controversies Regulation ofearlylungmorphogenesis:questions,factsand Development 133,1611-1624(2006)doi:10.1242/dev.02310 *Author forcorrespondence (e-mail:wcardoso@lung.bumc.bu.edu) Pulmonary Center, BostonUniversitySchoolofMedicine,Boston,MA 02118,USA Wellington V. Cardoso* andJiningLü early lungdevelopment. Topics discussedhereincludelung focuses onourcurrentknowledge ofthemolecularregulation of been thesubjectsofanincreasing numberofstudies.Thisreview demonstrated inthedeveloping lung. of thevasculature inepithelialpatterninghasstilltobeclearly (Lammert etal.,2001;Matsumoto2001).However, therole vessels serve asasourceofinductive signalstotheepithelium Shannon, 2000).Thereisevidence that,duringorganogenesis, blood vasculature (Wood etal.,1997;DemelloGebband and laterconnectstothelarger vessels togive risetothelung buds; arudimentarycapillarynetwork initially formsandexpands, vasculogenesis inthelungmesenchymeneardeveloping epithelial atrium tothelung(angiogenesis).Bloodvessels alsodevelop by migration ofbloodvessels fromtheaorticarchesandleft of thelung,pleura.Thelungvasculature forms,inpart,by the lymphatics,andmesothelialcellsthatcover theoutersurface that surroundsairways andbloodvessels, thecartilageoftrachea, connective tissue,endothelialcellprecursors,thesmoothmuscle gives risetomultiplecomponentsofthelung,includingits lung mesenchymeoriginatesfromthelateralplatemesodermand which linetheinnersurface ofthedeveloping lungandtrachea.The foregut endodermdifferentiates intovarious epithelialcelltypes, 1), whichultimatelygives risetothematureairways andalveoli. The tree-like systemofepithelialtubules andvascular structures(seeFig. in theprimitive foregut. Thisisfollowed bythedevelopment of a The processinitiateswiththeestablishmentofrespiratorycellfate is oxygenated. units closelyapposedtobloodvessels, wherethecirculating blood which airiscleaned,humidifiedanddelivered tonumerousalveolar here asthetracheaandlung,isthatofatreeepithelialtubules in The basicdesignofthemammalianrespiratorysystem,referredto Introduction regulation. questions andcontroversiesregardingtheirmolecular review earlystagesinlungdevelopmentandhighlight interact inthedevelopinglungissubjecttodebate.Here,we However, thepreciserolesofthesemoleculesandhowthey factors inregulatingtheinitialstagesoflungdevelopment. acid andWntsignalingpathways,varioustranscription factor, sonichedgehog,bonemorphogeneticprotein,retinoic alveoli. Recentstudieshaveimplicatedthefibroblastgrowth tubules formsanddifferentiates toproducetheairwaysand vascular development,atree-likestructureofepithelial Through branchingmorphogenesis,andincoordinationwith endoderm givesrisetothetrachealandlungcellprogenitors. During earlyrespiratorysystemdevelopment,theforegut The mechanismsthatcontrolrespiratorysystemformationhave The respiratorysystemarisesfromtheventral foregut endoderm. system From guttolungs:theoriginofrespiratory Bourbon etal.,2005). review (forreviews, seePauling andVu, 2004;Williams, 2003; such assacculationandalveoli formation,arenotdiscussedinthis data available (Table 1).Lungvascular development andlaterevents, described referstomouselungdevelopment becauseofthegenetic pathways andtargets arekey totheseprocesses?’.Mostofwhatis cellular diversity generatedinthedeveloping lung?’and‘which buds form?’,‘how arestereotypicalpatternsofairway branchingand ‘when andhow isrespiratorycellfate established?’,‘how dolung differentiation intheembryoniclung.We addressquestionssuchas regulation oftheinitialstagesbranchingmorphogenesisand endodermal specification,lungprimordiumformation,andthe transcription factor 1( homeodomain proteingene organ-specific domains(orfields;seeFig.1A).For example, the theanteroposterior(AP)axisof gutendodermmarks along 2005). ByE8.0-9.5,thelocalexpression oftranscription factors 1997; Morrisey etal.,1998;AngandRossant,1994; Wan etal., survival, differentiation and morphogenesisoftheforegut (Kuo etal., Gata6 1999). Transcription factor genessuchas regions, towards thecaudalendofembryo(Wells andMelton, midgut andhindgutarelocatedatprogressively moreposterior represents themostanterior(cranial)region ofthistube, whilethe ultimately leadtotheformationofprimitive guttube.Theforegut endoderm undergoes complex morphogeneticmovements that Following gastrulation(embryonicdayE7.5inmice),thedefinitive cell fate Foregut morphogenesis andestablishmentofendodermal pancreatic cellfates atmoreanteriordomains (Stafford and Prince, fates remainunaltered.Conversely, excess RAinduceshepaticand pancreatic (posterior)fates, whilethyroidandpharynx(anterior) signaling duringgastrulation results inthelossofliver and Prince, 2002;Wells andMelton,2000).Inzebrafish, disruptedRA structures toinitiatemorphogenesis (Tiso etal.,2002;Stafford and to respondsignalsfromtheadjacent mesodermorfromnearby identity totheearlyendoderm.They rendertheendodermcompetent (Bmp2) andretinoicacid(RA)areamongthesignalsthatconfer AP fate specification(Kumar andMelton,2003;Bortetal.,2004). at crucialdevelopmental windows isessentialforendodermalcell Exposure oftheendodermtodiffusible signalsfromthesestructures transversum (themesodermalcellsthatgive risetothediaphragm). neighboring structures,suchastheheart,notochordorseptum movements fosterdynamicinteractionsbetween theendodermand duodenal fields(Offield etal.,1996).Inaddition,morphogenetic associated homeoboxgene)isexpressed inthepancreaticand (Martinez Barberaetal.,2000),andthe expressed homeobox)isexpressed inthethyroidandliver fields respiratory fields(Kimuraetal.,1996), Fibroblast growth factor 4(Fgf4),bonemorphogeneticprotein2 whichareexpressed early intheendoderm,arecrucialfor , Titf1 ) or T/EBP Nkx2.1 ] isexpressed inthethyroidand Pdx1 [also known asthyroid Foxa1 Hex (pancreas-duodenal- (hematopoietically , Foxa2 REVIEW , Gata4 1611 and

DEVELOPMENT 1612 primordium, as the thyroidstartstodevelop atleast1daybefore the 2005). Thesesignals,however, probably originate fromthethyroid the foregut asearly E8-8.5 (eight-somitestage)(Serlsetal., Nkx2.1 lung/tracheal region of theforegut, at~E9(Minooetal.,1999). as agroupof Respiratory progenitorsarefirst visualizedbyinsituhybridization fate isestablishedintheforegut endodermisstillunclear. region ofthelung(Perletal.,2002).Preciselywhenrespiratorycell proximal lungdiffer inoriginfromthosethatwillformthedistal analysis suggeststhattheprogenitorcellsoftrachea and contrast, relatively fewer studieshave focusedonthelung.Lineage investigated (Rossietal.,2001;Kumar andMelton,2003).By The specificationoftheliver andpancreas hasbeenextensively When andhowislungcellfatespecifiedintheforegut? AP identityasitisinthezebrafish. (Rossant etal.,1991),but doesnotseemtobeascrucial forforegut 2002). Inmiceandrats,RAsignalinginitiatessoonaftergastrulation REVIEW transcripts have beenreportedbyRT-PCR tobepresentin Nkx2.1- expressing endodermal cells intheprospective are identifiedcollectivelyatE9.0by to theliverandpancreatic fields.Lungandtrachealprogenitors ventral foregut endoderm,whichisposteriortothethyroid butanterior and pancreas (Pa).Therespiratory progenitors (Tr, Lu) arisefrom the later giverisetothethyroid (Th),thymus,trachea(Tr), lung(Lu),liver(Li) into organ-specificdomainsalongitsanteroposterior (AP)axis,which depict primary, secondaryandtertiarygenerationsofbuds. branch andlatergiverisetothealveolarregion ofthelung.Numbers proximal airways(bronchi), whiledistalregions (green) continueto initially generated,yellow)stopbranchinganddifferentiate into established inthedevelopinglung.Proximal regions (where budsare respiratory (bronchial) tree. Asthisoccurs,aproximodistal axisis process isreiterated overseveralgenerationsofbranchestoformthe dichotomous subdivisionsandcleftformationatbranchingpoints.The morphogenesis, whichinvolvesbudoutgrowth andelongation, lobes. From E10.5toE16.5-E17.0,theepitheliumundergoesbranching has four:thecranial(cr),medial(me),caudal(ca)andaccessory(ac) lobes. AtE11.5-12.0(right),theleftlung(LLu)hasonelobeandRLu Fgfr2b at specificpositions(red arrowheads; theepitheliumislabeledby (left), secondarybudsariseasoutgrowths from theprimarylungbuds 1982; Sutliff andHutchins,1994;Ioannidesetal.,2002).