Nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number

Abstract

Heart formation is a complex morphogenetic process, and perturbations in cardiac morphogenesis lead to congenital heart disease. NKX2-5 is a key causative gene associated with cardiac birth defects, presumably because of its essential roles during the early steps of cardiogenesis. Previous studies in model organisms implicate NKX2-5 homologs in numerous processes, including cardiac progenitor specification, progenitor proliferation, and chamber morphogenesis. By inhibiting function of the zebrafish NKX2-5 homologs, nkx2.5 and nkx2.7, we show that nkx genes are essential to establish the original dimensions of the linear heart tube. The nkx-deficient heart tube fails to elongate normally: its ventricular portion is atypically short and wide, and its atrial portion is disorganized and sprawling. This atrial phenotype is associated with a surplus of atrial cardiomyocytes, whereas ventricular cell number is normal at this stage. However, ventricular cell number is decreased in nkx-deficient embryos later in development, when cardiac chambers are emerging. Thus, we conclude that nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number. Our data suggest that morphogenetic errors could originate during early stages of heart tube assembly in patients with NKX2-5 mutations.

Nkx genes are essential for maintenance of ventricular identity

“…Our previous studies in zebrafish have revealed roles for nkx2.5 and nkx2.7 in limiting atrial cell number and promoting ventricular cell number (Targoff et al, 2008). Here, through characterization of a newly identified nkx2.5 mutation, we find that loss of nkx2.5 function causes formation of a voluminous atrial chamber and a miniscule ventricular chamber despite normal specification of chamber-specific precursor populations.…”

“…Embryos were injected at the one-cell stage with 2 ng of an anti-nkx2.7 morpholino (MO) (5Ј-TGGAGGTCACAGGACTCGGAAGCAT-3Ј) (Targoff et al, 2008) along with 2 ng of an anti-p53 (tp53 -Zebrafish Information Network) MO (5Ј-GCGCCATTGCTTTGCAAGAATTG-3Ј) (Robu et al, 2007). For mosaic cell-labeling experiments, embryos were injected at the one-cell stage with 150 pg of Tg(myl7:EGFP) plasmid.…”

“…We counted cardiomyocytes in embryos carrying the transgene Tg , using immunofluorescence to detect DsRed in cardiomyocyte nuclei and to detect atrial myosin heavy chain (Amhc) in atrial cells, as previously described (Targoff et al, 2008). Embryos at 36 and 52 hours post-fertilization (hpf) were imaged with an Axioplan microscope, and embryos at 26 hpf were imaged with a Zeiss LSM510 confocal microscope.…”

“…Using morpholinos (MOs) to inhibit nkx2.5 and nkx2.7 function, our prior studies have indicated important roles of Nkx genes in controlling cardiomyocyte number and morphogenesis in each cardiac chamber (Targoff et al, 2008). However, in our previous work, it was difficult to decipher whether Nkx genes guide ventricular and atrial cell fate decisions, owing to the inherent variability and incomplete degree of MO-mediated gene knockdown.…”

“…In humans, mutations in NKX2-5 are associated with particular types of congenital heart defects (Benson et al, 1999;Elliott et al, 2003;Jay et al, 2003;McElhinney et al, 2003;Schott et al, 1998). Investigations in multiple model organisms have led to insights into the conserved roles of NKX2-5 homologs in cardiac specification, morphogenesis and maturation (Azpiazu and Frasch, 1993;Bodmer, 1993;Grow and Krieg, 1998;Lyons et al, 1995;Prall et al, 2007;Tanaka et al, 1999;Targoff et al, 2008;Tu et al, 2009). In addition, studies of the murine genes Nkx2-5 and Nkx2-6 have hinted at roles of Nkx genes in regulating chamber-specific gene expression.…”