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10 Molecular Regulation of Cardiogenesis

VISHAL NIGAM AND DEEPAK SRIVASTAVA

ongenital malformations, the most common of all human destined to contribute to speci( c chambers of the future heart (reviewed Cbirth defects, occur in almost 1% of the population worldwide, in Srivastava and Olson, 2000). However, such studies could not regardless of race (Hoffman and Kaplan, 2002). An additional determine the clonal contributions of individual cells (Meilhac 1%–2% of the population harbor more subtle cardiac developmental et al., 2004). More recent studies using Cre-lox technologies to mark anomalies that only become apparent as age-dependent phenomena progenitor cells and all their descendents indicate—in stark contrast reveal the underlying pathology. With more than 1 million survivors to previous models—that the heart tube derived from the FHF may of congenital heart disease (CHD) in the United States, it is becoming predominantly provide a scaffold which enables a second population apparent that genetic disruptions which predispose to developmental of cells to migrate and expand into cardiac chambers (Buckingham defects can have ongoing consequences in the maintenance of speci( c et al., 2005). These additional cells arise from an area often referred cell types and cellular processes over decades (Srivastava, 2004). A to as the “second heart ( eld (SHF)” or “anterior heart ( eld” based on more precise understanding of the causes of CHD is imperative for its location anterior and medial to the crescent-shaped primary heart the recognition and potential intervention of progressive degenerative ( eld (Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001; conditions among the survivors of CHD. Fig. 10–1). Both heart ( elds appear to be regulated by complex posi- Although genetic approaches have been important in understand- tive and negative signaling networks involving members of the bone ing human CHD, detailed molecular analysis of cardiac development morphogenetic protein (Bmp), sonic hedgehog (Shh), ( broblast growth in humans has been dif( cult. The recognition that genetic pathways factor (Fgf), Wnt and Notch proteins. Such signals often arise from the which dictate cardiac development are highly conserved across vastly adjacent endoderm, although the precise nature and role of these sig- diverse species ranging from * ies to man has resulted in a rapid nals remain unknown (reviewed in Schultheiss et al., 1997; Marvin expansion of information from the studies in more tractable biologi- et al., 2001; Schneider and Mercola, 2001; Zaffran and Frasch, cal models (Srivastava and Olson, 2000; Chien and Olson, 2002). 2002). SHF cells remain in an undifferentiated progenitor state until Despite the diversity of body plans adopted by different species, there incorporation into the heart, and this may in part be due to the closer seems to exist a common genetic program for the early formation of proximity to inhibitory Wnt signals emanating from the midline. a . Cardiovascular systems seem to have developed Recent work has raised the possibility that the Tbx18 may be required increasing complexity to adapt to speci( c environments. In a simpli- for the formation of a venous pole, which contributes portions of the ( ed view, it appears that higher organisms have retained the morpho- atria and venous structures (Christoffels et al., 2006). logic steps used by lower organisms and have built complexity into the As the heart tube forms, the SHF cells migrate into the midline and heart as needed. In particular, the speci( cation of chamber structures position themselves dorsal to the heart tube in the pharyngeal meso- and the advent of a parallel circulation through chamber duplication derm. Upon rightward looping of the heart tube, SHF cells cross the and out* ow tract division by neural crest derivatives have facilitated pharyngeal into the anterior and posterior portions, popu- the development of larger, air-breathing organisms using complex cir- lating a large portion of the out* ow tract, future right and culatory systems. In such a scheme, defects in particular regions of the atria (Cai et al., 2003; Fig. 10–1). Precursors of the left ventricle are heart may arise from speci( c genetic and environmental effects dur- sparsely