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Nkx2.7 and Nkx2.5 function redundantly and are required for cardiac morphogenesis of zebrafish embryos.

Tu CT, Yang TC, Tsai HJ - PLoS ONE (2009)

Bottom Line: Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage.We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20.Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Nkx2.7 is the tinman-related gene, as well as orthologs of Nkx2.5 and Nkx-2.3. Nkx2.7 and Nkx2.5 express in zebrafish heart fields of lateral plate mesoderm. The temporal and spatial expression patterns of Nkx2.7 are similar to those of Nkx2.5, but their functions during cardiogenesis remain unclear.

Methodology/principal findings: Here, Nkx2.7 is demonstrated to compensate for Nkx2.5 loss of function and play a predominant role in the lateral development of the heart, including normal cardiac looping and chamber formation. Knocking down Nkx2.5 showed that heart development was normal from 24 to 72 hpf. However, when knocking down either Nkx2.7 or Nkx2.5 together with Nkx2.7, it appeared that the heart failed to undergo looping and showed defective chambers, although embryos developed normally before the early heart tube stage. Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage. We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20. Although heart defects were induced by overexpression of an injection of 150-pg Nkx2.5 or 5-pg Nkx2.7 mRNA, either Nkx2.5 or Nkx2.7 mRNA rescued the defects induced by Nkx2.7-morpholino(MO) and Nkx2.5-MO with Nkx2.7-MO.

Conclusions and significance: Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage.

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The defective phenotypes of zebrafish embryo heart injected with Nkx2.5-MO, Nkx2.7-MO and Nkx2.5/2.7-MO.Eight nanograms of MO were injected into one-cell stage embryos derived from transgenic line Tg (cmlc2::GFP) to knock down the Nkx protein specifically. The embryos are shown at 36 hpf (A, B, C), 48 hpf (D, E, F), and 72 hpf (G, H, I). The heart phenotype of Nkx2.5-MO embryos was similar to that of control embryos whose ventricle is located at the right side of the atrium when embryos were observed at 36 hpf, 48 hpf and 72 hpf from the ventral view under fluorescence microscope (A, D, G). However, embryos injected with Nkx2.7-MO displayed an unlooping defect from 36 hpf to 72 hpf (B, E, H). Embryos injected with Nkx2.5/2.7-MO displayed a shrunken ventricle and an expanding atrium (C, F, I). v: ventricle; a: atrium.
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pone-0004249-g001: The defective phenotypes of zebrafish embryo heart injected with Nkx2.5-MO, Nkx2.7-MO and Nkx2.5/2.7-MO.Eight nanograms of MO were injected into one-cell stage embryos derived from transgenic line Tg (cmlc2::GFP) to knock down the Nkx protein specifically. The embryos are shown at 36 hpf (A, B, C), 48 hpf (D, E, F), and 72 hpf (G, H, I). The heart phenotype of Nkx2.5-MO embryos was similar to that of control embryos whose ventricle is located at the right side of the atrium when embryos were observed at 36 hpf, 48 hpf and 72 hpf from the ventral view under fluorescence microscope (A, D, G). However, embryos injected with Nkx2.7-MO displayed an unlooping defect from 36 hpf to 72 hpf (B, E, H). Embryos injected with Nkx2.5/2.7-MO displayed a shrunken ventricle and an expanding atrium (C, F, I). v: ventricle; a: atrium.

Mentions: We designed Nkx2.5- and Nkx2.7-MO to study whether Nkx2.5 or Nkx2.7 is required for heart development of zebrafish. Heart-specific-GFP transgenic line Tg (cmlc2::GFP) was used to monitor the cardiac morphology in zebrafish embryos. By fluorescence microscope, we observed that embryos injected with 10, 12, and 14 ng of Nkx2.5-MO developed normally from 24 to 72 hpf, although we occasionally observed a small percentage of injected embryos which had a slight degree of pericardial edema. This was also observed in the wild-type embryos and in the embryos injected with a high concentration of control MO (Table 1 and Fig. 1A). In contrast, embryos injected with Nkx2.7-MO at the same concentrations totally failed to complete heart looping during 30 to 72 hpf (Table 1 and Fig. 1B). Moreover, double knockdown of Nkx2.5 and Nkx2.7 (Nkx2.5/2.7-MO) by injection of 8 ng Nkx2.5-MO combined with 8 ng of Nkx2.7-MO into embryos displayed a shrunken ventricle and an expanded atrium with an incomplete heart looping. After 72 hpf, many symptoms of heart defects appeared in both Nkx2.7 and Nkx2.5/2.7 morphants, including regurgitation of blood, arrhythmia, string-like heart and pericardial edema (data not shown). The rates of defective heart occurrence in the Nkx2.7 and the Nkx2.5/2.7 morphants were dose-dependent (Table 1). Comparing the cardiac defects among Nkx2.5 morphants, Nkx2.7 morphants and Nkx2.5/2.7 morphants, we observed that the Nkx2.5 morphants did not have any obvious heart defects, while the Nkx2.7 morphants exhibited an unlooping defect with a low percentage of shrunken ventricles. The Nkx2.5/2.7 morphants displayed not only the unlooping defect, but also shrunken ventricles (Table 1).