( endodermal ridgesfrom thelateralwallsofforegut (Zaw-Tun, by growth ofanascendingtracheoesophagealseptumorbyfusion esophagus (Es),inapoorlyunderstoodprocess thatispossiblyinitiated forms ventrallyandseparatesfrom thedorsalforegut, theprimitive axis). ( primary buds(red arrows) oftherightandleftlung(V-D, ventral-dorsal which theninvadetheadjacentmesodermandelongatetoform arrows) are inducedfrom theventral-lateralaspectofforegut, also labelsthethyroid. ( respiratory system. Fig. 1.Keyeventsduringearlydevelopmentofthemouse et al.,1999).These lungs,however, arehighlyabnormal and respiratory region, Although Nkx2.1 anddistallungdevelopment ( lung (Lazzaroetal.,1991).Thesurfactant-associated proteinC factors thathave yettobeidentified. molecules, suchasFgfsfrom theheart,andlocaltranscription summary, lungspecificationislikely todependonsignaling the foregut, areinvolved inlungendodermspecification.In evidence thatsonichedgehog(Shh)orBmps,whicharepresentin Fgfr4 et al.,2005).Lungdevelopment, however, isapparently normalin induction oflungcellfate appearstoinvolve Fgfr4signaling(Serls 2001; Serlsetal.,2005;Jung1999).Inthissystem, the in inductionofliver, and thenoflungorthyroidfates (Rossietal., amounts ofcardiacmesodermorFgf2totheendodermresults first the endodermadoptsa‘default’ pancreaticfate; adding increasing derivatives, including theliver andlung(Fig.2A).Ifculturedalone, thresholds ofFgfspatterntheendodermintodifferent foregut model offoregut specificationhasbeenproposed,inwhich different ventral foregut endoderminaconcentration-dependentmanner. A emanating fromtheadjacentheartinfluenceAPfate ofthe after theprimarybuds form(Wert etal.,1993). lineage, but itsexpression isconsistentlydetected onlybyE10-10.5, Sftpc Studies inmouseforegut culturesstronglyindicatethat Fgfs C -null mice(Weinstein etal.,1998).Currently, there isno ) geneisthemostspecificmarker oflungepithelialcell ). Intherightlung(RLu),thesebudslaterdevelopintoseparate ) With primarylungbudformation,thetracheal(Tr)) With primordium Nkx2.1 is theearliestknown marker ofthepresumptive Nkx2.1 ( A B ) Theforegut (graytube)isinitiallyspecified ) AtE9.5,twoendodermallungbuds(black -null mutantmicedohave lungs(Minoo Nkx2.1 expression (purple),which Development 133(9) D ) At~E10.5

DEVELOPMENT Itga3 Pthlh Crh Adam17 Pcaf eesmo eenm xrsinptenPeoyeReference Phenotype Expressionpattern Tgfb3 Shh Hip1 Grem1 Fgf9 Genename Fgf18 Egfr Signaling molecule Gene symbol Table 1.Examplesofmutationsinmousegivingareported lungand/ortrachealphenotype Development 133(9) Cutl1 Lama5 Lmnb1 Eln Others Cebpa Transcription factor Nog Fgfr3/Fgfr4 Traf4 Lefty1 Nodal Acvr2b Fgf8 Fgfr2b Fgf10 Wnt5a Ltbp4 Ascl1 Gli2/Gli3 Gata6 Foxj1 Pitx2 Rarb/Rara Tcf21 Sox11 Nfib Titf1 Trp63 Mycn Klf2 Hoxa5 Foxf1a Foxa1/Foxa2 Catnnb1 Wnt7b aahri omn-ie pteimDfiin lelzto Rubinet al.(2004) Deficientalveolization Mugliaetal.(1999) Shikamaetal.(2003) Defectiveepithelialandmesenchymal Zhaoetal.(2001) Epithelium Impaired epithelialdifferentiation, Defectiveproximalanddistal Integrin Epithelium Parathyroid hormone-like Epitheliumand Corticotropin releasinghormone Epithelium A disintegrinandmetallopeptidase p300/CBP-associated factor oi eghg pteimIpie rnhn, Litingtungetal.(1998) Michosetal.(2004) Chuang etal.(2003) Impairedbranching, Deficientalveolization Usuietal.(2004) Epithelium Epitheliumand Impairedbranching Colvinetal.(2001) Deficientalveolization Transforming growthfactor, Mesenchyme Impairedbranching,reduced Miettinenetal.(1997) Epitheliumandpleura Sonic hedgehog Huntingtin-interacting protein1 Mesenchyme Impairedbranchinganddeficient Gremlin 1 Fibroblast growthfactor9 Fibroblast growthfactor18 Epithelium and Epidermal growthfactorreceptor u-ie1Eihlu mardeihla ifrnito Ellisetal.(2001) Impairedepithelialdifferentiation Wendel etal.(2000) Epithelium Vergnes etal.(2004) Deficientalveolization Deficientalveolization Mesenchyme Cut-like 1 Epitheliumand Laminin Lamin B1 Elastin Weaver etal.(2003) Loweetal.(2001) Lobationdefect Shielsetal.(2000) Sugaharaetal.(2001) Rightpulmonaryisomerism Mesenchyme OhandLi(1997) Sekineetal. (1999) Weinstein etal.(1998) HyperproliferationoftypeIIcells DeMoerlooze etal.(2000) Menoetal.(1998) Notreported Fischeretal.(2002) Rightpulmonaryisomerism Tracheal defect Epithelium Deficientalveolization Leftpulmonaryisomerism Rightpulmonaryisomerism CCAAT/enhancer bindingprotein Notreported Sterner-Kock etal.(2002) Lungagenesis Notreported Epitheliumand Noggin Lungagenesis Notreported Fibroblast growthfactorreceptor Notreported Tnf receptorassociatedfactor4 Left rightdeterminationfactor1 Mesenchyme Lietal.(2002) Nodal Activin receptorIIB Pulmonaryemphysema Increasedbranching,trachealdefect Epithelium Fibroblast growthfactor8 Fibroblast growthfactorreceptor2b Fibroblast growthfactor10 Mesenchymeand Wingless-related MMTVintegration Notreported Latent transforminggrowthfactor L-rpe aiymme Msnhm ugaeei Motoyama et al.(1998) Yang etal. (2002) Brody etal.(2000) Left-right asymmetry, lossofciliated Itoetal.(2000) Impairedsacculation Steele-Perkinset al.(2005) Socketal. (2004) Linetal.(1999) Lungagenesis Quaggin etal.(1999) Lossofneuroendocrinecells Epithelium Rightpulmonaryisomerism Epithelium Mesenchyme Neuroendocrinecells Moenset al.(1992) Sacculationdefect Achaete-scute complexhomolog- Hypoplasticlung Impairedbranching Mesenchyme Wani etal.(1999) GLI-Kruppel familymember Danielyetal.(2004) Kimuraetal.(1996) GATA-binding protein6 Aubin etal.(1997) Epitheliumand Forkhead boxJ1 Lossofdistallungfate,impaired Impairedbranching,trachealdefect Mesenchyme Limetal.(2002) Epithelium Paired-like homeodomain Impairedbranching Impairedbranching,lobationdefect Retinoic acidreceptor Tracheobronchial Wan defect etal.(2005) Transcription factor21(Pod1) Impairedsacculation Impairedbranching,reducedsmooth SRY-box-containing gene11 Mesenchyme Epithelium Nuclear factorI/B Mesenchyme Epithelium Epithelium Notreported Thyroid transcriptionfactor1 Epithelium Transformation-related protein63 Neuroblastoma myc-related Kruppel-like factor2(lung) Homeobox A5 Forkhead boxF1a Forkhead boxA1/A2 Shuetal.(2002) Vascular defect,reducedmesenchyme ␤ Epithelium Wingless-related MMTVintegration Ctnn pteimIpie rnhn,poia/itl Mucenskiet al.(2003) Impairedbranching,proximal/distal Epithelium -Catenin oan1 mardbacigPeschonetal.(1998) impairedbranching peptide domain 17 like 1 / mesenchyme (C/EBP), epithelium 3/4 site 5A ␤ GLI2/GLI3 transcription factor2 oncogene 1 site 7B binding protein4 ␣ ␣ pteimIpie rnhn Kreidbergetal.(1996) Impairedbranching Epithelium 3 pteimadper eetv oainNguyenetal.(2002) Defective lobation Epitheliumandpleura 5 ␣ ␣ / ␤ ␤ pteimadper mardbacigKaartinenetal.(1995) Impairedbranching Epitheliumandpleura 3 pteimad etln gnssadrgtln Mendelsohnetal.(1994) Leftlungagenesisandright Epithelium and eecyeepithelialcelldifferentiation mesenchyme eecyealveolization mesenchyme mesenchyme mesenchyme eecyehypoplasia mesenchyme mesenchyme maturation tracheoesophageal fistula mesenchyme specification cells fistula branching, tracheoesophageal muscle REVIEW 1613

