**2. Overview**

#### **2.1 Heart development**

During gastrulation, cardiac progenitors within the lateral plate mesoderm migrate in bilateral sheets of cells to the anterior of the embryo. Within the cardiac mesoderm, there are two populations of cells that contribute to the developing heart called the first and second heart fields (FHF and SHF, respectively). At embryonic (E) day 7.5 in mice, cells of the FHF form the cardiac crescent. At this stage, the second population of cardiac cells, the SHF, is medial and anterior to the FHF. As the embryo folds, at mouse E8.0, the cardiac crescent fuses along the midline and forms the heart tube while the SHF moves dorsally. The heart tube consists of an outer myocardial layer and an inner endocardial layer, separated by an extracellular matrix (ECM) called the cardiac jelly. SHF cells migrate through the pharyngeal mesoderm to populate the anterior and posterior regions of the heart tube. Starting from E8.5 in the mouse, the heart undergoes rightward looping. Regional proliferation along the myocardium of the outer curvature of the heart tube demarcates the future atrial and ventricular chambers. The myocardium of the inflow tract (IFT), outflow tract (OFT), atrioventricular canal (AVC) and inner curvature of the heart tube is characteristically nonproliferative. The FHF contributes primarily to the left ventricle as well as to part of the atrium, and the SHF contributes to the atria, right ventricle, and OFT. At about E9.5 in the

Bone Morphogenetic Protein Signaling Pathways in Heart Development and Disease 99

2007; Warner et al. 2004; Fukuda et al. 2007; Davis et al. 2010). *miR-21* is upregulated in

The timing, duration, and gradient of BMP ligands affect the outcomes and add to the complexity of BMP signaling pathways. After BMP processing and secretion, access to the receptors and retention in the ECM are inhibited by extracellular factors such as noggin, chordin, follistatin, cerberus, and gremlin (McMahon et al. 1998; Streit et al. 1998; Sasai et al. 1995; Hemmati-Brivanlou, Kelly, and Melton 1994; Fainsod et al. 1997; Bouwmeester et al. 1996; Hsu et al. 1998). These inhibitors bind BMP ligands, interfering with ligand-receptor interaction (Zimmerman, De Jesús-Escobar, and Harland 1996; Hsu et al. 1998; Yamashita et al. 1995; Piccolo et al. 1996; Iemura et al. 1998). BMP signaling is also regulated at the membrane, for instance by the pseudoreceptor BAMBI (BMP and Activin membrane bound inhibitor). BAMBI lacks the intracellular domain needed to transduce the signal and, upon binding BMP receptors, it inhibits the formation of an active BMP receptor complex (Onichtchouk et al. 1999; Grotewold et al. 2001). Alternatively, BMP signaling can be enhanced at the membrane level by modulators such as DRAGON, which acts as a coreceptor and presents BMPs to the receptors (Samad et al. 2005; Babitt et al. 2005; Babitt et al. 2006). Another example is endoglin, a transmembrane protein that binds to BMP ligands and enhances BMP signaling (Barbara, Wrana, and Letarte 1999; Scherner et al. 2007). Intracellularly, BMP signaling can be downregulated by SMURF, an E3 ubiquitin ligase that promotes R-SMAD degradation, receptor turnover, and facilitates inhibition by the inhibitory SMADs, SMAD6 and SMAD7 (Murakami et al. 2003; Kavsak et al. 2000; Ebisawa et al. 2001). SMAD6 and SMAD7 inhibit BMP signaling cascades through binding active type I BMP receptors and preventing R-SMAD activation, and by competing with SMAD4 for R-SMADs (Imamura et al. 1997; Hata et al. 1998; Hanyu et al. 2001). Lastly, crosstalk with other signaling pathways affects R-SMAD phosphorylation, activity, turnover and nuclear accumulation (Pera et al. 2003; Sapkota et al. 2007; Fuentealba et al. 2007; Suzawa et al. 2002).

In this section, we will review the functions of different components of BMP signaling

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

damaged cardiovascular tissue (Ji et al. 2007).

**3. BMP signaling in heart development** 

during the initial stages of heart development.

**3.1.1 BMP ligands** 

**3.1 Cardiac specification and heart tube formation** 

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

#### **2.2 BMP signaling pathways**

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 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. 2007; Warner et al. 2004; Fukuda et al. 2007; Davis et al. 2010). *miR-21* is upregulated in damaged cardiovascular tissue (Ji et al. 2007).

The timing, duration, and gradient of BMP ligands affect the outcomes and add to the complexity of BMP signaling pathways. After BMP processing and secretion, access to the receptors and retention in the ECM are inhibited by extracellular factors such as noggin, chordin, follistatin, cerberus, and gremlin (McMahon et al. 1998; Streit et al. 1998; Sasai et al. 1995; Hemmati-Brivanlou, Kelly, and Melton 1994; Fainsod et al. 1997; Bouwmeester et al. 1996; Hsu et al. 1998). These inhibitors bind BMP ligands, interfering with ligand-receptor interaction (Zimmerman, De Jesús-Escobar, and Harland 1996; Hsu et al. 1998; Yamashita et al. 1995; Piccolo et al. 1996; Iemura et al. 1998). BMP signaling is also regulated at the membrane, for instance by the pseudoreceptor BAMBI (BMP and Activin membrane bound inhibitor). BAMBI lacks the intracellular domain needed to transduce the signal and, upon binding BMP receptors, it inhibits the formation of an active BMP receptor complex (Onichtchouk et al. 1999; Grotewold et al. 2001). Alternatively, BMP signaling can be enhanced at the membrane level by modulators such as DRAGON, which acts as a coreceptor and presents BMPs to the receptors (Samad et al. 2005; Babitt et al. 2005; Babitt et al. 2006). Another example is endoglin, a transmembrane protein that binds to BMP ligands and enhances BMP signaling (Barbara, Wrana, and Letarte 1999; Scherner et al. 2007). Intracellularly, BMP signaling can be downregulated by SMURF, an E3 ubiquitin ligase that promotes R-SMAD degradation, receptor turnover, and facilitates inhibition by the inhibitory SMADs, SMAD6 and SMAD7 (Murakami et al. 2003; Kavsak et al. 2000; Ebisawa et al. 2001). SMAD6 and SMAD7 inhibit BMP signaling cascades through binding active type I BMP receptors and preventing R-SMAD activation, and by competing with SMAD4 for R-SMADs (Imamura et al. 1997; Hata et al. 1998; Hanyu et al. 2001). Lastly, crosstalk with other signaling pathways affects R-SMAD phosphorylation, activity, turnover and nuclear accumulation (Pera et al. 2003; Sapkota et al. 2007; Fuentealba et al. 2007; Suzawa et al. 2002).
