Uncovering the origins of myocardial cells is important for understanding and treating heart diseases. Cai et al. suggest that Tbx18-expressing epicardium provides a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Here we show that the T-box transcription factor gene 18 (Tbx18) itself is expressed in the myocardium, showing that their genetic lineage tracing system does not allow conclusions of an epicardial origin of cardiomyocytes in vivo to be drawn.

Abstract-The primary myocardium of the embryonic heart, including the atrioventricular canal and outflow tract, is essential for septation and valve formation. In the chamber-forming heart, the expression of the T-box transcription factor Tbx2 is restricted to the primary myocardium. To gain insight into the cellular contributions of the Tbx2 ϩ primary myocardium to the components of the definitive heart, genetic lineage tracing was performed using a novel Tbx2 Cre allele. These analyses revealed that progeny of Tbx2 ϩ cells provide an unexpectedly large contribution to the Tbx2-negative ventricles. Contrary to common assumption, we found that the embryonic left ventricle only forms the left part of the definitive ventricular septum and the apex. The atrioventricular node, but not the atrioventricular bundle, was found to derive from Tbx2 ϩ cells. The Tbx2 ϩ outflow tract formed the right ventricle and right part of the ventricular septum. In Tbx2-deficient embryos, the left-sided atrioventricular canal was found to prematurely differentiate to chamber myocardium and to proliferate at increased rates similar to those of chamber myocardium. As a result, the atrioventricular junction and base of the left ventricle were malformed. Together, these observations indicate that Tbx2 temporally suppresses differentiation and proliferation of primary myocardial cells. A subset of these Tbx2Cre -marked cells switch off expression of Tbx2, which allows them to differentiate into chamber myocardium, to initiate proliferation, and to provide a large contribution to the ventricles. These findings imply that errors in the development of the early atrioventricular canal may affect a much larger region than previously anticipated, including the ventricular base. Key Words: atrioventricular canal Ⅲ lineage Ⅲ fate Ⅲ patterning Ⅲ transgenic Ⅲ Cre T he embryonic heart tube is composed of "primary" myocardium and rapidly elongates by addition of progenitor cells to its poles. During looping, specific regions in the embryonic tubular heart differentiate to chamber myocardium and expand to form the future working myocardium of the ventricles and atria. In contrast, the region in between these expanding chambers does not differentiate or expand and becomes visible as an atrioventricular constriction. 1 During prenatal life, the atrioventricular canal (AVC) myocardium conducts the electric impulse between the atrial and ventricular chambers in a slow manner, reminiscent of the function of the mature atrioventricular (AV) node. This slow conducting feature allows the AVC to act as a sphincter preventing backflow from the ventricles to the atria, analogous to the AV valves. Furthermore, the primary myocardium provides the signals that initiate formation of the cushions, which subsequently will form the valves and partake in septation. 2,3 The primary myocardial AVC is extensively remodeled to properly connect and align both atria and ventricles and to coordinate the formation of the fibrous insulation. 4 Given all these roles of the AV...

Rationale:The clinically important atrioventricular conduction axis is structurally complex and heterogeneous, and its molecular composition and developmental origin are uncertain.Objective: To assess the molecular composition and 3D architecture of the atrioventricular conduction axis in the postnatal mouse heart and to define the developmental origin of its component parts. Methods and Results:We generated an interactive 3D model of the atrioventricular junctions in the mouse heart using the patterns of expression of Tbx3, Hcn4, Cx40, Cx43, Cx45, and Nav1.5, which are important for conduction system function. We found extensive figure-of-eight rings of nodal and transitional cells around the mitral and tricuspid junctions and in the base of the atrial septum. The rings included the compact node and nodal extensions. We then used genetic lineage labeling tools (Tbx2 ؉/Cre , Mef2c-AHF-Cre, Tbx18 ؉/Cre ), along with morphometric analyses, to assess the developmental origin of the specific components of the axis. The majority of the atrial components, including the atrioventricular rings and compact node, are derived from the embryonic atrioventricular canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium. No contributions to the conduction system myocardium were identified from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion. Key Words: atrioventricular canal Ⅲ atrioventricular node Ⅲ three-dimensional reconstruction Ⅲ lineage analysis Ⅲ heart development Ⅲ transgenic mice T he atria and ventricles are separated by the connective tissues of the atrioventricular junction that insulate the atrial and ventricular muscle masses. A small part of the musculature, however, the atrioventricular conduction axis, crosses the plane of insulation, thus allowing conduction of the impulse generated by the sinus node to the ventricles. The axis has atrial parts, including the atrioventricular node and atrioventricular ring bundles, and ventricular parts, the atrioventricular bundle and the bundle branches. 1,2 The atrioventricular node delays the electric impulse, thus permitting the ventricles to fill before ventricular contraction. The node can also function as a subsidiary pacemaker. Several arrhythmias, such as atrioventricular block and reentrant tachycardia, have their anatomic substrates within the axis, 2-5 which is complex and heterogeneous in terms of its morphology. [2][3][4][5][6] Insight into the mechanisms of the arrhythmias can be provided by an understanding of development. Of the transcription factors implicated in the regulation of the developmental process, transcription factor Tbx3 is expressed specifically in the central conduction system, thus providing a key marker with which to delineate these tissues throughout development and in the adult. 7-9 The origin and lineages of the atrioventricular junctions, however, have still to be clarified. It is currently thought that the atrial component...