DEVELOPMENT 2003) andRA(Dickmanetal.,1997). (Minoo etal.,1999;Litingtung1998).Thisdefecthasbeenalsoassociatedwithdeficienciesin normally present intheventralforegut endoderm,showtracheoesophagealfistula(incompleteseparationoftherespiratory andd Nkx2.1 interactions, areabsentorgreatly reducedinthelungepitheliumof several integrins, whicharerequiredforepithelial-mesenchymal epithelial-mesenchymal interactions. Indeed,collagentypeIVand controls theexpression ofmoleculesthatareimportantfor morphogenesis isstillunclear. Itispossiblethat,normally, Nkx2.1 from mesodermalinductionof Foregut mesodermisshowningray, endoderminblue,andtheofprospective tracheaandlunginred. Lungbudding Serls etal.(Serlsal.,2005)fordetails.]( specify theventralforegut endodermintoliver(Li)(blueline)orlung(Lu)andthyroid (Th)(red, endodermal competence.(b,c)Amodelofforegut specification,inwhichincreasing thresholds ofFgfs(purple),emanatingfrom t transcriptionfactorsgenes(yellow)are involvedinearlyevents,suchasforegut (Fg)tubec (a)TheFoxaandGata 8.0 toE9.0. dorsoventral (DV)differences ingeneexpression thatprobably influencethisprocess. Forexample,micedeficientin from theventralforegut anditsseparationfrom thedorsalguttube(Es,primitiveesophagus).Across-section through thefor and Tbxgenes( 1614 lung (Minooetal.,1999). the developmental programofprogenitor cells oftheproximal Nkx2.1 of relatively preserved featuresofproximaldifferentiation in Nkx2.1 identification ofadditionalearlymarkers oflungcellfate. Besides maintained inthesemicecannotbedeterminedwithout the lung. Whetherdistallungprogenitorsarespecifiedbut not the surfactant-protein genestypicallyfoundinthenormal distal marker analysisshows thattheepitheliumfails toexpress any of reminiscent ofthosefoundinproximalairways. Strikingly, lined bycolumnarcellswithscatteredcilia–featuresthat are consist oftwo mainbronchi,whichgive risetocystic structures, Fig. 2.Molecularregulation ofinitialeventsinlungandtrachealdevelopment. unknown intermediatefactor(X). Why thelackof -null mice(Yuan etal.,2000). , nootherearlymarker iscurrentlyavailable. Thepresence -null mutantssuggeststhat REVIEW TBX4 Nkx2.1 in chicks)regulate has suchanegative effect onbranching Fgf10 Bmp4 and from activationofFgfr2bsignalingintheendoderm(indicatedbyayellowbracket).Retinoicacid(RA) Nkx2.1 Fgf10 is expressed intheventralmesodermatlungfield,where itsrole isunknown.( B ) Regulationofprimarylungbudformation,basedondatafrom mouseandchick(seetextfordetails). expression. may notbecrucialfor Gli2 and Gli3 are bothrequired forprimarylungbudformation, presumably viaan or (Bellusci etal.,1997b;Park etal.,1998).Deletionofeither Fgf10 isachemotacticandproliferation factor fortheendoderm signaling byFgf10andFgfr2b iscrucialforlungbud formation. endoderm toinducebudding (Sutherlandetal.,1996).Inmammals, by thelocalactivation ofaFgfreceptor( ligand ( developing trachealsystemisinitiatedby theexpression ofaFgf 1). Asdeterminedbystudiesin In mice,lungprimordialbuds formatE9.5(~25-somite stage;Fig. Lung budmorphogenesis:therole ofFgf10andFgfr2b Sftpc Nkx2.1 isrequiredforexpression ofseveral lungmarkers, suchas developmental programofepithelialcells thedistallungandthat sequences thatdirect has beenstudied,littleisknown aboutthecis-active regulatory Primary lungbudformation 2004). These studiessuggestthatNkx2.1isessentialforthe Fgfr2b (Kelly etal.,1996).Althoughthepromoterregion of branchless ( A ) Thedevelopingmouseforegut from embryonicday(E) in miceresults inlungagenesisandmultiple ) atprospective sitesofbudding; thisisfollowed Nkx2.1 Nkx2.1 Foxf1 -expressing endoderm).[Seetextand (Lim etal.,2002), expression tothelung(Pan etal., Drosophila Shh or C Nkx2.1 ) Trachea (Tr) formation Development 133(9) losure andestablishing , thebudding ofthe egut shows Tbx4 breathless he heart(Ht), igestive systems) , whichare (Sakiyama etal., (red) results ) inthe Nkx2.1 Fgf10

DEVELOPMENT Tbx4 andFgf10arenotrequiredtoinitiate Fgf10 seen intheembryoniclung. recapitulate theFgf-dependentprogramofbudding andbranching develop postnatallyfromthetrachealepitheliumandprobably significantly reduced(Rawlins andHogan,2005).Thesestructures revealed thatthesizeandnumberoftrachealsubmucosalglandsare null miceatbirth,arecentanalysisof tracheal morphogenesishasbeenreportedtobenormalin normally carriedoutbyFgf10andFgfr2bsignaling.Although lung epithelialsurvival andthelungmorphogeneticprogramthatis much lower affinity (Luetal.,1999).Itisthusnotabletomaintain activation ofFgfr1b,areceptorthatalsobindstoFgf10,but with Moerlooze etal.,2000).Thishasbeenattributed toFgf10-mediated underdeveloped lungbud thatsoonundergoes apoptosis(De et al.,1999).Theoverlapping featuresof abnormalities (DeMoerloozeetal.,2000;Min1998;Sekine Development 133(9) 1996). redundant roleinforegut andlungmorphogenesis(Chapmanetal., and lungmesodermsuggests thatthesegenesmayhave a expression of formation (NaicheandPapaioannou, 2003).Theoverlapping genetic inactivation of and thelungprimordium(Sakiyama etal.,2003).However, the appear toplayaroleindefiningtheposteriorboundaryof of Studies inovo show thatmisexpression mesenchyme (Huietal.,1994).In that arepresentintheforegut mesodermandlaterin thelung and Gli3aretranscriptionaleffectors oftheShhsignalingpathway implicated intheformationoflungprimordium.Gli1,Gli2 mesoderm (Desaietal.,2006). 2004). Thisseemstoinvolve signalingbyRAreceptor Fgf10 lung buds fromforming.Inthismodel,RAselectively regulates foregut explants inthepresenceofaRAreceptorantagonistprevents agenesis (Wilson etal.,1953;Mollard2000).CulturingE8.5 most dramaticallyandleadstoseveral abnormalities,includinglung 2000). Yet, disruptionofRAsignalingintheforegut affects thelung prominent throughouttheE8.5-9.5mouseforegut (Malpel etal., dependent onRA(Desaietal.,2004).synthesisanduseare Fgf10 domain intheforegut (Fig.2B).Thereisevidence, however, that lung primordiumintheforegut ortheboundariesof the Little isknown aboutthegenesthatcontrolpositioningof tube? What controls lungprimordium positioninginthe foregut Interestingly, unlike mutants confirmFgfr2basthemajorreceptorforFgf10. that of foregut mesoderminthelungfieldadomainthatcoincideswith required forbud formation. currently unidentifiedmesodermaldiffusible signalthatis expressed inthelungfield.PresumablyGli2andGli3inducea during development theseGliproteins arepreferentially (Pepicelli etal.,1998),andalsobecausethereisnoevidence that