Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF

The atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. Mouse knockout and mammalian cell culture studies have shown that the ANF gene is regulated by combinatorial interactions between Nkx2-5, GATA-4, Tbx5, and SRF; however, the molecular mechanisms leading to chamber-specific expression are currently unknown. We have isolated the Xenopus ANF promoter in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, a T-box binding site (TBE), and two SRF binding sites (SREs). Our transgenic studies show that mutation of either SRE, the TBE or the distal GATA element, strongly reduces expression from the ANF promoter. However, mutations of the NKE, the proximal GATA, or both elements together, result in relatively minor reductions in transgene expression within the myocardium. Surprisingly, mutation of these elements results in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery-associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. We propose that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex that is required for atrial-specific expression of the ANF gene.     

AmphiNk2-tin,an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart

We isolated a full-length cDNA clone of amphioxus AmphiNk2-tin, an NK2 gene similar in sequence to vertebrate NK2 cardiac genes, suggesting a potentially similar function to Drosophila tinman and to vertebrate NK2 cardiac genes during heart development. During the neurula stage of amphioxus, AmphiNk2-tin is expressed first within the foregut endoderm, then transiently in muscle precursor cells in the somites, and finally in some mesoderm cells of the visceral peritoneum arranged in an approximately midventral row running beneath the midgut and hindgut. The peritoneal cells that express AmphiNk2-tin are evidently precursors of the myocardium of the heart, which subsequently becomes morphologically detectable ventral to the gut. The amphioxus heart is a rostrocaudally extended tube consisting entirely of myocardial cells (at both the larval and adult stages); there are no chambers, valves, endocardium, epicardium, or other differentiated features of vertebrate hearts. Phylogenetic analysis of the AmphiNk2-tin sequence documents its close relationship to vertebrate NK2 class cardiac genes, and ancillary evidence suggests a relationship with the Drosophila NK2 gene tinman. Apparently, an amphioxus-like heart, and the developmental program directing its development, was the foundation upon which the vertebrate heart evolved by progressive modular innovations at the genetic and morphological levels of organization.     

Cardioblast-intrinsic Tinman activity controls proper diversification and differentiation of myocardial cells in Drosophila

The NK homeobox gene tinman (tin) is required for the specification of the cardiac, visceral muscle and somatic muscle progenitors in the early dorsal mesoderm of Drosophila. Like its vertebrate counterpart Nkx2.5, the expression of tin is maintained in cardiac cells during cardiac maturation and differentiation; however, owing to the complete lack of a dorsal vessel in tin mutant embryos, the function of tin in these cells has not been defined. Here we show that myocardial cells and dorsal vessels can form even though they lack Tin, and that viable adults can develop, as long as Tin is provided in the embryonic precardiac mesoderm. However, embryos in which tin expression is specifically missing from cardial cells show severe disruptions in the normal diversification of the myocardial cells, and adults exhibit severe defects in cardiac remodeling and function. Our study reveals that the normal expression and activity of Tin in four of the six bilateral cardioblasts within each hemisegment of the heart allows these cells to adopt a cell fate as ;working' myocardium, as opposed to a fate as inflow tract (ostial) cells. This function of tin involves the repression of Dorsocross (Doc) T-box genes and, hence, the restriction of Doc to the Tin-negative cells that will form ostia. We conclude that tin has a crucial role within myocardial cells that is required for the proper diversification, differentiation, and post-embryonic maturation of cardiomyocytes, and we present a pathway involving regulatory interactions among seven-up, midline, tinman and Dorsocross that establishes these developmental events upon myocardial cell specification.     

The chicken NKX2.8 homeobox gene: A novel member of the NK-2 gene family

 We have isolated the chicken homeobox gene NKX2.8, which represents a novel member of the NK-2 gene family. Besides the homeodomain, the NKX2.8 protein contains two other conserved sequences, a TN and an NK2 domain. NKX2.8 is expressed in the ventral foregut, the developing heart, in the epithelial layers of the branchial arches and in the dorsal mesocardium. Thus, its expression overlaps partially, but also differs significantly from another chicken tinman orthologue, the NKX2.5 gene. It is suggestive that NKX2.8 and NKX2.5 play a cooperative role in early heart development. Received: 28 January 1997 / Accepted: 8 February 1997     

Vertebrate tinman homologues and cardiac differentiation.

In Drosophila, the homeobox gene tinman is required for specification of dorsal vessel and a number of mesodermal subtypes. Six tinman homologues have now been found in diverse vertebrate species: Nkx2-3, 2-5, 2-6, 2-7, 2-8 and 2-9. Of these, Nkx2-5 appears to be the mostly highly conserved among species, in terms of both primary protein sequence and mRNA expression pattern. Of the others, some have been found as yet only in a single species. Although expression patterns of vertebrate tinman homologues indicate that they may play a role in the specification of several mesodermal or endodermal tissues, to date most attention have been focussed on their role in cardiac development. Results of these studies indicate that, as for Drosophila tinman, vertebrate tinman homologues may be required for heart formation, but may not be sufficient. Studies in Drosophila are defining other pathways which are required in concert with tinman for dorsal vessel formation. Circumstantial evidence suggests that similar pathways may be operative in vertebrate heart formation. This review summarizes recent advances in our understanding of vertebrate tinman homologues and interacting genetic pathways.