populated by the SHF and appear to be largely derived from ing discrete developmental windows of time. To simplify the complex the FHF. In contrast to the FHF, SHF cells do not differentiate into events of cardiogenesis and CHD, different regions of the developing cardiac cells until they are positioned within the heart. Once within the heart will be considered individually in the context described earlier, heart, FHF and SHF cells appear to proliferate in response to endocar- weaving knowledge from model systems and human genetics when dial-derived signals such as neuregulin and epicardial signals depen- available. dent on retinoic acid, although the mechanisms through which these noncell autonomous events occur remain poorly understood (Garratt ORIGIN OF CARDIOMYOCYTE PRECURSORS et al., 2003; Stuckmann et al., 2003). Despite decades of cell lineage tracings and descriptive CARDIOMYOCYTE AND HEART TUBE FORMATION of the heart’s origins, a more complete and accurate picture of cardio- genesis emerged only recently (reviewed in Buckingham et al., 2005; Fruit* ies have a primitive heart-like structure known as the dorsal ves- Srivastava, 2006). Two distinct mesodermal heart ( elds that share a sel that is analogous to the straight heart tube of the vertebrate . common origin appear to contribute cells to the developing heart in a It contracts rhythmically and pumps hemolymph through an open temporally and spatially speci( c manner. The well-studied “( rst heart circulatory system. Formation of the dorsal vessel in * ies is depen- ( eld” (FHF) is derived from cells in the anterior lateral plate meso- dent on a protein, tinman, whose name is based on the Wizard of Oz derm, which align in a crescent shape at approximately embryonic day character that lacks a heart (Bodmer, 1993). Tinman belongs to the 7.5 (E7.5) in the mouse embryo, roughly corresponding to week 2 of homeodomain family of proteins, and was initially described to play human gestation (Fig. 10–1). By mouse E8.0, or 3 weeks in humans, a role in establishing the regional identity of cells and organs during these cells coalesce along the ventral midline to form a primitive heart embryogenesis. tube that consists of an interior layer of endocardial cells and an exte- In contrast to the requirement of tinman for heart formation in * ies, rior layer of myocardial cells, separated by the its mammalian ortholog, Nkx2.5, is not essential for speci( cation of the necessary for reciprocal signaling between the two layers. The tubular cardiac lineage in mice, suggesting either that other genes may share heart initiates rhythmic contractions at about day 23 in humans. functions with Nkx2.5 or that cardiogenesis in * ies and vertebrates dif- Previous lineage tracings using dye-labeling techniques suggested fers with respect to its dependence on this family of homeobox genes that cells along the anterior–posterior (AP) axis of the heart tube were (Lyons et al., 1995; Tanaka et al., 1999). The possibility of functional

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Figure 10–1. Illustration of cardiac development. Illustrations depict cardiac aortic arch arteries (III, IV, and VI) and , which together contrib- development, with morphologically related regions color-coded, seen from a ute to speci( c segments of the mature aortic arch, also color-coded. (C, D) ventral view. (A) Two distinct cardiogenic precursor ( elds form a crescent Mesenchymal cells form the cardiac valves from the conotruncal (CT) and that is speci( ed to form speci( c regions of the heart tube (A, artery; V, ven- atrioventricular valve (AVV) segments, which divide into separate left- and tricle), which is patterned to form the various regions and chambers of the right-sided valves. Corresponding days of human embryonic development looped and mature heart. (B) The secondary heart ( eld (SHF) contributes are indicated. Ao, ; DA, ; LA, left ; LCC, left to much of the right ventricle and out* ow tract as the heart loops. (C) Each common carotid; LSCA, left subclavian artery; LV, left ventricle; PA, pul- cardiac chamber balloons from the outer curvature of the looped heart tube monary artery; RA, right atrium; RCC, right common carotid; RSCA, right in a segmental fashion. Neural crest cells populate the bilaterally symmetric subclavian artery; RV, right ventricle.