intriguing, asitismoresevere thanthatof embryos diebyE10.5(Motoyama etal.,1998).Thisphenotypeis derivatives develop but aresmallerthannormal,andmost lung andtrachealprimordiumnever form;otherforegut Gli andT-box (Tbx)transcriptionfactors have beenalso In chickembryos, where thelungandneighboringstomachform(Desaietal., expression andbud formationinthelungfield arecrucially expression andectopicbuds thatexpress Nkx2.1 Tbx2 in theendoderm , Tbx3 Fgf10 Tbx4 Tbx4 , Tbx4 -null mutants, and in micedoesnotprevent lungbud , and except initsmostanteriorportion. Fgf10 Tbx5 Gli2/Gli3 Fgf10 Fgfr2b are co-expressed inthe in thedeveloping foregut Fgf10 Nkx.2.1 heterozygous micehas Tbx4 double-null mice, -null miceforman Shh - and Nkx 2.1 induces ectopic expression, but -null mutants Fgfr2b ␤ mRNA Nkx2.1 Fgf10 Fgf10 in the -null - . (E9.5). Therelatively lateappearanceof LR determinantsceasesbythetimelungprimordiumforms (Kitamura etal., 1999).Thus,duringnormal development, Pitx2 show rightpulmonary isomerism(four-lobed lungsbilaterally) pattern of highly stereotypicalfashion. Ithasbeenproposedthattheexpression At leastduringtheinitialgenerationofbranches,lungbuds ariseina remains toberigorouslytestedbyconditionalinactivation of budding, asitdoesduringprimarybudding (Fig.3A).This,however, remain unresolved. many questionsandcontroversies aboutitsmolecularregulation process hasbeenextensively studiedinthelung(seeTable 1),but morphogenesis toformtherespiratory(orbronchial)tree.This From E10.5-E17.0,thelungepitheliumundergoes branching Making therespiratory tree primary lungbud induction. lung development andapotential,ordemonstrated,involvement in have incommonexpression intheforegut mesodermattheonsetof Pitx2c (Kitamura etal.,1999).Among thethreeisoforms(a,b,c),only that controlasymmetryindifferent structuresoftheembryo hypothesis thatthisgeneactsasan executor ofearlygeneticprograms mesoderm onlytransiently(E8-8.5).Exceptfor Interestingly, mostofthesegenesareexpressed inthemouseforegut lobes (witheitherrightorleftpattern)onbothsides(Table 1). commonly manifestasisomerism,thepresenceofequalnumbers of laterality defectsinmultipleorgans. Inthelung,thesedefects et al.,2000;Lin1999).Disruptionofthesegenesresults in (Oh andLi,1997;Menoetal.,1998;Kitamura1999;Rankin differentiation factor 1,andbythebicoidtypehomeoboxgene molecules, suchasactivin receptor2,Lefty1,Lefty2andgrowth program ofaxisspecificationthatisregulated byseveral Tgfb-related on left-right(LR)determinants.Theprocessispartofanearlyglobal lobes ontheright(seeFig.1D).Asymmetryoflungisdependent Murine lungscharacteristicallyhave onelobe ontheleftandfour visceral pleura).Thenumberoflobesvaries indifferent species. side (lobesaremorphologicalunitsofthelungthatcovered bythe patterns ofbranchingandbythedifferent numberoflobesoneach Left andrightlungsareasymmetric,asisapparentbytheirdistinct Left-right asymmetryandbranching downregulation of thyroidepithelium hasledtothehypothesisthatlocal and HMG boxtranscriptionfactor role ofthesegenesinlungpatterningisstillunclear. axis ofthelunginsingleordoubleHox-nullmutantssuggeststhat Aubin etal.,1997).However, thelackofdramaticchangesinAP the mousedeveloping lung(Bogueetal.,1996;Volpe etal.,1997; are expressed inpartiallyoverlapping domainsalongtheAPaxisof (Metzger andKrasnow, 1999).Indeed,several Hoxfamily members set byglobalregulators ofaxisformation,suchastheHoxgenes in thelungisinvariant within a three-dimensionalgridandcouldbe morphogenesis. Duringlungbranchingmorphogenesis, inability ofthestemcellstoproliferate(Avilion etal.,2003). mice deficientinSox2diepriortoorganogenesis becauseofthe 1998). TheroleofSox2inthelungremainstobedemonstrated; and expression islostatsiteswherenascentbuds arise(Ishiietal., associated withtheepitheliumthatislessmorphogeneticallyactive, Fgf10 actsasthesignalthattriggerssecondaryandsubsequent RA, Fgf10,Gli2,Gli3,Tbx2,Tbx3andTbx4,discussedhere,all An intriguing,dynamicpatternofexpression ofthe Sry-like is asymmetricallyexpressed intheleftlung. branchless Sox2 in thedeveloping may berequiredforcommencementofbud Sox2 Drosophila in thedeveloping lung Pitx2 Pitx2 REVIEW trachea or Pitx2 has ledtothe , expression of -null mice Sox2 Fgf10 Fgf10 1615 Pitx2 is .

DEVELOPMENT lung. How Pitx2exerts itsfunctions,andwhether simpler patternofbranching(andlobation),characteristictheleft budding onlyatspecificsites.Bydoingso,Pitx2would generatea could presumablyinfluencegeneexpression intheleftlung,allowing 1616 bud). ( depicted ingrayandtheepitheliumblueorred (distal bud duringbranchingmorphogenesis.Mesenchymeis inthedevelopinglung. patterning Fig. 3.Modelsofbudformationandproximodistal line MLE15,Spry2inhibitsFgf10-Fgfr2signalingbybindingto and Egf(KimBar-Sagi, 2004).Inthemouselungepithelialcell with theintensityortimingofRtksignalingbyligandssuchasFgf receptor tyrosinekinaseRtk-Ras-Erk/Mapkcascadeandinterfere four membersinmice)thatinteractwithcrucialelementsofthe the sprouty(Spry)orShhpathways, respectively (Fig.3B-D). the mechanismsthatcontrolFgfr2bactivity or the sizeandshapeofbud duringbranching.Thisisillustratedby surrounding mesenchymeestablishesfeedbackresponsesthatcontrol The exchange ofsignalsbetweenthegrowing bud andthe Control ofbuddingbysprouty andShh on theseissues(Gangaetal.,2003). number of interact inthelungmesenchyme,areunknown. Onlyalimited Wnt) inhibits epithelium; mesenchymalWnt(aloneorwithepithelial signaling activates Control of and fibronectin, andTgfbiinstalkmesenchyme.( extracellular matrix(ECM)components,suchascollagen subepithelial mesenchyme;Tgfb1-inducedsynthesisof Tgfb activationintheepitheliumbyTgfb1from the mediated Fgfr2bsignalinginhibition(broken yellowline); inhibit ectopicbuddinginstalkregions include:netrin- signaling regulate Bmp4(seeF).Mechanismsthatmight Mesenchymal Pod1(Tcf21) (indirectly) andepithelialWnt yellow arrow), viaanunidentifiedmesenchymalsignal(X). can alsoenhancebuddinginaparacrinefashion(broken epithelium (Eblaghieetal.,2006)(broken yellowline)and distal buddingthrough autocrinesignalingfrom the Bmp4 signaling andinhibitsbudding,broken yellowline)and expression of arrow). ( epithelium) toactivatesignalingandinduceabud(white binds locallytoFgfr2b(expressed throughout thelung the distalmesenchyme.Fgf10diffuses (yellowarrow) and mesenchyme mayprevent Tbx5.Tgfb1signalinginstalk and Foxf1, Tbx4 ( signaling (broken yellowline)toallow Fgf10 also controls availabilityofFoxf1,apositiveregulator of distal mesenchymeinhibits proximal mesenchyme(boxinC).Shhsignalingthe timing oftheirdifferentiation (presumably bycontrolling progenitor cellsofthedistal lungepithelium. andFgf10(viaFgfr2bepithelialsignaling) maintai model ofproximodistal cell fateregulation inthelungbudepithelium.Mycn expression. Vegf regulates endothelialcelldifferentiation. RA(from thepleura)mayregulate adjacent mesenchyme,resulting inlaterinductionoflateralbuds.( differentiation ofproximal epithelium intociliatedcells.Seetext forreferences and Eblaghieetal.