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Early Events in Valvulogenesis:A Signaling Perspective

Joey V. Barnett* and Jay S. Desgrosellier

INTRODUCTIONThe continuing identification of themolecular cues that direct early val-vulogenesis furthers our under-standing of the biology behindvalve formation, and suggests pos-sible causes for congenital defectsin humans. Congenital heart de-fects represent the most commoncause of birth defects in humans,and occur at a rate of almost 1 in100 live births (Hoffman, 1990;Hoffman and Kaplan, 2002). Themajority of these defects includeabnormalities of valve formation.Heart defects are responsible forthe largest proportion of deaths dueto birth defects in the first year oflife, and remain a major cause ofdeath during childhood (Gallowayet al., 1999). Although congenitalheart disease is a major cause ofmorbidity and mortality in children,

over one-half million adults havecongenital heart disease in theUnited States (Foster, 1995). Inaddition, valvular injury in adultscan occur as a result of infection(Mylonakis and Calderwood, 2001),pharmacologic therapy (Connollyet al., 1997), or tumorigenesis (Ro-biolio et al., 1995). An understand-ing of the molecular cues that guidevalvulogenesis offers the possibilityto detect and repair valvular de-fects in embryos, children, andadults.

VALVULOGENESISAfter specification of myocardialand endothelial cell precursors atgastrulation, the cardiogenic fieldsform the cardiac crescent at thecranial end of the embryo. Theheart primordia become paired oneach side of the midline of the em-

bryo and form incomplete musculartubes surrounding an endothelialtube. These paired tubesmerge in acranial-to-caudal fashion to formthe tubular heart (Fig. 1A). Cardiaclooping brings the atrial region ofthe common heart tube into a posi-tion superior to the common ventri-cle. In the region between the com-mon atria and ventricle, and in thedistal aspect of the common out-flow tract, localized swellings of theinner heart wall arise due to pro-duction of extracellular matrix, andare termed the atrioventricular(AV) and outflow tract (OFT) cush-ions, respectively. Subsequent car-diac morphogenesis consists of theconversion of this simple tubularstructure, made up of two concen-tric cylinders of epithelia separatedby a gel-like matrix termed the car-diac jelly, into the adult organ. Acritical step in this process involvesthe transformation of a subpopula-tion of cells from the inner endocar-dial cell layer into mesenchymalcells that invade the cardiac jelly(Fig. 1B), and later contribute tothe connective tissue of the valvesand septa of the adult heart (Fig.1C) (Sadler, 1985). Transformationoccurs at the AV boundary to initi-ate formation of the mitral and tri-cuspid valves, and somewhat laterin the OFT to initiate aortic and pul-monary valve formation. This im-portant event is dependent on sol-uble factors derived from themyocardium that diffuse through

The proper formation and function of the vertebrate heart requires amultitude of specific cell and tissue interactions. These interactions drivethe early specification and assembly of components of the cardiovascularsystem that lead to a functioning system before the attainment of thedefinitive cardiac and vascular structures seen in the adult. Many of theseadult structures are hypothesized to require both proper molecular andphysical cues to form correctly. Unlike any other organ system in theembryo, the cardiovascular system requires concurrent function andformation for the embryo to survive. An example of this complexinteraction between molecular and physical cues is the formation of thevalves of the heart. Both molecular cues that regulate cell transformation,migration, and extracellular matrix deposition, and physical cuesemanating from the beating heart, as well as hemodynamic forces, arerequired for valvulogenesis. This review will focus on molecules andemerging pathways that guide early events in valvulogenesis. Birth De-fects Research (Part C) 69:58–72, 2003. © 2003 Wiley-Liss, Inc.

Joey V. Barnett and Jay S. Desgrosellier are from Department of Pharmacology, Vanderbilt University Medical Center, Nashville,Tennessee.

Grant sponsor: National Institutes of Health; Grant sponsor: American Heart Association.

*Correspondence to: Joey V. Barnett, Ph.D., Department of Pharmacology, Vanderbilt University Medical Center, Room 476 RobinsonResearch Building, 2220 Pierce Avenue, Nashville, TN 37232-6600. E-mail: [email protected]

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.10006

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WBirth Defects Research (Part C) 69:58–72 (2003)

© 2003 Wiley-Liss, Inc.

the cardiac jelly to reach the endo-cardium. After migration into thecardiac jelly mesenchymal cellsalign themselves in multiple layersresulting in the expansion of thecushion crests toward each other(Eisenberg and Markwald, 1995).

In the early heart tube, the AVand OFT cushions function as prim-itive valves, ensuring directionalblood flow. The crests of the oppos-ing cushions eventually contact andfuse, creating a partial separationbetween the atria and ventricles.This fusion leaves laterally placedconnections between the atria andventricles, thus initiating right andleft heart septation (Sadler, 1985).Cushion tissue also contributes tothe formation of the septa of theadult heart. The ventricular septumis composed of both AV cushion-de-rived membranous and non-cush-ion muscular components. As theprimitive ventricles expand due togrowth and trabeculation of themyocardium, the medial walls of thedeveloping ventricles fuse, formingthe muscular septum. An outgrowthof tissue from the inferior AV cushionfuses with the muscular septum toform the membranous septum. De-fects in the membranous septum

usually result from aberrations inAV cushion transformation (Sadler,1985; Keller and Markwald, 1998).The atrial septum is derived fromatrial muscle, and requires cushiontissue only in the most posterior as-pects. Therefore, the majority ofatrial septal defects are not due toabnormalities in the AV cushion,but in the formation of the primaryor secondary septa.

Septation of the OFT is a complexprocess involving coordination ofheart tube morphogenesis, epithe-lial–mesenchymal transformation(EMT), and ingrowth of neural crestcells. The origin of the various celltypes involved in OFT septation,and the relative contributions ofpreexisting structures and in-grow-ing cells, are controversial (Nodenet al., 1995). Cushion tissue in-growth, compaction of non-cardiacmesenchyme, and neural crest cellsall contribute to a complex spiralseparation of the truncus. Properheart tube looping is also critical, asleftward looped hearts often havetransposition of the aorta and pul-monary artery due to improper ori-entation of the heart tube relativeto the normal direction of septation,not a direct defect in the septation

process (Creazzo et al., 1998;Kirby, 2002). The unique cell con-tributions found in the OFT suggestthat additional molecules are re-quired for OFT cushion morphogen-esis that are not involved in the AVcushion.

