**3.2.1 Myocardial wall morphogenesis**

During early heart development, myocardial walls expand through cardiomyocyte proliferation and differentiation. The ventricle chamber myocardium develops a latticework of muscular projections on the subendocardial surface called trabeculae. Trabecular myocardium generates contractile force, coordinates intraventricular conduction, and helps diffuse nutrients to the cardiomyocytes within the expanding heart wall prior to vascularization. Later in heart development, the trabecular myocardium undergoes remodeling and is incorporated into the compact myocardium, the interventricular septum, and the papillary muscles of the atrioventricular valves. For a review, see Dunwoodie (2007). Proper formation of myocardial walls is essential for embryo viability and postnatal cardiac function. Abnormal myocardial wall morphogenesis can result in left ventricular noncompaction, which may lead to cardiomyopathy (Pignatelli et al. 2003; Xing et al. 2006). BMP10 is initially expressed in the looping mouse heart within regions destined to be the atrial and ventricular chambers, and its expression is maintained in the chamber myocardium during heart development. (Neuhaus, Rosen, and Thies 1999; Somi et al. 2004; Chen et al. 2004) Also, *Bmp10* is upregulated in mouse models of hypertrabeculation (Chen et al. 2004). Myocardial expression of BMP10 during chamber formation relies on

endocardial expression of notch (Grego-Bessa et al. 2007). Deleting *Bmp10* in mice causes embryonic lethality at E9.0 with decreased cardiomyocyte proliferation, downregulation of cardiac genes *Nkx2.5* and *Mef2c*, and loss of trabecular myocardium (Chen et al. 2004).

Bone Morphogenetic Protein Signaling Pathways in Heart Development and Disease 103

abnormal connection between the atria and ventricles (Gaussin et al. 2005). Recently, a heterozygous microdeletion was identified in a chromosomal region encompassing *BMP2* 

At E9.5 in mice, BMP2 has weak expression in OFT myocardium which disappears by E10.5 (Lyons, Pelton, and Hogan 1990). BMP2 is strongly and persistently expressed in AVC and atrial myocardium at E10.5 (Lyons, Pelton, and Hogan 1990; Abdelwahid et al. 2001). It is also expressed in the cushion mesenchyme during valve remodeling and in adult mouse valves (Sugi et al. 2004). In mice, BMP2 enhances cardiac jelly formation, endocardial EMT, and AVC myocardial patterning (Ma et al. 2005; Rivera-Feliciano and Tabin 2006; Sugi et al. 2004; Camenisch et al. 2002). BMP2 upregulates *Twist1*, an inducer of EMT, and *Has2*, a component of the cardiac jelly necessary for EMT (Camenisch et al. 2000; Ma et al. 2005; Yang et al. 2004). Myocardial deletion of *Bmp2* decreases ECM in the AVC cushions, however the OFT cushions develop normally (Ma et al. 2005; Rivera-Feliciano and Tabin 2006). This suggests a compensatory mechanism in the OFT such as BMP4 signaling. Data suggests that BMP2 signaling interacts with notch1 and TGFβ signaling pathways to

coordinate EMT (Luna-Zurita et al. 2010; Boyer et al. 1999; Yamagishi et al. 1999).

BMP4 is expressed in AVC myocardium in mice at E9.5, but at E10.5 its expression is largely restricted to the myocardium of the OFT (Jones, Lyons, and Hogan 1991; Abdelwahid et al. 2001). It is also expressed in the chicken OFT (Somi et al. 2004). BMP4 is 92% identical in the C-terminus to BMP2, and they have overlapping functions (Goldman, Donley, and Christian 2009; Uchimura et al. 2009). Conditional deletion of *Bmp4* from mouse myocardium causes atrioventricular septation defects, double outlet right ventricle (DORV, both arteries are connected to the right ventricle), and aortic arch artery malformations (Jiao et al. 2003; Liu et al. 2004). Mouse models with myocardial-specific deletion of *Bmp4* or with hypomorphic *Bmp4* alleles have impaired AVC cushion mesenchymal cell proliferation (Jiao et al. 2003; Kulessa and Hogan 2002). Mice compound heterozygous for *Bmp2*-null and *Bmp4-*null or *Bmp4-*hypomorphic alleles have VSD (Goldman, Donley, and Christian 2009). Decreased

