilaterallyasymmetricvisceralorgans, suchasthe contribute torightside-specificmorphology. Thus,intheabsenceof cells intherightLPM,whichdonotreceive theNodalsignal, various visceralorgans (asdiscussedinmoredetailbelow), whereas the Nodalsignalcontribute totheleftside-specificmorphologyof as aleft-sidedeterminant.CellsintheleftLPMthathave received the nodeatE7.5and( Fig. 6.Dynamicexpressionof pattern expression of and hascompletelyceasedbythesix-somitestage.Asymmetric genes, possess two Lefty posterior (B)viewsofmouseembryos.( at thecorrecttimeandplace. et al.,2000).ThissystemensuresthepresenceofNodalsignal et al.,1999;NorrisandRobertson,Saijoh Lefty2 and theLeftyproteins.Thus,expression ofboth positive andnegative regulatory loopsthataremediatedbyNodal dynamic expression of The expression of Lefty1 isafeedbackinhibitorofNodalthatrestrictsthearea (as wellasthatof is regulated bytheNodalresponsive enhancerASE(Adachi Lefty1 Nodal expression (Yamamoto etal.,2003).Expressionof E ) disappearsbythe seven-somitestage(red arrow B in LPMthuspersistsforonly~6hours.Such ) developsasymmetricallyafewhours later Nodal C Nodal Lefty1 Lefty1 ) Nodal Nodal D ) Itthenexpandsrapidlywithinthe left n et ee isdynamicand and Leftygenes Nodal Lefty2 and and and Leftygenesisachieved by expression intheleftLPM begins in expression (Menoetal.,1998; Nodal Lefty2 A Lefty2 Nodal expression intheLPMbegins ) Nodal expression intheleftLPM Nodal expression intheLPM , whichareexpressed in ) thenstartstodecrease Development 133(11) expression. Mammals . expression beginsin (A,B) Ventral (A)and Nodal and

DEVELOPMENT et al.,2002). and Schier, 2001;ChenandSchier, 2002; Menoetal.,2001;Sakuma evidence supportsthatNodaland Leftyfulfilltheserequirements (Chen an inhibitordiffuses faster than anactivatordoes.Circumstantial that bothanactivatorandinhibitor traveloveralongdistanceandthat ( diffusion (RD)systemcomprising NodalandLeftyproteins. difference isthenconverted toarobust asymmetrybyareaction- difference (pink)betweenthetwosidesofLPM. ( endogenous Nodal expression vector intotherightLPMactivates expression of is connectedattheposteriorend.Given thattheintroductionofa patterning intheLPMremainunanswered.First,onbothsides 7). robust asymmetrybyareaction-diffusion system(Fig. nodal flow generatesonlyasmalldifference, whichisconverted to Yahsiro, C.MenoandH.H.,unpublished).Itis,thus,possiblethat embryos (T. Nakamura,N.Mine,E.Nakaguchi,M.Yamamoto, K. that cansimulateinvivo dataforwild-typeandvarious mutant over alongdistance;andtheconstructionofmathematicalmodel both Leftygenes;theabilityofNodalandproteinstoact further supportedby:thedynamicexpression patternsof inhibition (Turing, 1990;MeinhardtandGierer, 2000).Thisnotionis into arobust difference throughlocalactivation andlong-range is abletoconvert asmalldifference betweentwo separatedregions factors constituteareaction-diffusion system,atheoreticalmodelthat al., 2000;BranfordandYost, 2004)have suggestedthatthesesecreted Schier, 2002;Hamadaetal.,JuanandHamada,2001;Saijoh mediated byNodalandLeftyproteins,weothers(Chen Development 133(11) generated intwosteps.( robust LRasymmetry(asymmetric asymmetry inthelateralplatemesoderm(LPM). Fig. 7.Two stepsinthegenerationofrobust left-right(LR) D Photomicrograph illustratingthisasymmetry. ARD systemrequires ) Despite suchprogress,importantquestionsrelatedtoasymmetric Given theoperationofpositive andnegative regulatory loops Nodal D RL C B A (Yamamoto etal.,2003),leftLPMandright A , B ) First,nodalflowgeneratesasmall difference bynodalflow Generate asmall Nodal asymmetry byRDsystem Convert torobust expression) intheLPMis C In thismodel, ) Thissmall Nodal and Utah, USA). mechanism are shown.Figure