(Eblaghieal.,2006) depends onShhandWnt7bsignals from thedistalepitheliumandonFgf9from thepleura (purple).Foxa1andFoxa2regulate immediately adjacentregions, where ShhsignalingisunopposedbyHhip.Lowlevels inmore proximal budregions allow D ) Atthebudtips,highShh(distalepithelium)andHhipmesenchyme) levelsresult inoveralllessShhsignalingandmo Spry genesencodeafamily ofcysteine-rich proteins (thereare . Shhinductionof A in thedistalepithelium.Bmp4possiblyalsoinhibits REVIEW Branchinginitiateswithlocal ) B ) Asthebudelongates,Fgfr2bsignalinginduces Fgf10 Pitx2 Fgf10 Spry2 and targets have beenreported,andthey shedlittlelight Fgfr2b . Positiveregulators of (which negativelyregulates Fgf Fgfr2b Hhip expression inthelung Fgf10 Fgf10 expression. CanonicalWnt expression inhibitsShh expression inthe expression, butviaGli3 Fgf10 Fgf10 A developinglung Fgf10 expression. expression in Bmp4 include C Fgf10 ) prevents distalepithelialcells from assumingaproximal phenotype.Wntsignaling regulates the Pitx2 Bmp4 expression by and and Fgf10 E Mycn ) Theproliferation ofmultipotent mesenchymalprogenitors whilethelunggrows xrsin n sngtvl euae ydckp Dk) oj induces 1(Dkk1). Foxj1 expression) andisnegatively regulated bydickkopf impairs branching(Mailleuxetal.,2001;Perl2003). Spry4 1998). Conversely, overexpression of tracheal systemof increased branching(Tefft etal.,1999),asalsoreportedforthe tips ofthegrowing epithelialbuds, while phosphatase) (Tefft etal.,2002). protein 1)andShp2(Srchomology2-containingphosphotyrosine oncogene 1),andbydisassociatingfromGap1(GTPase-activating factor receptorboundprotein2)andRaf(v-raf-leukemia viral Frs2 (fibroblastgrowth factor receptorsubstrate2),Grb2(growth this role,reducing and, ultimately, toinhibitbranching morphogenesis.Consistentwith mechanism tocontrolthesizeofbud ortostopbud formation lung epitheliumwhenbuds areforming.Thiscouldbepartofa Fgfr2b activity, Spry2limitstheproliferationormigrationof (Mailleux etal.,2001).ByactingasaFgf10-dependentinhibitorof targets tobeinducedinthelungepitheliumresponseFgf10 surrounding distallungmesenchyme. In thedeveloping E11-12mouselung, in thedistallungepitheliumoftransgenicmiceseverely Fgf9 expression butthisremains tobeshown.( Spry2 Drosophila activity inlungorgan culturesresultsin in Spry . Spry2 Spry2 mutants (Hacohenetal., Spry2 Spry4 Fgf10 re re n theproliferation of Development 133(9) Shh is oneoftheearliest or misexpression of Fgf10 is expressed atthe and is presentinthe expression inthe than inthe Wnt7b F ) A

DEVELOPMENT locally (seeFig.3D).Inthelungsof sequestration and,thus,releasesShh-mediatedrepressionof in thedistalmesenchyme.Hhip1inhibitsShhsignalingbyligand involves Shhinductionof thehedgehoginteractingprotein spatial patternof (Pepicelli etal.,1998).Preciselyhow Shhhelpstocontrolthe structures andbranchingmorphogenesisisseverely disrupted forming typicalbuds, theepitheliumdevelops aslarge cystic found diffusely expressed inthelungmesenchyme.Insteadof epithelium. Inlungsfrom mesenchyme andthegeneralizedactivation ofFgfr2binthedistal by preventing thewidespreadexpression of bud size.Interestingly, Shhmayalsocontroltheshapeofbud bud outgrowth. Bydoingso,Shhwould contribute tocontrolling towards the progressively downregulates These findingshave ledtotheproposalthatShhatbud tips expression inthelung(Belluscietal.,1997a;Lebeche1999). vivo studiessupporttheideathatShhnegatively regulates Gli3 (Belluscietal.,1997a).Datafromlungorgan cultureandin smoothened (Smo)andtheirtranscriptionaleffectors Gli1,Gli2and signaling inthecorrespondingmesenchymeviapatched(Ptch1)/ expressed inthedistalepithelium,fromwhereitdiffuses toactivate ultimately influenceFgfsignalingintheepithelium.Shhishighly may alterlevels ordistribution of originating fromepithelialcellsatthetipsofnascentbuds, which Development 133(9) initiates. A earliest stages,but itisnotpresentintheepitheliumuntilbranching studied. Bmp ligandspresentintheembryoniclung,Bmp4isbest Wnt signalinginfluencelungbranchingmorphogenesis.Amongthe There have beenapparentlyconflictingreports abouthow Bmpand branching? Bmp andWntsignaling:positiveornegativeregulators of appears later, oncethebud iselongating.Inmesenchyme-free lung induced intheepitheliumofE11.5 lungsduringbud initiationbut highest indistalbuds, near morphogenesis show that Weaver etal.,2000).AnalysesofE11.5 lungsundergoing branching supported bythefollowing observations (Lebecheetal.,1999; of distalbuds tolimit Fgf10-mediated bud outgrowth. Thismodel is during branching,Bmp4isinducedandactivated intheepithelium mice (Winnier etal.,1995).Ithasbeenpreviously proposedthat, unclear, inpartbecauseoftheearlyembryonic deathof epithelium) andinaparacrinefashion (inthemesenchyme). signaling canbeactivated bothin an autocrinefashion (inthe is notcurrentlyavailable. Thepatternsabove suggestthatBmp4 detailed analysisofBmpr2distribution intheearlyembryonic lung of theembryoniclung(Belluscietal.,1996;Chen2005). A Smad1 proteinarepresentbothintheepitheliumandmesenchyme The Bmp4receptor(typeI,orAlk3),andtheBmptransducing proximal mesenchyme(Weaver etal.,1999;Weaver etal.,2003). Bmp4 significance ofthisfindingremainstobedetermined.ByE11-12, region asearlyE9(Weaver etal.,1999).Thebiological of al., 2003). repression of inhibited becauseofincreasedShhactivity andthenearlycomplete Bud formationcanbealsocontrolledbydiffusible signals The preciseroleofBmp4inthedeveloping lunginvivo remains Bmp4 transcripts arefoundinthedistallungepitheliumand Bmp4 in theventral foregut mesodermattheprospective lung Bmp4 Fgf10 Fgf10 is transcribedinthelungmesenchymefromits Fgf10 lacZ -expressing mesenchyme,thuslimitingfurther expression inthedeveloping lung(Chuang et reporter mousereveals astrikingdistribution expression isunknown. Onepotentialway Bmp4 Shh Fgf10- Fgf10 expression inthelungepithelium is -null mice, Fgf10 expressing cells. Hhip1 expression asabud grows in themesenchyme,and -null mice,branchingis Fgf10 transcripts are Fgf10 Bmp4 Bmp4 in the Hhip1 Fgf10 Fgf10 is not -null transcripts and where the responsive reportermouse(TOPGAL), inwhich Bmp4 an autocrineoraparacrinemechanism(Fig.3B).Regulation of branching would dependonwhetherBmpsignalingisactivated via buds (Braggetal.,2001).Inthisway, negative or positive effects on mesenchymal signalthatenhancesproliferationofdistalepithelial fashion). Bmp4,then,inducesacurrentlyunidentifieddistal also activate Bmpsignalingintheadjacentmesenchyme(paracrine the intactlung,however, Bmp4presentinthedistalepitheliummay fashion, proliferationisinhibited(but seeEblaghieetal.,2006).In Bmp4 signalingisactivated intheepitheliumanautocrine influences theabilityofepitheliumtorespondBmp4.When negative effects inthelung.Themodelpredictsthatmesenchyme been proposedtoexplain how Bmp4canhave bothpositive and branching isenhanced(Braggetal.,2001).Analternative modelhas lung explants inwhichtheepitheliumandmesenchymearepresent, budding inthesecultures. expression, whilerecombinantBmp4inhibitstheFgf10-mediated epithelial cultures,recombinantFgf10inducesbudding and part, fromfailure toinduceproperlevels of branching morphogenesis.Thedefectappearstoresult,atleastin distal lungbuds fromformingandmarkedly interfereswith 1in vivo, prevents targeted expression of theWntinhibitordickkopf signaling atthesesitesbytargeted deletionof Hogan, 2004;DeLangheetal.,2005).DisruptionofcanonicalWnt lung epithelium,thesitesthatareactively branching(Okuboand epithelium. However, nuclear-localized In