redundancy between Nkx2.5 and other cardiac-expressed homeobox outer curvature of the heart (Biben and Harvey, 1997; Thomas et al., genes in vertebrates is supported by the ability of dominant-negative 1998). Remodeling of the inner curvature occurs, allowing migration versions of Nkx2.5 to block cardiogenesis in frog and zebra( sh of the in* ow tract to the right and out* ow tract to the left, facilitating (Fu et al., 1998; Grow and Krieg, 1998). Similarly, the transcriptional proper alignment and separation of right- and left-sided circulations. co-activator, myocardin, is necessary and suf( cient in frogs for cardiac Defects of inner curvature remodeling may underlie a host of human gene expression, likely through the activation of serum response fac- congenital heart malformations that involve improper alignment of the tor (SRF)-dependent genes (Wang et al., 2001; Small et al., 2005). atria, ventricles and out* ow tract, and are often observed in the setting Combinations of these transcription factors along with Mef2, Gata, of abnormalities of left–right (LR) asymmetry. Other cardiac defects Hand, and Tbx family members appear to form core regulatory circuits are a result of genetic defects that cause disruption of discrete devel- that control early events during cardiogenesis (reviewed in Srivastava opmental events, making it useful to consider the molecular processes et al., 2006). governing central morphogenetic aspects of cardiogenesis. The cardiac out* ow tract (conotruncus) and parts of the right ven- tricle are the last segments to form and are derived from SHF cells COMPLEX REGULATION OF CARDIAC as described earlier. The transcription factor, Tbx1, which appears to be a cause of cardiac and craniofacial disorders in humans (Lindsay et al., 1999; Jerome and Papaioannou, 2001; Lindsay et al., 2001; While the pathways regulating individual cell lineages contributing Merscher et al., 2001), is a major transcriptional regulator of the SHF to the heart are deeply understood, the subsequent complex events and is necessary for proper development of conotruncal myocardium involved in integrating multiple cell types, formation of chambers and and Fgf secretion (Yamagishi et al., 2003; Hu et al., 2004; Xu et al., patterning of the distinct regions of the heart are also being elucidated 2004). Islet1 (Isl1), a transcription factor involved in pancreatic devel- now. Some aspects of these morphogenetic events are described below, opment, also marks this population and is necessary for its develop- while others have been reviewed in depth elsewhere (Olson, 2004; ment (Cai et al., 2003). Interestingly, Isl1-positive cells mark niches of Parmacek and Epstein, 2005; Srivastava, 2006). Notably, develop- cardiac progenitor cells in the postnatal heart (Laugwitz et al., 2005), ment of the cardiac electrical conduction system, derived in part from suggesting that understanding the regulation of SHF-derived progeni- specialized cells that respond to secreted signals from tor pools may be useful in developing approaches for cardiac repair. the vascular and (Gourdie et al., 1998; In addition to the AP segmentation, a discrete dorsal–ventral (DV) Rentschler et al., 2003), is an area not discussed here but which is polarity is present in the primitive heart tube. As the heart tube loops essential to normal cardiac function (reviewed in Mikawa et al., 2003). to the right, the ventral surface of the tube rotates, becoming the The recent ( nding that Irx5-mediated regulation of key ion channels outer curvature of the looped heart with the dorsal surface forming in the myocardium patterns the propagation wave of electrical activity the inner curvature. The outer curvature becomes the site of active highlights the importance of integration between specialized conduc- growth, while remodeling of the inner curvature is essential for ulti- tion cells and the myocardium (Costantini et al., 2005). mate alignment of the in* ow and out* ow tracts of the heart. A model in which individual chambers “balloon” from the outer curvature in a DV Polarity segmental fashion has been proposed (Moorman and Christoffels, Consistent with the “balloon” model of chamber formation described 2003). Consistent with this model, numerous genes, including the tran- earlier, numerous genes, including the transcription factor Hand1 and scription factor Hand1, are expressed speci( cally on the ventral and the sarcomeric protein Serca2, are expressed speci( cally on the outer

