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In recent years, insights into the role of the epicardium in cardiac development have significantly changed. An important contribution to this increasing knowledge comes from the availability of mouse models that facilitate the study of the fate of the epicardial cell lineage and that allow epicardial-specific manipulation of expression of genes involved in regulation of epicardial cell behavior. In this contribution we will discuss our growing understanding of the role of the epicardium and epicardially derived cells in the formation of the atrioventricular valve leaflets. We will illustrate how epicardially derived cells specifically contribute to the development of the leaflets that derive from the lateral atrioventricular cushions, and we will discuss the role of Bmp signaling, through the Bmp receptor BmpR1A/Alk3, in the regulation of the preferentially migration of EPDCs into the parietal AV valve leaflets.

Among members of the TGFβ superfamily, bone morphogenetic protein (BMP) 9 and BMP10 regulate vascular endothelium differentiation and morphogenesis by activating the specific receptor complex, which consists of ALK1 (or ACVRL1), BMPR2, and endoglin. Mutations in , , or are associated with hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension in humans [1, 2]. We previously identified as a downstream target gene of BMP9/BMP10-ALK1 signaling pathway [3]. TMEM100 is a novel intracellular protein with two putative transmembrane domains, and its amino acid sequence is highly conserved from fish to humans.

Distal outgrowth and fusion of the mesenchymalized AV endocardial cushions are essential morphogenetic events in AV valvuloseptal morphogenesis. BMP-2 myocardial conditional knockout (cKO) mice die early by embryonic day (ED) 10.5 [1], hampering investigation of the role of BMP-2 in AV valvuloseptal morphogenesis after this stage. In our previous study, we localized BMP-2 and type I BMP receptors, and , in AV endocardial cushions [2, 3]. Based on their expression patterns, we hypothesize that autocrine signaling by BMP-2 within mesenchymalized AV cushions plays a critical role during AV valvuloseptal morphogenesis. To test this hypothesis, we employed recently generated endocardial/endocardial cushion-specific cre-driver line . Unlike a previously generated line whose cre-mediated recombination is restricted to AV and OT endocardium, this line confers cre-mediated recombination within the endocardial cells as well as their mesenchymal progeny. Using the driver line, we disrupted BMPR1A (Alk3) and BMP-2 specifically from AV endocardium and endocardial cushions. endocardial cushion cKO () mouse embryos died by ED 12.5 and exhibited failure of cellularization of AV cushions (Fig. 22.1a–c) and disruption of extracellular matrix (ECM) protein deposition in the cushion mesenchyme. On the other hand, AV cushion formation occurred in the BMP-2 mice that survived beyond the AV cushion formation stage because BMP-2 expression remained intact in the AV myocardium during AV cushion formation. - mice exhibited perimembranous ventricular septal defects (VSDs) (Fig. 22.1d, e), defective deposition of ECMs in the membranous septum, and AV mitral valve dysplasia, suggesting the cell autonomous requirement of BMP-2 in AV endocardial cushions. - did not exhibit muscular VSDs. These data strongly support our hypothesis that cell autonomous signaling by BMP-2 in the endocardial lineage plays a significant role in mesenchymalized AV cushions during AV valvuloseptal morphogenesis.

Heart tube elongation occurs by progressive addition of cells from pharyngeal mesoderm to the poles of the heart. These progenitor cells, termed the second heart field, contribute to right ventricular and outflow tract myocardium at the arterial pole of the heart and to atrial myocardium at the venous pole. Perturbation of this process results in congenital heart defects. Since the discovery of this progenitor cell population, much has been learned about the signaling pathways and transcription factors regulating second heart field deployment. However, fundamental questions about the molecular and cellular mechanisms underlying heart tube elongation remain. Here we briefly review a selection of recent findings in the area of second heart field biology and discuss the clinical implications of these new studies for our understanding of the etiology of congenital heart defects.

Nodal is a TGF-β family member ligand that is critical for early embryonic patterning in vertebrates. Nodal signaling functions through core TGF-β receptors to activate a Smad transcription factor signaling cascade. However, unlike other TGF-β ligands, Nodal signaling requires an additional co-receptor of the EGF-CFC family to activate intracellular signaling. Nodal signaling is also subject to extensive negative regulation by Lefty and other factors. Work in numerous model organisms, including mouse, chicken, and zebrafish, established that Nodal signaling plays an essential role during germ layer formation, anterior-posterior axis patterning, and left-right axis determination. Incomplete or delayed loss of Nodal signaling results in defective organogenesis and birth defects, including congenital heart defects, and clinical studies have linked aberrant Nodal signaling in humans with many common congenital malformations, including congenital heart defects. Congenital heart defects associated with disrupted Nodal signaling in mammals include those that arise due to global defects in left-right patterning of the embryo, such as heterotaxy. Other Nodal-associated heart defects appear to occur as more subtle isolated malformations of the great arteries and atrioventricular septum, which may not be related to overall perturbations in laterality. A more detailed understanding of the Nodal signaling pathway and its targets in the heart is required to more fully understand the etiology of Nodal signaling pathway-associated congenital heart defects.

