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Wnt6 signaling regulates heart muscle development during organogenesis.

Lavery DL, Martin J, Turnbull YD, Hoppler S - Dev. Biol. (2008)

Bottom Line: We find, however, that Wnt6 is not required as expected during gastrulation stages, but later during organogenesis stages just before cells of the cardiogenic mesoderm begin to differentiate into heart muscle (myocardium).Our gain-of-function experiments show that Wnt6 and also activated canonical Wnt/beta-catenin signaling are capable of restricting heart muscle development at these relatively late stages of development.This repressive role of Wnt signaling is mediated initially via repression of cardiogenic transcription factors, since reinstatement of GATA function can rescue expression of other cardiogenic transcription factors and downstream cardiomyogenic differentiation genes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, Cell and Developmental Biology Research Programme, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK.

ABSTRACT
Mesodermal tissue with heart forming potential (cardiogenic mesoderm) is induced during gastrulation. This cardiogenic mesoderm later differentiates into heart muscle tissue (myocardium) and non-muscular heart tissue. Inhibition of Wnt/beta-catenin signaling is known to be required early for induction of cardiogenic mesoderm; however, the identity of the inhibiting Wnt signal itself is still elusive. We have identified Wnt6 in Xenopus as an endogenous Wnt signal, which is expressed in tissues close to and later inside the developing heart. Our loss-of-function experiments show that Wnt6 function is required in the embryo to prevent development of an abnormally large heart muscle. We find, however, that Wnt6 is not required as expected during gastrulation stages, but later during organogenesis stages just before cells of the cardiogenic mesoderm begin to differentiate into heart muscle (myocardium). Our gain-of-function experiments show that Wnt6 and also activated canonical Wnt/beta-catenin signaling are capable of restricting heart muscle development at these relatively late stages of development. This repressive role of Wnt signaling is mediated initially via repression of cardiogenic transcription factors, since reinstatement of GATA function can rescue expression of other cardiogenic transcription factors and downstream cardiomyogenic differentiation genes.

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Overexpression of Wnt6 during organogenesis stages inhibits heart muscle development. (A) Schematic representation of transgene for concomitant overexpression of xWnt6 and GFP in transgenic Xenopus embryos. (B-E) Identification of a non-transgenic control embryo (B, D) versus a transgenic embryo (C, E) induced at stage 22 by heat shock treatment to overexpress concomitantly GFP and xWnt6 viewed at stage 28 under UV light (D, E; compare with the same embryos viewed under visible light in panels B and C, respectively). Note only faint background fluorescence (mainly from yolk) in non-transgenic embryo (D), but strong fluorescence due to GFP expression in transgenic embryo (E). External morphology of whole embryos (F, G) and of the heart forming regions of the same embryos (H, J) at stage 40; TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (I, K); and analysis of marker gene expression with whole-mount RNA in situ hybridization at stage 32 (L–U) in non-transgenic control (F, H, I, L, N, P, R, T) and xWnt6-overexpressing transgenic embryos (G, J, K, M, O, Q, S, U). Note abnormal morphology in xWnt6-overexpressing embryos, particularly in the eye (G) and the heart-forming region (G, J). Note much reduced TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (panel K, see also panel V). Note significantly reduced and restricted GATA4 (M) and GATA6 (O) expression, dramatic reduction of Nkx2.5 expression (panel Q, but see panel W and panel X) and restricted domains of TroponinIc (TnIc) (S) and Myosin Light Chain 2 (MLC2) (U) expression in xWnt6-overexpressing embryos. (V) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in non-transgenic control and xWnt6-overexpressing transgenic embryos; note much smaller TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (see also panels I, K). (W) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in non-transgenic control (NT) and weak or strong xWnt6 overexpression in transgenic embryos. Note reduction of GATA4, GATA6, TroponinIc (TnIc), myosin light chain 2 (MLC2) and generally Nkx2.5 expression in embryos with xWnt6 overexpression. (X) Bar chart of quantitative RT-PCR (qPCR) analysis of heart development marker gene expression in stage 32 embryos. Note reduced expression in xWnt6-overexpressing embryos, apart from Nkx2.3 and Nkx2.5.
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fig3: Overexpression of Wnt6 during organogenesis stages inhibits heart muscle development. (A) Schematic representation of transgene for concomitant overexpression of xWnt6 and GFP in transgenic Xenopus embryos. (B-E) Identification of a non-transgenic control embryo (B, D) versus a transgenic embryo (C, E) induced at stage 22 by heat shock treatment to overexpress concomitantly GFP and xWnt6 viewed at stage 28 under UV light (D, E; compare with the same embryos viewed under visible light in panels B and C, respectively). Note only faint background fluorescence (mainly from yolk) in non-transgenic embryo (D), but strong fluorescence due to GFP expression in transgenic embryo (E). External morphology of whole embryos (F, G) and of the heart forming regions of the same embryos (H, J) at stage 40; TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (I, K); and analysis of marker gene expression with whole-mount RNA in situ hybridization at stage 32 (L–U) in non-transgenic control (F, H, I, L, N, P, R, T) and xWnt6-overexpressing transgenic embryos (G, J, K, M, O, Q, S, U). Note abnormal morphology in xWnt6-overexpressing embryos, particularly in the eye (G) and the heart-forming region (G, J). Note much reduced TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (panel K, see also panel V). Note significantly reduced and restricted GATA4 (M) and GATA6 (O) expression, dramatic reduction of Nkx2.5 expression (panel Q, but see panel W and panel X) and restricted domains of TroponinIc (TnIc) (S) and Myosin Light Chain 2 (MLC2) (U) expression in xWnt6-overexpressing embryos. (V) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in non-transgenic control and xWnt6-overexpressing transgenic embryos; note much smaller TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (see also panels I, K). (W) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in non-transgenic control (NT) and weak or strong xWnt6 overexpression in transgenic embryos. Note reduction of GATA4, GATA6, TroponinIc (TnIc), myosin light chain 2 (MLC2) and generally Nkx2.5 expression in embryos with xWnt6 overexpression. (X) Bar chart of quantitative RT-PCR (qPCR) analysis of heart development marker gene expression in stage 32 embryos. Note reduced expression in xWnt6-overexpressing embryos, apart from Nkx2.3 and Nkx2.5.

