Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. By combining use of a hypomorphic Bone morphogenetic protein 4 (Bmp4) allele with conditional gene inactivation, we here identify Bmp4 as a signal from the myocardium directly mediating atrioventricular septation. Defects in this process cause one of the most common human congenital heart abnormalities, atrioventricular canal defect (AVCD). The spectrum of defects obtained through altering Bmp4 expression in the myocardium recapitulates the range of AVCDs diagnosed in patients, thus providing a useful genetic model with AVCD as the primary defect. Congenital heart malformation is the most common human birth defect and the leading cause of death in the first year of life (Hoffman 1995). One of the most frequently diagnosed disorders is atrioventricular canal defect (AVCD), which accounts for 7.3% of all congenital heart abnormalities (Pierpont et al. 2000). During normal development, septation of the AV canal (AVC) is initiated with the formation of the inferior and superior endocardial cushions through epithelial-mesenchymaltransformation (EMT) by some endocardial cells invading into cardiac jelly, the extracellular matrix between the endocardium and myocardium (Nakajima et al. 2000;Markwald and Wessels 2001). Subsequent growth and fusion of the AV cushions produce the central mesenchymal mass, which further develops into the mature AV septum and valves through complex remodeling processes. The central mesenchymal mass also fuses with the atrial septum primum (ASP) and the inlet portion of the ventricular septum to prevent abnormal blood flow between chambers (Marino and Digilio 2000; Markwald and Wessels 2001). AVCD covers a spectrum of abnormalities, from the partial form with defects in the lower part of the ASP to the complete form in which absence of the AV septum results in a single common AVC (Marino and Digilio 2000).The complex cushion morphogenesis during AV septation depends both on signals released from the overlying myocardium and on proper responses of the endocardial and mesenchymal cells (Nakajima et al. 2000;Markwald and Wessels 2001). The molecular pathways for the initiation of cushion formation (EMT) have been studied extensively. In both chicken and mouse explant culture assays, transforming growth factor ␤2 (TGF␤2) is able to replace the overlying myocardium to activate EMT, and inhibition of TGF␤2 activity blocks EMT (Boyer et al. 1999;Nakajima et al. 2000;Camenisch et al. 2002a). Consistent with these in vitro studies, TGF␤2-deficient mice show defects in valvulogenesis (Bartram et al. 2001). In addition to the TGF␤ signaling pathway, the Hyaluronic acid (Ha) synthase 2 (Has2)/ErbB2,ErbB3/Ras pathway has been recently shown to play essential roles in EMT (Camenisch et al. 2000(Camenisch et al. , 2002b. Ha is a prominent component of cardiac jelly in embryonic day 9.5 (E9.5) embryos and its production depends on Has2. In addition to serving as a substrate for migrati...

Rationale Formation of heart valves requires early endocardial to mesenchymal transformation (EMT) to generate valve mesenchyme and subsequent endocardial cell proliferation to elongate valve leaflets. Nfatc1 (nuclear factor of activated T cells, cytoplasmic 1) is highly expressed in valve endocardial cells and is required for normal valve formation, but its role in the fate of valve endocardial cells during valve development is unknown. Objective Our aim was to investigate the function of Nfatc1 in cell-fate decision making by valve endocardial cells during EMT and early valve elongation. Methods and Results Nfatc1 transcription enhancer was used to generate a novel valve endocardial cell–specific Cre mouse line for fate-mapping analyses of valve endocardial cells. The results demonstrate that a subpopulation of valve endocardial cells marked by the Nfatc1 enhancer do not undergo EMT. Instead, these cells remain within the endocardium as a proliferative population to support valve leaflet extension. In contrast, loss of Nfatc1 function leads to enhanced EMT and decreased proliferation of valve endocardium and mesenchyme. The results of blastocyst complementation assays show that Nfatc1 inhibits EMT in a cell-autonomous manner. We further reveal by gene expression studies that Nfatc1 suppresses transcription of Snail1 and Snail2, the key transcriptional factors for initiation of EMT. Conclusions These results show that Nfatc1 regulates the cell-fate decision making of valve endocardial cells during valve development and coordinates EMT and valve elongation by allocating endocardial cells to the 2 morphological events essential for valve development.

Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal-spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.