Our data indicate that the cardiogenic mesoderm contains an additional progenitor subpopulation that contributes to the sinus venosus myocardium. After patterning of the cardiogenic mesoderm, this progenitor population remains spatially separated and genetically distinctive from the second heart field subpopulation.

Notch signaling is a crucial regulator of SM differentiation of EPDCs, and thus, of formation of a functional coronary system.

Rationale: T-box transcription factors play critical roles in the coordinated formation of the working chambers and the atrioventricular canal (AVC). Tbx2 patterns embryonic myocardial cells to form the AVC and suppresses their differentiation into chamber myocardium. Tbx20-deficient embryos, which fail to form chambers, ectopically express Tbx2 throughout the entire heart tube, providing a potential mechanism for the function of Tbx20 in chamber differentiation. Objective: To identify the mechanism of Tbx2 suppression by Tbx20 and to investigate the involvement of Tbx2 in Tbx20-mediated chamber formation. Methods and Results: We generated Tbx20 and Tbx2 single and double knockout embryos and observed that loss of Tbx2 did not rescue the Tbx20-deficient heart from failure to form chambers. However, Tbx20 is required to suppress Tbx2 in the developing chambers, a prerequisite to localize its strong differentiation-inhibiting activity to the AVC. We identified a bone morphogenetic protein (Bmp)/Smad-dependent Tbx2 enhancer conferring AVC-restricted expression and Tbx20-dependent chamber suppression of Tbx2 in vivo. Unexpectedly, we found in transfection and localization studies in vitro that both Tbx20 and mutant isoforms of Tbx20 unable to bind DNA attenuate Bmp/Smad-dependent activation of Tbx2 by binding Smad1 and Smad5 and sequestering them from Smad4. Key Words: cardiac Ⅲ T-box factors Ⅲ differentiation Ⅲ interaction Ⅲ repression T he complex multichambered heart of vertebrates arises from a simple, rapidly elongating tubular structure through a coordinated program of cellular differentiation and proliferation and tissue morphogenesis. Highly localized processes of further myocardial differentiation and increased proliferation within the growing heart tube mediate the formation of the atrial and ventricular chambers. Regions separating and bordering the developing chambers, the atrioventricular canal (AVC) and the outflow tract (OFT), retain low proliferation rates and slow impulse conduction and resist differentiation in chamber myocardium. 1 Functional analyses in the mouse revealed that members of the T-box family of transcription factors participate in myocardial patterning and cardiac compartmentalization 2,3 Both Tbx5 and Tbx20 are activated in the early cardiac field by bone morphogenetic protein (Bmp) signaling 4 and act as transcriptional activators that cooperate with other conserved cardiac transcription factors including Nkx2.5 and Gatabinding proteins to activate expression of chamber-specific genes such as ANF (Nppa) and connexin40 (Cx40). 3,[5][6][7] Mice homozygous mutant for Tbx20 establish a heart tube with a primary myocardial phenotype but fail to undergo looping morphogenesis and to initiate chamber formation. 5,8 -10 Tbx5 acts independently of Tbx20 and maintains posterior domains of the heart. 7 Conclusions: Our data suggest that Tbx20 directly interferes withTbx2 encodes a transcriptional repressor that suppresses differentiation and the chamber-specific gene program. 2 Individual ...

