Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning

Cardiac cushion development provides a valuable system to investigate epithelial to mesenchymal transition (EMT), a fundamental process in development and tumor progression. In the atrioventricular (AV) canal, endocardial cells lining the heart respond to a myocardial-derived signal, undergo EMT, and contribute to cushion mesenchyme. Here, we inactivated bone morphogenetic protein 2 (Bmp2) in the AV myocardium of mice. We show that Bmp2 has three functions in the AV canal: to enhance formation of the cardiac jelly, to induce endocardial EMT and to pattern the AV myocardium. Bmp2 is required for myocardial expression of Has2, a crucial component of the cardiac jelly matrix. During EMT, Bmp2 promotes expression of the basic helix-loop-helix factor Twist1, previously implicated in EMT in cancer metastases, and the homeobox genes Msx1 and Msx2. Deletion of the Bmp type 1A receptor, Bmpr1a, in endocardium also resulted in failed cushion formation, indicating that Bmp2 signals directly to cushion-forming endocardium to induce EMT. Lastly, we show that Bmp2 mutants failed to specify the AV myocardium with loss of Tbx2 expression uncovering a myocardial, planar signaling function for Bmp2. Our data indicate that Bmp2 has a crucial role in coordinating multiple aspects of AV canal morphogenesis.     

Essential functions of Alk3 during AV cushion morphogenesis in mouse embryonic hearts

Accumulated evidence has suggested that BMP pathways play critical roles during mammalian cardiogenesis and impairment of BMP signaling may contribute to human congenital heart diseases (CHDs), which are the leading cause of infant morbidity and mortality. Alk3 encodes a BMP specific type I receptor expressed in mouse embryonic hearts. To reveal functions of Alk3 during atrioventricular (AV) cushion morphogenesis and to overcome the early lethality of Alk3(-/-) embryos, we applied a Cre/loxp approach to specifically inactivate Alk3 in the endothelium/endocardium. Our studies showed that endocardial depletion of Alk3 severely impairs epithelium-mesenchymal-transformation (EMT) in the atrioventricular canal (AVC) region; the number of mesenchymal cells formed in Tie1-Cre;Alk3(loxp/loxp) embryos was reduced to only approximately 20% of the normal level from both in vivo section studies and in vitro explant assays. We showed, for the first time, that in addition to its functions on mesenchyme formation, Alk3 is also required for the normal growth/survival of AV cushion mesenchymal cells. Functions of Alk3 are accomplished through regulating expression/activation/subcellular localization of multiple downstream genes including Smads and cell-cycle regulators. Taken together, our study supports the notion that Alk3-mediated BMP signaling in AV endocardial/mesenchymal cells plays a central role during cushion morphogenesis.     

Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.     

Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice

Transformation of endocardial endothelial cells into invasive mesenchyme is a critical antecedent of cardiac cushion tissue formation. The message for bone morphogenetic protein (BMP)-2 is known to be expressed in myocardial cells in a manner consistent with the segmental pattern of cushion formation [Development 109(1990) 833]. In the present work, we localized BMP-2 protein in atrioventricular (AV) myocardium in mice at embryonic day (ED) 8.5 (12 somite stage) before the onset of AV mesenchymal cell formation at ED 9.5. BMP-2 protein expression was absent from ventricular myocardium throughout the stages examined. After cellularization of the AV cushion at ED 10.5, myocardial BMP-2 protein expression was diminished in AV myocardium, whereas cushion mesenchymal cells started expressing BMP protein. Expression of BMP-2 in cushion mesenchyme persisted during later stages of development, ED 13.5-16, during valuvulogenesis. Intense expression of BMP-2 persisted in the valve tissue in adult mice. Based on the expression pattern, we performed a series of experiments to test the hypothesis that BMP-2 mediates myocardial regulation of cardiac cushion tissue formation in mice. When BMP-2 protein was added to the 16-18 somite stage (ED 9.25) AV endocardial endothelium in culture, cushion mesenchymal cells were formed in the absence of AV myocardium, which invaded into collagen gels and expressed the mesenchymal marker, smooth muscle (SM) alpha-actin; whereas the endothelial marker, PECAM-1, was lost from the invaded cells. In contrast, when noggin, a specific antagonist to BMPs, was applied together with BMP-2 to the culture medium, AV endothelial cells remained as an epithelial monolayer with little expression of SM alpha-actin, and expression of PECAM-1 was retained in the endocardial cells. When noggin was added to AV endothelial cells cocultured with associated myocardium, it blocked endothelial transformation to mesenchyme. AV endothelium treated with BMP-2 expressed elevated levels of TGFbeta-2 in the absence of myocardium, as observed in the endothelium cocultured with myocardium. BMP-2-supported elevation of TGFbeta-2 expression in endocardial cells was abolished by noggin treatment. These data indicated that BMP signaling is required in and BMP-2 is sufficient for myocardial segmental regulation of AV endocardial cushion mesenchymal cell formation in mice.     

