Disease relevance of Nkx2-5

• To define these pathways, we generated mice with a ventricular-restricted knockout of Nkx2-5, which display no structural defects but have progressive complete heart block, and massive trabecular muscle overgrowth found in some patients with Nkx2-5 mutations [1].
• In order to establish an efficient in vivo system for mapping the Nkx2-5 genomic locus for regulatory regions, we developed improved homologous recombination technology for use in Escherichia coli and then knocked an IRES-hrGFP reporter gene into Nkx2-5 gene in a 120 kb Nkx2-5 bacterial artificial chromosome (BAC) clone [2].
• Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause congenital heart disease [3].
• We discovered that a family of transcriptional coactivators, called CAMTAs, promotes cardiomyocyte hypertrophy and activates the ANF gene, at least in part, by associating with the cardiac homeodomain protein Nkx2-5 [4].
• p204 is required for the differentiation of P19 murine embryonal carcinoma cells to beating cardiac myocytes: its expression is activated by the cardiac Gata4, Nkx2.5, and Tbx5 proteins [5].
• In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion [6].

High impact information on Nkx2-5

• Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5 [7].
• Our data provide a potential mechanism for chamber-restricted gene activity in which the cooperative action of Tbx2 and Nkx2.5 inhibits expression in the AVC [8].
• In hearts of embryos lacking the homeobox gene Nkx2-5, which do not loop, left-sided eHand expression was abolished [9].
• The murine homeo box gene Nkx2-5 is expressed in precardiac mesoderm and in the myocardium of embryonic and fetal hearts [10].
• The data demonstrate that Nkx2-5 is essential for normal heart morphogenesis, myogenesis, and function [10].

Chemical compound and disease context of Nkx2-5

• The transcripts of this gene and Nkx-2.5/Csx were detected in the early stage of cardiac differentiation of P19CL6 embryonal carcinoma cells treated with 1% dimethyl sulfoxide [11].

Biological context of Nkx2-5

• We describe a cardiac enhancer, located about 9 kilobases upstream of the Nkx2-5 gene, that fully recapitulates the expression pattern of the endogenous gene in cardiogenic precursor cells from the onset of cardiac lineage specification and throughout the linear and looping heart tube [12].
• The homeobox gene Nkx2-5 is the earliest known marker of the cardiac lineage in vertebrate embryos [12].
• Finally, a carp promoter-lacZ transgene, which displays cardiac-specific expression in wild-type and Nkx2-5(+/-) background, was also significantly reduced in Nkx2-5(-/-) embryos, indicating that Nkx2-5 either directly or indirectly regulates carp promoter activity during in vivo cardiogenesis as well as in cultured cardiac myocytes [13].
• These results reveal a novel GATA-dependent mechanism for activation of Nkx2-5 transcription in the developing heart and indicate that regulation of Nkx2-5 is controlled in a modular manner, with multiple regulatory regions responding to distinct transcriptional networks in different compartments of the developing heart [12].
• To test for an early cardiogenic function for Nkx2-5 and to examine whether cardiogenic mechanisms are conserved, we introduced the mouse Nkx2-5 gene and various mutant and chimeric derivatives into the Drosophila germline, and tested for their ability to rescue the tin mutant phenotype [14].

Anatomical context of Nkx2-5

• Co-transfection assays using a dominant negative mutant Nkx2-5 construct with CARP promoter-luciferase reporter constructs in cardiac myocytes confirms that Nkx2-5 either directly or indirectly regulates carp at the transcriptional level [13].
• While tin itself strongly rescued both heart and visceral mesoderm, Nkx2-5 rescued only visceral mesoderm [14].
• In addition to the heart, Nkx2-5 is transiently expressed in the developing pharynx, thyroid and stomach [12].
• Complete Tbx20 knockdown resulted in defects in heart formation, including hypoplasia of the outflow tract and right ventricle, which derive from the anterior heart field (AHF), and decreased expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation [15].
• We observed the absence of myogenic alpha-actins, SM22alpha, and myocardin expression and the failure to form beating cardiac myocytes in aggregated SRF null embryonic stem cells, whereas the appearance of transcription factors Nkx2-5 and GATA4 were unaffected [16].