**3.2.3 Valvulo-septal development of the atrioventricular canal and outflow tract**  The AVC and OFT are septated by endocardial cushion maturation into valvulo-septal structures. Cushions develop in the AVC and OFT through the expansion of the ECM. Induction of cushion formation occurs within the looped heart, when the myocardium signals through the cardiac jelly to the endocardium. Endocardial cells then delaminate and invade the cardiac jelly to form the mesenchymal cells of the endocardial cushions. For reviews, see Person, Klewer, and Runyan (2005) and Butcher and Markwald (2007). The AVC cushions form earlier than the OFT cushions and develop into the mitral (left) and tricuspid (right) valves at the junction of the atria and ventricles. The OFT cushions, but not the AVC cushions, have a CNCC contribution (Kirby, Gale, and Stewart 1983; Waldo et al. 1998; Jiang et al. 2000). The cushions in the OFT develop into the semilunar valves in the aorta (left) and pulmonary artery (right). Congenital defects in valve formation and septation comprise the most common CHDs, while defects involving the OFT are found in 4 per 10,000 live births and are often lethal (Hoffman 1995; Edmonds and James 1993). Pathological mutations in the BMP receptor *ALK2* have been found in patients with congenital defects in atrioventricular septum development, providing evidence for the importance of BMP signaling pathways in human heart development (Smith et al. 2009;

that is associated with predisposition to WPWS (Lalani et al. 2009).

Joziasse et al. 2011).

Removing both *Bmp6* and *Bmp7* in mice causes embryonic lethality at midgestation with hypoplastic ventricles and reduced trabeculations (Kim, Robertson, and Solloway 2001). Mice with conditional deletion of the BMP receptor *Alk3* from the myocardium die during embryogenesis and display underdeveloped myocardial walls and ventricle septal defects (VSD) (Gaussin et al. 2002). Specific inactivation of the common Smad, *Smad4*, from the myocardium likewise causes embryonic lethality at midgestation and disrupts myocardial wall formation and ventricle septation (Azhar et al. 2010; Song, Yan, et al. 2007; Qi et al. 2007; Wang, Xu, et al. 2005). Myocardial deletion of *Smad4* causes downregulation of genes encoding cell cycle regulators, cardiac structural proteins, and transcription factors. Together, these studies provide multiple lines of evidence that show BMP signaling is required for ventricular myocardial wall morphogenesis through regulation of cardiomyocyte proliferation, differentiation, and gene expression.

#### **3.2.2 Conduction system development**

In vertebrates, regional differentiation of the myocardium allows for development of slowconducting, nonchamber myocardium (IFT, AVC, and OFT) and fast-conducting chamber myocardium (atria and ventricles). Proper formation of the AVC is important for establishment of the primary conduction system. The primary conduction system includes the atrioventricular node (AVN) and its associated structures. In mice, AVN precursor cells are observed in the AVC at E9.5. The AVN subsequently extends into the left ventricle and connects with the trabecular myocardium and the interventricular septum. (See reviews, (Christoffels et al. 2010; Moorman and Christoffels 2003)). The electrical impulse is carried from the atria, across the AVC to the ventricles (Valderrábano et al. 2006; Rentschler et al. 2002; de Jong et al. 1992). The AVC has a slower conduction rate than the atria and delays the atrial-ventricular electrical impulse (de Jong et al 1992).

