Cardiovascular development and morphogenesis is a complex process, involving a number of highly conserved transcription factors and signaling pathways 
. KLF2 plays a multi-faceted role in cardiovascular development. It is expressed in the endocardium of the developing heart. The accumulation of endothelial-like cells lining the AV canal, reduced EMT, delayed atrial septal formation, and the absence of normal cardiac jelly composition are novel phenotypes for FVB/N KLF2−/− mice, and therefore may be related to the earlier embryonic death in the FVB/N genetic background. Our studies also identify putative effectors downstream of KLF2 that may impact each of these processes in the embryonic heart.
KLF2 is important for vascular integrity 
. Kuo et al.
and Wani et al
reported hemorrhaging in the abdominal and cardiac outflow tract region in KLF2−/− embryos. To the contrary, no hemorrhaging was observed in the KLF2−/− embryos examined by Lee et al. 
. This discrepancy may be partially explained by the current study. Erythroid cells were observed outside E10.5 KLF2−/− dorsal aortas in FVB/N but not in mixed genetic background embryos, indicating that this phenotype is genetic background-specific. The genetic background of the KLF2−/− mice used in the previous studies was not well defined. It is theoretically possible that the dorsal aorta defect in FVB/N KLF2−/− embryos causes shear stress, resulting in the AV cushion defect. However, this hypothesis is not favored because the AV cushion defect is observed by E9.5, whereas the dorsal aorta defect is first apparent at E10.5.
The importance of genetic background in cardiac development has been demonstrated in a number of studies. Sakata et al. studied Hey2 deficient mice and observed a spectrum of cardiovascular anomalies that varied in the BALB/c and C57BL/6 genetic backgrounds 
. Astrof et al. studied the role of fibronectin in heart development; a null mutation in the gene results in arrested heart development earlier in 129S4 than in C57BL/6 embryos 
. The current study shows that the role of KLF2 in the morphology and function of the developing heart is also genetic background specific. In the FVB/N background, loss of KLF2 results in an EMT defect in the AV cushion region, delayed formation of the atrial septum, myocardial thinning and death by E10.5. In the C57BL/6 background, KLF2−/− shows delayed atrial septation and myocardial thinning. In a mixed background the major defect in KLF2−/− hearts is myocardial thinning 
In this work, we have demonstrated that KLF2 binds the promoters of, and positively regulates, the Tbx5 and Gata4 genes in the mouse E10.5 AV region. An endocardial specific Gata4 KO has multiple layers of endocardium in the AV canal, and hypocellular AV cushions at E10.5 
, similar to FVB/N KLF2−/−. Tbx5 KO embryos have hypoplastic endocardial cushions 
, like FVB/N KLF2−/−. These phenotypes correlate with our observation that Gata4 and Tbx5 expression is reduced 3-fold in the absence of KLF2 in FVB/N AV canals. Interestingly, the Tbx5 and Gata4 proteins physically interact during cardiac development. A heterozygous mutation (mG295S) in the Gata4 gene disrupts these protein interactions, resulting in cardiac defects like atrial septal defects (ASD), AV septal defects (AVSD) and myocardial thinning beginning at E11.5 
. The AVSD and ASD in these mice are known to result from abnormal EMT and remodeling of endocardial cushions. Gata4 mG295S is a missense mutation resulting in diminished DNA binding affinity and transcriptional activity, making it similar to a null allele. In mice with this Gata4 mutation and a null allele for Tbx5, Gata4+/−Tbx5+/−, there is normal EMT but defective remodeling, resulting in septal defects 
. Like these double heterozygotes, FVB/N KLF2−/− embryos have about 50% less Gata4 and Tbx5 mRNA than normal, and similarly remodeling is affected. However, EMT is affected, indicating that the KLF2−/− embryos are more severely affected than Gata4+/−Tbx5+/−, likely because KLF2 also controls other cardiac genes.
KLF2 binds to and positively regulates the UDP-glucose dehydrogenase (Ugdh) gene. UGDH is expressed in the endocardium and catalyzes conversion of UDP-Glucose to UDP-Glucuronic acid (UDP-GA) 
. UDP-GA is further converted to hyaluronic acid and other glycosaminoglycans by hyaluronan synthase 2 (Has2) 
. There is an approximately 2-fold reduction in Ugdh mRNA in the FVB/N KLF2−/− AV canal. This may result in decreased production of UDP-GA and consequently reduced glycosaminoglycans in the cardiac jelly. Interestingly, two missense mutations in the Ugdh gene were recently identified in 3 patients with congenital valve defects 
. These mutations result in structural defects in UGDH that significantly compromise enzyme function 
. These findings support our hypothesis that reduced UGDH contributes to the cushion defects in KLF2−/− mice.
Sox9 is a cardiovascular transcription factor expressed in endothelial and mesenchymal cells in the endocardial cushion region 
. In our study, the Sox9 gene is positively controlled by KLF2, but the lack of evidence for KLF2 promoter binding suggests that the regulation is indirect, or controlled by a more distant DNA element. Sox9 KO results in hypoplastic endocardial cushions and abnormal valve formation 
. A study by Lincoln et al. showed that ablation of Sox9 in endocardial cells results in reduced EMT 
. These phenotypes are similar to FVB/N KLF2−/−. The reduced expression of Sox9 in KLF2−/− hearts could be attributed to a reduced number of mesenchymal cells in the AV cushion. However, this seems unlikely because Has2 mRNA, which is also expressed in endocardial and mesenchymal cells, does not show reduced expression in KLF2−/− AV regions.
The current study indicates that KLF2 plays an important role in the synthesis of cardiac jelly, AV endocardial cushion EMT and atrial septation, by regulating several important cardiac genes. These genes include but are probably not limited to Ugdh (cardiac jelly), Sox9 (EMT), and Tbx5 and Gata4 (EMT, AV cushion development, and septation). KLF2 influences the development of the AV cushions and atrial septum, and is likely required for normal cardiac function. Future studies of KLF2 variants may be relevant to the better understanding of human heart defects.