**3. BMP signaling in heart development**

#### **3.1 Cardiac specification and heart tube formation**

In this section, we will review the functions of different components of BMP signaling during the initial stages of heart development.

#### **3.1.1 BMP ligands**

98 Congenital Heart Disease – Selected Aspects

AVC, the endocardial cells respond to signals from the myocardium and undergo epithelial to mesenchymal transition (EMT) to form the cushions, the primordial valve structures. Cushions are also formed in the proximal region of the OFT at a slightly later stage. Around E10.0, another population of cells called the cardiac neural crest cells (CNCC) migrates from the dorsal neural tube and contributes to the developing OFT. By E11.5, the proepicardial cells have migrated around and enveloped the heart, forming the epicardium. Finally, septation and valve development result in a four-chambered heart with right, pulmonary,

BMP ligands are conserved growth factors that belong in the Transforming Growth Factor-β (TGFβ) superfamily. More than twenty BMPs have been identified and they have a myriad of functions during development. BMP precursor proteins are activated via endoproteolytic cleavage, glycosylated, and then secreted as homo- or hetero-dimers (Derynck et al. 1985; Derynck et al. 1986; Wozney et al. 1990). Once processed and secreted, BMP ligands relay their signal to the nucleus through signaling cascades that utilize unique combinations of serine threonine kinase receptors which respond to specific ligand combinations. There are three type I receptors (out of seven) and three type II receptors (out of five) that transduce the BMP signals. The type I receptors are ALK2 (ACVRI, ACTRI), ALK3 (BMPRIA/BRK-1), and ALK6 (BMPRIB, BRK-2) (Macías-Silva et al. 1998; Koenig et al. 1994; ten Dijke et al. 1994). The type II receptors are BMPR2 (BMPRII, BRK-3), ACVR2A (ACTRIIA), and ACVR2B (ACTRIIB) (Yamashita et al. 1995; Nohno et al. 1995; Rosenzweig et al. 1995; Kawabata, Chytil, and Moses 1995). The BMP dimer binds a type II receptor, which recruits and phosphorylates a type I receptor in its intracellular kinase domain (Yamashita et al. 1995). The type I receptor then phosphorylates an intracellular receptor-regulated SMAD protein (R-SMAD). SMAD1, SMAD5, and SMAD8 are activated specifically by BMP signals (Cárcamo, Zentella, and Massagué 1995; Wieser, Wrana, and Massagué 1995; Hoodless et al. 1996; Nishimura et al. 1998; Chen, Bhushan, and Vale 1997). After phosphorylation, activated R-SMADs form a complex with the common SMAD, SMAD4 (Zhang, Musci, and Derynck 1997). The R-SMAD-SMAD4 complex translocates to the nucleus, where it cooperates with other cofactors to regulate gene transcription; an example is illustrated elsewhere (Jiao, Zhou, and Hogan 2002). BMP signaling can occur independently of SMAD proteins in non-canonical pathways. For example, BMPs can activate MAP kinase pathways, resulting in the activation of p38 MAPK, PI3K, ERK, and JNK with downstream effects on cell proliferation and differentiation (Yamaguchi et al. 1995; Shibuya et al. 1998; Kimura et

and left, systemic, halves by mouse E14.5. For a review, see (Evans et al. 2010).

al. 2000; Lou et al. 2000; Lai and Cheng 2002; Yanagisawa et al. 2001; Xu et al. 1996).

It has recently been demonstrated that BMP signaling can regulate microRNA (miRNA) biosynthesis. miRNAs are short non-coding RNA that target messenger RNA (mRNA) in a sequence-specific manner for post-transcriptional degradation and translational inhibition. miRNAs are transcribed as primary miRNAs (pri-miRNAs), which are processed by the Drosha complex within the nucleus. pri-miRNA processing results in a shorter pre-miRNA, which is exported from the nucleus to the cytoplasm where it is cleaved into its mature miRNA structure by Dicer. For a review on miRNA during cardiovascular development and disease, the reader is referred to Liu and Olson (2010). Activated R-SMADs can directly interact with the microprocessor complex, Drosha, independent of the common SMAD, SMAD4, to promote the biosynthesis of miRNAs such as *miR-21* (Davis et al. 2008; Ji et al.

**2.2 BMP signaling pathways** 

Initial insight into the roles of BMP signaling pathways in cardiac specification came from studying the *BMP2/4* ortholog, *Dpp*, in *Drosophila melanogaster*. *Dpp*-deficient larva did not form the precursor cells for the heart organ, the dorsal vessel, while ectopic DPP caused ectopic formation of the dorsal vessel precursor cells (Xu et al. 1998; Frasch 1995; Yin and Frasch 1998). In the anterior region of chick embryos, the endoderm expresses BMP2 and 5, and the ectoderm expresses BMP4 and BMP7 (Schultheiss, Burch, and Lassar 1997; Somi et al. 2004). *In vivo* and *in vitro* experiments using chicken embryos revealed that both the FHF and the SHF pre-cardiac mesodermal cells differentiate in response to BMP signals (Waldo et al. 2001; Tirosh-Finkel et al. 2006). In mice, BMP2, BMP4, BMP5, and BMP7 are expressed in the anterior mesoderm (Zhang and Bradley 1996; Dudley and Robertson 1997; Solloway and Robertson 1999). Regardless of the differences in BMP expression patterns between species, it has been well-established that BMP signaling pathways induce precardiac

Bone Morphogenetic Protein Signaling Pathways in Heart Development and Disease 101

and failure to gastrulate (Sirard et al. 1998). Conditional deletion of *Smad4* from the epiblast causes embryonic lethality by E8.5, but the heart tube forms and *Nkx2.5* is expressed (Chu et al. 2004). Heart tube formation and cardiac gene expression may occur in these mice because canonical BMP signaling occurs before *Smad4* deletion or because other R-SMAD

Inhibition of BMP during gastrulation restricts the heart forming fields to discrete territories in the anterior of the embryo. Noggin, chordin, and follistatin are secreted from the notochord and bind BMP ligands, preventing receptor activation (McMahon et al. 1998; Streit et al. 1998; Sasai et al. 1995; Hemmati-Brivanlou, Kelly, and Melton 1994; Fainsod et al. 1997). The responsiveness of pre-cardiac mesoderm to inhibitory signals from the notochord is developmentally regulated. Ectopic application of noggin to stage 4 chick mesendoderm prevents the initiation of the cardiac gene expression and development of the contracting cardiomyocytes (Schultheiss, Burch, and Lassar 1997; Schlange et al. 2000). If noggin is applied to explants a stage later, the cardiac gene expression is initiated without spontaneous contraction of myocytes. If noggin is applied at stage 6, differentiation occurs normally (Nakajima et al. 2002). In mice, deletion of noggin or follistatin individually does not cause heart defects, but deletion of both reverses heart looping (Bachiller et al. 2000; McMahon et al. 1998; Matzuk, Lu, et al. 1995). Deleting chordin causes defects

In this section, we will discuss BMP signaling during different cardiogenic processes after

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).

transcriptional cofactors compensate for the loss of SMAD4.

phenocopying those in DiGeorge syndrome (Bachiller et al. 2003).

**3.2 Cardiogenesis after heart tube formation** 

**3.2.1 Myocardial wall morphogenesis** 

**3.1.4 BMP inhibitors** 

heart tube formation.

mesoderm to undergo cardiac differentiation (Alsan and Schultheiss 2002; Barron, Gao, and Lough 2000; Tirosh-Finkel et al. 2010). *Bmp2* deletion in mice causes embryonic lethality between E7.5-E9.0 (Zhang and Bradley 1996). Some mutant embryos lack hearts altogether and others develop ectopic heart tubes in the exocoelomic cavity, suggesting a critical role of BMP signaling for heart formation (Zhang and Bradley 1996).