BMP2 is necessary for AVC specification and expression of *Tbx2* (Yamada et al. 2000; Ma et al. 2005). TBX2 is a transcriptional repressor of chamber-specific genes and is specifically expressed in nonchamber myocardium of the IFT and the AVC (Aanhaanen et al. 2009; Habets et al. 2002; Yamada et al. 2000; Christoffels et al. 2004; Harrelson et al. 2004). In the AVC, BMP2 activates *Tbx2* transcription to suppress proliferation and inhibit the expression of chamber-specific genes *Nppa*, *Cx40*, *Cx43*, and *Chisel* (Ma et al. 2005; Shirai et al. 2009; Christoffels et al. 2004). BMP2 can directly regulate *Tbx2* through a SMAD-dependent enhancer upstream of its transcription start site (Singh et al. 2009). BMP signaling also promotes *Tbx2* transcription through SMAD1 inhibition of TBX20, a *Tbx2* repressor (Singh et al. 2009). The BMP2-TBX2 pathway is restricted to the AVC region by notch/HEY signaling in the developing heart chambers (Rutenberg et al. 2006; Kokubo et al. 2005).

Deletion of *Bmp2* from mouse myocardium decreases *Tbx2* expression and results in the expansion of chamber myocardium into the AVC region (Ma et al. 2005). Inactivation of the BMP receptor *Alk3* specifically in the AVC myocardium disrupts AV valve development and AVN morphogenesis, resulting in ventricular pre-excitation (Gaussin et al. 2005; Stroud et al. 2007). Lastly, removal of myocardial *Tbx2* results in abnormal AVC patterning and ventricular pre-excitation (Aanhaanen et al. 2011). Taken together, these data suggest that BMP2 regulation of *Tbx2* expression and AVC myocardial patterning is important for development of the AVN and proper atrial-ventricular conduction. Indeed, the phenotype resulting from AVC-depletion of *Alk3* resembles Wolff-Parkinson-White syndrome (WPWS, OMIM 224700), a pre-excitation syndrome that can present as tachycardia due to an

Removing both *Bmp6* and *Bmp7* in mice causes embryonic lethality at midgestation with hypoplastic ventricles and reduced trabeculations (Kim, Robertson, and Solloway 2001). Mice with conditional deletion of the BMP receptor *Alk3* from the myocardium die during embryogenesis and display underdeveloped myocardial walls and ventricle septal defects (VSD) (Gaussin et al. 2002). Specific inactivation of the common Smad, *Smad4*, from the myocardium likewise causes embryonic lethality at midgestation and disrupts myocardial wall formation and ventricle septation (Azhar et al. 2010; Song, Yan, et al. 2007; Qi et al. 2007; Wang, Xu, et al. 2005). Myocardial deletion of *Smad4* causes downregulation of genes encoding cell cycle regulators, cardiac structural proteins, and transcription factors. Together, these studies provide multiple lines of evidence that show BMP signaling is required for ventricular myocardial wall morphogenesis through regulation of

In vertebrates, regional differentiation of the myocardium allows for development of slowconducting, nonchamber myocardium (IFT, AVC, and OFT) and fast-conducting chamber myocardium (atria and ventricles). Proper formation of the AVC is important for establishment of the primary conduction system. The primary conduction system includes the atrioventricular node (AVN) and its associated structures. In mice, AVN precursor cells are observed in the AVC at E9.5. The AVN subsequently extends into the left ventricle and connects with the trabecular myocardium and the interventricular septum. (See reviews, (Christoffels et al. 2010; Moorman and Christoffels 2003)). The electrical impulse is carried from the atria, across the AVC to the ventricles (Valderrábano et al. 2006; Rentschler et al. 2002; de Jong et al. 1992). The AVC has a slower conduction rate than the atria and delays

BMP2 is necessary for AVC specification and expression of *Tbx2* (Yamada et al. 2000; Ma et al. 2005). TBX2 is a transcriptional repressor of chamber-specific genes and is specifically expressed in nonchamber myocardium of the IFT and the AVC (Aanhaanen et al. 2009; Habets et al. 2002; Yamada et al. 2000; Christoffels et al. 2004; Harrelson et al. 2004). In the AVC, BMP2 activates *Tbx2* transcription to suppress proliferation and inhibit the expression of chamber-specific genes *Nppa*, *Cx40*, *Cx43*, and *Chisel* (Ma et al. 2005; Shirai et al. 2009; Christoffels et al. 2004). BMP2 can directly regulate *Tbx2* through a SMAD-dependent enhancer upstream of its transcription start site (Singh et al. 2009). BMP signaling also promotes *Tbx2* transcription through SMAD1 inhibition of TBX20, a *Tbx2* repressor (Singh et al. 2009). The BMP2-TBX2 pathway is restricted to the AVC region by notch/HEY signaling