Endocardial to Myocardial Notch-Wnt-Bmp Axis Regulates Early Heart Valve Development

Endocardial to mesenchymal transformation (EMT) is a fundamental cellular process required for heart valve formation. Notch, Wnt and Bmp pathways are known to regulate this process. To further address how these pathways coordinate in the process, we specifically disrupted Notch1 or Jagged1 in the endocardium of mouse embryonic hearts and showed that Jagged1-Notch1 signaling in the endocardium is essential for EMT and early valvular cushion formation. qPCR and RNA in situ hybridization assays reveal that endocardial Jagged1-Notch1 signaling regulates Wnt4 expression in the atrioventricular canal (AVC) endocardium and Bmp2 in the AVC myocardium. Whole embryo cultures treated with Wnt4 or Wnt inhibitory factor 1 (Wif1) show that Bmp2 expression in the AVC myocardium is dependent on Wnt activity; Wnt4 also reinstates Bmp2 expression in the AVC myocardium of endocardial Notch1 null embryos. Furthermore, while both Wnt4 and Bmp2 rescue the defective EMT resulting from Notch inhibition, Wnt4 requires Bmp for its action. These results demonstrate that Jagged1-Notch1 signaling in endocardial cells induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT.     

Activin receptor-like kinase 2 and Smad6 regulate epithelial-mesenchymal transformation during cardiac valve formation

Epithelial-mesenchymal transformation (EMT) occurs during both development and tumorigenesis. Transforming growth factor beta (TGFbeta) ligands signal EMT in the atrioventricular (AV) cushion of the developing heart, a critical step in valve formation. TGFbeta signals through a complex of type I and type II receptors. Several type I receptors exist although activin receptor-like kinase (ALK) 5 mediates the majority of TGFbeta signaling. Here, we demonstrate that ALK2 is sufficient to induce EMT in the heart. Both ALK2 and ALK5 are expressed throughout the heart with ALK2 expressed abundantly in endocardial cells of the outflow tract (OFT), ventricle, and AV cushion. Misexpression of constitutively active (ca) ALK2 in non-transforming ventricular endocardial cells induced EMT, while caALK5 did not, thus demonstrating that ALK2 activity alone is sufficient to stimulate EMT. Smad6, an inhibitor of Smad signaling downstream of ALK2, but not ALK5, inhibited EMT in AV cushion endocardial cells. These data suggest that ALK2 activation may stimulate EMT in the AV cushion and that Smad6 may act downstream of ALK2 to negatively regulate EMT.     

Msx1creERT2 knock‐In allele: A useful tool to target embryonic and adult cardiac valves

Summary : Heart valve development begins with the endothelial‐to‐mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate‐mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock‐in tamoxifen‐inducible Cre line was recently generated (Msx1^CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock‐in allele and track the Msx1‐expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1^CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling. genesis 53:337–345, 2015. © 2015 Wiley Periodicals, Inc.     

Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.     

Transforming growth factor-beta-stimulated endocardial cell transformation is dependent on Par6c regulation of RhoA

Valvular heart disease due to congenital abnormalities or pathology is a major cause of mortality and morbidity. Understanding the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. In the developing heart, epithelial-mesenchymal transformation (EMT) in a subpopulation of endocardial cells in the atrioventricular cushion (AVC) is an important step in valve formation. Transforming growth factor-beta (TGFbeta) has been shown to be an important regulator of AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Recently Par6c (Par6) has been shown to function downstream of TGFbeta to recruit Smurf1, an E3 ubiquitin ligase, which targets RhoA for degradation to control apical-basal polarity and tight junction dissolution. We tested the hypothesis that Par6 functions in a pathway that regulates endocardial cell EMT. Here we show that the Type I TGFbeta receptor ALK5 is required for endocardial cell EMT. Overexpression of dominant negative Par6 inhibits EMT in AVC endocardial cells, whereas overexpression of wild-type Par6 in normally non-transforming ventricular endocardial cells results in EMT. Overexpression of Smurf1 in ventricular endocardial cells induces EMT. Decreasing RhoA activity using dominant negative RhoA or small interfering RNA in ventricular endocardial cells also increases EMT, whereas overexpression of constitutively active RhoA in AVC endothelial cells blocks EMT. Manipulation of Rac1 or Cdc42 activity is without effect. These data demonstrate a functional role for Par6/Smurf1/RhoA in regulating EMT in endocardial cells.     