Mentions: The ventral Wnt6 mRNA injection experiments illustrate the effects Wnt6 overexpression has on early embryonic development (see above and Fig. 1). However, endogenous xWnt6 is not expressed at higher levels until organogenesis stages (Lavery et al., 2008), and our loss of function experiments (see above) indicate that xWnt6 is not required until these later stages of development. Wnt6 mRNA injection experiments are unsuitable for studying Wnt function at later stages since the effects Wnt6 overexpression has on early embryonic development would obscure any direct effect Wnt6 might have on later stages of development. In order to study xWnt6 functional activity during organogenesis stages, we used an inducible DNA construct in transgenic Xenopus embryos (see Materials and methods), which allows for stage-specific overexpression of xWnt6 during organogenesis stages (i.e. stage 22), when Wnt6 function was found to be required. The full-length xWnt6 was expressed under control of the Xenopus heat shock protein 70 promoter (Fig. 3A). The transgenic Wnt6-overexpressing embryos developed reduced eyes when compared to the non-transgenic control embryos (Figs. 3F, G), but also an enlarged cavity surrounding a much smaller developing heart (Figs. 3F, G, H, J, see also movies 9–11 in Supplementary data). Analysis of TroponinT-expressing heart muscle tissue shows development of a smaller heart with less myocardial tissue (Figs. 3K, V). The overall shape and morphogenetic looping of the heart is also affected (Figs. 3J, K). When transgenic Wnt6-overexpressing embryos were analyzed by whole-mount in situ hybridization at stage 32, there was a clear reduction in expression of all cardiogenic markers tested, including both genes that are usually associated with cardiac fate specification, such as Nkx2.5, GATA4 and GATA6 (Figs. 3L–Q, W), as well as later heart muscle differentiation genes, such as MLC2 and TroponinIc (Figs. 3R–U, W). Markers for other tissues were found to be unaffected or even up-regulated (e.g. the kidney tubule marker NKCC2, data not shown). The reduction of Nkx2.5, GATA4 and GATA6 expression is not confined to the prospective myocardium tissue but is more general, affecting the wider expression domain of these transcription factor genes (Figs. 3L–Q).


Wnt6 signaling regulates heart muscle development during organogenesis.