110-Epstein-Chap10.indd0-Epstein-Chap10.indd 112525 88/21/2007/21/2007 88:35:16:35:16 PPMM 126 PATTERNS OF DEVELOPMENT curvature of the heart (Biben and Harvey, 1997; Thomas et al., 1998). and cause out* ow tract defects similar to those observed in humans Also, through a complex transcriptional network, the unique identity (Hutson and Kirby, 2003). Embryos de( cient in of inner curvature cells is determined by Tbx2-mediated repression of cell migration or differentiation display a variety of cardiac out* ow genes typically found on the outer curvature (Harrelson et al., 2004). tract and aortic arch defects resembling those in humans. These Another Tbox transcription factor, Tbx20, serves to repress Tbx2 activ- include tetralogy of Fallot, persistent , double-outlet ity in the outer curvature as it expands into the cardiac chambers, thereby right ventricle, ventricular septal defects and defects of aortic arch pat- establishing the regional patterning of expanding or remodeling myo- terning. Thus, abnormalities in neural crest migration or differentia- cardium (Singh et al., 2005; Stennard et al., 2005; Takeuchi et al., tion likely underlie many of the conotruncal and aortic arch defects 2005). Remodeling of the inner curvature allows migration of the in* ow seen in humans. Indeed, human mutations of the neural crest–enriched tract to the right and the out* ow tract to the left, facilitating proper align- transcription factor, TFAP2β result in persistent patency of the ductus ment and separation of right- and left-sided circulations. In addition to its arteriosus, a specialized aortic arch vessel essential for fetal cardiac role in repressing Tbx2, Tbx20 affects expansion of both the FHF- and physiology (Satoda et al., 2000; Fig. 10–1). It is likely that other the SHF-derived cells and is necessary for out* ow tract development, genetic mutations affect speci( c regions of the aortic arch. possibly via regulation of Nkx2.5 and Mef2c (Takeuchi et al., 2005). Disruption of SHF development by mutation of genes such as Tbx1, Expansion of the ventricular chambers and the regulation of number of Fgf8 , and Isl1 results in the defects similar to those observed with myocytes may also be regulated by the homeodomain-only protein, Hop, neural crest disruption, including persistent truncus arteriosus (failure a downstream target of Nkx2.5 (Chen et al., 2002; Shin et al., 2002). of out* ow septation), malalignment of the out* ow tract of the heart with the ventricular chambers and ventricular septal defects (Jerome LR Asymmetry and Papaioannou, 2001; Abu-Issa et al., 2002; Frank et al., 2002; Defects of inner curvature remodeling may underlie a host of human Cai et al., 2003). Since SHF-derived myocardial cells neighbor neural congenital heart malformations that involve improper alignment of the crest–derived cells and secrete growth factors such as Fgf8 in a Tbx1- atria, ventricles and out* ow tract. These include situations where both dependent manner that in* uence neural crest cells (Hu et al., 2004), atrioventricular valves empty into the left ventricle (double-inlet left reciprocal interactions between the SHF- and neural crest–derived ventricle), or when the aorta and both exit from the cells in the out* ow tract are likely essential for normal development. right ventricle (double-outlet right ventricle), similar to those observed Consistent with this, humans with deletion or mutation of Tbx1 (Yagi in ma ny muta nt mouse models (reviewed inKathiriya and Srivastava, et al., 2003), expressed in the SHF, appear to have cell-autonomous 2000; Franco and Campione, 2003). Such defects are often observed defects of SHF development and noncell-autonomous anomalies of in the setting of abnormalities of LR asymmetry determination. Thus, neural crest–derived tissues. It will be interesting to determine if a LR decisions may affect not only the direction of cardiac looping but large number of human cardiac out* ow tract defects is a direct result also the proper alignment of chambers, likely through the regulation of SHF migration, differentiation or proliferation. of gene expression along the inner versus outer curvature and ventral versus dorsal surface of the developing heart. Cardiac Valve Formation The elegant molecular network regulating LR asymmetry of the body Appropriate placement and function of cardiac valves is essential for plan has been reviewed (Palmer, 2004) and will not be summarized chamber septation and for unidirectional * ow of blood through the here. However, it is worth highlighting several clues about the rela- heart. A molecular network involving Bmp2 and Tbx2 de( nes the tionship between LR asymmetry and proper alignment of the cardiac position of the valves relative to the chambers (Harrelson et al., 2004; chambers. The cascade of LR signals, including Shh and Nodal, con- Beis et al., 2005; Ma et al., 2005). During early heart tube formation, verge on the transcription factor Pitx2 (Piedra et al., 1998). Pitx2 is “cushions” of extracellular matrix between the endocardium and initially LR asymmetric in the linear heart tube, but this asymmetry is myocardium presage valve formation at each end of the heart tube. translated into a DV polarity in the looped heart tube. Because Pitx2 Reciprocal signaling, mediated in part by transforming growth factor- regulates cell proliferation via cyclin D2 and also controls cell migra- βTGF-β family