Mesenchymal tissue derived fromthe fused AV cushions serves as ru-dimentary valves before definitivemitral and tricuspid valve forma-tion. Cushion mesenchyme com-prises only a small fraction of themature valves, the bulk of whichare derived from muscle. The pre-sumptive valves are eroded fromthe ventricular myocardial wall. Asthe valves are eroded, long thinstrands of muscle remain attachedto the valve tissue on one end, andthickened trabecular outpocketingsof the myocardium on the other(Fig. 2). These strands and special-ized trabeculae become the chor-dae tendonae and papillary mus-cles of the mature valve (Fig. 3).Although much of the later mecha-nism of valve erosion and matura-tion remains poorly understood, itis clear that AV cushion mesen-chyme is critical to the process ofvalve formation (Sadler, 1985;Keller and Markwald, 1998). Our

Figure 1. Development from tubular to four-chambered heart.A: Tubular heart.B: Looped heart with well-formed atrioventricular andoutflow tract cushions seeded with mesenchymal cells. A-atrium, V-ventricle. C: Adult four-chambered heart depicting AV and OFTvalves in the left ventricle. Ao-aorta, LA-left atrium, LV-left ventricle, RV-right ventricle, P-papillary muscle. In each depiction,myocardium is red, atrial region is yellow, ventricular region is brown, mesenchymal cells are blue, and outflow region is white. Valvularregions are orange.

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Birth Defects Research (Part C) 69:58–72, (2003)

discussion will focus on moleculesthat are involved in generating andregulating AV cushion mesen-chyme.

AV cushion transformation hasbeen studied extensively in aviansystems using an in vitro assay inwhich the AV cushion is excised andplaced on a collagen gel. The myo-cardium compacts and beats,whereas the endothelial cells form

a monolayer on the surface of thegel. If the culture is allowed toprogress undisturbed, some endo-thelial cells undergo EMT and enterthe collagen gel. Transformationcan be divided into three stepsbased on gross cellular morphol-ogy. Monolayer endothelial cellsseparate from the endothelial sheetand elongate in a step termed acti-vation. Next, these elongated mes-

enchymal cells enter the underlyingcardiac jelly, a step termed inva-sion. Finally, cells move throughthe gel in the migration step. Thus,these three steps, activation, inva-sion, and migration, constitute EMT(Fig. 4). Transformation is quanti-tated by measuring mesenchymeproduction, i.e., counting the num-ber of cells that enter the gel, or bydetermining the migration rate ofindividual cells in the gel. Studies inthe chick have demonstrated thatthe ability of endothelial cells to un-dergo EMT is tightly restricted inthe developing heart. If AV cushionmyocardium is replaced with ven-tricular myocardium, EMT fails tooccur. In the obverse experimentusing ventricular explants, ventric-ular myocardium is removed andreplaced with inductive AV cushionmyocardium and EMT fails to occur.Therefore, there is restriction ofboth the endothelial cell populationthat transforms and restriction ofthe myocardial cell population thatsignals EMT. AV cushion explantshave been used to identify mole-cules that play a role in transforma-tion. Members of the transforminggrowth factor � (TGF�) family havebeen shown to play a significantrole in stimulating EMT. TGF�, incombination with ventricular myo-cardium, can induce EMT in AV en-docardial cells. Further, neutraliz-ing antisera (Potts and Runyan,1989) or antisense oligonucleotides(Potts et al., 1991) to TGF� ligands

Figure 2. Stylized depiction of AV cushion remodeling intomature valves. Looped heart is depicted with the AV cushion region exposed.A:Well formed AV cushion separates atrium (A) and ventricle (V).B: Erosion of cushion tissue begins. C: Valve leaflets are taking shapeand are attached to developing papillary muscles in the ventricle by chordae tendonae.D:Mature valve anchored via chordae tendonaeto papillary muscle.

Figure 3. Adult human heart AV valves. Left side mitral valve and right side tricuspidvalves are evident. Chordae tendonae anchor valve leaflets to papillary muscles in theventricles. RA-right atrium, LA-left atrium, RV-right ventricle, LV-left ventricle, P-papil-lary muscle. Modified with permission of Anderson and Becker, Cardiac Anatomy: AnIntegrated Text and Color Atlas. London: Grower Medical Publishing, 1980.

60 BARNETT AND DESGROSELLIER


inhibit transformation. These ob-servations established TGF� as aleading candidate molecule regu-lating transformation in the AVcushion.

TGF� SIGNALING IN THEAV CUSHION

TGF� controls cell growth and dif-ferentiation, regulating processesas diverse as development, woundhealing, atherosclerosis, and tumorprogression (Jessell and Melton,1992). The TGF� family is composedof three 25-kDa homodimeric pro-teins: TGF�1, TGF�2, and TGF�3.TGF�1 and TGF�3 share identical li-gand binding and biological activitieswhereas TGF�2 has distinct biologi-cal activities (Roberts and Sporn,1990; Sanford et al., 1997; Hu etal., 1998). Activation of ligand re-quires proteolytic cleavage of thesecreted precursor, and dissocia-tion of the C-terminal portion fromthe latency-associated protein(Gentry and Nash, 1990). The re-leased C-terminal fragment bindsto specific receptors on the cell sur-face.

Four cell surface proteins dividedinto two classes bind TGF� with pi-comolar affinity. The first class con-sists of two transmembrane serine/threonine kinase receptors termedthe type I TGF� receptor (TBRI) and

the type II TGF� receptor (TBRII)(Lin et al., 1992; Ebner et al.,1993b; Bassing et al., 1994). TBRIIhas a constitutively active cytoplas-mic kinase domain and an extracel-lular domain that binds TGF�1 andTGF�3 with high affinity (Lin andMoustakas, 1994). TBRII has a lowaffinity for binding TGF�2, althougha splice variant of TBRII, TBRII-B,has been described that bindsTGF�2 with high affinity (Rotzer etal., 2001). Ligand binding results inTBRII phosphorylating TBRI andsubsequent activation of TBRI ki-nase activity (Wrana et al., 1994).TBRIs activate a signaling cas-cade, known to include the Smadfamily of transcription factors(Kretzschmar and Massague, 1998),that results in specific cellular re-sponses. A number of type I recep-tors, termed “activin receptor-likekinases (ALKs),” are capable of in-teracting with TBRII. ALK5, in asso-ciation with TBRII, activates PAI-1expression (ten Dijke et al., 1994a)via TGF� response elements in thePAI-1 promoter (Bassing et al.,1994). Formation of an ALK5/TBRIIcomplex and subsequent phos-phorylation of ALK5 by TBRII, me-diates growth arrest (Wrana et al.,1994). Based on the effects of TGF�on mink lung epithelial cells, thesedata establish ALK5 as the proto-typic TBRI. ALK2 is capable of inter-

acting with TBRII (Attisano et al.,1993; Ebner et al., 1993b), as wellas activin and bone morphogeneticprotein (BMP) type II receptors (At-tisano et al., 1993; Ebner et al.,1993a; ten Dijke et al., 1994b; Ya-mashita et al., 1995; Macias-Silvaet al., 1998). ALK2 does not medi-ate TGF� signaling in mink lung ep-ithelial cells, but has been postu-lated to play a role in TGF�-mediated EMT in the mammarygland (Miettinen et al., 1994). Ex-periments carried out in a mam-mary epithelial cell line indicateALK5 is absent, but ALK2 ispresent. When ALK2 is targeted byantisense or dominant negative re-ceptor constructs, the TGF�-in-duced progression of the mammarycells from an epithelial to a mesen-chymal morphology is inhibited.The regulation of TGF� receptorsignaling by selective interactionswith ALKs is an intriguing mecha-nism to explain the pleiotropic ef-fects of TGF�.