theE11-13lung, Wnt targets (NelsonandNusse,2004;DasGuptaFuchs,1999). of nucleartranslocated Activation ofcanonicalWntsignalingcanbemonitoredbydetection 1996; Lako etal.,1998;ZakinTebar etal.,2001). wntwindow.html) arepresentinthedeveloping lung(Belluscietal., Tcf/Lef transcriptionfactors (seehttp://www.stanford.edu/~rnusse/ components oftheWntcanonicalpathway, suchas also beendebated.Several Wntligands,frizzledreceptorsand present inthemesenchyme(Quagginetal.,1999). as Wnt(seebelow), Fgf10andPod1(Tcf21),atranscriptional factor Indeed, related toWntsignaling,maybealsoaffected (Deanetal.,2005). canonical signaling,other although al., 2003;Shuet2005).Ithasbeenpointedout,however, that epithelium whereWnt/ morpholino and that thisdiscrepancy in resultsisduetothefact thatinthe Fgf10 and mesenchyme)(Lietal.,2002; Deanetal.,2005).Inbothcases, gene (anon-canonicalWnt,normally presentinthelungepithelium catenin; theotherisageneticmodel inwhichmicelackthe explants weretreatedwith morpholinooligonucleotidesagainst consequence ofdisruptedWntsignaling.Inonemodel, lung models thatshow increasedbranchingmorphogenesisasa both itsWntandnon-Wntfunctions(Deanetal.,2005). deleted fromtheepitheliumcouldhave resultedfromchangesto branching defectreportedinthemodelswhich bound formthatregulates celladhesion.Thus,itispossiblethatthe Paradoxically, whenrecombinantBmp4isadministeredtointact The roleofWntsignalinginlungbranchingmorphogenesishas Interestingly, theresultsabove conflictwiththatoftwo other expression was locally increasedintheselungs.Itispossible in theepitheliumiscomplex anddependentonsignalssuch ␤ ␤ -catenin isalsofoundincellmembranesacadherin- ␤ -catenin deletionisagoodmethodfordisruptingallWnt -catenin-Lef1/Tcf complex activates thetranscriptionof lacZ Wnt5 ␤ -TOPGAL expression areincreasedinthedistal -catenin isexpressed throughouttheentirelung a models,Wnt signaling was inactivated in ␤ -catenin signalingisinhibited(Mucenskiet ␤ -catenin, andbyanalysisofaWnt ␤ -catenin functions,notnecessarily Fgfr2b ␤ -catenin, Tcf/Lef lacZ ␤ REVIEW in thedistallung -catenin, orby ␤ ␤ -catenin, and -catenin was is expressed Wnt5a Bmp4 1617 ␤ -

DEVELOPMENT the lung(seebelow). Wnt arerequiredfortheestablishmentofdistalepithelialcellfate in epithelium andmesenchyme.Inaddition,bothBmp4canonical taking intoaccounttheoverall balanceofthesesignalsinthe Bmp4 andWntsignalingduringbranching,theimportanceof clarify theseissues. have distinctfunctions(Fig.3C,E,F).Furtherstudiesarerequiredto non-canonical Wnts(andeven different Wntfamily members)may both theepitheliumandmesenchyme.Inaddition,canonical 1618 2001). Thepresence oflungdefectsthatresemble thosefrom lobes, amongotherabnormalities (Limetal.,2002;Mahlapuu and displayalteredlungbud orientationandectopicbudding inthese right lung.Thesemiceshow low levels of budding selectively inthecranial,middleandaccessorylobes of the suggests thatFoxf1 is required forpropergeneexpression and stages. However, analysisof the mesenchymeofbothlungsfromearliestdevelopmental For example, thetranscriptionfactor regulate bud formationatspecificlocationsinthedeveloping lung. Branching patternscanbefurtherrefinedbymechanisms that andpreventingRefining patterning ectopicbudding epithelium, while (Heine etal.,1990). components collagenI,IIIandfibronectinarealsopresent stalks andinregions inbetweenbuds, where extracellular matrix (Lebeche etal.,1999).However Tgfb1proteinaccumulatesin to theepithelium,withoutanobvious proximodistal gradient morphogenesis, (reviewed byMassague,2000).Duringlungbranching effects theseTgfbproteinsdiffer onlyintheintensityoftheir of in thedeveloping mouselung.Many ofthebiologicalactivities Tgfbr2) andtransducingproteinsSmad2Smad3,areexpressed morphogenesis. TheseTgfbligands,theirreceptors(Tgfbr1and also beenimplicatedinthecontroloflungbranching Tgfb1, Tgfb2andTgfb3,membersoftheTgfbsubfamily, have morphogenesis Tgfb signalingasanegativeregulator ofbranching affected bythelackof or mesenchyme andpleura(Peltonetal.,1991).Analysisof et al.,1996).Interestingly, transgenic micemisexpressing 1994; Zhaoetal.,1996).Thiseffect hasbeenalsoreportedin differentiation inculturedmouseembryoniclungs(Serraetal., et al.,1997;Kaartinen1995). 3B). extracellular matrixcomponentsandprevent budding locally(Fig. lung.Atthesesites,Tgfb1couldalsoinducesynthesisof the the mesenchymeofbud stalksorinmoreproximal regions of be partofamechanismthatprevents al., 1999;Tomlinson et al.,2004).Inthedeveloping lung,Tgfb1may markedly inhibits indicate thatactivation ofTgfb1signalinginmesenchymalcells Studies inNIH3T3fibroblasts,lungandprostateorgan cultures phenotype mayhave beenrescuedbymaternal transferofTgfb1. lung morphogenesishasbeendebated,asthereisevidence thatthe syndrome (Letterioetal.,1994).WhetherTgfb1isdispensablefor structural lungdefectanddieperinatallyofadiffuse inflammatory The discussioninthissectionunderscoresthecomplexity ofthe Exogenous Tgfb1inhibitsbranchingmorphogenesis,growth and Tgfb3 REVIEW -null micereveals thatlungbranchingmorphogenesisis Tgfb1 Tgfb3 Fgf10 Tgfb3 is transcribedinthemesenchymeadjacent is presentintheproximallungepithelium, Tgfb2 expression (Beeretal.,1997;Lebeche , but notby Tgfb1 Foxf1 Tgfb1 is expressed inthedistallung -null miceshow noobvious heterozygous miceatE10-11 Fgf10 Foxf1 in thedistalepithelium(Zhou Tgfb2 Fgf10 from beingexpressed in is expressed throughout deficiency (Sanford and Gli3 mRNAs, Tgfb2 Gli3 - - branching morphogenesisiscurrentlyunknown. Whether Tgfbirestrictsbud formationinstalksduringlung molecules, suchasfibronectinandcollagen(Billingsetal.,2002). is stillunclear(Kimetal.,2003).Tgfbibindstoavariety ofmatrix mediate Tgfbresponsesinsomecellsystemsbyamechanismthat signaling viaGli3(Fig.3C). null mutantssuggestageneticinteractionbetween negative required forbranching,asshown byexpression ofadominant- both aredownstream oftheFgfr2bpathway, they donotseemtobe respectively), have beenidentifiedinthedistalepithelium.Although mouse lung,two Etsfamily members,Pea3andErm (Etv4 andEtv5, factor (Samakovlis etal.,1996;Sutherland1996).IntheE11.5 pointed epithelial migration.Thisisinpartpromotedbytheinduction of filopodial extensions atthetipofprimarybranches,leadingto Drosophila, bud formationandofthetargets ofFgf10inthisprocess.In There isstilllittleunderstandingofthecellularmechanismslung formation? What isdownstream of Fgf signalingduringlungbud Lu etal.,2004). proliferation (Serraetal.,1994;Zhao1996;Heine1990; deposition ofextracellular matrixanddecreasedepithelial cell mesenchyme, atsitestraditionallyassociatedwithTgfb-induced interbud regions ofthedeveloping lung,ispresentinthe families ofgenes(Stricklandetal.,2006). inactivation ofmorethanonememberthesefunctionallyrelated axon guidancemoleculesinthelungwillrequiresimultaneous gland ductalmorphogenesissuggeststhatdecipheringtheroleof demonstrating thatSlit2andnetrin1actsynergistically inmammary branching morphogenesis(Hinck,2004).Recentevidence proteins) inmicehasnotresultedobvious defectsinlung axon guidancemolecules,suchassemaphorins(orSlitandRobo been confirmedinvivo, asgeneticinactivation ofnetrinsorother Fgf signaling(Liuetal.,2004).Thishypothesis,however, hasnot from formingintheseregion