pubmed.ncbi.nlm.nih.gov

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

  • ️Tue Jan 01 2008

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

Kimara L Targoff et al. Dev Biol. 2008.

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Knockdown of nkx2.5 and/or nkx2.7 causes prominent defects in cardiac chamber morphogenesis. (A–D) Frontal views, dorsal to the top at 52 hpf. MF20/S46 immunofluorescence distinguishes ventricular myocardium (red) from atrial myocardium (yellow). In comparison to wild-type embryos (A), injection of anti-nkx2.5 ATG MO (B) or anti-nkx2.7 ATG MO (C) causes subtle abnormalities in chamber size and shape. (D) Injection of anti-nkx2.5 and anti-nkx2.7 ATG MOs together reveals more striking defects in both ventricular and atrial morphology. (E,F) Lateral views of live embryos, anterior to the right, at 52 hpf. Other than their cardiac defects, pericardial edema, brain ventricle swelling, and jaw hypoplasia, nkx-deficient embryos appear morphologically normal.

Figure 2
Figure 2

Splice MOs confirm specificity and efficacy of nkx2.5 and nkx2.7 knockdown. (A,B) Frontal views, dorsal to the top at 52 hpf. MF20/S46 immunofluorescence distinguishes ventricular myocardium (red) from atrial myocardium (yellow). Injection of anti-nkx2.5 and anti-nkx2.7 splice MOs has effects similar to that of injection of anti-nkx2.5 and anti-nkx2.7 ATG MOs (Fig. 1D). (C,D) MO injection impairs splicing of nkx2.5 and nkx2.7. Schematics depict the structures of the nkx2.5 (C) and nkx2.7 (D) pre-mRNA and mRNA. Locations of splice MOs are indicated in red. Primer pairs used for RT-PCR are indicated in green. Gels document RT-PCR amplification products, comparing detection of spliced (lanes 2 and 4) and unspliced (lanes 3 and 5) messages in cDNA from 26 hpf control embryos and embryos injected with anti-nkx2.5 splice MO or anti-nkx2.7 splice MO, respectively. Primer pair F1R1 amplifies 189 bp of unspliced nkx2.5 pre-mRNA, primer pair F1R2 amplifies 230 bp of spliced nkx2.5 mRNA, primer pair F3R3 amplifies 229 bp of unspliced nkx2.7 pre-mRNA, and primer pair F4R3 amplifies 323 bp of spliced nkx2.7 mRNA. In both gels, lane 1 contains a 100 bp molecular weight ladder (NEB, Ipswich, MA, USA). Embryos injected with anti-nkx2.5 splice MO exhibit a loss of spliced nkx2.5 mRNA, and embryos injected with anti-nkx2.7 splice MO demonstrate a partial reduction in spliced nkx2.7 mRNA with a concomitant increase in unspliced pre-mRNA. Note that the predicted PCR products with intronic inclusion for the unspliced nkx2.5 (1.4 kb) and nkx2.7 (2.2 kb) pre-mRNAs are absent, most likely because PCR conditions did not favor amplification of larger products.

Figure 3
Figure 3

Bilateral populations of cardiomyocytes are normal in nkx-deficient embryos. In situ hybridization depicts expression of cmlc2 (A,B) and vmhc (C,D) in wild-type (A,C) and nkx-deficient (B,D) embryos. Embryos viewed dorsally, anterior to the top, at the 18-somite stage. Aside from a mild delay in cardiomyocyte migration toward the midline, nkx-deficient embryos appear to have the same patterns of cmlc2 and vmhc expression as are observed in wild-type embryos.

Figure 4
Figure 4

Cardiac cone formation occurs normally in nkx-deficient embryos. In situ hybridization depicts expression of cmlc2 (A,B), vmhc (C,D), and amhc (E,F) in wild-type and nkx-deficient embryos. Embryos viewed dorsally, anterior to the top, at the 22-somite stage. (A,C,E) Wild-type embryos exhibit normal fusion of the bilateral cardiac precursors, creating an intact cardiac cone. (B,D,F) nkx-deficient embryos mirror the expression patterns observed in wild-type, with the exception of a slightly enlarged and elongated lumen within the cardiac cone.

Figure 5
Figure 5

nkx genes play a critical role in early steps of heart tube extension. In situ hybridization depicts expression of cmlc2 (A,B), vmhc (C,D), and amhc (E,F) in wild-type (A,C,E) and nkx-deficient (B,D,F) embryos. Embryos viewed dorsally, anterior to the top, at 26 hpf. (A,B) nkx-deficient embryos exhibit prominent defects in heart tube extension, including a splayed and disorganized inflow region and an unusually compact outflow region. (C,D) In nkx-deficient embryos, the ventricular portion of the heart tube is abnormally short and wide. (E,F) In nkx-deficient embryos, the atrial portion of the heart tube is broad and sprawling.

Figure 6
Figure 6

nkx genes limit atrial cell number during heart tube assembly. (A,B) Immunofluorescence indicates nuclear localization of Mef2 (green) throughout the heart tube, facilitating counting of differentiated cardiomyocytes at 26 hpf. Atrial cells are indicated by the anti-Amhc antibody, S46 (red). Hearts are flattened with a cover slip to improve visualization of cardiomyocyte nuclei. (C) Quantification of cardiomyocyte nuclei in wild-type (n=25) and nkx-deficient (n=11) embryos reveals a statistically significant increase in atrial cell number in nkx-deficient embryos (p<0.001, Student’s t-test) and no significant difference in ventricular cell number. Bar graph indicates mean and standard error of each data set; asterisk indicates statistically significant difference from wild-type.

Figure 7
Figure 7

nkx genes are necessary to establish sufficient ventricular cell number during cardiac chamber formation. (A,B) Immunofluorescence indicates that both chambers express the transgene Tg(cmlc2:DsRed2-nuc) (red), facilitating cardiomyocyte counting at 52 hpf. Atria are labeled with the anti-Amhc antibody, S46 (green). Hearts are flattened with a cover slip to improve visualization of cardiomyocyte nuclei. (C) Quantification of cardiomyocyte nuclei in wild-type (n=10) and nkx-deficient (n=13) embryos reveals a statistically significant increase in atrial cell number in nkx-deficient embryos (p<0.001, Student’s t-test) and a statistically significant decrease in ventricular cell number in nkx-deficient embryos (p<0.001, Student’s t-test). Bar graph indicates mean and standard error of each data set; asterisks indicate statistically significant differences from wild-type.

Similar articles

Cited by

References

    1. Alexander J, et al. Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning. Dev Genet. 1998;22:288–299. - PubMed
    1. Allen BG, et al. Reduction of XNkx2-10 expression leads to anterior defects and malformation of the embryonic heart. Mech Dev. 2006;123:719–729. - PMC - PubMed
    1. Azpiazu N, Frasch M. tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev. 1993;7:1325–1340. - PubMed
    1. Baird GS, et al. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A. 2000;97:11984–11989. - PMC - PubMed
    1. Bartlett HL, et al. Transient early embryonic expression of Nkx2-5 mutations linked to congenital heart defects in human causes heart defects in Xenopus laevis. Dev Dyn. 2007;236:2475–2484. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources