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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.

Show MeSH
Abnormal cardiac differentiation occurred in the Nkx2.7-knockdown zebrafish embryos.The expressions of bmp4 (A–F) and versican (G–L) in hearts were compared between wild-type (WT) (A, D, G, J), Nkx2.7-MO- (B, E, H, K) and Nkx2.5/2.7-MO- injected embryos (C, F, I, L) at 48 (A–C, G–I) and 72 hpf (D–F, J–L). In WT embryos, bmp4 was expressed in the ventricle and inflow tract at 48 hpf (A), and then bmp4 was restricted in its expression at the AV boundary at 72 hpf (D). However, in the Nkx2.7-MO (B, E) and Nkx2.5/2.7-MO (C, F) embryos, bmp4 was still expressed predominantly in the ventricle and atrium from 48 to 72 hpf. Similarly, in WT embryos, the versican expression was more predominant in ventricle than in atrium, at about 31 to 33 hpf, and then versican was confined in its expression at the AV boundary after 33 hpf (G, J). In contrast, in the Nkx2.7-MO (H, K) and Nkx2.5/2.7-MO (I, L) embryos, the versican was significantly expressed in the atrium and ventricle. In addition, the versican expression pattern in otoliths remained unchanged (J, K, and L). All images are ventral views, anterior to the top. a: atrium; v: ventricle; i: inflow tract; av: atrioventricular boundary; ot: otoliths.
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pone-0004249-g005: Abnormal cardiac differentiation occurred in the Nkx2.7-knockdown zebrafish embryos.The expressions of bmp4 (A–F) and versican (G–L) in hearts were compared between wild-type (WT) (A, D, G, J), Nkx2.7-MO- (B, E, H, K) and Nkx2.5/2.7-MO- injected embryos (C, F, I, L) at 48 (A–C, G–I) and 72 hpf (D–F, J–L). In WT embryos, bmp4 was expressed in the ventricle and inflow tract at 48 hpf (A), and then bmp4 was restricted in its expression at the AV boundary at 72 hpf (D). However, in the Nkx2.7-MO (B, E) and Nkx2.5/2.7-MO (C, F) embryos, bmp4 was still expressed predominantly in the ventricle and atrium from 48 to 72 hpf. Similarly, in WT embryos, the versican expression was more predominant in ventricle than in atrium, at about 31 to 33 hpf, and then versican was confined in its expression at the AV boundary after 33 hpf (G, J). In contrast, in the Nkx2.7-MO (H, K) and Nkx2.5/2.7-MO (I, L) embryos, the versican was significantly expressed in the atrium and ventricle. In addition, the versican expression pattern in otoliths remained unchanged (J, K, and L). All images are ventral views, anterior to the top. a: atrium; v: ventricle; i: inflow tract; av: atrioventricular boundary; ot: otoliths.

Mentions: We also observed myocardial differentiation markers, such as bmp4, versican, tbx5, and tbx20. BMP4 is one of the TGF-β superfamily proteins and is involved in valve development during the cardiac maturation stage. A ventricle-enriched expression of bmp4 was the same between wild-type and Nkx-deficient embryos from 31 to 33 hpf (data not shown). Moreover, in wild-type embryos, bmp4 was found to express in the ventricle and inflow tract at 48 hpf (Fig. 5A), and then it was expressed exclusively in the atrioventricular junction at 72 hpf (Fig. 5D). However, unlike the dynamic expression of bmp4 in the wild-type embryos, bmp4 in the Nkx2.7 and in the Nkx2.5/2.7 morphants failed to change its predominant ventricular and atrial expression pattern (Figs. 5B and 5E; 5C and 5F). Similarly, versican was expressed broadly in the atrium and weakly in the ventricle of the wild-type embryos at 33 hpf. This atrium-enriched expression of versican was the same between wild-type and Nkx-deficient embryos (data not shown). After 36 hpf, the versican expression of wild-type was restricted to the AV boundary (Figs. 5G and 5J). However, Nkx2.7 and the Nkx2.5/2.7 morphants still expressed a high level versican in the atrium of the heart from 48 to 72 hpf (Figs. 5H and 5K; 5I and 5L). Nevertheless, we noticed that the versican was expressed in otoliths of both the wild-type embryos and the Nkx-deficient embryos by 72 hpf (Figs. 5J, 5K and 5L), indicating that the development of Nkx-deficient embryos was not delayed and that the defects caused by Nkx-MO were specific in the heart, not in otoliths (Figs. 5J, 5K and 5L).