byinterferingwithlocalactivation of branching morphogenesis,netrinexpression instalksprevents buds al., 2004).Theseobservations favor thehypothesisthat,during bud formationinmesenchyme-freelungepithelialexplants (Liuet exogenous netrininhibitsFgf-mediatedErkphosphorylationand presentinthelungepitheliumatthesestages.Moreover, are and theirreceptorsUnc5bDcc(deletedincolorectalcancer) or and netrin4,theextracellular matrixproteinTgfbinduced( (Fig. 3B).Thisisthecaseofaxonguidancemoleculesnetrin1 tubules inapatternthatsuggestsrolepreventing localbudding 2004). Shh signalingthroughShh-dependentprocessingofGli3(Lietal., [ traditionally associatedwith:cell rearrangementandcellmigration stages ofbudding arecharacterizedbyupregulation ofgenes mesenchyme-free lungepithelialbuds hasshown thattheinitial transducer 2),but notcellproliferation (Luetal.,2005a).This hormone-sensitive lipase( inflammatory processes(annexins); lipidmetabolism[ Notch signalingantagonist and metastaticbehavior ( Tm4sf3 Tgfbi Other genesaredynamicallyexpressed atthestalksofbranching A microarray-basedscreentoidentifyFgf10targets in ßig-h3 , anothermoleculedynamicallyexpressed instalksand in ( , transmembrane4superfamily member3( Erm Spdef ) intheE11-13mouselung.Thereisevidence that netrins Fgf signalingactivation causesdynamicactin-based targeted tothelunginvivo (Liuetal.,2003). os eoeIfrais anEtstranscription – MouseGenomeInformatics) Tgfbi is known tobeinducedbyTgfb1and Tacstd2 Lipe Numb Foxf1 )]; proteolysis(cathepsinH, , tumor-associated calcium signal ; and expression isinpartregulated by Lmo7 , LIMdomainonly7]; Development 133(9) Foxf1 Tspan8 and Shh Timp3 ); the Tgfbi HSL ); ,

DEVELOPMENT probably signaling viaFgfr1c,actsasatrophic factor forthedistal pulmonary hypoplasia.Fgf9(also presentinthedistalepithelium), 2000). Miceinwhichthesepathways have beeninactivated have activities (Colvin et al.,2001;BraggMalpel molecules suchas Indeed, mesothelialcellsofthepleura express anumberofsignaling differentiation status ofthedistalmesenchyme(Weaver etal.,2003). been proposedthatthepleuracontrolsproliferationand the smooth muscledifferentiation (Fig.3E)(Wan etal.,2005).Ithas expression andresultsindisruptedbranchingmorphogenesis 2004; DelMoraletal.,2006). interactions duringbranchingmorphogenesis(Pauling andVu, aspects ofvascular development, andforepithelial-endothelial severely hypoplastic(Shuetal.,2002).Vegf iscrucial forseveral defective vascular smoothmuscledifferentiation andarealso (Pepicelli etal.,1998). hypoplasia, aconditioncharacterizedbyabnormalsmalllungs proliferation andsmoothmuscledifferentiation, andsevere others. Lungsfrom Development 133(9) lung epitheliumoftransgenicmiceinhibits and mesenchymal programsbyregulating thelevels ofexpression of epithelium andhave beenshown toinfluencebothepithelialand signals forthemesenchyme,suchas et al.,2003).Epithelialbuds areasourceoftrophicordifferentiation proliferating multipotentprogenitorswhilethelunggrows (Weaver concert,appeartomaintainabalanceofdifferentiated and in from theepitheliumandpleura(Fig.3E,Box1,Table 1)that, Lung mesenchymedevelopment iscruciallyinfluencedbysignals Integrating epithelialandpleuralsignals themesenchyme Patterning budding (Nogawa etal.,1998). changes inproliferationarenotthetriggeringevent thatinitiateslung observation isconsistentwithprevious studiesthatshow local once theseprogenitor cellsare locatedalongproximal airways.Thismodelissupportedbyanalysisofan u ugot lrewiearw )ado yatv eecya elmgain(ml ht ros ) ( bud outgrowth (largewhitearrow, B)and/orbyactivemesenchymalcellmigration(smallwhitearrows, B). ( isexposedtohighBmp4(inducedintheepitheliumbyFgf10)andShhlevelsthatdiffusein turn, from thetipsofgrowing buds figure) (Mailleuxetal.,2005;Weaver etal.,2003).( distal signalsinfluencedifferentiation ofaproximal mesenchymalderivative?Amodelhasbeenproposed toresolve thisapparen undergoes aprogram ofdifferentiation thatinitiallydependsonShhandBmp4,signalsexpressed mostlybydistalepithelialbu al., 2001).Interestingly, airwaysmoothmuscleappearspartlytooriginatefrom apoolofprogenitor cellsinthedistallung and primarylungbudsispresent atE11andcontinuestoformaround proximal, butnotdistal,epithelialtubulesasbranching Airway smoothmusclestartstoformrelatively earlyinthedevelopingmouselung.Asmoothmusclelayeradjacenttoepithe Box 1.Myogenesisandbudmorphogenesis Fgf10 signaling-mediated inductionoffibronectin (Yang etal.,2000;DeLanghe2005;Jakkaraju2005). muscle celldifferentiation alsodependsonphysicalstretch, serumresponse factor, laminin2,tension-inducedproteins sucha in whichlowFgf10levelsresult inreduced The transcriptionfactors B Wnt7b ) Thistriggersamyogenicprogram indistalmesenchymalcellsthatcontinueswhiletheyare relocated tomore proximal regions -expressing distalmesenchymalcellsandairwaysmoothmuscle(Mailleuxetal.,2005).Furthermore, ahypomorphic . Theconditionaldeletionof Fgf9, Tgfb3 Shh -null miceshow inhibitedmesenchymalcell Wnt7b Foxa1 and RA,whichcanmediatethese -null mousemutantsalsohave and Shh, Vegf Foxa2 Foxa1 Bmp4 are presentinthelung and and expression andepithelialbranching,showeddecreased smoothmuscledifferentiation. Smooth Foxa2 A Shh ) Initially, lungepithelialbuds(blue)are inducedbyFgf10from thedistalmesenchyme,which, Wnt7b and in thedistal , among Wnt7b Shh Generating celldiversity (Wilson etal.,1953). hypoplasia hasbeenalsoassociatedwithdisruptionofRAsignaling expansion (Lavine etal.,2005).Asin activates anFgfpathway thatisessentialforcardiomyoblast mesothelial cellsoftheepicardiuminduces 2000; Colvinetal.,1999).Inthedeveloping heart,RAfrom expression isregulated byRAinmesothelialcells(Malpeletal., agtn oiatngtv m eetr( targeting adominant-negative Bmpreceptor are crucial.WhenBmpsignalingisinhibitedintransgenicmice by distal signalingcenterinwhichBmp4andWntcanonical epithelium aslungbuds formandbranch,seems todependona is establishedalongtheproximodistalaxisofrespiratory cells thataretypicalofthedistalalveolar region of thelung.Cellfate proximal regions oftherespiratorysystem,andtypeIII including ciliated,neuroendocrineandsecretorycellspresent in The maturerespiratoryepitheliumconsistsofmultiplecelltypes, epithelial differentiation Mechanisms influencingproximal-distal cellfateand is co-expressed with into smoothmuscleinvitro(Weaver etal.,2003). Fgf9 prevents Shh-induceddifferentiation ofthelungmesenchyme overall toinducenormalepithelialbranching(Colvinetal.,2001). mesenchymal cells,whichresultsinless mesenchyme. 2001). Thus,agradient ofBmp4andWntsignaling, withthehighest epithelium (Weaver etal.,1999;Mucenski etal.,2003;Lu results fromthetargeted deletionof a proximalphenotypeandstop branching.Proximalizationalso Bmp signalingmakes lung epithelialcellsfromdistalbuds acquire In themodelproposedby authors ofthesestudies,disrupted lung becomes‘proximalized’(Weaver etal.,1999;Lu2001). development ofthedistalepitheliumisseverely impairedandthe tothedistallungepithelium, antagonists (noggin,gremlin) The RAsynthesizingenzyme Fgf9 mutants have amarkedly reducednumberof Fgf9 in thepleura,anditispossiblethat Fgf10 C ) Themyogenicprogram completes Raldh2 lacZ reporter mouse,whichlabels ␤ -catenin inthedistallung (Niederreither etal.,1999) Fgf10 Fgf9 Fgf10 lium ofthetrachea s Tip1 andonWnt s Tip1 ds. Howcanthese mesenchyme that proceeds (Tollet et (red region, A-C). Fgf9 -null mice,lung dnAlk6 REVIEW mouse mutant, being available t paradox(see bycontinued , whichthen ) orBmp 1619 Fgf9