Periostin regulates atrioventricular valve maturation

Cardiac valve leaflets develop from rudimentary structures termed endocardial cushions. These pre-valve tissues arise from a complex interplay of signals between the myocardium and endocardium whereby secreted cues induce the endothelial cells to transform into migratory mesenchyme through an endothelial to mesenchymal transformation (EMT). Even though much is currently known regarding the initial EMT process, the mechanisms by which these undifferentiated cushion mesenchymal tissues are remodeled “post-EMT” into mature fibrous valve leaflets remains one of the major, unsolved questions in heart development. Expression analyses, presented in this report, demonstrate that periostin, a component of the extracellular matrix, is predominantly expressed in post-EMT valve tissues and their supporting apparatus from embryonic to adult life. Analyses of periostin gene targeted mice demonstrate that it is within these regions that significant defects are observed. Periostin null mice exhibit atrial septal defects, structural abnormalities of the AV valves and their supporting tensile apparatus, and aberrant differentiation of AV cushion mesenchyme. Rescue experiments further demonstrate that periostin functions as a hierarchical molecular switch that can promote the differentiation of mesenchymal cells into a fibroblastic lineage while repressing their transformation into other mesodermal cell lineages (e.g. myocytes). This is the first report of an extracellular matrix protein directly regulating post-EMT AV valve differentiation, a process foundational and indispensable for the morphogenesis of a cushion into a leaflet.     

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.Download video file.^{(86M, mov)}     

Signaling via the Tgf-beta type I receptor Alk5 in heart development

Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelial (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-βs (Tgf-β) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-βs in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-β type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and in vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell-cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-β-induced EMT in vitro. Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that Alk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-β signaling via Alk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells.     

Endocardial cell epithelial-mesenchymal transformation requires Type III TGFβ receptor interaction with GIPC

An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFβR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFβR3-mediated endothelial cell EMT.     

TGFbeta2 and TGFbeta3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart

Heart valve formation is initiated by an epithelial-mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor beta (TGFbeta) mediates EMT in the AV canal, and TGFbeta1 and 2 isoforms are expressed in the mouse heart while TGFbeta 2 and 3 are expressed in the avian heart. Depletion of TGFbeta3 in avian or TGFbeta2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFbeta isoforms participate in valve formation. In this study, we examined the localization and function of TGFbeta2 and TGFbeta3 in the chick heart during EMT. TGFbeta2 was present in both endothelium and myocardium before and after EMT. TGFbeta2 antibody inhibited endothelial cell-cell separation. In contrast, TGFbeta3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFbeta3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFbeta2 and 3 increased fibrillin 2 expression. However, only TGFbeta2 treatment increased cell surface beta-1,4-galactosyltransferase expression. These data suggest that TGFbeta2 and TGFbeta3 are sequentially and separately involved in the process of EMT. TGFbeta2 mediates initial endothelial cell-cell separation while TGFbeta3 is required for the cell morphological change that enables the migration of cells into the underlying ECM.     

VEGF signaling has distinct spatiotemporal roles during heart valve development

Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.     

Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field

A hallmark of heart-valve development is the swelling and deposition of extracellular matrix in the heart-valve region. Only myocardium overlying this region can signal to underlying endothelium and cause it to lose cell-cell contacts, delaminate, and invade the extracellular space abutting myocardium and endocardium to form endocardial cushions (EC) in a process known as epithelial to mesenchymal transformation (EMT). The heart-valve myocardium expresses bone morphogenetic protein-2 (Bmp2) coincident with development of valve mesenchyme. BMPs belong to the transforming growth factor beta superfamily (TGF-beta) and play a wide variety of roles during development. We show that conditional ablation of Bmp2 in cardiac progenitors results in cell fate changes in which the heart-valve region adopts the identity of differentiated chamber myocardium. Moreover, Bmp2-deficient hearts fail to induce production and deposition of matrix at the heart-valve-forming region, resulting in the inability of the endothelium to swell and impairing the development of ECs. Furthermore, in collagen invasion assays, Bmp2 mutant endothelium is incapable of undergoing EMT, and addition of BMP2 protein to mutant heart explants rescues this phenotype. Our results demonstrate that Bmp2 is both necessary and sufficient to specify a field of cardiac progenitor cells as the heart-valve-inducing region amid developing atria and ventricles.     

BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFβ receptor to promote invasion

Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor β Receptor (TGFβR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFβR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFβR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFβR3 in BMP2 signaling in epicardial cells. Whereas TGFβ induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFβ-stimulated differentiation. Tgfbr3(-/-) cells respond to BMP2 indicating that TGFβR3 is not required. However, Tgfbr3(-/-) cells show decreased invasion in response to BMP2 and overexpression of TGFβR3 in Tgfbr3(-/-) cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFβR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3(+/+) or Tgfbr3(-/-) cells rescued with TGFβR3 decreased BMP2-stimulated invasion confirming a requirement for TGFβR3/GIPC interaction. Our results reveal the relative roles of TGFβR3-dependent and TGFβR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFβR3 in mediating BMP2-stimulated invasion.