Deletion of *Bmp2* from mouse myocardium decreases *Tbx2* expression and results in the expansion of chamber myocardium into the AVC region (Ma et al. 2005). Inactivation of the BMP receptor *Alk3* specifically in the AVC myocardium disrupts AV valve development and AVN morphogenesis, resulting in ventricular pre-excitation (Gaussin et al. 2005; Stroud et al. 2007). Lastly, removal of myocardial *Tbx2* results in abnormal AVC patterning and ventricular pre-excitation (Aanhaanen et al. 2011). Taken together, these data suggest that BMP2 regulation of *Tbx2* expression and AVC myocardial patterning is important for development of the AVN and proper atrial-ventricular conduction. Indeed, the phenotype resulting from AVC-depletion of *Alk3* resembles Wolff-Parkinson-White syndrome (WPWS, OMIM 224700), a pre-excitation syndrome that can present as tachycardia due to an

in the developing heart chambers (Rutenberg et al. 2006; Kokubo et al. 2005).

cardiomyocyte proliferation, differentiation, and gene expression.

the atrial-ventricular electrical impulse (de Jong et al 1992).

**3.2.2 Conduction system development** 

abnormal connection between the atria and ventricles (Gaussin et al. 2005). Recently, a heterozygous microdeletion was identified in a chromosomal region encompassing *BMP2*  that is associated with predisposition to WPWS (Lalani et al. 2009).

#### **3.2.3 Valvulo-septal development of the atrioventricular canal and outflow tract**

The AVC and OFT are septated by endocardial cushion maturation into valvulo-septal structures. Cushions develop in the AVC and OFT through the expansion of the ECM. Induction of cushion formation occurs within the looped heart, when the myocardium signals through the cardiac jelly to the endocardium. Endocardial cells then delaminate and invade the cardiac jelly to form the mesenchymal cells of the endocardial cushions. For reviews, see Person, Klewer, and Runyan (2005) and Butcher and Markwald (2007). The AVC cushions form earlier than the OFT cushions and develop into the mitral (left) and tricuspid (right) valves at the junction of the atria and ventricles. The OFT cushions, but not the AVC cushions, have a CNCC contribution (Kirby, Gale, and Stewart 1983; Waldo et al. 1998; Jiang et al. 2000). The cushions in the OFT develop into the semilunar valves in the aorta (left) and pulmonary artery (right). Congenital defects in valve formation and septation comprise the most common CHDs, while defects involving the OFT are found in 4 per 10,000 live births and are often lethal (Hoffman 1995; Edmonds and James 1993). Pathological mutations in the BMP receptor *ALK2* have been found in patients with congenital defects in atrioventricular septum development, providing evidence for the importance of BMP signaling pathways in human heart development (Smith et al. 2009; Joziasse et al. 2011).

At E9.5 in mice, BMP2 has weak expression in OFT myocardium which disappears by E10.5 (Lyons, Pelton, and Hogan 1990). BMP2 is strongly and persistently expressed in AVC and atrial myocardium at E10.5 (Lyons, Pelton, and Hogan 1990; Abdelwahid et al. 2001). It is also expressed in the cushion mesenchyme during valve remodeling and in adult mouse valves (Sugi et al. 2004). In mice, BMP2 enhances cardiac jelly formation, endocardial EMT, and AVC myocardial patterning (Ma et al. 2005; Rivera-Feliciano and Tabin 2006; Sugi et al. 2004; Camenisch et al. 2002). BMP2 upregulates *Twist1*, an inducer of EMT, and *Has2*, a component of the cardiac jelly necessary for EMT (Camenisch et al. 2000; Ma et al. 2005; Yang et al. 2004). Myocardial deletion of *Bmp2* decreases ECM in the AVC cushions, however the OFT cushions develop normally (Ma et al. 2005; Rivera-Feliciano and Tabin 2006). This suggests a compensatory mechanism in the OFT such as BMP4 signaling. Data suggests that BMP2 signaling interacts with notch1 and TGFβ signaling pathways to coordinate EMT (Luna-Zurita et al. 2010; Boyer et al. 1999; Yamagishi et al. 1999).

