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.

The transforming growth factor␤ (Tgf␤) signaling pathway plays crucial roles in many biological processes. To understand the role(s) of Tgf␤ signaling during cardiogenesis in vivo and to overcome the early lethality of Tgfbr2 -/-embryos, we applied a Cre/loxp system to specifically inactivate Tgfbr2 in either the myocardium or the endothelium of mouse embryos. Our results show that Tgfbr2 in the myocardium is dispensable for cardiogenesis in most embryos. Contrary to the prediction from results of previous in vitro collagen gel assays, inactivation of Tgfbr2 in the endocardium does not prevent atrioventricular cushion mesenchyme formation, arguing against its essential role in epithelium-mesenchyme transformation in vivo. We further demonstrate that Tgf␤ signaling is required for the proper remodeling of the atrioventricular canal and for cardiac looping, and that perturbation in Tgf␤ signaling causes the double-inlet left ventricle (DILV) defect. Thus, our study provides a unique mouse genetic model for DILV, further characterization of which suggests a potential cellular mechanism for the defect.