Smads are downstream of ALKsand mediate many of the effects ofreceptor activation (Massague etal., 1997). The Smad family in ver-tebrates can be divided into threeclasses. The first class are receptor-activated Smads that are phos-phorylated directly by specificALKs. For example, Smad2 andSmad3 are phosphorylated by ALK5and Smad1, Smad5, and Smad8are phosphorylated by ALK1 (chenand Massague, 1999), ALK2 (Ma-cias-Silva et al., 1998), and ALK3(Lux et al., 1999). Receptor-acti-vated Smads share a phosphoryla-tion domain containing an SSXSmotif that is absent in the otherSmad family members (Heldin et al.,1997; Kretzschmar and Massague,1998). The second group contains asingle member, Smad4, termed thecommon mediator Smad, which in-teracts with phosphorylated recep-tor-activated Smads to form an ac-tive complex that is translocated tothe nucleus to modulate transcrip-tion. There are also two inhibitorySmads, Smad6 and Smad7 (Ima-mura et al., 1997; Nakao et al.,1997). Smad6 selectively inhibitssignaling through Smad1 by com-peting directly with Smad4 forbinding to phosphorylated Smad1,

Figure 4. In vitro AV cushion collagen gel assay. Stage 18 chick embryo with the AVcushion region demarcated is shown. AV cushion explants placed on a collagen gel forman endothelial cell monolayer and a portion of the endothelial cells undergo EMT. Stagesof transformation are indicated.



and has little or no effect on Smad2(Hata et al., 1998). Smad7, how-ever, inhibits signaling of all recep-tor-activated Smads throughmechanisms that include competi-tion for Smad binding sites on ALKs(Nakao et al., 1997).

The second class of TGF� bindingproteins contains two transmem-brane proteins termed the type IIITGF� receptor (TBRIII), or Betagly-can, and endoglin. Both contain ashort, highly conserved intracellu-lar tail with no apparent signalingfunction (Lopez-Casillas et al.,1991; Wang et al., 1991; Cheifetzet al., 1992). A single PDZ protein–protein interaction domain found inTBRIII was demonstrated to regu-late receptor trafficking mediatedby GTPase-activated protein for G�isubunits interacting protein C-ter-minus (GIPC) (Blobe et al., 2001).TBRIII binds all three TGF� iso-forms, as well as inhibin (Lewis etal., 2000), and has been proposedto function in ligand presentation tothe TBRII/TBRI complex (Lopez-Casillas et al., 1991, 1993, 1994).Although the TBRII/TBRI complexbinds TGF�1 and TGF�3 with highaffinity, this complex binds TGF�2poorly (Lopez-Casillas et al., 1991).The addition of TBRIII to the TBRII/TBRI signaling complex allows forthe binding of TGF�2 with picomo-lar affinity. Recently, we have dem-onstrated that TBRIII is essentialfor AV cushion transformation, thefirst step in heart valve formation.These data supply the first biologi-cal role for TBRIII and suggest theexistence of a unique signalingpathway in the heart (Brown et al.,1999a). Endoglin binds TGF�1 andTGF�3 in the presence of TBRII(Cheifetz et al., 1992; Barbara etal., 1999), and is expressed at highlevels by endothelial cells (Gougosand Letarte, 1988, 1990; Gougos etal., 1992). A role for endoglin inTGF� signal transduction is unclear,but mutations in the endoglin geneare linked to human hereditaryhemorrhagic telangiectasia (HHT)(McAllister et al., 1994). The bio-logical significance of the forms ofTGF� and how they may interactdifferentially with TGF� receptorsduring embryonic development islargely unknown.

All three TGF�s have been identi-fied in cardiac tissue. In situ hybrid-ization studies demonstrated TGF�2expression in chick heart AV cush-ion and OFT during EMT (Dickson etal., 1993; Barnett et al., 1994).Studies of active ligand in the chickdemonstrate a correlation betweenlevels of active TGF�2 and EMT (Mc-Cormick, 2001). Active TGF�3 ispresent in relatively low levels dur-ing EMT (Ghosh and Brauer, 1996).In the mouse, cardiac mesenchy-mal induction begins around E9.0and TGF�1 expression is restrictedto endocardial cells that contributeto mesenchymal cushion tissue.TGF�2 is restricted to the myocar-dial cells of the AV canal and OFT,which underlie the mesenchymalcushion, and is expressed onlytransiently. By E12.5, septation isnearly complete, and myocardialexpression of TGF�2 is absent(Dickson et al., 1993). TGF�3 is ex-pressed only later in mouse cardiacdevelopment (Letterio et al., 1994),and studies of EMT using mouse AVcushion explants revealed a re-quirement for TGF�2 but not TGF�3(Camenisch et al., 2002a). Thecomplex patterns of TGF� ligandexpression in the heart and thedemonstration of a functional rolefor TGF� ligands in mediating AVcushion transformation, suggestthat this peptide growth factor fam-ily plays an important role in valvu-logenesis.

Targeted inactivation of genesencoding TGF� ligands revealedspecific roles for each in embryo-genesis. Mice lacking either tgf�1 ortgf�3 do not exhibit cardiac defects.Disruption of tgf�1 is associatedwith specific immune defects (Shullet al., 1992) and defects in vascu-logenesis (Dickson et al., 1995).Cleft palate and a delay in lung de-velopment are reported in tgf�3null mice (Kaartinen et al., 1995).Because TGF�1 and TGF�3 arepharmacologically identical anddisplay overlapping patterns of ex-pression, functional redundancy inthe single null animals is likely.Therefore, signaling by TGF�1 orTGF�3 in cardiovascular develop-ment is not excluded by the pheno-type of the single null animals. Par-tially penetrant cardiac defects,

including, double inlet left ventricle,double outlet right ventricle, atrialand ventricular septal defects, andfailure of muscularization of theOFT, are evident in tgf�2 null mice(Sanford et al., 1997; Bartram etal., 2001). Further, a failure of en-docardial cushion volume to de-crease, and hyperplastic valve leaf-lets were noted in some animals.Transformation does occur, but thespecific defects noted suggest a re-quirement of TGF�2 for proper re-modeling of the cushions into val-vular and septal tissue. Therefore,data from expression studies, cush-ion explant studies in chick andmouse, and targeted gene inactiva-tion in the mouse all suggest a rolefor TGF�2 in valvulogenesis.

To date, inactivation of the genesencoding TBRII, ALK5, and ALK2have been uninformative, becauseeach results in early embryonic le-thality (Oshima et al., 1996; Gu etal., 1999; Larsson et al., 2001). En-doglin is expressed in endothelialand mesenchymal cells in bothmouse and chick (St.-Jacques etal., 1994; Vincent et al., 1998).Targeted disruption of Endoglin re-sults in numerous vascular defects,as well as defects in trabeculationand EMT in the heart (Bourdeau etal., 1999). Targeting of TBRIII hasnot been reported. Thus, the role ofspecific TGF� receptors in cushiontransformation and valvulogenesisis not revealed by these studies inthe mouse.