BMP4 is expressed in AVC myocardium in mice at E9.5, but at E10.5 its expression is largely restricted to the myocardium of the OFT (Jones, Lyons, and Hogan 1991; Abdelwahid et al. 2001). It is also expressed in the chicken OFT (Somi et al. 2004). BMP4 is 92% identical in the C-terminus to BMP2, and they have overlapping functions (Goldman, Donley, and Christian 2009; Uchimura et al. 2009). Conditional deletion of *Bmp4* from mouse myocardium causes atrioventricular septation defects, double outlet right ventricle (DORV, both arteries are connected to the right ventricle), and aortic arch artery malformations (Jiao et al. 2003; Liu et al. 2004). Mouse models with myocardial-specific deletion of *Bmp4* or with hypomorphic *Bmp4* alleles have impaired AVC cushion mesenchymal cell proliferation (Jiao et al. 2003; Kulessa and Hogan 2002). Mice compound heterozygous for *Bmp2*-null and *Bmp4-*null or *Bmp4-*hypomorphic alleles have VSD (Goldman, Donley, and Christian 2009). Decreased

Bone Morphogenetic Protein Signaling Pathways in Heart Development and Disease 105

al. 2007; Ko et al. 2007; Nie et al. 2008). Deletion of *Smad8* does not affect viability or heart development, but mice display defects in pulmonary vascular remodeling (Huang et al.

BMP signaling regulates SHF myocardialization and OFT morphogenesis in part by promoting *miR-17-92* cluster transcription (Wang et al. 2010). The *miR-17-92* cluster has roles in lung and heart development (Lu et al. 2007; Ventura et al. 2008). It is expressed as a primary transcript that encodes six miRNAs *(miR-17*, -*18a*, -*19a*, -*20a*, -*19b-1*, and -*92a-1*). BMP regulates the transcription of *miR-17-92* through SMAD binding sites in the 5' region (Wang et al. 2010). In turn, *miR-17-92* negatively regulates *Isl1* and *Tbx1* mRNA stability and translation (Wang et al. 2010). Deleting BMP reduces *miR-17-92*, causes misexpression of *Isl1* 

Inhibition of BMP signaling is also critical for normal valvulo-septal formation. For example, *Nkx2.5* is required for OFT development, in part by repressing BMP signaling (Prall et al. 2007). Deleting *Nkx2.5* results in expansion of SHF specification due to increased BMP expression, decreased proliferation, and failed OFT truncation. Disrupting the misregulated BMP signaling in the *Nkx2.5* mutants by deleting *Smad1* effectively rescues the proliferation and the OFT defects. Mutations in the BMP-inhibitor *Smad6* cause hyperplasia of cardiac valves and OFT septation defects, due to unregulated BMP signaling (Galvin et al. 2000). Noggin blocks EMT in mouse explants and overexpression of noggin in chicken embryos causes OFT septation defects (Sugi et al. 2004; Allen et al. 2001). Mutations in chordin cause abnormal OFT septation, resembling syndromes associated with loss of CNCC (Bachiller et

and *Tbx1*, and leads to defects in proximal OFT septation (Wang et al. 2010).