members, between the myocardium and endocardium tion events (Kioussi et al., 2002), it is a potential link between the in the cushion region induces a transformation of endocardial cells into signals regulating the direction and process of cardiac looping. Within mesenchymal cells that migrate into the extracellular matrix cushion certain subdomains, regulation of Pitx2 by Tbx1 integrates the tran- (Brown et al., 1999; Kim et al., 2001; Gaussin et al., 2002). These mes- scriptional pathways controlling morphogenesis and LR asymmetry enchymal cells differentiate into the ( brous tissue of the valves and (Nowotchin et al., 2006). are involved in septation of the common into right- and left-sided ori( ces. Cardiac Outfl ow Tract Regulation The Smad proteins are intracellular transcriptional mediators of sig- Congenital cardiac defects involving the cardiac out* ow tract, aortic naling initiated by TGF-β ligands. Smad6 is speci( cally expressed in arch, ductus arteriosus and proximal pulmonary arteries account for the atrioventricular cushions and out* ow tract during cardiogenesis, 20%–30% of all CHD. This region of the heart undergoes extensive and is a negative regulator of TGF-β signaling. Targeted disruption of and rather complex morphogenetic changes with contributions from Smad6 in mice results in thickened and gelatinous atrioventricular and neural crest cells and the SHF, as discussed earlier. Mesenchymal cells semilunar valves, comparable to those observed in human aortic and originating from the crest of the neural folds are essential for proper pulmonary valve disease (Galvin et al., 2000). Similarly, the absence of septation and remodeling of the out* ow tract and aortic arch (reviewed PTPN11, which encodes the protein tyrosine phosphatase Shp-2, results in Hutson and Kirby, 2003). Such neural crest–derived cells migrate in dysplastic out* ow valves through its involvement in a signaling path- away from the neural folds and retain the ability to differentiate into way mediated by epidermal growth factor receptor (Chen et al., 2000). multiple cell types. The migratory path and ultimate fate of these cells The importance of PTPN11 in CHD was shown by the presence of point depends on their relative position of origin along the AP axis and are mutations in PTPN11 in patients with Noonan’s syndrome, who com- partly regulated by the Hox code (LeDourin et al., 2004). Neural crest monly have pulmonic valve stenosis ( Tartaglia et al., 2001). Finally, cells differentiate and contribute to diverse embryonic structures, mice lacking Ephrin B2 also have thickened valves and, although the including the cranial ganglia, peripheral nervous system, adrenal mechanism for this remains unclear, it will be interesting to determine glands and melanocytes. Neural crest cells that arise from the otic pla- how these signaling pathways intersect (Cowan et al., 2004). code to the third somite migrate through the developing pharyngeal In contrast to the thickened lea* ets described earlier, disruption arches and populate the mesenchyme of each of the aortic arch arter- of signaling pathways converging on the transcription factor Nfatc ies and the mesenchyme necessary to septate the out* ow tract septum revealed a requirement of this calcium-activated regulator. Nfatc is (Fig. 10–1). Because of their migratory path and role, this segment of expressed speci( cally in the forming embryonic valves, and targeted the neural crest is often referred to as the cardiac neural crest. deletion of Nfatc in mice results in the absence of cardiac valve forma- Mutations in many signaling cascades affect neural crest migration tion (de la Pompa et al., 1998; Ranger et al., 1998). Signaling via the or development, including the endothelin and semaphorin pathways, phosphatase, calcineurin results in nuclear translocation of Nfatc and

110-Epstein-Chap10.indd0-Epstein-Chap10.indd 112626 88/21/2007/21/2007 88:35:19:35:19 PPMM Molecular Regulation of Cardiogenesis 127 is similarly involved in cardiac valve formation, in part through the structural motifs in the protein. One potential cofactor is NKX2-5, as regulation of vascular endothelial growth factor (Vegf ) expression in the two physically interact and cooperate to activate common target the endocardium (Chang et al., 2004). genes (Hiroi et al., 2001). The Notch signaling pathway is required for cell fate and differ- Like the NKX2.5 and TBX5 mutations, mutations in the zinc- entiation decisions throughout the embryo (Artavanis-Tsakonas ( nger-containing protein GATA4 cause similar atrial and ventricular et al., 1999), but only recently have Notch proteins been implicated in septal defects in autosomal dominant nonsyndromic human pedi- vertebrate cardiac development. In ( sh and frogs, Notch appears to be grees (Garg et al., 2003). GATA4 or related proteins are essential for involved in the development of that contribute cardiogenesis in * ies, ( sh, and mice (Kuo et