Other members of the TGF� su-perfamily, including activins andBMPs, are expressed in the heart.Activin �A is expressed in mesen-chymal cells within the AV cushion(Moore et al., 1998). Addition of ac-tivin �A to AV cushion endocardiuminduced transformation, and anti-sense oligonucleotides to activin �Ainhibited transformation, suggest-ing a role for activin �A in AV cush-ion transformation in the chick. Inboth the AV cushion and OFT,BMP2, 4, 5, 6, and 7 are expressedin the myocardium (Lyons et al.,1990, 1995; Jones et al., 1991;Eisenberg and Markwald, 1995;Solloway and Robertson, 1999).BMP2-deficient mice have cardiacdefects that include failure of theheart to form, formation of the



heart outside of the coelom, and re-tardation of cardiac development(Zhang and Bradley, 1996). De-fects in the AV cushion were notedin Bmp2 null mice, but these maybe secondary to the primary defectin cardiogenesis. In an in vitromodel of AV cushion transformationantisense oligonucleotides to BMP2inhibited EMT (Yamagishi et al.,1999). BMP2 alone was unable tostimulate EMT, although BMP2 en-hanced transformation in the pres-ence of TGF�3. Therefore, anessential role for BMP2 in transfor-mation remains to be demon-strated. Mice with a targeted dele-tion of Bmp4 die early in utero,making a cardiac phenotype impos-sible to discern (Winnier et al.,1995). However, overexpression ofnoggin, a soluble inhibitor of BMP2and BMP4, in chick embryos yieldsa spectrum of OFT defects that in-cludes cushion hypoplasia, doubleoutlet right ventricle, truncus arte-riosus, and ventricular septal de-fects (Allen et al., 2001). In miceBMP5 is expressed in the myocar-dium beginning at E8.5, and per-sists through at least E10.5 (Sollo-way and Robertson, 1999). BMP6 isexpressed in themyocardium of theOFT at E9.5 (Jones et al., 1991;Dudley and Robertson, 1997), andat E11.5 is abundant in AV cushionmesenchyme, but never observedin OFT mesenchyme (Kim et al.,2001). Expression is also observedin valve leaflets, cushion mesen-chyme of the aortico-pulmonaryseptum, and endothelial cells in theaortic and pulmonary trunks (Kimet al., 2001). BMP7 is expressedabundantly throughout the myo-cardium (Dudley and Robertson,1997; Solloway and Robertson,1999), and at E12.5 is evident inmesenchyme directly under thevalve leaflets (Kim et al., 2001).Mice with a targeted deletion ofBmp5, Bmp6, or Bmp7 lack cardiacdefects (Kingsley et al., 1992; Dud-ley and Robertson, 1997). Bmp5;Bmp7 double nulls demonstrate adelay in cardiac development, how-ever, accompanied by a lack of en-docardial cushion formation (Sollo-way and Robertson, 1999) andBmp6;Bmp7 double-nulls have de-layed OFT cushion morphogenesis

with less affected AV cushions (Kimet al., 2001).

At least two BMP receptors areimplicated in valvulogenesis. Micecarrying a hypomorphic allele ofBmpr2 exhibit persistent truncusarteriosus with absent semilunarvalves (Delot et al., 2003), al-though the AV valves appear nor-mal. Targeted deletion of Alk3, aBMP type I receptor, in myocardiumresults in hypoplastic AV cushionsand defects in the interventricularseptum and trabeculation. Thesedata suggest a requirement forALK3 in the crosstalk betweenmyo-cardium and endocardium (Gaussinet al., 2002). Of note, TGF�2 ex-pression was downregulated signif-icantly in Alk3-null mice. BMP fam-ily members are clearly importantin cardiogenesis and valvulogen-esis; however, a specific role forBMPs in AV cushion transformationremains to be elucidated.

A major focus of our laboratoryhas been to determine the TGF� re-ceptor complex that mediatestransformation in the AV cushion.Because AV cushion endothelialcells transform in response toTGF�, and adjacent ventricular en-dothelial cells do not, we chose tocompare receptor expression andfunction in these two populations.Endothelial cells throughout theembryo (Brown et al., 1999b), in-cluding AV cushion endocardialcells, express TBRII. Further, anti-TBRII antisera inhibit both mesen-chyme production and cell migra-tion in AV cushion explants (Brownet al., 1996). These data demon-strate that TBRII is required for AVcushion transformation, but TBRIIlocalization is not responsible forrestricting transformation to AVcushion endocardial cells. TBRIII isrestricted to specific endothelial cellpopulations in the embryo includingthose in the AV cushion and OFT.Anti-TBRIII antisera that blocked li-gand binding inhibited both mesen-chyme production and cell migra-tion in AV cushion explants. Thesedata implicated TBRIII in transfor-mation, and suggested that thelocalization of TBRIII restrictedtransformation to the AV cushion.To address this question, we usedretroviral vectors to misexpress

TBRIII in explanted ventricular en-docardial cells. The addition ofTGF�2 to infected ventricular ex-plants resulted in EMT (Brown etal., 1999a). Although ventricularendothelial cells express both TBRIIand TBRI, they do not transform inresponse to ligand. These data sug-gest that a unique and nonredun-dant signaling pathway is used byTBRIII to mediate transformation.Together, these data suggest that aminimal receptor complex of TBRIIand TBRIII is required for AVcushion transformation, and thatTGF�2 activates this complex.

Current models of TGF� signaltransduction require that a TBRI bea component of the TBRII/TBRIIIsignaling complex. Numerous ALKsare components of the activin, BMP,and TGF� signaling pathways (tenDijke et al., 2000). ALK5 mediatesgrowth arrest and PAI-1 production(ten Dijke et al., 1994a; Wrana etal., 1994). ALK2 interacts with ac-tivin (Ebner et al., 1993a), BMP(Macias-Silva et al., 1998), andTGF� receptors (Ebner et al.,1993a), and is a functional compo-nent of BMP7 signal transductioncomplexes (Macias-Silva et al.,1998). In breast epithelial cells,ALK2 mediates EMT in response toTGF� (Miettinen et al., 1994).Therefore, we chose to examine theroles of ALK2 and ALK5 in AV cush-ion transformation. Anti-ALK2 anti-sera inhibited mesenchymeproduction whereas neutralizingantisera to ALK5 was without effect(Lai et al., 2000). This suggeststhat ALK2, not ALK5, signals trans-formation. ALK2 may interact di-rectly with TBRII/TBRIII, or maysignal transformation via an alter-native pathway. The presence ofBMP ligands in the myocardium,and known interactions betweenALK2 and BMPs, raise the possibilitythat ALK2 may be involved in BMP-mediated signals that result intransformation. Therefore, TBRIIand TBRIII might lie in a pathwayparallel to a BMP pathway that ac-cesses ALK2 to modulate AV cush-ion transformation. We postulatethat TGF� may stimulate ALK2 inendothelial cells in the AV cushionand that TBRIII regulates signalingvia this pathway (Fig. 5).