**4. BMP induction of stem cells and progenitor cells to a cardiac fate** 

Controlled differentiation of stem cells has relevance in translational research for pre-clinical cell grafting and for establishing cardiomyocyte cultures for drug discovery and toxicology. As primary inducers of cardiac differentiation, BMP cytokines have important roles in growth factor-based stem cell therapies for cardiac tissue repair. Use of growth factor peptides to induce cardiac muscle formation from embryonic stem (ES) cells has been researched for over a decade. BMP ligands have been proven to be important inductors of cardiac fate in multiple ES cell types. In mouse ES cells (mESC), BMP2 or BMP4 can activate cardiac differentiation, in combination with other factors such as Activin A or fibroblast growth factor 2 (FGF2) (Johansson and Wiles 1995; Kawai et al. 2004; Behfar et al. 2002). In the pluripotent mouse embryonal carcinoma cell line, P19C16, treatment with BMP4 promotes cardiomyocyte formation and expression of α-MHC (Monzen et al. 1999; Monzen et al. 2001). Addition of noggin, a BMP inhibitor, prevents differentiation and this can be abolished by overexpressing BMP2, or SMAD1 and SMAD4 (Monzen et al. 1999; Monzen et al. 2001). In human ES cells (hESC), BMP4 promotes a cardiac fate (Takei et al. 2009; Kattman et al. 2011). BMP stimulation induces *Sox17* expression, which is important for directing mesoderm toward a cardiac fate (Stefanovic et al. 2009). Addition of BMP2 or BMP4 in hESC, along with Activin A and other factors, can reliably induce multipotent cardiovascular progenitors that can generate multiple cell lineages such as cardiomyocytes, smooth muscle cells, and endothelial cells *in vitro* and *in vivo* (Laflamme et al. 2007; Tomescot et al. 2007; Yang et al. 2008; Liu et al. 2007). Human induced pluripotent stem cells (iPSC) can also be induced to undergo cardiomyogenesis using similar multistep additions of factors, including BMP4 (Takahashi et al. 2007; Carvajal-Vergara et al. 2010; Kattman et al.

2009).

al. 2003).

expression of myocardial BMP4 does not affect OFT development, but it increases BMP7 expression (Liu et al. 2004). On a *Bmp7*-null background, BMP4 reduction causes a shortened OFT with hypoplastic OFT cushions, revealing dose-dependence and functional redundancy of BMP signaling in the OFT morphogenesis (Liu et al. 2004).

Despite being expressed during early heart development, single gene deletions of *Bmp5*, *Bmp6*, or *Bmp7* do not cause heart defects, likely due to redundancy of the BMP signaling family members (Kingsley et al. 1992; Jena et al. 1997; Dudley and Robertson 1997; Luo et al. 1995; Solloway et al. 1998; Kim, Robertson, and Solloway 2001). BMP5 is expressed throughout the heart tube myocardium and later becomes restricted to the myocardium of the AVC and OFT in mouse and chicken embryos (Yamagishi et al. 2001; Solloway and Robertson 1999; Somi et al. 2004). In mice, BMP6 is expressed in OFT endocardium and myocardium, and within the OFT and AVC mesenchyme (Kim, Robertson, and Solloway 2001; Jones, Lyons, and Hogan 1991; Solloway and Robertson 1999; Yamagishi et al. 2001). BMP6 is not expressed in the developing chicken heart (Somi et al. 2004). BMP7 is robustly expressed throughout the myocardium of the developing hearts of mice and chickens (Solloway and Robertson 1999; Lyons, Hogan, and Robertson 1995; Somi et al. 2004). Combinations of gene deletions in mouse models reveal their essential roles in chamber formation and septal-valvulogenesis. *Bmp5* and *Bmp7* double deletion causes embryonic lethality at E10.5, with delayed heart development, no endocardial cushion formation or chamber septation, and abnormal pericardium (Solloway and Robertson 1999). Removal of *Bmp6* and *Bmp7* results in defects in OFT cushion development, chamber septation, and myocardial wall formation (Kim, Robertson, and Solloway 2001). Deletion of *Bmp5* and *Bmp6* does not cause heart defects (Solloway et al. 1998).

