Foxp1 coordinates cardiomyocyte proliferation through both cell-autonomous and nonautonomous mechanisms.

Cardiomyocyte proliferation is high in early development and decreases progressively with gestation, resulting in the lack of a robust cardiomyocyte proliferative response in the adult heart after injury. Little is understood about how both cell-autonomous and nonautonomous signals are integrated to regulate the balance of cardiomyocyte proliferation during development. In this study, we show that a single transcription factor, Foxp1, can control the balance of cardiomyocyte proliferation during development by targeting different pathways in the endocardium and myocardium. Endocardial loss of Foxp1 results in decreased Fgf3/Fgf16/Fgf17/Fgf20 expression in the heart, leading to reduced cardiomyocyte proliferation. This loss of myocardial proliferation can be rescued by exogenous Fgf20, and is mediated, in part, by Foxp1 repression of Sox17. In contrast, myocardial-specific loss of Foxp1 results in increased cardiomyocyte proliferation and decreased differentiation, leading to increased myocardial mass and neonatal demise. We show that Nkx2.5 is a direct target of Foxp1 repression, and Nkx2.5 expression is increased in Foxp1-deficient myocardium. Moreover, transgenic overexpression of Nkx2.5 leads to increased cardiomyocyte proliferation and increased ventricular mass, similar to the myocardial-specific loss of Foxp1. These data show that Foxp1 coordinates the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation of Fgf ligand and Nkx2.5 expression.

Foxp1 regulates cardiac outflow tract, endocardial cushion morphogenesis and myocyte proliferation and maturation.

We have recently described a new subfamily of Fox genes, Foxp1/2/4, which are transcriptional repressors and are thought to regulate important aspects of development in several tissues, including the lung, brain, thymus and heart. Here, we show that Foxp1 is expressed in the myocardium as well as the endocardium of the developing heart. To further explore the role of Foxp1 in cardiac development, we inactivated Foxp1 through gene targeting in embryonic stem cells. Foxp1 mutant embryos have severe defects in cardiac morphogenesis, including outflow tract septation and cushion defects, a thin ventricular myocardial compact zone caused by defects in myocyte maturation and proliferation, and lack of proper ventricular septation. These defects lead to embryonic death at E14.5 and are similar to those observed in other mouse models of congenital heart disease, including Sox4 and Nfatc1 null embryos. Interestingly, expression of Sox4 in the outflow tract and cushions of Foxp1 null embryos is significantly reduced, while remodeling of the cushions is disrupted, as demonstrated by reduced apoptosis and persistent Nfatc1 expression in the cushion mesenchyme. Our results reveal a crucial role for Foxp1 in three aspects of cardiac development: (1) outflow tract development and septation, (2) tissue remodeling events required for cardiac cushion development, and (3) myocardial maturation and proliferation.

Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions.

Foxp1, Foxp2, and Foxp4 are large multidomain transcriptional regulators belonging to the family of winged-helix DNA binding proteins known as the Fox family. Foxp1 and Foxp2 have been shown to act as transcriptional repressors, while regulatory activity of the recently identified Foxp4 has not been determined. Given the importance of this Fox gene subfamily in neural and lung development, we sought to elucidate the mechanisms by which Foxp1, Foxp2, and Foxp4 repress gene transcription. We show that like Foxp1 and Foxp2, Foxp4 represses transcription. Analysis of the N-terminal repression domain in Foxp1, Foxp2, and Foxp4 shows that this region contains two separate and distinct repression subdomains that are highly homologous termed subdomain 1 and subdomain 2. However, subdomain 2 is not functional in Foxp4. Screening for proteins that interact with subdomains 1 and 2 of Foxp2 using yeast two-hybrid analysis revealed that subdomain 2 binds to C-terminal binding protein 1, which can synergistically repress transcription with Foxp1 and Foxp2, but not Foxp4. Subdomain 1 contains a highly conserved leucine zipper similar to that found in N-myc and confers homo- and heterodimerization to the Foxp1/2/4 family members. These interactions are dependent on the conserved leucine zipper motif. Finally, we show that the integrity of this subdomain is essential for DNA binding, making Foxp1, Foxp2, and Foxp4 the first Fox proteins that require dimerization for DNA binding. These data reveal a complex regulatory mechanism underlying Foxp1, Foxp2, and Foxp4 activity, demonstrating that Foxp1, Foxp2, and Foxp4 are the first Fox proteins reported whose activity is regulated by homo- and heterodimerization.

The WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in lung epithelium.

In this study, we find that WNT7b is the only member of the WNT family of autocrine/paracrine signaling molecules whose expression in the lung is restricted to the airway epithelium during embryonic development. To study the transcriptional mechanisms that underlie this restricted pattern of WNT7b expression, we isolated the proximal 1.0-kb mouse WNT7b promoter and mapped the transcriptional start sites. Transfection of the lung epithelial cell line MLE-15, which expresses WNT7b, shows that the 1.0-kb mouse WNT7b promoter is highly active in lung epithelial cells. This region of the WNT7b promoter contains several DNA binding sites for the important lung-restricted transcription factors TTF-1, GATA6, and Foxa2. Electrophoretic mobility shift assays showed that TTF-1, GATA6, and Foxa2 can bind to a specific subset of their consensus DNA binding sites within the WNT7b promoter. Using cotransfection assays, we demonstrate that TTF-1, GATA6, and Foxa2 can trans-activate the WNT7b promoter in NIH-3T3 cells. Truncation of GATA6 or Foxa2 binding sites reduced the ability of these transcriptional regulators to trans-activate the WNT7b promoter. Finally, the minimal 118-bp region of the mouse WNT7b promoter containing only TTF-1 binding sites was synergistically activated by TTF-1 and GATA6, and we show that TTF-1 and GATA6 physically interact in vivo. Together, these results suggest that WNT7b gene expression in the lung epithelium is regulated in a combinatorial fashion by TTF-1, GATA6, and Foxa2.