This model of TGF� signaling viaan alternative TBRI, ALK2, is sup-ported by emerging data that TGF�may signal through a number of dif-ferent ALKs. For example, a similarmodel of receptor interactions withalternate ALKs is emerging with en-doglin. Endoglin is related closely toTBRIII and binds TGF�1 and TGF�3.Mutations in endoglin are found inthe majority of cases of HHT (McAl-lister et al., 1994), but mutationsalso have been noted in ALK1(Johnson et al., 1996), suggestingthat endoglin and ALK1 might inter-act on the surface of endothelialcells. Biochemical studies havedemonstrated a ligand-dependentactivation of ALK1 in the presenceof endoglin (Lux et al., 1999). Acti-vation of ALK1 with TGF�1 andTGF�3, but not TGF-�2, was consis-tent with the binding specificity ofendoglin. The addition of BMP2,BMP7, or activin A did not result inreceptor activation, supporting aspecific effect of TGF�. These datademonstrate that endoglin inter-acts with ALK1 in a TGF-�-depen-dent manner suggesting that en-doglin influences the composition of

TGF� signaling complexes. Similarto the phenotypes of mice deficientin TGF�1, TBRII, or endoglin, tar-geted disruption of Alk1 in themouse generates severe vascularabnormalities including excessivelylarge diameter vessels (Urness etal., 2000). Analysis of ALK1 in hu-man umbilical vein endothelial cells(HUVECs) revealed that ALK1 canbind TGF�1, activate Smad1 andSmad5, and inhibit Smad2 down-stream of ALK5 (Goumans et al.,2002). Differential activation ofALK1 and ALK5 is proposed to ex-plain the apparent contradictory ef-fects of TGF� as being both pro- andanti-angiogenic. TGF� at low dosesactivates ALK1 and subsequentlySmad1 and Smad5 to promote cellmigration and proliferation. TGF�

at high doses activates Smad2 viaALK5 to inhibit endothelial cell mi-gration and proliferation. There-fore, the balance of TGF� activationof ALK5 and ALK1 may determinethe specific endothelial cell re-sponse and angiogenic conse-quences. It is intriguing to postu-late that endoglin may play a role in

regulating the balance betweenthese two ALK signaling pathways.

Differential signaling of TGF� viadistinct ALKs is not restricted to en-dothelial cells. Recently, we havedemonstrated a switch in TGF� acti-vation of ALK2 and ALK5 during car-diomyocyte differentiation (Ward etal., 2002). Signaling of TGF� in myo-cytes before vagal innervation ispredominantly via ALK5, which stim-ulates G�i2 expression and para-sympathetic responsiveness. Asdevelopment proceeds, ALK2 lev-els increase markedly, and TGF�addition stimulates an ALK2 re-porter construct. ALK2 stimulationacts to decrease G�i2 expression.These data suggest that the bal-ance of ALK2 and ALK5 signalingin myocytes regulates parasym-pathetic responsiveness via ef-fects on G�i2 expression. WhetherTBRIII or endoglin is involvedin regulating TGF� activation ofALK5 and ALK2 is unknown.

Taken together, these data impli-cate TBRIII, TBRII, and ALK2 in AVcushion transformation. The dem-onstration of a role for TBRIII in AVcushion transformation is signifi-cant for two reasons. First, thesedata supply the first biological rolefor TBRIII. Second, the TBRIII geneis contained within a linkage regionfor AV cushion defects on humanchromosome 1 (Johnson et al.,1995; Sheffield et al., 1997), mak-ing TBRIII a candidate gene for thiscongenital defect in humans.

Little is known concerning thedownstream signaling pathwaysthat connect TGF� receptor activa-tion with altered gene expressionand transformation in the heart. Toaddress this issue, we tested ifSmads, specific downstream medi-ators of TGF� signaling, are in-volved in AV cushion transforma-tion. To define initially a role forSmads in transformation, we choseto overexpress Smad6, which spe-cifically inhibits Smad1 with limitedeffect on TGF� signaling throughSmad2 (Hata et al., 1998). Inhibi-tion of mesenchyme productionwas seen in AV cushion explantsthat overexpress Smad6 (Barnettand Desgrosellier, 2002). In bothchick and mouse, Smad6 isexpressed in the myocardium,

Figure 5. Possible TGF� signaling pathways in AV cushion endocardial cells. The ligandsTGF�1 or TGF�3 directly interact with TBRII/ALK5, and signal via Smad2 and Smad3. Arole for this well-characterized pathway is not supported. BMP ligands interact withBMPRII and ALKs, such as ALK2, to activate Smad 1, Smad5, or Smad8. ALK2 andSmads, inhibited by the Smad inhibitor Smad6, are implicated. TGF�2 binds to TBRIIIand may present ligand to the TBRII/ALK5 complex to augment signaling via this path-way. Alternatively, TBRIII/TBRII may activate ALK2. TBRIII may signal independent ofan ALK, as suggested by direct interaction of TBRIII with proteins such as GTPaseActivated Protein for G�i subunits-interacting protein C-terminus (GIPC).



endocardium, and cushion tissue(Yamada et al., 1999; Galvin et al.,2000). In the mouse, expressionbecomes restricted to the cardio-vascular system after birth, espe-cially endothelial and endocardialcells (Galvin et al., 2000). Smad6null mice have hyperplastic valves,and an increase in EMT in the devel-oping cushions was suggested toexplain this phenotype (Galvin etal., 2000). In our experiments, wedetermined a direct effect of Smad6on EMT. Because the Smad6 nullmouse results in valvular hyperpla-sia, whereas Smad6 overexpres-sion results in a decrease in EMT,this is consistent with Smad6 regu-lating EMT in the AV cushion.

Slug is a zinc finger protein be-longing to the Snail family of tran-scription factors that plays a role inEMT in both the neural crest andduring gastrulation. Slug is ex-pressed in AV cushion myocardium,endocardium, and transformedmesenchymal cells (Romano andRunyan, 1999). Slug expression inAV cushion endocardium is upregu-lated by an inductive signal producedonly by AV cushion myocardium.Further, antisense oligodeoxynucle-otides to Slug inhibited transforma-tion in AV cushion explant assays.Analysis of the molecules that mayinfluence Slug expression duringAV cushion transformation re-vealed that anti-TGF�2 antibody in-hibited Slug expression (Romanoand Runyan, 2000). Inhibition oftransformation caused by anti-TGF�2 antibody was rescued bySlug. These data suggest that Slugis downstream of TGF�2/TBRIIIsignaling.