Deletion of *Alk3* from the myocardium or the endocardium disrupts endocardial cushion formation (Gaussin et al. 2002; Song, Fässler, et al. 2007; Ma et al. 2005). Myocardial deletion of *Alk3* causes VSD and hypoplastic AVC cushions, with decreased TGFβ signaling in the AVC myocardium (Gaussin et al. 2002). Deleting *Alk3* specifically from the AVC myocardium disrupts AV valve maturation (Gaussin et al. 2005). Endocardial deletion of *Alk3* causes hypoplastic cushions with reduced cushion mesenchyme to about 20% of normal (Ma et al. 2005; Song, Fässler, et al. 2007; Park et al. 2006; Rivera-Feliciano and Tabin 2006). Endocardial deletion of *Alk2* causes failure of EMT in AVC cushions along with decreased expression of EMT proteins MSX1 and SMAD2, an intracellular modulator of TGFβ signaling (Wang, Sridurongrit, et al. 2005). The role of ALK2 in cushion formation appears to be specific to the endocardium, as conditional deletion of *Alk2* from the myocardium has no effect on cushion development (Wang, Sridurongrit, et al. 2005). Ectopic expression of active ALK2 in the chicken ventricle endocardium induces EMT (Desgrosellier et al. 2005). CNCC-depletion of *Alk3* or *Alk2* disrupts CNCC invasion, resulting in a shortened OFT with defective proximal septation (Stottmann et al. 2004; Kaartinen et al. 2004). Hypomorphic *Bmpr2* alleles cause defects in proximal OFT septation and loss of semilunar valve formation, while AVC cushions form normally (Délot et al. 2003). However, completely abrogating *Bmpr2* in mouse hearts causes an array of CHDs, such as DORV, VSD, and AVC cushion defects (Beppu et al. 2009). Disruption of *Acvr2b* causes postnatal death with abnormal cardiac septation (Oh and Li 1997). Deletion of *Smad4* from the myocardium affects OFT positioning, with a DORV phenotype in one mouse model (Azhar et al. 2010). Conditional deletion of *Smad4* in CNCC reduced the contribution of CNCC to OFT, causing defects in OFT cushion formation, septation, elongation, and positioning (Jia et

expression of myocardial BMP4 does not affect OFT development, but it increases BMP7 expression (Liu et al. 2004). On a *Bmp7*-null background, BMP4 reduction causes a shortened OFT with hypoplastic OFT cushions, revealing dose-dependence and functional redundancy

Despite being expressed during early heart development, single gene deletions of *Bmp5*, *Bmp6*, or *Bmp7* do not cause heart defects, likely due to redundancy of the BMP signaling family members (Kingsley et al. 1992; Jena et al. 1997; Dudley and Robertson 1997; Luo et al. 1995; Solloway et al. 1998; Kim, Robertson, and Solloway 2001). BMP5 is expressed throughout the heart tube myocardium and later becomes restricted to the myocardium of the AVC and OFT in mouse and chicken embryos (Yamagishi et al. 2001; Solloway and Robertson 1999; Somi et al. 2004). In mice, BMP6 is expressed in OFT endocardium and myocardium, and within the OFT and AVC mesenchyme (Kim, Robertson, and Solloway 2001; Jones, Lyons, and Hogan 1991; Solloway and Robertson 1999; Yamagishi et al. 2001). BMP6 is not expressed in the developing chicken heart (Somi et al. 2004). BMP7 is robustly expressed throughout the myocardium of the developing hearts of mice and chickens (Solloway and Robertson 1999; Lyons, Hogan, and Robertson 1995; Somi et al. 2004). Combinations of gene deletions in mouse models reveal their essential roles in chamber formation and septal-valvulogenesis. *Bmp5* and *Bmp7* double deletion causes embryonic lethality at E10.5, with delayed heart development, no endocardial cushion formation or chamber septation, and abnormal pericardium (Solloway and Robertson 1999). Removal of *Bmp6* and *Bmp7* results in defects in OFT cushion development, chamber septation, and myocardial wall formation (Kim, Robertson, and Solloway 2001). Deletion of *Bmp5* and