Rationale: The Slit–Roundabout (Robo) signaling pathway has pleiotropic functions during Drosophila heart development. However, its role in mammalian heart development is largely unknown. Objective: To analyze the role of Slit–Robo signaling in the formation of the pericardium and the systemic venous return in the murine heart. Methods and Results: Expression of genes encoding Robo1 and Robo2 receptors and their ligands Slit2 and Slit3 was found in or around the systemic venous return and pericardium during development. Analysis of embryos lacking Robo1 revealed partial absence of the pericardium, whereas Robo1/2 double mutants additionally showed severely reduced sinus horn myocardium, hypoplastic caval veins, and a persistent left inferior caval vein. Mice lacking Slit3 recapitulated the defects in the myocardialization, alignment, and morphology of the caval veins. Ligand binding assays confirmed Slit3 as the preferred ligand for the Robo1 receptor, whereas Slit2 showed preference for Robo2. Sinus node development was mostly unaffected in all mutants. In addition, we show absence of cross-regulation with previously identified regulators Tbx18 and Wt1 . We provide evidence that pericardial defects are created by abnormal localization of the caval veins combined with ectopic pericardial cavity formation. Local increase in neural crest cell death and impaired neural crest adhesive and migratory properties underlie the ectopic pericardium formation. Conclusions: A novel Slit–Robo signaling pathway is involved in the development of the pericardium, the sinus horn myocardium, and the alignment of the caval veins. Reduced Slit3 binding in the absence of Robo1, causing impaired cardiac neural crest survival, adhesion, and migration, underlies the pericardial defects.

Rationale: The cardiac venous pole is a common focus of congenital malformations and atrial arrhythmias, yet little is known about the cellular and molecular mechanisms that regulate its development. T he systemic venous return of the mature mammalian heart, which terminates in the right atrium, consists of multiple anatomic components including the myocardial sleeves of the right superior and inferior caval veins, the sinoatrial node (SAN), the coronary sinus (persisting left caval vein in the mouse), and the sinus venarum. 1 The systemic venous return is a focus of congenital malformations and atrial arrhythmias, 2,3 necessitating insight into the cellular and molecular programs by which it arises during cardiac development. Most myocardial components of the heart are not represented in its initial anlage, but are continuously added by recruitment and differentiation of precursor cells. 4 The sinus horns, the myocardial parts of the common cardinal veins upstream of the venous valves that bulge into the pericardial cavity, form from pericardial precursors that differentiate into sinus venosus myocardium around the systemic venous connection to the atrium. 5 They form only after embryonic day (E)9.5, when outflow tract, left and right ventricle, and the common atrium have already been established. In adults, most of the right sinus horn myocardium is incorporated into the right atrium to form the sinus venarum. In humans, the left sinus horn will lose its connection to the body and form the coronary sinus, whereas in mouse it will persist as the left superior caval vein.Few genes regulating venous pole development have been characterized, including the Tbx18 (T-box transcription factor 18) that marks the sinus horn lineage. 14 which harbors a tetrameric repeat of the RAR␤2 RARE linked to the Hsp68 minimal promoter used to determine RA signaling, were all described before. For the Wt1 BAC-IRES-EGFPCre transgenic line, the BAC clone RP23-266M16 was modified by inserting an IRES/EGFP-Cre cassette 17bp downstream of the translation stop site of the Wt1 gene. (The generation and evaluation of this line will be described elsewhere.) In Wt1 tTA mice, the coding sequence of an improved tetracyclinedependent transactivator tTA2S 15 was introduced into the Wt1 locus by gene targeting (E Lausch, S Fees, C Steinwender, C Spangenberg, L Eshkind, E Bockamp, B Zabel, manuscript in preparation). All mouse lines were maintained on an outbred (NMRI or FvB) background.An expanded Materials and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Results Defects in the Systemic Venous Return of Wt1-Deficient Hearts Wt1Ϫ/Ϫ mice maintained on an NMRI outbred background died during midgestation presenting defects reported on earlier, including lack of kidneys, diaphragmatic hernia, and defects in coronary vessel formation the latter of which may underlie embryonic lethality. Yet, histological inspection of surviving embryos at E14.5 revealed an undescribed variation in the systemic venous ...