Heart formation relies on two sources of cardiomyocytes: the first heart field (FHF), which gives rise to the linear heart tube, and the second heart field (SHF), which gives rise to the right ventricle, the outflow tract, parts of the atria, and the inflow tract. The development of the SHF is of particular importance due to its relevance to common congenital heart defects. However, it remains unclear how the SHF is maintained in a progenitor state while the FHF differentiates. Likewise, the factors that trigger SHF differentiation into specific cardiac cell types are poorly understood. Investigation of SHF development can benefit from the utilization of multiple model organisms. Here, we review the experiments that have identified the SHF in zebrafish and investigated its contribution to the poles of the zebrafish heart. Already, zebrafish research has illuminated novel positive and negative regulators of SHF development, cementing the utility of zebrafish in this context.

Progenitor cells, derived from the cardiac neural crest (CNC) and the second heart field (SHF), play key roles in development of the cardiac outflow tract (OFT), and their interaction is essential for establishment of the separate pulmonary and systemic circulation in vertebrates. 22q11.2 deletion syndrome (22q11DS) or Takao syndrome is the most common human chromosomal deletion syndrome that is highly associated with OFT defects. Historically, based on the observations in animal models, OFT defects implicated in the 22q11/Takao syndrome are believed to result primarily from abnormal development of CNC that populate into the conotruncal region of the heart. In the twenty-first century, elegant efforts to model 22q11/Takao syndrome in mice succeeded in the identification of T-box-containing transcription factor, Tbx1, as an etiology of OFT defects in this syndrome. Subsequent investigations of the Tbx1 expression pattern revealed that Tbx1 was surprisingly not detectable in CNC but was expressed in the SHF and provided a new concept of molecular and cellular basis for OFT defects associated with 22q11/Takao syndrome. More recently, it was reported that mutations in the gene encoding the transcription factor GATA6 caused CHD characteristic of OFT defects. Genes encoding the neurovascular guiding molecule semaphorin 3C (SEMA3C) and its receptor plexin A2 (PLXNA2) appear to be regulated directly by GATA6. Elucidation of molecular mechanism involving GATA6, SEMA3C, PLXNA2, and TBX1 in the interaction between the CNC and the SHF would provide new insights into the OFT development.

Congenital heart disease is the most common birth defects, affecting 1 % live births [1]. The cardiovascular system undergoes a series of morphogenetic events to form a heart and an aorta in fetuses. Formation of the heart and aorta requires migration, differentiation, and precise interactions among multiple cells from several embryonic origins [2]. Forkhead box2 (Foxc2) encodes a transcription factor and is expressed in mesodermal tissues, such as the pharyngeal artery, outflow tract endothelial/surrounding mesenchyme, bone, and kidney [3]. Simple knockout of Foxc2 in mouse causes an interrupted aortic arch, ventricular septal defect, cleft palate, and skeletal malformation [4]. The heart is made from primary and secondary heart field progenitors. The primary heart field gives rise to the left ventricle and atria, while the secondary heart field contributes mainly to the right ventricle and outflow tract [5] (Fig. 27.1).

Congenital heart disease is still the leading cause of death within the first year of life. Our lab forces on understanding the morphology of congenital heart disease. Outflow tract anomalies, including abnormal alignment or septation, account for 30 % of all congenital heart disease. To solve the developmental problem of these defects, we are interested in the role of the second heart field (SHF) that gives rise to the outflow tract structure.

Blood vessels constantly subjected to mechanical stress have well-developed elastic fiber-rich frameworks, which contribute to the elasticity and distensibility of the vascular wall. Destruction of the fibrous structure due to genetic predisposition as well as acquired disorders such as Kawasaki disease often induces irreversible dilation of blood vessels, e.g., aneurysm formation. In addition to their structural role, extracellular matrix molecules also provide important biological signaling, which influences various cellular functions. Among them, increased attention has been focused on matricellular proteins, a group of nonstructural extracellular matrix (ECM) proteins highly upregulated in active tissue remodeling, serving as biological mediators by interacting directly with cells or regulating the activities of growth factors, cytokines, proteases, and other ECM molecules. Tenascin-C (TNC) is a typical matricellular protein expressed during embryonic development and tissue repair/regeneration in a spatiotemporally restricted manner. Various growth factors, pro-inflammatory cytokines, and mechanical stress upregulate its expression. TNC controls cell adhesion, migration, differentiation, and synthesis of ECM molecules. Our recent results suggest that TNC may not only play a significant role in the recruitment of smooth muscle/mural cells during vascular development, but also regulate the inflammatory response during pathological remodeling. TNC may be a key molecule during vascular development, adaptation, and pathological tissue remodeling.