Deletion of *Alk3* from the myocardium or the endocardium disrupts endocardial cushion formation (Gaussin et al. 2002; Song, Fässler, et al. 2007; Ma et al. 2005). Myocardial deletion of *Alk3* causes VSD and hypoplastic AVC cushions, with decreased TGFβ signaling in the AVC myocardium (Gaussin et al. 2002). Deleting *Alk3* specifically from the AVC myocardium disrupts AV valve maturation (Gaussin et al. 2005). Endocardial deletion of *Alk3* causes hypoplastic cushions with reduced cushion mesenchyme to about 20% of normal (Ma et al. 2005; Song, Fässler, et al. 2007; Park et al. 2006; Rivera-Feliciano and Tabin 2006). Endocardial deletion of *Alk2* causes failure of EMT in AVC cushions along with decreased expression of EMT proteins MSX1 and SMAD2, an intracellular modulator of TGFβ signaling (Wang, Sridurongrit, et al. 2005). The role of ALK2 in cushion formation appears to be specific to the endocardium, as conditional deletion of *Alk2* from the myocardium has no effect on cushion development (Wang, Sridurongrit, et al. 2005). Ectopic expression of active ALK2 in the chicken ventricle endocardium induces EMT (Desgrosellier et al. 2005). CNCC-depletion of *Alk3* or *Alk2* disrupts CNCC invasion, resulting in a shortened OFT with defective proximal septation (Stottmann et al. 2004; Kaartinen et al. 2004). Hypomorphic *Bmpr2* alleles cause defects in proximal OFT septation and loss of semilunar valve formation, while AVC cushions form normally (Délot et al. 2003). However, completely abrogating *Bmpr2* in mouse hearts causes an array of CHDs, such as DORV, VSD, and AVC cushion defects (Beppu et al. 2009). Disruption of *Acvr2b* causes postnatal death with abnormal cardiac septation (Oh and Li 1997). Deletion of *Smad4* from the myocardium affects OFT positioning, with a DORV phenotype in one mouse model (Azhar et al. 2010). Conditional deletion of *Smad4* in CNCC reduced the contribution of CNCC to OFT, causing defects in OFT cushion formation, septation, elongation, and positioning (Jia et

of BMP signaling in the OFT morphogenesis (Liu et al. 2004).

*Bmp6* does not cause heart defects (Solloway et al. 1998).

al. 2007; Ko et al. 2007; Nie et al. 2008). Deletion of *Smad8* does not affect viability or heart development, but mice display defects in pulmonary vascular remodeling (Huang et al. 2009).

BMP signaling regulates SHF myocardialization and OFT morphogenesis in part by promoting *miR-17-92* cluster transcription (Wang et al. 2010). The *miR-17-92* cluster has roles in lung and heart development (Lu et al. 2007; Ventura et al. 2008). It is expressed as a primary transcript that encodes six miRNAs *(miR-17*, -*18a*, -*19a*, -*20a*, -*19b-1*, and -*92a-1*). BMP regulates the transcription of *miR-17-92* through SMAD binding sites in the 5' region (Wang et al. 2010). In turn, *miR-17-92* negatively regulates *Isl1* and *Tbx1* mRNA stability and translation (Wang et al. 2010). Deleting BMP reduces *miR-17-92*, causes misexpression of *Isl1*  and *Tbx1*, and leads to defects in proximal OFT septation (Wang et al. 2010).

Inhibition of BMP signaling is also critical for normal valvulo-septal formation. For example, *Nkx2.5* is required for OFT development, in part by repressing BMP signaling (Prall et al. 2007). Deleting *Nkx2.5* results in expansion of SHF specification due to increased BMP expression, decreased proliferation, and failed OFT truncation. Disrupting the misregulated BMP signaling in the *Nkx2.5* mutants by deleting *Smad1* effectively rescues the proliferation and the OFT defects. Mutations in the BMP-inhibitor *Smad6* cause hyperplasia of cardiac valves and OFT septation defects, due to unregulated BMP signaling (Galvin et al. 2000). Noggin blocks EMT in mouse explants and overexpression of noggin in chicken embryos causes OFT septation defects (Sugi et al. 2004; Allen et al. 2001). Mutations in chordin cause abnormal OFT septation, resembling syndromes associated with loss of CNCC (Bachiller et al. 2003).