courtesyofYukio Saijoh(Universityof regression. Examples ofanatomicalstructures generatedbyeach in theLPMbynegatively regulating midline (Leftyproteins)establishesasymmetric 2000) alsosuggeststhatadiffusible inhibitor produced atthe (Meno etal.,1998).Theoreticalsimulation(MeinhardtandGierer, component ofthemidlinebarrierinregion anteriortothenode Nodal crossing themidline.Inabsenceofafunctionalmidlinebarrier, prevent theNodalsignalthatoriginatedinleftLPMfrom establishing theasymmetric structures, suchasthefloorplateandnotochord,areessentialfor mechanism would notbeexpected tobeerrorfree. expression asitapproachestheposteriormidline,but sucha Nodal andLeftyproteinsmayoperatetoreducethelevel of The autonomouspositive-negative regulatory loopsmediatedby or isthereanadditionalmechanismthatrestrictsNodalsignaling? the inhibitorLefty2sufficient toprevent leakageoftheNodalsignal, extends totheposteriorregion ofrightLPM(Menoetal.,2001).Is the LPM,Nodalsignal(asrevealed bytheexpression of across themidlineatposteriorend?InabsenceofLefty2in then isthemechanismthatprevents theNodalsignalfromleaking LPM areequallycompetenttorespondtheNodalsignal.What ( different mechanisms forthegenerationofmorphologicalasymmetries: Fig. 8.Generationofanatomical asymmetries. organogenesis. Anatomicasymmetriesbecomerecognizablein asymmetric informationisconveyed togive risetoasymmetric specific organogenesis. Itisnotcurrentlyknown how LR Less clearlyunderstoodisthefinalstepofLRpatterning,situs- signal interpretation Situs-specific organogenesis:thefinalstepofLR Nodal signalexclusively totheleft sideisthusnotfullyunderstood. where What isthenatureofmidlinebarrierinposteriorregion 5B, step4).However, isLefty1alonesufficient forbarrierfunction? A ) directional looping,( It isalsonotclearhow themidlinebarrierfunctions. Themidline C B A Lefty1 expression intheLPMbecomesbilateral.Lefty1ismajor expression isabsent?Themechanismthatrestrictsthe B ) differential branching and( Nodal Nodal expression intheLPMandto over alongdistance(Fig. (A-C) Three Nodal REVIEW C Blood vessels ) one-sided Intestine Stomach Heart Liver Lung expression Nodal Pitx2 2101 )

DEVELOPMENT regulatory elementshasindicatedthatasymmetric does thatoftheseothertwo genes.Ananalysisoftranscriptional asymmetric expression of and factor Pitx2(Loganetal., 1998; Yoshioka etal.,1998).Like organogenesis downstream oftheNodalsignal isthetranscription pancreas bud. may inducetheformationofbothsplenicmesenchymeand the asymmetry intheSMP(Hecksher-Sorensen etal.,2004). TheSMP proliferation isatleastoneofthemechanismsresponsiblefor the laterally whiletherightSMPdisappears.Differential cell Under thecontrolofLRsignal,however, theleftSMPgrows stages, surroundingtheprospective stomach locatedatthemidline. (Hecksher-Sorensen etal.,2004).TheSMPisbilateral atearly derived celllayerknown asthesplanchnicmesodermalplate(SMP) factors Hox11andNkx2.5,dependsonacolumnarmesoderm- which comprisesagroupofcellspositive forthetranscription mesogastrium dorsaltothestomach.Theformationofthisrudiment, condensation ofmesenchymealongtheleftside splanchnopleure istherudimentofspleenthatformsasasingle that exerted bythesplanchnopleure(Voronov etal.,2004).The Alternatively, theloopingmaybedriven byexternal forces,suchas intracellular actinbundles (Itasakietal.,1991;Itasaki1989). cell shape(Manaseketal.,1972)orinthearrangementof to thehearttube,suchasthosegeneratedbychangesinmyocardial remain unknown. Theloopingmaybeachieved byforcesintrinsic However, thecellularandbiophysicalbasesofcardiaclooping looping resultsincongenitalheartmalformationshumans. morphogenesis, hasreceived muchattentionbecauseimpaired Cardiac looping,thefirstvisibleindicationofasymmetric remains