Complex modular cis-acting elements regulate expression of the cardiac specifying homeobox gene Csx/Nkx2.5

The murine homeobox gene Csx/Nkx2.5 is an evolutionarily highly conserved g ene related to the Drosophila tinman gene, which specifies cardiac and visc eral mesoderm, Since Csx/Nkx2.5 plays an essential role in heart developmen t, studying its regulation is essential for the better understanding of mol ecular mechanisms of cardiogenesis and the pathogenesis of congenital heart disease in humans. In this study, we characterized the murine Csx/Nkx2.5 g ene and identified two novel untranslated exons, la, and Ib, resulting in t hree different Csx/Nkx2.5 transcripts. To examine the tissue-specific trans criptional regulation in vivo, we analyzed a total of 23 kb of Csx/Nkx2.5 u pstream and downstream sequences by generating transgenic embryos carrying lacZ reporter constructs containing various lengths of flanking sequence, W ith 14 kb of 5' flanking sequence, lacZ expression was observed in the card iac crescent at E7.5, and in the outflow tract, the interatrial groove, the atrioventricular canal and right and left ventricles, as well as in pharyn geal floor, thyroid primordia, and stomach at E10.5, In adult animals, lacZ expression of the transgene was limited to the atrioventricular junction a nd the subendocardium of the ventricular septum, Reducing the size of flank ing sequence to 3.3 kb of intron 2 restricted lacZ expression to the outflo w tract and the basal part of the right ventricle in E10.5 embryos. In cont rast, the addition of 6 kb of 3' flanking sequence caused strong expression of the reporter gene in the entire right ventricle, Interestingly, Csx/Nkx 2.5 seems to be negatively regulated by its own gene product, because when lacZ was "knocked-in" to replace the entire coding exons, lacZ expression w as much higher in the heart of homozygous embryos than that in the heterozy gote. These results indicate that the transcriptional regulatory elements o f Csx/Nkx 2.5 seems unexpectedly highly modular, and is temporally regulate d in a dynamic manner by different enhancer regions. Since Csx/Nkx2.5-like genes are expressed in all species having a heart, their complex modular or ganization with multiple enhancers probably reflects progressive addition o f regulatory elements during the evolution from a simple heart tube to a co mplex four-chambered organ.