DEVELOPMENT successful targeting ofthetransgeneto Although itisnotclearexactly whenthetransgenestartstoact, genes involved intheestablishmentofintestinalcell lineages. a hyperproliferative epitheliumthatexpresses Remarkably, theselungslackdifferentiated lung celltypesandshow ␤ of thisprogramtotake placeinthedistallung. toallowprogram, but subsequentstages laterithastobeturned‘off’ switch. RAisinitially‘on’toactivate anearlydevelopmental Thus, inthedeveloping lung,RAseemstoactasadevelopmental differentiation andremainimmature(Wongtrakool etal.,2003). result, distallungprogenitorsarepresentbut donotundergo further in thedistalepithelium,asduringearlydevelopmental stages.Asa Interestingly, inthetransgenic modelabove, RAsignalingpersists secondary budding andbranchinginitiates(Malpeletal.,2000). bud formation,but isdownregulated intheepitheliumonce al., 2003).EndogenousRAsignalingisactive duringprimarylung epithelium throughoutbranchingmorphogenesis(Wongtrakool et in whichRAsignalingisconstitutively activated inthedistallung required toformciliatedcells(Chenetal.,1998). proximal lung,theforkheadboxtranscriptionfactor of theepitheliumintospecificcelltypes.For example, inthe subsequent stages,othermoleculescontribute tothedifferentiation distal cellsfromassumingaproximalphenotype(Fig.3F).At levels ofactivation indistalepithelialbuds, isthoughttoprevent 1620 vivo intransgenic miceharboringa et al.,2001).Lungepithelialreprogramminghasalsobeenshown in of thelungepithelium(Linetal.,2003;Hyatt2004;Ohtsuka molecules thathave beenimplicatedinthevitroreprogramming proteins, collagenXVIII,Wnt2,Shhandtransferrinaresomeof the specific informationthatradicallychangesepithelialcellfate. Fgf local inductive signalsfromthemesenchyme confernovel position- Shannon, 1994).Theseobservations areconsistentwiththeideathat present intrachealoruretericprogenitorcells(Linetal.,2003; specific patternofbranchinganddifferentiation thatisnotnormally mesenchyme hasbeendemonstratedbytheinductionofalung- Reprogramming oftrachealepitheliumoruretericbud bylung vitro. documented intissuerecombinationexperiments in type oramountofsignalsinthelocalenvironment. Thishasbeen embarked onaspecificlineagepathway, simplybychangingthe developmental programscanbealteredincellsthathave initially There isaccumulatedevidence invitroandvivo that Cell plasticity:reprogramming cellfates and shows thatWntcontrolslevels of canonical signalinginmutantmicecausesasimilarlungphenotype premature differentiation (Okuboetal.,2005).InhibitionofWnt of mutantmiceinhibitsdistallungproliferationandinduces Targeted disruptionof cells thatexpresses highlevels of theproto-oncogene in thedistallung,asapopulationofproliferatingimmatureepithelial cells forcontinuedgrowth. Ithasbeenproposedthatthispoolresides is crucialtomaintainandexpand apoolofuncommitted progenitor As epithelialcellsinbranchingairways continuetodifferentiate, it Maintaining progenitor cells whiledifferentiating al., 2002;Bhushanet2001;Norgaard etal.,2003). pool ofepithelialprogenitorcellsduringorganogenesis (Haradaet tooth, skinandthelungmayberequiredtoexpand or maintaina activation ofFgf10andFgfr2bsignalinginthedeveloping pancreas, the distallung(Shuetal.,2005)(Fig.3F).Thereisalsoevidence that ctnnLffusionproteinconstruct (OkuboandHogan,2004). -catenin/Lef Disrupted distallungdevelopment isalsoseenintransgenicmice REVIEW Mycn expression indistallungepithelialcells Sfptc- Mycn driven constitutively active and Sftpc Cdx1, Atoh1 Bmp4 -expressing cells expression in and other Foxj1 Mycn is . mechanisms bywhichthelungdevelops. these issueswillprovide insightsintothemolecularand cellular niches inthedeveloping lung?How canthey beidentified?Tackling debated issueofstemcells.Whatarethesecells?Wheretheir such asFgf10,areestablishedinthelung.Finally, thereisthemuch coordinates thatsetupthethree-dimensionalpatternofmorphogens, array ofmarkers, whichmaybealreadyavailable. These questionsrequirepowerful imageanalysissystemsandan Fgf10 activates Fgfr2b. How docellsrearrangetoformnew buds? in themilieuaroundnascentlungbud andinthebud itselfwhen functional studies. targeting genestotheseearlyprogenitorsintheforegut infuture progenitor cells.Alsocrucialwillbethedevelopment oftoolsfor the developing foregut willbeuseful tofindothermarkers oflung microdissection approachesanddetailedgeneprofilinganalysisof issue isthatthisgenealsoexpressed bythethyroid.Laser capture only earlymarker currentlyavailable forthesecells.Aconfounding be identifiedpriortotheemergence ofprimarybuds, clear gapofknowledge inthefield.Because (Izvolsky etal.,2003;Shannon2003). mammalian lung,but themechanismsremaintobeunderstood et al.,2001).Thereisevidence thatthisisalso trueforthe essential forFgfsignalingandtrachealmorphogenesis(Kamimura systems. In organogenesis hasbeenwelldocumentedinseveral developing modulators ofgrowth factor distribution andsignalingin crucial roleforheparanandchondroitinsulfate proteoglycansas mechanisms (Luetal.,2005b;Harris2006;Li2004).A methylation, endogenousmicroRNAs andproteolysis,amongother activation ofthesemoleculesiscontrolled;includegene gained byexploring thedifferent ways bywhichexpression or molecules (Fgfs,Tgfb,Shh,Wntproteins).Novel insightswillbe morphogenesis arecontrolledbyarelatively limitedgroupof Overall, thestudiessofar suggestthatthemajorevents inearlylung Conclusion primitive foregut, remainstobeinvestigated. signaling influencestheinitialspecificationoflungfieldin change theirfate intoanintestinalsecretorycellfate. WhetherWnt suggests thatincreasedWntsignalingleadslungprogenitorcellsto Aubin, J.,Lemieux,M.,Tremblay, M.,Berard, J.andJeannotte,L. Ang, S.L.andRossant,J. References required fordistallungepithelialcellproliferationandsurvival. genetic evidence thatBmpr1amediatesanautocrinesignaling A recentreportbyEblaghieetal.(Eblaghieal.,2006)provides Note addedinproof manuscript. Theauthorsresearch is,inpart,supportedbyNIH/NHLBI. cited here. We are gratefultoBrigidHoganforcriticalreading ofthe Wongtrakool andSarahMalpel) forvaluableinsightsandsomeofthework (Felicia Chen,KonstantinIzvolsky, Jeff Sedita,Tushar Desai,Cherry discussions. We alsothank present andformermembersofthelaboratory We thankMarkKrasnow, forhelpful JerryBrody andMaryWilliams Avilion, A.A.,Nicolis,S.K.,Pevny, L.H.,Perez, N.andLovell- L.,Vivian, formation inmousedevelopment. defects. Early postnatallethalityinHoxa-5mutant miceisattributabletorespiratory tract on SOX2function. Badge, R. A rathermorecomplex problemistheunderstandingof how the Still relatively littleisknown aboutthechangesthatareinduced The lackofearlymarkers oflungprogenitorcellsrepresentsa Dev. Biol. (2003). Multipotent celllineagesinearlymousedevelopment depend Drosophila 192 Genes Dev , 432-445. (1994). HNF-3betaisessentialfornode andnotochord , integrity andpropersulfation ofheparanare . 17 , 126-140. Cell 78 , 561-574. Sftpc Development 133(9) expression cannot Nkx2.1 (1997). is the

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