al., 1997; Molkentin to valve tissue (Timmerman et al., 2004). In humans, heterozygous et al., 1997; Gajewski et al., 2001; Reiter et al., 2001). Like NKX2.5, NOTCH1 mutations disrupt normal development of the aortic valve GATA4 and TBX5 also form a complex to regulate downstream genes, and, occasionally, the mitral valve ( Garg et al., 2005). The severity such as myosin heavy chain (MHC). Consistent with an important role of valve disease associated with NOTCH1 mutations in humans varies for such combinatorial interactions, a familial GATA4 point mutation widely from the mild disease in which the aortic valve has two, rather disrupts GATA4’s ability to interact with TBX5 ( Garg et al., 2005). than three, lea* ets (bicuspid aortic valve) to the severe defects in valve Conversely, several human TBX5 mutations disrupt TBX5 interaction patency in utero, resulting in left ventricular growth failure. Consistent with GATA4, suggesting that the two cooperate in cardiac septation with this genetic ( nding, 15% of “normal” relatives of children with events (Garg et al., 2003). Con( rming a genetic interaction between hypoplastic left heart syndrome (HLHS) have subclinical bicuspid the two proteins, mice heterozygous for Gata4 and Tbx5 are embryonic aortic valves (Cripe et al., 2004; Loffredo et al., 2004), suggesting lethal from a myocardial defect and severe hypoplasia of the endocar- that disruption of the NOTCH signaling cascade may underlie a spec- dial cushion tissue necessary for valvuloseptal development (V. Garg trum of aortic valve disease. While not speci( cally affecting valves, and D. Srivastava, unpublished observations). GATA4, TBX5, and human mutations in JAGGED1, a NOTCH ligand, also cause out* ow NKX2-5 may form a common complex that is necessary for proper tract defects associated with the autosomal dominant disease, Alagille cardiac septation. Disruption of any one of the three proteins or their syndrome (Li et al., 1997; Oda et al., 1997; Krantz et al., 1999). interactions can result in atrial or ventricular septal defects. Although The hairy-related family of transcriptional repressors (Hrt1, Hrt2, the compendium of septal genes regulated by these transcription fac- and Hrt3) may mediate the Notch signal during valve and myocardial tors is unknown, it is intriguing that mutations in human MHC, a direct development; however, their targets for repression remain unknown target of GATA4, TBX5, and NKX2-5, also cause atrial septal defects (Nakagawa et al., 1999; reviewed in Kokubo et al., 2005). (Ching et al., 2005). This observation suggests a possible mechanism by which these genes cause septation defects. MOLECULAR REGULATION OF SEPTAL FORMATION Recent ( ndings with the cardiac transcription factors NKX2.5, TBX5, microRNA REGULATION OF CARDIOMYOCYTE and GATA4 exemplify the synergy between human genetics and stud- DIFFERENTIATION ies of model organisms for understanding the etiology of human CHD. Numerous point mutations have been identi( ed in Nkx2.5 in families While transcriptional and epigenetic events regulate many critical with atrial septal defects and progressive cardiac conduction abnormal- cardiac genes, translational control by small noncoding RNAs, such as ities (Schott et al., 1998). Retrospective analysis of mice heterozygous microRNAs (miRNAs), has recently emerged as another mechanism to for Nkx2.5 disruption revealed a similar phenotype and progressive “( ne-tune” dosages of key proteins during cardiogenesis (Kwon et al., apoptotic loss of conduction cells, suggesting a likely mechanism for 2005; Zhao et al., 2005). miRNAs are genomically encoded 20–22 the human phenotype (Biben et al., 2000; Jay et al., 2004). nucleotide RNAs that target messenger RNAs (mRNAs) for translational Humans with Holt–Oram syndrome caused by mutations in Tbx5 inhibition or degradation by many of the same pathways as small interfer- have cardiac anomalies, similar to those with Nkx2.5 mutations ing RNA (siRNA) (Ambros, 2004; He and Hannon, 2004). More than 400 (atrial and ventricular septal defects), as well as limb abnormalities human miRNAs have been identi( ed (Cummins et al., 2006), but in only (Basson et al., 1997; Mori and Bruneau, 2004). Intriguingly, muta- a few cases are the biological function and mRNA targets known. tions responsible for defects in the heart and limbs are clustered in dif- The miRNA-1 family (miR-1-1 and miR-1-2) is highly conserved ferent regions of the protein, suggesting that TBX5 engages different from worms to humans and is speci( cally expressed in the develop- downstream genes or cofactors in these tissues that depend on unique ing cardiac and skeletal muscle progenitor cells as they differentiate

Figure 10–2. miR-1-1 and miR-1-2 enhancer-driven lacZ expression. The expression patterns of miR-1-1 (A) and miR-1-2 (B) are demonstrated by the β-gal (blue) staining in embryonic day 11.5 mouse embryos. h, head; ht, heart; arrowhead indicates somites.