unknown how thedirectionofcellmigrationisregulated. dependent onLRsignaling(Horne-Badovinac etal.,2003), but it left. Geneticevidence indicatesthatasymmetricLPMmigrationis ventrolaterally, resultinginashiftofthedeveloping intestineto the dorsally totheendoderm,whilerightLPMmigrates LPM (Horne-Badovinac etal.,2003).TheleftLPMthus migrates however, resultsfromtheasymmetricmigrationofleftandright elongation (Mulleretal.,2003).Loopingofthezebrafishgut, chick embryoisthoughttobemediatedbydifferential cell arteries isunknown. Bycontrast,directionalloopingofthegutin in developing arteries,but how theLRsignalisinterpretedby (Navaratnam, 1963).LRsignalingmayinducedifferential celldeath component disappearswhiletheleftcounterpartpersists case ofthesixthbranchialarchartery, forexample, theright complex remodelingthatincludesasymmetricregression. Inthe sided regression ofbloodvessels. Branchialarcharteriesundergo differently. Itislikely thatdifferential celldeathcontributes toone- each organ primordiumseemstointerpretLRinformation and disappears,leaving onlytheotherside(Fig.8C). symmetric structure,suchasabloodvessel, undergoes regression as isthecaseforlungs(Fig.8B).Andinthird,onesideofa subsequently develop differences intheirsizeorbranchingpattern, primordial organs thatformsymmetricallyonbothsides positioning withinthebody(Fig.8A).Insecond,apairof looping, bendingandrotationstepsthatresultintheircorrect formed asatube,includingtheheartandgut,undergo aseriesof (Fig. 8).Thefirstisdirectionallooping:organs thatareinitially mechanisms areresponsibleforgeneratingasymmetricstructures the LPMhasdisappeared.Macroscopically, atleastthreedifferent various visceralorgans onlyafterasymmetric 2102 At themolecularlevel, themainplayerthatregulates asymmetric The cellularbasisofasymmetricorganogenesis isnotclear, but Lefty2 REVIEW , Pitx2 is expressed asymmetrically inleftLPM,but Pitx2 persists untilmuchlaterstagesthan Nodal Pitx2 expression in expression Nodal Nodal discovery ofLRasymmetricallyexpressed genes,suchas The fieldonLRasymmetryhasdeveloped rapidlysincethe Perspectives a similarmechanismoperatesinLPM-derived cells. cyclin genes(Kioussietal.,2002).However, itisnotknown whether stimulates cellproliferationinthepituitaryglandbyregulating is relevant toasymmetricmorphogenesishasbeenidentified.Pitx2 of Pitx2activity alsodiffers. Currently, notarget geneofPitx2that regulate distinctsetsofgenesindifferent organs, sothatthereadout or decreasedcellproliferation,deathandmigration?Itmay Pitx2 induceseeminglydifferent cellularevents, suchasincreased dependent and-independentmechanismsareoperative. How can normally intheabsenceofPitx2,suggestingthatbothPitx2- events, suchascardiacloopingandembryonicturning,take place as thelungs,exhibit rightisomerism.However, certainlaterality morphologies. Thus,intheabsenceofPitx2,bilateralorgans, such LPM-derived cellsthatexpress Pitx2activity develop left-side exhibit lateralitydefectsinmostvisceralorgans (Liuetal.,2001). (specifically, Pitx2c,theisoform thatisasymmetricallyexpressed) Nodal signal(Shiratorietal.,2001).MicethatlackPitx2 is inducedbyNodalandmaintainedNkx2intheabsenceof Adachi, H.,Saijoh,Y., Mochida,K.,Ohishi,S.,Hashiguchi,H.,Hirao,A.and References performed intheauthors’laboratory has beensupportedbyCREST. work wewere unabletocite becauseoflengthconstraint.Thework for theircriticalyetconstructivecomments.We apologizecolleagueswhose discussions andforproviding illustrations,andwethankunknownreviewers We thankmembersofthe Hamadalaboratoryforproposing variousideas,for Branford, W. W. andYost, H.J. process ofLRasymmetryinthenearfuture. breakthrough ortwo, weshouldbeabletounderstandthe whole certainly importantissues.With steady