BMP signaling pathways induce cardiac differentiation through upregulation of cardiogenic genes. Expression of the cardiac transcription factors *Nkx2.5* and *Gata4* is initiated by BMP signaling (Frasch 1995; Schultheiss, Burch, and Lassar 1997; Andrée et al. 1998; Schlange et al. 2000; Jamali et al. 2001; Liberatore et al. 2002; Shi et al. 2000; Lien et al. 2002; Reiter, Verkade, and Stainier 2001; Schultheiss, Xydas, and Lassar 1995). The *Nkx2.5* promoter region contains evolutionary conserved BMP-response elements that are necessary for its cardiac expression (Lien et al. 2002; Liberatore et al. 2002; Brown et al. 2004). BMP signaling also activates the expression of myocardin, a cardiac and smooth muscle-specific transcriptional cofactor for serum response factor, a regulator of cardiac differentiation (Arsenian et al. 1998; Wang et al. 2001; Callis, Cao, and Wang 2005). SMAD1 is also a transcriptional cofactor for myocardin to activate downstream gene expression (Callis, Cao, and Wang 2005).

### **3.1.2 BMP receptors**

The BMP type I receptor ALK3 is widely expressed in mouse embryos and *Alk3* deletion causes embryonic lethality at E8.0 with no mesoderm formation (Mishina et al. 1995; Dewulf et al. 1995). ALK2, another type I receptor, is expressed in Hensen's node and in the primitive streak (Gu et al. 1999; Mishina et al. 1999). Deleting *Alk2* in mouse embryos results in gastrulation defects and embryonic lethality before E9.5 (Gu et al. 1999; Mishina et al. 1999). The third type I receptor, ALK6, is not expressed during early heart development and disrupting its function does not affect mouse cardiogenesis or viability (Dewulf et al. 1995; Yi et al. 2000). Knockout of the type II receptor, BMPR2, which is expressed widely throughout chicken embryos and during mouse cardiomyogenesis, causes embryonic lethality at gastrulation (Ehrman and Yutzey 1999; Stern et al. 1995; Feijen, Goumans, and van den Eijnden-van Raaij 1994; Beppu et al. 2000). In mice, ACVR2A is expressed after cardiomyocyte formation at E9.5 and ACVR2B is ubiquitously expressed during cardiomyogenesis (Feijen, Goumans, and van den Eijnden-van Raaij 1994; Beppu et al. 2000). Disruption of *Acvr2a* alone does not cause heart defects and disruption of *Acvr2b* causes heart defects later in development (Matzuk, Kumar, et al. 1995; Oh and Li 1997). However, deletion of both *Acvr2a* and *Acvr2b* results in embryonic death at gastrulation, suggesting functional redundancy of these type II receptors (Song et al. 1999).

#### **3.1.3 SMAD proteins**

In chicken embryos, the receptor-regulated SMAD proteins, SMAD1, SMAD5, and SMAD8, are enriched in the heart forming region (Faure et al. 2002). In mice, *Smad1* and *Smad5*  mRNA are expressed in the mesoderm during cardiomyocyte formation (Tremblay, Dunn, and Robertson 2001). *Smad1* disruption in mice results in embryonic lethality at E10.5 from failure of umbilical-placental connections to form (Tremblay, Dunn, and Robertson 2001). Germline deletion of *Smad5* results in defective left-right symmetry with a heart looping abnormality and defective angiogenesis (Chang et al. 2000; Yang et al. 1999). Deletion of *Smad4,* the gene encoding the common SMAD, causes death before E7.5, with reduced size and failure to gastrulate (Sirard et al. 1998). Conditional deletion of *Smad4* from the epiblast causes embryonic lethality by E8.5, but the heart tube forms and *Nkx2.5* is expressed (Chu et al. 2004). Heart tube formation and cardiac gene expression may occur in these mice because canonical BMP signaling occurs before *Smad4* deletion or because other R-SMAD transcriptional cofactors compensate for the loss of SMAD4.