110-Epstein-Chap10.indd0-Epstein-Chap10.indd 112727 88/21/2007/21/2007 88:35:20:35:20 PPMM 128 PATTERNS OF DEVELOPMENT (Zhao et al., 2005). Enrichment of miR-1-1 is initially observed in well-understood organs in biology. This knowledge is now being used the atrial precursors before becoming ubiquitous in the heart, while to discover the underlying genetics of CHD and the basis for many adult- miR-1-2 is speci( c for the ventricle throughout development, sug- onset diseases that have their origin in the mutations of developmental gesting that the two may have chamber-speci( c effects in vivo. Both genes. What remains lacking is a coherent picture of the hierarchical are highly expressed in the SHF-derived cells of the cardiac out* ow pathways that govern most developmental processes and the mechanisms tract (Fig. 10–2). Interestingly, expression of these miRNAs is directly through which such pathways regulate the cell biology of morphogenesis. controlled by well-studied transcriptional regulatory networks that Such knowledge will require a better understanding of the target genes of promote muscle differentiation. Cardiac expression is dependent on critical transcriptional and signaling pathways that control cardiogenesis. SRF, and skeletal muscle expression requires the myogenic transcrip- This will be an essential step as the targets will be the most amenable sites tion factors, MyoD and Mef2. SRF recruits the potent co-activator, of intervention, both in a therapeutic sense and for prevention. For exam- myocardin, to cardiac and smooth muscle–speci( c genes that control ple, the identi( cation of dietary substances, such as the folic acid used to differentiation (Wang et al., 2001). Consistent with a role in differen- prevent neural tube defects, that modulate key developmental pathways tiation, overexpression of miR-1 in the developing mouse heart results will be necessary for preventive efforts. In addition, genetic identi( cation in a decrease in ventricular myocyte expansion, with fewer proliferat- of those at risk for adult-onset disease, which has its origin in a cardiac ing cardiomyocytes remaining in the cell cycle. In vivo validation of developmental defect such as the age-related calci( cation that occurs in Hand2, a transcription factor that regulates ventricular expansion, as bicuspid aortic valve, will provide ample time for intervention to slow a miR-1 target suggests that tight regulation of Hand2 protein levels the progression of disease. The convergence of addition developmental AQ1 may be involved in controlling the balance between cardiomyocyte knowledge and improved genetic tools should make this vision a reality proliferation and differentiation. Disruption of the single * y ortholog in the coming years. of miR-1 had catastrophic consequences resulting in uniform lethality at embryonic or larval stages with a frequent defect in maintaining cardiac gene expression (Kwon et al., 2005). In a subset of * ies lacking ACKNOWLEDGMENTS miR-1, a severe defect of cardiac progenitor cell differentiation pro- The authors thank B. Taylor for editorial assistance. V.N. is supported by an vided loss-of-function evidence that miR-1 was involved in muscle dif- NIH T32 grant; D.S. is supported by grants from NHLBI/NIH, March of Dimes ferentiation events, similar to the gain-of-function ( ndings in mice. Birth Defects Foundation, and is an Established Investigator of the American Heart Association. ENVIRONMENTAL FACTORS ASSOCIATED WITH CHD References Abu-Issa R, Smyth G, Smoak I, Yamamura K, Meyers EN (2002). Fgf8 is required for pharyn- A number of maternal conditions and/or exposures have been associ- geal arch and cardiovascular development in the mouse. Development 129: 4613–4625. ated with congenital cardiac defects. We will brie* y highlight a few Artavanis-Tsakonas S, Rand MD, Lake RJ (1999). Notch signaling: cell fate control and examples of maternal in* uences below. signal integration in development. Science 284: 770–776. Both maternal systemic lupus erythematosus and diabetes mellitus Basson CT, Bachinsky DR, Lin RC, Levi T, Elkins JA, Soults J, Grayzel D, Kroumpouzou E, Traill TA, Leblanc-Stracesk i J, et al. (1997). Mutations in human TBX5 cause limb and have well-established linkages to congenital heart defects. Maternal cardiac malformation in Holt-Oram syndrome. Nat Genet 15: 30–35. systemic lupus is a cause of congenital heart block. 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AQ1: Did you mean “additional”?

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