progressanda during LRpatterning).They maylooklesschallengingbut are Nodal andLeftyproteins;how andwhereBMPsignalingacts (such astheexact roleofthemidline;behaviors ofsecreted taken forgrantedbut thatneedtobeexamined inthenearfuture manipulations. Inaddition,therearemany moreissuesthatare theoretical modeling,andmoresophisticatedgenetic imaging, invitrocultureandtime-lapseobservation systems, necessary todevelop andintegrate new techniquesincluding morphogenesis. To addresstheseoutstandingissues,itmaybe to theLPM,andunderstandcellularbasisofasymmetric asymmetric signalgeneratedinthenodeandhow itistransferred are toclarifyhow nodalflow works, toknow thenatureof despite recentprogress.Key challengesintheimmediatefuture challenging topic!Many key questionsremaintobeanswered such abnormalities. models willthereforecertainlycontribute toourunderstandingof defective LRpatterning.Theknowledge obtainedfromanimal that many humancongenitalcardiacmalformations are dueto problem tosolve, ithasapracticalapplicationaswell, itislikely LR patterning.Althoughasymmetryisaninterestingscientific mutants willfurtheridentifythosegenesthatarerequiredfornormal generating LRasymmetricorgans. ThesystematicscreeningofLR We now know thatalarge numbersofgenesareinvolved in mouse (Collignonetal.,1996;Lowe etal.,1996;Meno1996). Genes Dev. by aleftside-specificenhancerwithsequence similaritytoalefty-2enhancer. Hamada, H. of antagonismdecoded. As isclearfromthisreview, LRasymmetryisstilla in thechick(Levin etal.,1995),and 13 (1999). Determinationofleft/rightasymmetric expression ofnodal , 1589-1600. Curr. Biol. (2004). Nodalsignaling: CrypticLeftymechanism 14 , R341-R433. Nodal Development 133(11) and Lefty1 Shh in the and

DEVELOPMENT Kioussi, C.,Briata,P., Baek,S.H.,Rose,D.W., Hamblet,N.S.,Herman,T., Kawakami, Y., Raya,A.,R.M.,Rodriguez-Esteban,C.andBelmonte,J. Klein, T. J.andMlodzik,M. Juan, H.andHamada, Chen, Y. andSchier, A.F. Capdevila, J.,Vogan, K.J.,Tabin, C.J.andIzpisuaBelmonte, Levin, M. Lee, G.,Abdi,K.,Jiang,Y., Michaely, P., Bennett,V. andMarszalek,P. E. Kramer-Zucker, A.G.,Olale,F., Haycraft, C.J.,Yoder, B.K.,Schier, A.F. and Itasaki, N.,Nakamura,H.,Sumida,H.andYasuda, M. Itasaki, N.,Nakamura,H.andYasuda, M. Huangfu, D.,Liu,A.,Rakeman,A.S.,Murcia, N.S.,Niswander, L.and Hoodless, P. A.,Pye,M.,Chazaud,C.,Labbe,E.,Attisano,L.,Rossant, J.and Hecksher-Sorensen, J.,Watson, R.P., Lettice,L.A.,Serup,P., Eley, L.,De Hamada, H.,Meno,C.,Watanabe, D.andSaijoh,Y. Garcia-Garcia, M.J.,Eggenschwiler, J.T., Caspary, T., H.L.,Wyler, Alcorn, M. Essner, J.J.,Amack,D.,Nyholm,M.K.,Harris,E.B.andYost, H.J. Cheng, S.K.,Olale,F., Bennett,J.T., Brivanlou,A.H.and Schier, A.F. Chen, Y. andSchier, A.F. Chang, H.,Zwijsen,A.,Vogel, H.,Huylebroeck, D.andMatzuk,M. Cartwright, J.H.,Piro, O.andTuval, I. Brown, N.A.,McCarthy, A.andWolpert, L. Brennan, J.,Norris,D.P. andRobertson,E.J. Development 133(11) Liron, N. C.D.,Kuehn,M.andTabin,Levin, M.,Johnson,R.L.,Stern, C. Huangfu, D.andAnderson,K.V. S.,Rebagliati,M.andStainier,Horne-Badovinac, D.Y. Essner, J.J.,Vogan, K.J.,Wagner, M.K.,Tabin, C.J.,Yost, H.J.and Collignon, J.,Varlet, I.andRobertson,E.J. during development. Wnt/Dvl/beta-Catenin Ohgi, K.A.,Lin,C.,Gleiberman,Wang, J.etal. 171. bilaterally symmetricsomitogenesisinthezebrafishembryo. C. embryonic patterning ofvertebrates. embryonic patterning points intherightdirection. looping intheembryonicchickheart. on therightsideincaudalpartofhearttubeplayarole indextro- vertebrate left-rightasymmetry. squint-mediated nodalsignaling. Mechanisms ofleft-rightdeterminationinvertebrates. body asymmetryinmammals. governs left-rightasymmetry.governs Cell molecular pathwaydeterminingleft-right asymmetryinchickembryogenesis. comprehensive review. (2006). Nanospringbehaviourofankyrin repeats. 