Associations of Nkx2-5 with chemical compounds

• Thus, the HF-1 element, directs chamber-specific transcription of a transgene reporter independently of retinoid X receptoralpha and Nkx2-5, and defines a minimal combinatorial pathway for ventricular chamber gene expression [17].
• These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment [18].
• DMSO-primed GATA-4 and Nkx-2.5 gene expression was preceded by a marked increase in prodynorphin gene expression and dynorphin B synthesis and secretion [19].
• JMJ physically associates with Nkx2.5 and GATA4 in vitro and in vivo as determined by glutathione S-transferase pull-down and immunoprecipitation assays [20].
• Finally, TSA facilitated the expression of green fluorescence protein under the control of the cardiac-specific Nkx-2.5 promoter and of endogenous cardiac beta-myosin heavy chain during the differentiation [21].

Physical interactions of Nkx2-5

• Smad4 and GATA4 were also found to bind in vivo with the Nkx2-5 composite enhancer, as revealed by chromatin immunoprecipitation analysis of differentiated P19 cells [22].
• An enhancer located between exons 4 and 5 in which a putative T-half site was identified near an Nkx2.5-binding site regulates asymmetric expression of Pitx2 [23].
• The GATA6 cardiac enhancer contained a binding site for Nkx2.5 that was essential for cardiac-specific expression in transgenic mice [24].

Regulatory relationships of Nkx2-5

• Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner [25].
• Myocardin expression is regulated by Nkx2.5, and its function is required for cardiomyogenesis [26].
• In addition, the nuclear translocation of NFATc1 in the endocardial endothelial cells is inhibited in the Nkx2-5-/- embryos [27].
• Complex cardiac Nkx2-5 gene expression activated by noggin-sensitive enhancers followed by chamber-specific modules [28].
• During heart formation, Lbh is expressed as early as Nkx2.5 and dHand in the bilateral heart primordia, with the highest levels in the anterior promyocardium [29].

Other interactions of Nkx2-5

• Interestingly, in mutant embryos homozygous for Nkx2.6, Nkx2.5 expression extended to the lateral side of the pharynx, suggesting a compensatory function of Nkx2.5 in the mutant pharyngeal pouches [30].
• Expression of Nkx2.6 overlaps that of Nkx2.5 in the pharynx and heart and that of Nkx2.3 in the pharynx [30].
• The cardiac determination factor, Nkx2-5, is activated by mutual cofactors GATA-4 and Smad1/4 via a novel upstream enhancer [22].
• Mice heterozygous for mutant alleles of Nkx2.5 and dHAND were viable [31].
• Early cardiac development might therefore be regulated by other genes, which may act either independently or in concert with Csx/Nkx2 [32].

Analytical, diagnostic and therapeutic context of Nkx2-5

• In Nkx2-5(-/-)embryos, carp expression was found to be significantly and selectively reduced as assessed by both whole-mount in situ hybridizations and RNase protection assays, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory network [13].
• This composite Nkx2-5 enhancer was a direct target of BMP signaling via cooperative interactions between the downstream transducers Smad1/4 and GATA-4 [22].
• Sequence analysis reveals remarkable conservation between the distalmost 300 bp of the Xenopus promoter and a portion of the AR2 element upstream of the mouse and human Nkx2-5 genes [33].
• 5. Gene targeting of Csx/Nkx2.5 showed that this gene is required for completion of the looping morphogenesis of the heart [32].
• Nkx-2.5 expression will therefore be a valuable marker in the analysis of mesoderm development and an early entry point for dissection of the molecular basis of myogenesis in the heart [34].

References