Transformation is a multistepprocess involving activation, inva-sion, and migration through matrix.Studies have attempted to identifyspecific roles for TGF� ligands andreceptors in these steps. In explantassays, TGF�3 is upregulated bymesenchymal cells and has beenproposed to support cell migration(Potts et al., 1991; Boyer et al.,1999). This effect of TGF�3 may becomplemented by BMP signaling asdescribed earlier (Yamagishi et al.,1999). These studies in the chickare not supported by studies in themouse, where TGF�2 seems to play

a major role in explant assays (Ca-menisch et al., 2002a) and in thewhole animal (Sanford et al.,1997). Our observations in thechick support a major role forTGF�2, but do not rule out contri-butions by either TGF�1 or TGF�3.As noted earlier, the single nulltgf�1 or tgf�3mice do not elucidatethe role of either ligand in valvulo-genesis. In fact, TGF�2 and TGF�3have been suggested to mediatespecific steps in transformation andmodulate distinct genes in the chick(Boyer et al., 1999). The assign-ment of specific roles to each ligandin transformation and valvulogen-esis remains to be determined.

In summary, the identification ofunique TGF� receptor signalingcomponents contained in trans-forming versus non-transformingendocardial cells has allowed forthe identification of the first biolog-ical role of TBRIII, the identificationof TBRIII as a candidate gene formediating AV cushion defects, andimplicated ALK2 and downstreamSmad signaling pathways in AVcushion transformation.

ADDITIONAL MOLECULESINVOLVED INVALVULOGENESIS

Although much effort has focusedon the role of TGF� in EMT in the AVcushion and valvulogenesis, addi-tional molecules have been impli-cated in both processes. Analysis ofthese molecules suggests otherpathways regulate EMT and valvedevelopment that may or may notimpinge upon TGF� signaling. Inthe following section, we discusssome of these molecules with aneffort to integrate them into possi-ble pathways or relationships.Other molecules that may play arole in valve development are listedin Table 1. A major effort should bedirected at integrating these pres-ently disconnected molecules andpathways into a cogent picture ofsignaling in the developing valves.

Hyaluronic acid (HA) is a glycos-aminoglycan that is a prominentcomponent of cardiac jelly in E9.5mice. Production of HA in the heartis due entirely to HA synthase 2(Has2) (Camenisch et al., 2000).

Mice with a targeted deletion ofHas2 lack AV cushion transforma-tion, demonstrating a role for HA inthe transformation process (Cam-enisch et al., 2000). Addition of HAor constitutively active ras rescuedHas2 null AV cushion explants indi-cating that HA may act to signaltransformation by activating ras.Rescue of transformation by exog-enous HA could be prevented byaddition of a dominant negativeras, indicating further that ras liesdownstream of HA. Addition of adominant negative ras to wild-typeexplants recapitulated the Has2null phenotype by inhibiting EMT;however, unlike explants from theHas2 null, endothelial cells main-tained the ability to migrate on topof the collagen gel. These data sug-gest that HA produced by Has2 mayhave two functions during AV cush-ion transformation: to act as a sub-strate for endothelial cell migration,and to signal transformation via rasactivation.

HA may interact with other ma-trix molecules in the cardiac jelly.At least two other molecules in thecardiac jelly, versican and fibulins,have HA-binding domains. Thephenotype of Has2 null mice is verysimilar to mice lacking the extra-cellular matrix molecule versican(Mjaatvedt et al., 1998). Versican isa chondroitin sulfate proteoglycanexpressed in the heart in a patternsimilar to Has2. The similarities inthe phenotype of Has2- and versi-can-deficient mice, and the coex-pression of these molecules in theheart, suggest that versican may liein the same pathway as HA. Fibu-lin-2 is expressed in the AV cushion(Zhang et al., 1995), and can bindto versican (Olin et al., 2001), aswell as another extracellular matrixmolecule, perlecan (Fig. 6) (Hopf etal., 1999). Perlecan-deficient miceexhibit cardiac defects includingcomplete transposition of the greatarteries and hyperplastic semilunarvalves (Costell et al., 2002). There-fore, loss of HA, versican, or perle-can can alter valvulogenesis. Addi-tional data to support a role forHA in AV cushion transformationcomes from zebrafish. The jekyllmutation is characterized by a lackof myocardial proliferation and hy-



poplastic endocardial cushions. Thegene responsible for the jekyll phe-notype was identified recently asUDP-glucose dehydrogenase (UDP-GD) (Walsh and Stainer, 2001). Be-cause UDP-GD is a required compo-nent of HA synthesis, the phenotypeobserved may be related to a defectin HA production.

Further insight into the molecularpathway through which HA signalsAV cushion transformation comesfrom studies of the ErbB family ofreceptors. Both ErbB2 and ErbB3 areexpressed in the endocardium andinvading mesenchyme of mouse AVcushions during transformation, andmice deficient in either ErbB2 orErbB3 produce few transformed cells(Camenisch et al., 2002b). Phos-phorylation of ErbB2 and ErbB3 re-sults in activation of ras, and is de-pendent upon HA because neitherreceptor is phosphorylated in Has2

null mice. Addition of HA or heregu-lin, the ligand for ErbB3, restoresphosphorylation of both ErbB2 andErbB3 in Has2 null AV cushion ex-plants. Because ErbB2 can only bephosphorylated when present in aheterodimer with other ErbB recep-tors, these data suggest that anErbB2/ErbB3 heterodimer is respon-sible for mediating the effects of HAon AV cushion transformation. An-tagonists to ErbB2 and ErbB3 inhibittransformation in wild-type AV cush-ion explants, further supporting arole for these receptors. HA does notseem to directly stimulate the ErbBreceptors. Rather, HA may act tomodulate signaling by other growthfactors such as heregulin. The proto-typical receptor for HA, CD44, is notrequired for transformation, thus it isnot known how HA leads to phos-phorylation of ErbB2 and ErbB3 andactivation of ras.

The importance of ras activationin AV cushion transformation is em-phasized further by the activity ofneurofibromin. Neurofibromin isexpressed in the AV cushion endo-cardium near the end of transfor-mation, E11.5 in the mouse. Inmice lacking the gene for neurofi-bromin, Nf1, there is an abundanceof mesenchymal cells in the cardiacjelly of the AV cushion resulting inhyperplastic valves (Lakkis and Ep-stein, 1998). Normally, trans-formed cells undergo proliferationand apoptosis that lead to an opti-mal number of cells needed to forma valve. Mice that lack Nf1, how-ever, have enhanced proliferationand decreased apoptosis of trans-formed cells within the AV cush-ions. These results could explainthe enhanced cell number observedin AV cushions from Nf1 null mice.Neurofibromin is known to act as aras GTPase-activating protein, andthus can downregulate ras signal-ing. Dominant negative ras inhib-ited transformation in both Nf1 nulland wild-type AV cushion explants.Constitutively active ras, however,stimulated transformation in wild-type explants and recapitulated thephenotype observed in Nf1 null ex-plants.

These experiments demonstratethat ras activation is regulated neg-atively by neurofibromin, suggest-ing that precise regulation of rasactivity is required for transforma-tion to occur properly. Further,these data support a role for HA insignaling AV cushion transforma-tion by ErbB2 and ErbB3 phosphor-ylation and subsequent activationof ras (Fig. 6). Whether TGF� is in-volved in the HA pathway is cur-rently unknown. Studies haveshown that TGF� is capable of sig-naling through activation of ras(Hartsough and Mulder, 1997). Thissuggests that TGF� may crosstalkwith ErbB receptor signaling at thelevel of ras activation to influenceAV cushion transformation.

A requirement for urokinaseduring AV cushion transformationin vitro was demonstrated usingurokinase-neutralizing antisera thatsignificantly decreased the rate ofcell migration in transformed cellswith no effect on cell number (Gorny

TABLE 1. Other Molecules Implicated in Valvulogenesis

Molecule Expression pattern or phenotype

EGF receptor/Shp2 Hyperplastic semilunar valves (Chen et al., 2000)FKBP12 Expressed in endocardium; treatment with FK506

causes enlargement of the heart (Obata et al.,2001)

bFGF (FGF4) Myocardial expression decreases while expressionin endocardium increases from Stages 9–15 inthe chick (Choy et al., 1996)

Smooth muscle alphaactin

Expressed in AV cushion mesenchymal cells(Nakajima et al., 1997)

Fibrillin-2 Labels endocardium and mesenchyme in AVcushions of chick embryos (Rongish et al., 1998)

Periostin Labels only mesenchymal cells within outflow tractand AV cushion; expression is induced by TGF�and BMPs (Kruzynska-Frejtag et al., 2001)

Pitx2 Outflow and AV cushion defects (Liu et al., 2002)GATA4 Mice with a mutant GATA4 unable to bind Fog-2

have semilunar valve defects, double-outlet rightventricle, ventricular septal defects and acommon AV valve, similar to phenotype of Fog-2null mice (Crispino et al., 2001)

Fog-2 Multi-zinc finger protein that interacts with GATA4and represses GATA-dependent transcription.Mice deficient for Fog-2 have tricuspid atresiaamong other defects described for GATA4deficient mouse (Svensson et al., 2000; Tevosianet al., 2000)

Nkx2-5 Nkx2-5 deficient mice exhibit an enhancedfrequency of atrial septal defects compared towild-type mice, supporting the finding thatmutations in Nkx2-5 in humans may beresponsible for familial atrial septal defects(Biben et al., 2000)

Msx2 A homeobox gene downstream of Pax3 that isrequired for proper migration of neural crest(Kwang et al., 2002)



and Brauer, 1999). Urokinase inhi-bition by phosphatidylinositol-spe-cific PLC, which causes release ofurokinase from the cell surface,or antisense oligonucleotides de-creased cell outgrowth and motilityin AV cushion and OFT explants aswell as cultured endocardium-de-rived mesenchymal cells (McGuireand Alexander, 1993a). These datademonstrate that urokinase activitymodulates transformation. Endocar-dial-derived cells in culture exposedto fibronectin upregulated urokinaseexpression and increased cell motil-ity, showing that urokinase ex-pression is regulated in responseto extracellular matrix composition(McGuire and Alexander, 1993b).The transcription factors Ets-1(Wernert et al., 1992) and Ets-2(Majka and McGuire, 1997) havebeen shown to alter urokinase ex-pression. Ets-2 is expressed in cush-ion mesenchyme, binds to the uroki-nase promoter, and deletion of theEts-2 binding sites in the promotermarkedly decreases urokinase ex-pression (Majka andMcGuire, 1997).These data suggest that Ets-2 regu-lates urokinase expression in AV

cushion and OFT mesenchyme. He-patocyte growth factor (HGF) hasalso been implicated in regulatingthe expression of urokinase. HGF up-regulates urokinase in AV cushionendocardiumandmesenchymal cells(Song et al., 1999). Further, activeHGF is detected during transforma-tion, and the HGF receptor is ex-pressed by both AV cushion endo-cardial cells and mesenchyme.HGF activates Ets-1 (Paumelle etal., 2002), so it is possible that theeffects of HGF on urokinase expres-sion are mediated by members ofthe Ets family of transcription fac-tors. Additionally, urokinase is re-quired for cleavage of pro-HGF toits active form, thereby providing apotential positive feedback loop forHGF-mediated urokinase expres-sion. A well-known proteolytic func-tion of urokinase is to convert plas-minogen to plasmin; however, thisconversion is not required for trans-formation (Gorny and Brauer,1999). Therefore, the mechanismby which urokinase regulates EMTin the AV cushion is unclear.

Matrix metalloproteinase-2(MMP-2) is a proenzyme present in

the endocardium of both the OFTand AV cushions as well as cushionmesenchyme (Alexander et al.,1997; Cai et al., 2000). MMP-2 de-grades extracellular matrix to sup-port cell migration and tissue re-modeling. Although MMP-2 mRNAlevels exhibit a small increase dur-ing AV cushion transformation, lev-els of membrane-type matrix met-alloproteinase (MT-MMP), whichproteolytically cleaves pro-MMP-2to yield active MMP-2, increasemarkedly (Alexander et al., 1997).Endogenous tissue inhibitors ofMMPs (TIMPs), are expressed alsoin the AV cushion, suggesting thatMMP regulation may be importantduring valvulogenesis (Brauer andCai, 2002). During EMT, MMP-2 andMT-MMP are the only MMPs presentin the developing quail heart (Alex-ander et al., 1997). Functional ex-periments show that MMP inhibitorsblock transformed cells from mi-grating in intact quail embryos(Song et al., 2000). Because onlyMMP-2 and MT-MMP are expressedin the heart, these data implicatethese two MMPs in cushion trans-formation. The expression of MMPsmay be important in allowing cellsto migrate on specific substrates.The ability of AV cushion explantsto transform on a type IV collagenmatrix was impaired in the pres-ence of MMP inhibitors (Song et al.,2000). Type IV collagen is presentin cardiac jelly. Therefore, the ex-pression of MMPs may be requiredto degrade type IV collagen and al-low cell migration. Further data de-fining a role for MMPs in AV cushiontransformation come from studiesof the platelet-derived growth fac-tor (PDGF) receptor. Patch micelacking the � subunit of the PDGFreceptor die in utero from incom-plete heart septation and heartvalve abnormalities. These miceare deficient in MMP-2 and MT-MMP(Robbins et al., 1999). The additionof PDGF-AA to branchial arch ex-plants from wild-type animals up-regulated MMP-2, indicating thatPDGF can regulate MMP expression.MMP-2 is also expressed in thevalves at Day 7 of chick heart de-velopment, indicating that MMP-2may be involved in valve develop-

Figure 6. Hyaluronic acid and ErbB signaling in the AV cushion. Enzymes essential forhyaluronic acid (HA) synthesis include UDP-glucose dehydrogenase and hyaluronic acidsynthase 2 (Has2). HA released into the cardiac jelly may bind to versican or fibulin2 inthe matrix. Versican, fibulin, and perlecan may interact as depicted. HA stimulatesErbB2/ErbB3 phosphorylation by an unknown mechanism leading to ras activation andsubsequent transformation. Neurofibromin negatively regulates ras. Regulation of rasactivity by molecules such as ErbB and neurofibromin modulates EMT. Gray arrowsindicate potential interactions based on the presence of predicted HA binding domains.



ment post-EMT (Alexander et al.,1997).