Lavery DL, Martin J, Turnbull YD, Hoppler S - Dev. Biol. (2008)

Overexpression of Wnt6 during organogenesis stages inhibits heart muscle development. (A) Schematic representation of transgene for concomitant overexpression of xWnt6 and GFP in transgenic Xenopus embryos. (B-E) Identification of a non-transgenic control embryo (B, D) versus a transgenic embryo (C, E) induced at stage 22 by heat shock treatment to overexpress concomitantly GFP and xWnt6 viewed at stage 28 under UV light (D, E; compare with the same embryos viewed under visible light in panels B and C, respectively). Note only faint background fluorescence (mainly from yolk) in non-transgenic embryo (D), but strong fluorescence due to GFP expression in transgenic embryo (E). External morphology of whole embryos (F, G) and of the heart forming regions of the same embryos (H, J) at stage 40; TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (I, K); and analysis of marker gene expression with whole-mount RNA in situ hybridization at stage 32 (L–U) in non-transgenic control (F, H, I, L, N, P, R, T) and xWnt6-overexpressing transgenic embryos (G, J, K, M, O, Q, S, U). Note abnormal morphology in xWnt6-overexpressing embryos, particularly in the eye (G) and the heart-forming region (G, J). Note much reduced TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (panel K, see also panel V). Note significantly reduced and restricted GATA4 (M) and GATA6 (O) expression, dramatic reduction of Nkx2.5 expression (panel Q, but see panel W and panel X) and restricted domains of TroponinIc (TnIc) (S) and Myosin Light Chain 2 (MLC2) (U) expression in xWnt6-overexpressing embryos. (V) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in non-transgenic control and xWnt6-overexpressing transgenic embryos; note much smaller TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (see also panels I, K). (W) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in non-transgenic control (NT) and weak or strong xWnt6 overexpression in transgenic embryos. Note reduction of GATA4, GATA6, TroponinIc (TnIc), myosin light chain 2 (MLC2) and generally Nkx2.5 expression in embryos with xWnt6 overexpression. (X) Bar chart of quantitative RT-PCR (qPCR) analysis of heart development marker gene expression in stage 32 embryos. Note reduced expression in xWnt6-overexpressing embryos, apart from Nkx2.3 and Nkx2.5.
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Related In: Results  -  Collection