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)

Abnormal cardiac differentiation occurred in the Nkx2.7-knockdown zebrafish embryos.The expressions of bmp4 (A–F) and versican (G–L) in hearts were compared between wild-type (WT) (A, D, G, J), Nkx2.7-MO- (B, E, H, K) and Nkx2.5/2.7-MO- injected embryos (C, F, I, L) at 48 (A–C, G–I) and 72 hpf (D–F, J–L). In WT embryos, bmp4 was expressed in the ventricle and inflow tract at 48 hpf (A), and then bmp4 was restricted in its expression at the AV boundary at 72 hpf (D). However, in the Nkx2.7-MO (B, E) and Nkx2.5/2.7-MO (C, F) embryos, bmp4 was still expressed predominantly in the ventricle and atrium from 48 to 72 hpf. Similarly, in WT embryos, the versican expression was more predominant in ventricle than in atrium, at about 31 to 33 hpf, and then versican was confined in its expression at the AV boundary after 33 hpf (G, J). In contrast, in the Nkx2.7-MO (H, K) and Nkx2.5/2.7-MO (I, L) embryos, the versican was significantly expressed in the atrium and ventricle. In addition, the versican expression pattern in otoliths remained unchanged (J, K, and L). All images are ventral views, anterior to the top. a: atrium; v: ventricle; i: inflow tract; av: atrioventricular boundary; ot: otoliths.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626283&req=5

pone-0004249-g005: Abnormal cardiac differentiation occurred in the Nkx2.7-knockdown zebrafish embryos.The expressions of bmp4 (A–F) and versican (G–L) in hearts were compared between wild-type (WT) (A, D, G, J), Nkx2.7-MO- (B, E, H, K) and Nkx2.5/2.7-MO- injected embryos (C, F, I, L) at 48 (A–C, G–I) and 72 hpf (D–F, J–L). In WT embryos, bmp4 was expressed in the ventricle and inflow tract at 48 hpf (A), and then bmp4 was restricted in its expression at the AV boundary at 72 hpf (D). However, in the Nkx2.7-MO (B, E) and Nkx2.5/2.7-MO (C, F) embryos, bmp4 was still expressed predominantly in the ventricle and atrium from 48 to 72 hpf. Similarly, in WT embryos, the versican expression was more predominant in ventricle than in atrium, at about 31 to 33 hpf, and then versican was confined in its expression at the AV boundary after 33 hpf (G, J). In contrast, in the Nkx2.7-MO (H, K) and Nkx2.5/2.7-MO (I, L) embryos, the versican was significantly expressed in the atrium and ventricle. In addition, the versican expression pattern in otoliths remained unchanged (J, K, and L). All images are ventral views, anterior to the top. a: atrium; v: ventricle; i: inflow tract; av: atrioventricular boundary; ot: otoliths.
Mentions: We also observed myocardial differentiation markers, such as bmp4, versican, tbx5, and tbx20. BMP4 is one of the TGF-β superfamily proteins and is involved in valve development during the cardiac maturation stage. A ventricle-enriched expression of bmp4 was the same between wild-type and Nkx-deficient embryos from 31 to 33 hpf (data not shown). Moreover, in wild-type embryos, bmp4 was found to express in the ventricle and inflow tract at 48 hpf (Fig. 5A), and then it was expressed exclusively in the atrioventricular junction at 72 hpf (Fig. 5D). However, unlike the dynamic expression of bmp4 in the wild-type embryos, bmp4 in the Nkx2.7 and in the Nkx2.5/2.7 morphants failed to change its predominant ventricular and atrial expression pattern (Figs. 5B and 5E; 5C and 5F). Similarly, versican was expressed broadly in the atrium and weakly in the ventricle of the wild-type embryos at 33 hpf. This atrium-enriched expression of versican was the same between wild-type and Nkx-deficient embryos (data not shown). After 36 hpf, the versican expression of wild-type was restricted to the AV boundary (Figs. 5G and 5J). However, Nkx2.7 and the Nkx2.5/2.7 morphants still expressed a high level versican in the atrium of the heart from 48 to 72 hpf (Figs. 5H and 5K; 5I and 5L). Nevertheless, we noticed that the versican was expressed in otoliths of both the wild-type embryos and the Nkx-deficient embryos by 72 hpf (Figs. 5J, 5K and 5L), indicating that the development of Nkx-deficient embryos was not delayed and that the defects caused by Nkx-MO were specific in the heart, not in otoliths (Figs. 5J, 5K and 5L).

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