The composition of the extracel-lular matrix in which cells migratealso seems to be critical for properAV cushion transformation. Thegenes encoding collagen VI �1 and�2 chains are found within the re-gion of chromosome 21 thought tobe critical for defects in patientswith trisomy 21 (Down Syndrome)(Klewer et al., 1998). One defectassociated with trisomy 21 is ab-normal development of the valves.Both chains of type VI collagen arefound in mouse hearts beginning atE11.0–11.5. The transcripts areboth localized in the AV cushion andare expressed predominantly bytransformed cells at the interfacebetween the endocardium and thecardiac jelly. Beyond E13.0, colla-gen VI chains are expressed on theventricular side of the developingvalve leaflets in the fibrous com-pact tissue layer. Skin fibroblastsfrom trisomy 21 patients displayedan increased adhesion to collagenVI compared to cells from euploidindividuals (Jongewaard et al.,2002). This has been suggested toexplain the valve defects associ-ated with trisomy 21 patients.Transformed cells in the AV cushionmay have an increased adhesive-ness to the cardiac jelly that altersmigration. The binding of cells tocollagen VI is dependent largely on�1 integrins, and activation of �1integrins increases the adhesive-ness of euploid cells with no effecton trisomy 21 cells. Therefore, itseems that euploid skin fibroblastspossess a �1 integrin receptor sig-naling pathway lacking in trisomy21 cells.

Retinoic acid (RA) treatment ofE9.0, 9.5, and 10.0 mouse embryosresulted in cardiac malformationsthat included ventricular septal de-fects, double outlet right ventricle,and transposition of the great arter-ies (Davis and Sadler, 1981). BothOFT and AV cushions were reducedin size with fewer mesenchymalcells. Proliferation of mesenchymalcells and glycosaminoglycan produc-tion in the cardiac jelly were de-creased in RA-treated embryos,suggesting that RA blocks transfor-mation by decreasing the substrate

required for cell migration or by in-hibiting cell proliferation (Davis andSadler, 1981). Treatment of mouseE9.5 OFT and AV cushion explantswith RA reduced transformation,with the OFT much more sensitiveto RA treatment than the AV cush-ion (Nakajima et al., 1996). RA alsoreduced production of extracellularmatrix proteins such as fibronectinand type I collagen. Mice deficientin the retinoic acid receptor RXR-�have both OFT and AV cushion de-fects including hypoplastic cushionsand cleft mitral and tricuspid valves(Gruber et al., 1996). These micealso have increased apoptosis inthe OFT cushions whereas prolifer-ation is unchanged. TGF�2 was up-regulated in the hearts of the RXR-�null mice (Kubalak et al., 2002).Addition of TGF�2 to E11.5 mouseembryos increased apoptosis inboth the OFT and AV cushions andresulted in OFT and aortic sac de-fects consistent with the RXR-�null mouse. Embryos deficient inRXR� and heterozygous null fortgf�2 exhibit a partial restorationof apoptosis levels in the cush-ions, and rescue of the OFT andaortic sac malformations. Thesedata support a role for TGF�2 inregulating apoptosis in the devel-oping endocardial cushions down-stream of RA.

NF-ATc, a transcription factorregulated by calcium signaling, ispresent in endothelial cells of theheart, and plays a role in endocar-dial cushion formation. Expressionof NF-ATc in the heart is most abun-dant in the endocardium of the OFTand AV cushions, and mice deficientin NF-ATc lack semilunar valves (dela Pompa et al., 1998; Ranger etal., 1998). At present, it is unclearwhether there are defects observedin the mitral or tricuspid valves.Transformation occurs normallywithin the OFT cushions of null miceindicating that NF-ATc plays no rolein transformation. Because nuclearlocalization of NF-ATc is dependentupon the activation of the calcium-sensitive phosphatase calcineurin,the defects observed in NF-ATc-de-ficient mice suggest that calciumregulation may be important in val-vulogenesis. Connexins act as gapjunctions to allow the flow of small

molecules and ions, such as cal-cium, between adjacent cells. Arole for connexins in heart develop-ment, other than myocardial con-traction, has been elucidated re-cently. Connexin45 is expressedthroughout the myocardium andendocardium of E9.5 mouse em-bryos, and is upregulated in the en-docardium of the AV cushion andOFT during transformation (Kumaiet al., 2000). Connexin45 defi-cient mice form cardiac jelly, butthe cells do not undergo EMT.Measurement of NF-ATc in con-nexin45-deficient mice showed adecrease in levels of active NF-ATc1 compared to wild-type miceindicating a defect in Ca2�/cal-cineurin signaling. This is consis-tent with observations that cal-cium levels increase in the AVcushion during EMT (Runyan etal., 1990). A dependence of activeNF-ATc levels on functional con-nexin45 suggests that NF-ATcmay be acting downstream of con-nexin45 to regulate valve forma-tion.

Although much of this review hasfocused on molecules that stimu-late or support transformation, theinhibition of transformation also isnecessary for proper cushion mor-phogenesis. Transformation in theAV cushion is tightly regulated, witha defined number of cells enteringthe matrix. Therefore, a number ofsignals must be present that limittransformation. One such signaldescribed earlier is Smad6. A sec-ond negative regulator that mayhave physiological relevance isVEGF (Dor et al., 2001). VascularEndothelial Growth Factor (VEGF) isexpressed throughout the myocar-dium of the developing heart, but isupregulated locally in the myocar-dium of the AV cushion soon aftertransformation ends. Endocardialcells express VEGF receptors 1 and2, and both are downregulatedupon transformation. Experimentsusing AV cushion explants demon-strated that addition of exogenousVEGF inhibits transformation. Thesame result was achieved by induc-ing premature expression of VEGFin the developing myocardium ei-ther by expression of a transgeneor by hypoxia. The inhibition of



transformation caused by hypoxiawas reversed by adding a solubleVEGF receptor that acts as an an-tagonist. How VEGF signals endo-cardial cells to maintain their phe-notype remains unknown.

CONCLUSIONS

Much has been learned concerningthe signaling molecules that regu-late EMT in the AV cushion ofthe developing heart. Transgenicmouse models have complementedexperimental embryology in thisregard and have extended thescope of these studies to identifymolecules that regulate valvulo-genesis post EMT. Major issues re-maining to be resolved include theintegration of molecules into path-ways that regulate valvulogenesis,an understanding of the cellularand molecular events that controlremodeling of the cushion into ma-ture valves, and how both of theseprocesses are related to physicalforces generated by heart beat andblood flow during valvulogenesis.

ACKNOWLEDGMENTSWe thank H. Olivey, L. Manderfield,and members of the Barnett Labora-tory for critical reading of the manu-script. J.S.D. was supported by aPharmaceutical Research and Manu-facturers of America Foundation Pre-doctoral Fellowship. J.V.B. wishes toacknowledge the support of the Na-tional Institutes of Health, AmericanHeart Association, and March ofDimes for past and present support.

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