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Background
Congenital heart disease (CHD) is the leading non-infectious cause of death in children, affecting 1 in 100 newborn babies. Twice as many children die from CHD as die from all forms of childhood cancer. In the United States, CHD affects more than 2 million children (AHA, 2001). Formation of the heart is a complex process that involves a precisely controlled series of morphogenetic events. Human genetic linkage analyses together with studies in vertebrate model organisms have shown that a variety of CHD's is caused by mutations in transcriptional regulatory factors. The cardiac transcription factor Nkx2.5 is the mammalian homologue of the Drosophila tinman genen (Bodmer, 1993). Human NKX2-5 has been identified as a dominant diseases locus for a variety of human congenital heart disease (Benson et al., 1999; Schott et al., 1998). Ten mutations have been described so far, which may account for a significant portion of patients with atrioventricular (AV) block, and defects in atrial and ventricular septation [Benson et al., 1999]. Other notable defects were Tetralogy of Fallot, double outlet right ventricle (DORV), and Ebstein's anomaly and other tricuspid valve abnormalities were also present (Goldmuntz et al., 2001).
Over the last years, our laboratory has made significant progress towards the understanding of the role of Nkx2.5 in the complex regulatory network of cardiac development (Lee et al, 1998; Kasahara et al., 1998; 1999; 2001). We have developed gene-targeted mice with deletion of Nkx2.5 (Tanaka et al., 1999). Heterozygous mutant mice reflect many aspects of the human phenotype such as a high incidence of secundum ASD and AV conduction abnormalities (Biben et al., 2000; Tanaka et al., unpublished). These mouse models provide valuable tools to examine the role of Nkx2.5 in human congenital heart disease.
Homozygous mutant mice die around embryonic day 10.5 and show an arrest of cardiac development after looping. Histological analysis of mutant hearts revealed that formation of trabeculae is very poor and endocardial cushion is absent in the mutant hearts In addition, blood vessels, such as intersomitic arteries, pharyngeal arch arteries and the dorsal aorta, are poorly developed in homozygous mutant embryos. Moreover, there are severe defects in vascular formation and hematopoiesis in the mutant yolk sac. TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 is disturbed in the mutant heart. Taken together, homozygous mutant embryos show that Nkx2.5 regulates the expression of several transcription factors in the developing heart, and that Nkx2.5 is required for later differentiation of cardiac myocytes (Tanaka et al., 1999). The identification of additional downstream target genes and affected pathways will help to further define the function of Nkx2.5 within the regulatory pathway leading to cardiac differentiation.
Experimental Design
Embryos were isolated at embryonic day 9.5. The middle third of the embryos containing the heart was isolated in ice-cold PBS and immediately frozen on dry-ice. Total RNA was extracted from pooled samples of wildtype (4-5 samples), heterozygous (4-5 samples) and mutant embryos (6-7 samples) as described in Methodology.
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Page last modified: 11-Jun-2002
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