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fig3: Overexpression of Wnt6 during organogenesis stages inhibits heart muscle development. (A) Schematic representation of transgene for concomitant overexpression of xWnt6 and GFP in transgenic Xenopus embryos. (B-E) Identification of a non-transgenic control embryo (B, D) versus a transgenic embryo (C, E) induced at stage 22 by heat shock treatment to overexpress concomitantly GFP and xWnt6 viewed at stage 28 under UV light (D, E; compare with the same embryos viewed under visible light in panels B and C, respectively). Note only faint background fluorescence (mainly from yolk) in non-transgenic embryo (D), but strong fluorescence due to GFP expression in transgenic embryo (E). External morphology of whole embryos (F, G) and of the heart forming regions of the same embryos (H, J) at stage 40; TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (I, K); and analysis of marker gene expression with whole-mount RNA in situ hybridization at stage 32 (L–U) in non-transgenic control (F, H, I, L, N, P, R, T) and xWnt6-overexpressing transgenic embryos (G, J, K, M, O, Q, S, U). Note abnormal morphology in xWnt6-overexpressing embryos, particularly in the eye (G) and the heart-forming region (G, J). Note much reduced TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (panel K, see also panel V). Note significantly reduced and restricted GATA4 (M) and GATA6 (O) expression, dramatic reduction of Nkx2.5 expression (panel Q, but see panel W and panel X) and restricted domains of TroponinIc (TnIc) (S) and Myosin Light Chain 2 (MLC2) (U) expression in xWnt6-overexpressing embryos. (V) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in non-transgenic control and xWnt6-overexpressing transgenic embryos; note much smaller TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (see also panels I, K). (W) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in non-transgenic control (NT) and weak or strong xWnt6 overexpression in transgenic embryos. Note reduction of GATA4, GATA6, TroponinIc (TnIc), myosin light chain 2 (MLC2) and generally Nkx2.5 expression in embryos with xWnt6 overexpression. (X) Bar chart of quantitative RT-PCR (qPCR) analysis of heart development marker gene expression in stage 32 embryos. Note reduced expression in xWnt6-overexpressing embryos, apart from Nkx2.3 and Nkx2.5.
Mentions: The ventral Wnt6 mRNA injection experiments illustrate the effects Wnt6 overexpression has on early embryonic development (see above and Fig. 1). However, endogenous xWnt6 is not expressed at higher levels until organogenesis stages (Lavery et al., 2008), and our loss of function experiments (see above) indicate that xWnt6 is not required until these later stages of development. Wnt6 mRNA injection experiments are unsuitable for studying Wnt function at later stages since the effects Wnt6 overexpression has on early embryonic development would obscure any direct effect Wnt6 might have on later stages of development. In order to study xWnt6 functional activity during organogenesis stages, we used an inducible DNA construct in transgenic Xenopus embryos (see Materials and methods), which allows for stage-specific overexpression of xWnt6 during organogenesis stages (i.e. stage 22), when Wnt6 function was found to be required. The full-length xWnt6 was expressed under control of the Xenopus heat shock protein 70 promoter (Fig. 3A). The transgenic Wnt6-overexpressing embryos developed reduced eyes when compared to the non-transgenic control embryos (Figs. 3F, G), but also an enlarged cavity surrounding a much smaller developing heart (Figs. 3F, G, H, J, see also movies 9–11 in Supplementary data). Analysis of TroponinT-expressing heart muscle tissue shows development of a smaller heart with less myocardial tissue (Figs. 3K, V). The overall shape and morphogenetic looping of the heart is also affected (Figs. 3J, K). When transgenic Wnt6-overexpressing embryos were analyzed by whole-mount in situ hybridization at stage 32, there was a clear reduction in expression of all cardiogenic markers tested, including both genes that are usually associated with cardiac fate specification, such as Nkx2.5, GATA4 and GATA6 (Figs. 3L–Q, W), as well as later heart muscle differentiation genes, such as MLC2 and TroponinIc (Figs. 3R–U, W). Markers for other tissues were found to be unaffected or even up-regulated (e.g. the kidney tubule marker NKCC2, data not shown). The reduction of Nkx2.5, GATA4 and GATA6 expression is not confined to the prospective myocardium tissue but is more general, affecting the wider expression domain of these transcription factor genes (Figs. 3L–Q).

Bottom Line: We find, however, that Wnt6 is not required as expected during gastrulation stages, but later during organogenesis stages just before cells of the cardiogenic mesoderm begin to differentiate into heart muscle (myocardium).Our gain-of-function experiments show that Wnt6 and also activated canonical Wnt/beta-catenin signaling are capable of restricting heart muscle development at these relatively late stages of development.This repressive role of Wnt signaling is mediated initially via repression of cardiogenic transcription factors, since reinstatement of GATA function can rescue expression of other cardiogenic transcription factors and downstream cardiomyogenic differentiation genes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, Cell and Developmental Biology Research Programme, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK.

ABSTRACT
Mesodermal tissue with heart forming potential (cardiogenic mesoderm) is induced during gastrulation. This cardiogenic mesoderm later differentiates into heart muscle tissue (myocardium) and non-muscular heart tissue. Inhibition of Wnt/beta-catenin signaling is known to be required early for induction of cardiogenic mesoderm; however, the identity of the inhibiting Wnt signal itself is still elusive. We have identified Wnt6 in Xenopus as an endogenous Wnt signal, which is expressed in tissues close to and later inside the developing heart. Our loss-of-function experiments show that Wnt6 function is required in the embryo to prevent development of an abnormally large heart muscle. We find, however, that Wnt6 is not required as expected during gastrulation stages, but later during organogenesis stages just before cells of the cardiogenic mesoderm begin to differentiate into heart muscle (myocardium). Our gain-of-function experiments show that Wnt6 and also activated canonical Wnt/beta-catenin signaling are capable of restricting heart muscle development at these relatively late stages of development. This repressive role of Wnt signaling is mediated initially via repression of cardiogenic transcription factors, since reinstatement of GATA function can rescue expression of other cardiogenic transcription factors and downstream cardiomyogenic differentiation genes.

Show MeSH
Related in: MedlinePlus