132 brain andKupffer’s vesicleisrequired fornormalorganogenesis. Drummond, I.A. 413-420. of actinbundlesduringheartloopinginthechickembryo. intraflagellar transportproteins. Anderson, K.V. the mouse. streak inthemouse. Wrana, J.L. Development plate directs spleenandpancreatic laterality, andisregulated byBapx1/Nkx3.2. Angelis, C.,Ahlgren, U.andHill,R.E. forward genetics. al. R., Huangfu,D.,Rakeman,A.S.,Lee,J.Feinberg,E.H.,Timmer, J.R.et 1247-1260. initiates left-rightdevelopmentofthebrain,heartandgut. Kupffer’s vesicleisaciliatedorganofasymmetryinthezebrafishembryothat 418 GDF1. EGF-CFC proteins are essentialcoreceptors fortheTGF-betasignalsVg1and morphogen. 78. (2000). Smad5isessentialforleft-rightasymmetryinmice. Sci. USA embryonic developmentofleft-rightasymmetryinvertebrates. domension. framework forgut-loopingmorphogenesisinzebrafish. Brueckner, M. 381 asymmetric nodalexpression andthedirection ofembryonicturning. (2005). Retinoic acid signalling links left-right asymmetric patterning and (2005). Retinoicacidsignallinglinksleft-rightasymmetricpatterning (2005). Analysis of mouse embryonic patterning andmorphogenesisby (2005). Analysisofmouseembryonicpatterning , 1907-1921. , 37-38. , 155-158. 82 , 803-814. Genes Dev. (1996). Strokes flowduetoinfinitearraysofstrokeslets inthree (2005). Left-rightasymmetryinembryonic development:a 101 Proc. Natl.Acad.Sci.USA J. Eng.Math. , 7234-7239. Nature (2001). FoxH1(Fast)functionstospecifytheanteriorprimitive 131 (2002). Conservedfunctionforembryonicnodalcilia. (2003). Hedgehogsignallinginthemouserequires , 4665-4675. Proc. Natl.Acad.Sci.USA 17 (2005). Cilia-drivenfluidflowinthezebrafishpronephros, 411 Genes Dev. , 31-36. Cell r Mech. Dev. , 607-610. (2002). Leftyproteins are long-rangeinhibitorsof (2001). ThezebrafishNodalsignalSquintfunctionsasa Pitx2 pathwaymediatingcell-type-specificproliferation (2001). Rolesofnodal-leftyregulatory loopsin 111 30 (2005). Planarcellpolarization:anemergingmodel Annu. Rev. CellDev. Biol. , 267-297. , 673-685. Genes Dev. Ciba FoundSymp. Nat. Rev. Genet. Nature 15 Curr. Biol. (2005). CiliaandHedgehogresponsiveness in 122 , 1257-1271. Genes Cells Anat. Embryol. Anat. Embryol. 102 , 3-25. (2004). Fluid-dynamicalbasisofthe 426 (2004). Thesplanchnicmesodermal 16 , 11325-11330. (1989). Changesinthearrangement , 83-87. 12 , 2339-2344. (1996). Relationshipbetween 102 (1991). Developmentofhanded , 2124-2128. (2002). Nodalactivityinthenode , 5913-5919. 3 162 6 Nature , 103-113. , 923-930. , 182-196. 183 (2002). Establishmentof 21 Cell (2002). Identificationofa (1991). Actinbundles Science , 155-176. (2003). Acellular 440 , 29-39. Anat. Embryol. Anat. Embryol. 101 Development Dev. Biol. Nature , 246-249. , 9-21. Proc. Natl.Acad. 302 Development (1995). A 435 , 662-665. (2000). 219 Nature Nature (2005). , 165- (2003). , 71- 132 180 , , Logan, M.,Pagan-Westphal, S.M.,Smith,D.Paganessi,L.andTabin, C. Liu, C.,W., Lu,M.F., Brown, N.A.andMartin,J.F. Murcia, N.S.,Richards, W. G.,Yoder, B.K.,Mucenski,M.L.,Dunlap, J.R. Nauli, S.M.andZhou,J. Muller, J.K.,Prather, D.R.andNascone-Yoder, N.M. Luo, Y., Vassilev, P. M.,Li,X.,Kawanabe,Y. andZhou,J. Lowe, L.A.,Supp,D.M.,Sampath,K.,Yokoyama, T., Wright, C.V., Potter, Navaratnam, V. Nauli, S.M.,Alenghat,F. J.,Luo,Y., Williams,E.,Vassilev, P., Li,X.,Elia,A.E., Manasek, F. M.B.andWaterman, J.,Burnside, R.E. Nonaka, S.,Tanaka, Y., Okada, Y., Takeda, S.,Harada,A.,Kanai,Y., Kido,M. Norris, D.P. andRobertson, E.J. Nonaka, S.,Yoshiba, S.,Watanabe, D.,Ikeuchi,S.,Goto,T., Marshall,W. F. Meinhardt, H.andGierer, A. Marques, S.,Borges,A.C.,Silva,Freitas, S.,Cordenonsi, M.andBelo, Nonaka, S.,Shiratori,H.,Saijoh,Y. andHamada,H. Nurnberger, J.,Bacallao,R.L.andPhillips,C. Meno, C.,Shimono,A.,Saijoh,Y., Yashiro, K.,Mochida,Ohishi,S.,Noji, Meno, C.,Saijoh,Y., Fujii,H.,Ikeda,M.,Yokoyama, T., Yokoyama, M., McGrath, J.,Somlo,S.,Makova,Tian,X.andBrueckner, M. Okada, Y., Nonaka, S.,Tanaka, Y., Saijoh,Y., Hamada,H.andHirokawa, N. Meyers, E.N.andMartin,G.R. Meno, C.,Takeuchi, J.,Sakuma,R.,Koshiba-Takeuchi, K.,Ohishi,S.,Saijoh, Morgan, D.,Eley, L.,Sayer, J.,Strachan,T., Yates, L.M.,Craighead,A.S.and