Nkx2.7 and Nkx2.5 function redundantly and are required for cardiac morphogenesis of zebrafish embryos.

Tu CT, Yang TC, Tsai HJ - PLoS ONE (2009)

The defective phenotypes of zebrafish embryo heart injected with Nkx2.5-MO, Nkx2.7-MO and Nkx2.5/2.7-MO.Eight nanograms of MO were injected into one-cell stage embryos derived from transgenic line Tg (cmlc2::GFP) to knock down the Nkx protein specifically. The embryos are shown at 36 hpf (A, B, C), 48 hpf (D, E, F), and 72 hpf (G, H, I). The heart phenotype of Nkx2.5-MO embryos was similar to that of control embryos whose ventricle is located at the right side of the atrium when embryos were observed at 36 hpf, 48 hpf and 72 hpf from the ventral view under fluorescence microscope (A, D, G). However, embryos injected with Nkx2.7-MO displayed an unlooping defect from 36 hpf to 72 hpf (B, E, H). Embryos injected with Nkx2.5/2.7-MO displayed a shrunken ventricle and an expanding atrium (C, F, I). v: ventricle; a: atrium.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626283&req=5

pone-0004249-g001: The defective phenotypes of zebrafish embryo heart injected with Nkx2.5-MO, Nkx2.7-MO and Nkx2.5/2.7-MO.Eight nanograms of MO were injected into one-cell stage embryos derived from transgenic line Tg (cmlc2::GFP) to knock down the Nkx protein specifically. The embryos are shown at 36 hpf (A, B, C), 48 hpf (D, E, F), and 72 hpf (G, H, I). The heart phenotype of Nkx2.5-MO embryos was similar to that of control embryos whose ventricle is located at the right side of the atrium when embryos were observed at 36 hpf, 48 hpf and 72 hpf from the ventral view under fluorescence microscope (A, D, G). However, embryos injected with Nkx2.7-MO displayed an unlooping defect from 36 hpf to 72 hpf (B, E, H). Embryos injected with Nkx2.5/2.7-MO displayed a shrunken ventricle and an expanding atrium (C, F, I). v: ventricle; a: atrium.
Mentions: We designed Nkx2.5- and Nkx2.7-MO to study whether Nkx2.5 or Nkx2.7 is required for heart development of zebrafish. Heart-specific-GFP transgenic line Tg (cmlc2::GFP) was used to monitor the cardiac morphology in zebrafish embryos. By fluorescence microscope, we observed that embryos injected with 10, 12, and 14 ng of Nkx2.5-MO developed normally from 24 to 72 hpf, although we occasionally observed a small percentage of injected embryos which had a slight degree of pericardial edema. This was also observed in the wild-type embryos and in the embryos injected with a high concentration of control MO (Table 1 and Fig. 1A). In contrast, embryos injected with Nkx2.7-MO at the same concentrations totally failed to complete heart looping during 30 to 72 hpf (Table 1 and Fig. 1B). Moreover, double knockdown of Nkx2.5 and Nkx2.7 (Nkx2.5/2.7-MO) by injection of 8 ng Nkx2.5-MO combined with 8 ng of Nkx2.7-MO into embryos displayed a shrunken ventricle and an expanded atrium with an incomplete heart looping. After 72 hpf, many symptoms of heart defects appeared in both Nkx2.7 and Nkx2.5/2.7 morphants, including regurgitation of blood, arrhythmia, string-like heart and pericardial edema (data not shown). The rates of defective heart occurrence in the Nkx2.7 and the Nkx2.5/2.7 morphants were dose-dependent (Table 1). Comparing the cardiac defects among Nkx2.5 morphants, Nkx2.7 morphants and Nkx2.5/2.7 morphants, we observed that the Nkx2.5 morphants did not have any obvious heart defects, while the Nkx2.7 morphants exhibited an unlooping defect with a low percentage of shrunken ventricles. The Nkx2.5/2.7 morphants displayed not only the unlooping defect, but also shrunken ventricles (Table 1).

Bottom Line: Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage.We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20.Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Nkx2.7 is the tinman-related gene, as well as orthologs of Nkx2.5 and Nkx-2.3. Nkx2.7 and Nkx2.5 express in zebrafish heart fields of lateral plate mesoderm. The temporal and spatial expression patterns of Nkx2.7 are similar to those of Nkx2.5, but their functions during cardiogenesis remain unclear.

Methodology/principal findings: Here, Nkx2.7 is demonstrated to compensate for Nkx2.5 loss of function and play a predominant role in the lateral development of the heart, including normal cardiac looping and chamber formation. Knocking down Nkx2.5 showed that heart development was normal from 24 to 72 hpf. However, when knocking down either Nkx2.7 or Nkx2.5 together with Nkx2.7, it appeared that the heart failed to undergo looping and showed defective chambers, although embryos developed normally before the early heart tube stage. Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage. We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20. Although heart defects were induced by overexpression of an injection of 150-pg Nkx2.5 or 5-pg Nkx2.7 mRNA, either Nkx2.5 or Nkx2.7 mRNA rescued the defects induced by Nkx2.7-morpholino(MO) and Nkx2.5-MO with Nkx2.7-MO.

Conclusions and significance: Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage.

Show MeSH