Morgan, D.,Turnpenny, L.,Goodship,J.,Dai,W., Majumder, K.,Matthews, Mochizuki, T., Saijoh,Y., Tsuchiya, K.,Shirayoshi,Y., Takai, S.,Taya, C., response toleft-rightasymmetricsignals. J. 2048. left-right asymmetrybythresholds ofPitx2cactivity. is required forleft-rightaxisdetermination. and Woychik, R.P. 672-682. asymmetric morphogenesisintheXenopusdigestivesystem. nodal cilia. renal epithelia. polycystin 2functionsasaplasmamembraneCa2+-permeablecationchannelin 161. of nodalexpression andalterationsinmurinesitusinversus. S. S.,Overbeek,P. andKuehn,M.R. nerve supplyinhumanembryo. mechanosensation intheprimaryciliumofkidneycells. Lu, W., Brown, E.M.,Quinn,S.J.etal. shape changeasamechanismofembryonicheartlooping. KIF3B motorprotein. nodal ciliageneratingleftward flowofextraembryonicfluidinmicelacking and Hirokawa, N. Genes Dev. expression are patterns controlled bytwodistinctcis-actingregulatory elements. posterior tiltofnodalcilia. and Hamada,H. 96-99. ofthemouseembryobyartificialnodalflow.left-right patterning and lateralinhibition. 114 populations ofnodemonociliainitiateleft-rightasymmetryinthemouse. required forcorrect L/Rbodyaxis. J. A. 371. 13 complex withcateninsandN-cadherin in polarizedepithelialcells. determination asaregulator oflefty-2andnodal. S., Kondoh,H.andHamada, TGF beta-familymemberleftyinmouseembryos. Toyoda, Y. andHamada,H. 4 (1999). Abnormalnodal flowprecedes situsinversus inivandinvmice. mouse andchick:functionsofFGF8SHH. 1 nodal signalingactivityintheabsenceoffeedbackinhibitorLefty2. Y., Miyazaki,J.,tenDijke,P., Ogura,T. andHamada,H. involvement inthecellcycle. promoting complexprotein Apc2suggestarole forinversininprimaryciliaand Goodship, J.A. mouse. novel geneinthevertebrateleft-rightaxispathway, ispartiallydeletedintheinv L., Gardner, A.,Schuster, L.,Harrison,W. G.,Vien, etal. 395 inv, agenethatcontrols left/rightasymmetryandkidneydevelopment. Yonekawa, H.,Yamada, K.,Nihei,H.,Nakatsuji,N.etal. , 459-468. , 127-138. (1998). ThetranscriptionfactorPitx2mediatessitus-specificmorphogenesisin , 3096-3106. , 61-73. , 177-181. (2004). TheactivityoftheNodalantagonistCerl-2inmousenodeis Nat. Genet. BioEssays 13 , 1575-1588. (1963). Observationsontherightpulmonaryarch ateryandits Mol. Cell.Biol. (2002). Expression analysesandinteractionwiththeanaphase (2005). Denovoformationofleft-rightasymmetryby (1998). Randomizationofleft-rightasymmetryduetoloss 20 (2000). TheOakRidgePolycysticKidney(orpk)diseasegene 26 Cell BioEssays , 149-156. , 844-856. (2004). Polycystinsandmechanosensationinrenal and 95 PLoS Biol. , 829-837. (2000). Pattern formationbylocalself-activation (2000). Pattern Hum. Mol.Genet. (1996). Left-rightasymmetricexpression ofthe 23 22 (1999). Differences inleft-rightaxispathways J. Anat. (1999). Asymmetricandnode-specificnodal , 2600-2607. Genes Dev. , 753-760. (1998). lefty-1isrequired forleft-right 3 , e268. (1996). Conservedleft-rightasymmetry 97 Cell (2003). Polycystins1and2mediate , 569-573. Development 94 18 Science 11 , 307-317. , 2342-2347. (2002). Inversinformsa Nature Cell , 3345-3350. Development (2002). Determinationof (1972). Myocardial cell 94 285 Nat. Genet. (2003). Left-right (2001). Regulationof , 287-297. 381 127 , 403-406. (2003). Native Dev. Biol. REVIEW (2001). Diffusion of (1998). Inversin,a Nature Dev. Dyn. , 151-155. , 2347-2355. (1998). Cloningof (2003). Two 128 Nature Mol. Biol.Cell 33 381 29 , 129-137. , 2039- 228 Dev. Cell Nature Mol. Cell , 349- , 158- 418 Cell 2103 , ,

DEVELOPMENT Rundle, D.R.,Gorbsky, G.andTsiokas, L. Scheffers, M.S.,Le,H.,vanderBent,P., Leonhard, W., Prins,F., Spruit,L., Saijoh, Y., Adachi,H.,Sakuma,R.,Yeo, C. Y., Yashiro, K.,Watanabe, M., Saijoh, Y., Adachi,H.,Mochida,K.,Ohishi,S.,Hirao, A.andHamada,H. Rodriguez Esteban,C.,Capdevila,J.,Economides,A.N.,Pascual,Ortiz, Rankin, C.T., Bunton,T., Lawler, A.M.andLee,S.J. Praetorius, H.A.andSpring,K.R. Praetorius, H.A.andSpring,K.R. Pennekamp, P., Karcher, C.,Fischer, A.,Schweickert,Skryabin,B., Sakuma, R.,OhnishiYi, Y., Meno, C.,Fujii,H.,Juan,Takeuchi, J.,Ogura, Saijoh, Y., Oki,S.,Tanaka, C.,Nakamura,T., Adachi,H.,Yan, Y. T., Shen,M. Saijoh, Y., Oki,S.,Ohishi,S.andHamada,H. Pedersen, S.F., Owsianik,G.andNilius,B. Okada, Y., Takeda, S.,Tanaka, Y., Belmonte,J.C.andHirokawa, N. 2104 Simons, M.,Gloy, J.,Ganner, A.,Bullerkotte,Bashkurov, M.,Kronig, C., Pearce, J.J.,Penny, G.andRossant,J. Orr, A.W., Helmke,B.P., Blackman,B.R.andSchwartz,M.A. Sulik, K.,Dehart,D.B.,Iangaki,T., Carson,J.L.,Vrablic, T., Gesteland,K.and Shiratori, H.,Sakuma,R.,Watanabe, M.,Hashiguchi,H.,Mochida,K.,Sakai, localization tomitoticspindles. localizes withmDia1tomitoticspindlesofdividingcells:role ofmDia1INPKD2 251. Breuning, M.H.,deHeer, E.andPeters,D.J. signaling pathwaythatincludesthetranscriptionfactorFAST2. Left-right asymmetricexpression oflefty2andnodalisinducedbya Hashiguchi, H.,Mochida,K.,Ohishi,S.,Kawabata,M.etal. asymmetric expression oflefty-1andlefty-2. (1999). Distincttranscriptionalregulatory mechanismsunderlieleft-right mediates theestablishmentofembryonicleft-rightasymmetry. and IzpisuaBelmonte,J.C. 265. inmicebygrowth/differentiationright patterning factor-1. cilium abolishesflowsensing. cilium increases intracellularcalcium. 938-943. polycystin-2 isrequired forleft-rightaxisdeterminationinmice. B. Horst, J.,Blum,M.andDworniczak, Cell Calcium apparatus ofMDCKcells. expression ofendogenouspolycystin-2intheplasmamembraneandGolgi diffusion. by Leftymediatedthrough interactionwithcommonreceptors andefficient T., Li,E.,Miyazono,K.andHamada,H. asymmetric expression ofNodal. M. andHamada,H. 160-172. the mouselateralplaterequires nodalproduced inthenode. 35-47. gene family. determination. Mechanism ofnodalflow:aconservedsymmetrybreaking eventinleft-rightaxis switch betweenWntsignalingpathways. the geneproduct mutatedinnephronophthisis typeII,functions asamolecular Schermer, B.,Benzing,T., Cabello,O.A., Jenny, A.etal. Mechanisms ofmechanotransduction. signaling andmaintenancebyNkx2. regulation ofleft-rightasymmetricexpression ofPitx2:initiation bynodal Y., Nishino,J.,Saijoh,Y., Whitman,M.andHamada,H. REVIEW Genes Cells Dev. Biol. 38 Cell , 233-252. 121 (2005). Two nodal-responsive enhancerscontrol left-right 7 209 , 401-412. , 633-644. Hum. Mol.Genet. , 98-110. (1999). ThenovelCer-like protein Caronte J. Membr. Biol. J. Biol.Chem. Dev. Dyn. (2003). RemovaloftheMDCKcellprimary (2001). BendingtheMDCKcellprimary Mol. Cell J. Membr. Biol. (1999). Amousecerberus/Dan-related Dev. Cell Nat. Genet. (2004). PKD2interactsandco- (2002). InhibitionofNodalsignalling 232 (2002). Theionchannel (2005). TRPchannels:anoverview. 11 Genes Dev. 191 279 (2003). Left-right patterning of (2003). Left-rightpatterning 7 , 1031-1036. , 59-67. , 137-149. 10 (2002). Distinctsubcellular , 69-76. , 29728-29739. , 11-20. 184 37 (2000). Regulationofleft- , 71-79. , 537-543. 13 Nat. Genet. , 259-269. (2001). Two-step (2005). Inversin, Dev. Biol. Nature (2006). Curr. Biol. Mol. Cell (2000). (2005). 401 256 24 , 262- , 243- , 12 5 , , Supp, D.M.,Brueckner, M.,Kuehn,M.R.,Witte,D.P., Lowe,L.A.,McGrath, Supp, D.M.,Witte,P., Potter, S.andBrueckner, M. Vermot, J.,GallegoLlamas,Fraulob,V., Niederreither, K.,Chambon,P. Tabin, C. Tabin, C.J.andVogan, K.J. Yoshioka, H.,Meno,C.,Koshiba,K.,Sugihara,M.,Itoh,Ishimaru,Y., Yokoyama, T., Copeland,N.G.,Jenkins, N.A.,Montgomery, C.A.,Elder, F. F. Voronov, D.A.,Alford, P. W., Xu,G.andTaber, L.A. Vermot, J.andPourquie, O. Turing, A.M. Tanaka, Y., Okada,Y. andHirokawa, N. Yamamoto, M.,Meno,C.,Sakai,Y., Shiratori, H.,Mochida,K.,Ikawa,Y., Watanabe, D.,Saijoh, Y., Nonaka,S.,Sasaki,G.,Ikawa,Y., Yokoyama, T. and Wall, N.A.,Craig, E.J.,Labosky, P. A.andKessler, D.S. Zhang, X.M.,Ramalho-Santos,M.andMcMahon,A.P. Yasuhiko, Y., Imai,F., Ookubo,K.,Takakuwa, Y., Shiokawa,K.and Yamamoto, M.,Mine,N.,Mochida,K.,Sakai,Y., Saijoh,Y., Meno,C.and Yokouchi, Y., Vogan, K.J.,Pearse,R.V., 2ndandTabin, C.J. Yoder, B.K.,Hou,X.andGuay-Woodford, L.M. plate. Schoenwolf, G.C. left-right asymmetricgeneexpression. Antagonistic signalingbyCaronte, anovelCerberus-related gene,establishes invertebrateembryos. left-right patterning Biol. established inthevertebrateembryo? right asymmetries. domain ofleft-rightdyneinconfirmsitsrole inspecifyingdevelopmentofleft- J., Corrales,J.andPotter, S. Nature an axonemaldyneinaffects left-rightasymmetryininversusviscerummice. formation inthemouseembryo. and Dolle,P. Dev. Biol. induction andreversalbymouseGdf1,aVg1-related ofleft-rightpattern gene. Dev. Biol. mechanical forces indextralrotation duringcardiac loopinginthechickembryo. axis specification. L/R symmetrybythemousenode. mutants reveal redundant roles forShhandIhhsignalingincludingregulation of determination ofleft-rightasymmetry. bicoid-type homeoboxgene,isinvolvedinalefty-signalingpathway Inoue, T., Ohuchi,H.,Semina,E.V., Murray, J.C.etal. mutation. and Overbeek,P. A. determination. Sonic hedgehogandretinoic acidinleftward nodalflowiscriticalforleft-right formation inthemouse. mediates Nodalsignalingduringanterior-posterior andnode patterning Saijoh, Y. andHamada,H. monocilia andother9+0cilia. Hamada, H. renal cilia. disease proteins, polycystin-1,polycystin-2,polaris,andcystin,are co-localizedin regulation ofinvfunction. Yokoyama, T. Nodal expression inthelateralplate. Hamada, H. 52 Dev. Dyn. (2005). Doweknowanythingabouthowleft-rightasymmetryisfirst 389 , 153-197. 227 272 Science J. Am.Soc.Nephrol. , 963-966. (1990). Thechemicalbasisofmorphogenesis.1953. (2003). Theleft-rightdeterminantInversinisacomponentofnode (2003). Nodalsignalinginducesthemidlinebarrierbyactivating , 495-509. , 339-350. (2005). Retinoicacidcontrols thebilateralsymmetryofsomite (2001). Calmodulinbindstoinvprotein: implicationforthe Nature 201 Genes Dev. 260 Development (1994). Morphogenesisofthemurinenodeandnotochordal , 260-278. (1993). Reversalofleft-rightasymmetry:asitusinversus , 679-682. 435 Genes Dev. Dev. GrowthDiffer. , 172-177. (2003). Atwo-ciliamodelforvertebrateleft-right (2005). Retinoicacidcoordinates somitogenesisand (2001). ThetranscriptionfactorFoxH1(FAST) 17 Development 13 , 1-6. 126 Science (1999). Targeted deletionoftheATP binding , 2508-2516. Cell 15 , 5495-5504. Development J. Mol.Histol. Cell Cell 106 , 1242-1256. (2005). FGF-inducedvesicularrelease of 308 , 781-792. 98 94 Nature 130 , 563-566. , 573-583. , 299-305. 43 , 1725-1734. , 671-681. (2002). Thepolycystickidney 130 435 Development 133(11) 36 (2004). Therole of , 1795-1804. , 215-220. , 317-323. (2000). Mesendoderm (2001). Smoothened (1997). Mutationof (1998). Pitx2,a (1999). Bull. Math.

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