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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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xWnt6 is required for regulating heart muscle development. Morphology of the head and heart forming region at stage 42 (A, B), TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (C, D) and whole-mount RNA in situ hybridization analysis of marker gene expression at stage 32 (E–N) or at stage 40 (O, P) in embryos that were injected into the marginal zone of each blastomere at the two-cell stage with 20 ng of either the Control MO (A, C, E, G, I, K, M, O) or Wnt6MO3 (B, D, F, H, J, L, N, P). Note enlarged tissue mass in Wnt6 morphants in the heart forming region (panel B, compare to panel A) and more TroponinT-expressing myocardial tissue (panel D, compare to panel C, scale bar indicates 100 μm, see also panel Q). Also note slightly stronger and extended expression of GATA4 (panel F, compare to panel E) and GATA6 (panel H, compare to panel G), and much stronger and extended expression of Nkx2.3 (panel J, compare to panel I), Nkx2.5 (panel L, compare to panel K), TnIc (panel N, compare to panel M) and MLC2 (panel P, compare to panel O) (see also panel R). (Q) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in Wnt6 MO- and control MO-injected embryos at stage 42 (see also panels C, D). Note larger TroponinT-expressing tissue in Wnt6 MO-injected embryos. (R) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in xWnt6 MO3- and Control MO-injected embryos at stage 32 (see also panels E–P). Note slightly increased expression of GATA4, and GATA6; and strongly increased expression of Nkx2.3, Nkx2.5, TroponinIc (TnIc) and myosin light chain 2 (MLC2) in xWnt6 MO3-injected embryos compared to controls. (S) Bar chart of qPCR analysis of cardiogenic transcription factor gene expression and heart muscle differentiation markers in xWnt6 MO3- and Control MO-injected embryos at stage 20 and stage 32. Note increased expression of GATA4 in particular at stage 20 and increased expression of heart muscle differentiation markers (MLC2, TnIc) in particular at stage 32 of development.
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fig2: xWnt6 is required for regulating heart muscle development. Morphology of the head and heart forming region at stage 42 (A, B), TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (C, D) and whole-mount RNA in situ hybridization analysis of marker gene expression at stage 32 (E–N) or at stage 40 (O, P) in embryos that were injected into the marginal zone of each blastomere at the two-cell stage with 20 ng of either the Control MO (A, C, E, G, I, K, M, O) or Wnt6MO3 (B, D, F, H, J, L, N, P). Note enlarged tissue mass in Wnt6 morphants in the heart forming region (panel B, compare to panel A) and more TroponinT-expressing myocardial tissue (panel D, compare to panel C, scale bar indicates 100 μm, see also panel Q). Also note slightly stronger and extended expression of GATA4 (panel F, compare to panel E) and GATA6 (panel H, compare to panel G), and much stronger and extended expression of Nkx2.3 (panel J, compare to panel I), Nkx2.5 (panel L, compare to panel K), TnIc (panel N, compare to panel M) and MLC2 (panel P, compare to panel O) (see also panel R). (Q) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in Wnt6 MO- and control MO-injected embryos at stage 42 (see also panels C, D). Note larger TroponinT-expressing tissue in Wnt6 MO-injected embryos. (R) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in xWnt6 MO3- and Control MO-injected embryos at stage 32 (see also panels E–P). Note slightly increased expression of GATA4, and GATA6; and strongly increased expression of Nkx2.3, Nkx2.5, TroponinIc (TnIc) and myosin light chain 2 (MLC2) in xWnt6 MO3-injected embryos compared to controls. (S) Bar chart of qPCR analysis of cardiogenic transcription factor gene expression and heart muscle differentiation markers in xWnt6 MO3- and Control MO-injected embryos at stage 20 and stage 32. Note increased expression of GATA4 in particular at stage 20 and increased expression of heart muscle differentiation markers (MLC2, TnIc) in particular at stage 32 of development.

Mentions: In order to understand what function xWnt6 may normally play in development; we performed loss of function studies. A knockdown strategy was developed involving anti-sense morpholinos (MO) (see Materials and methods). Three different MOs were tested to examine which would be the most effective and specific. Using an in vitro transcription/translation assay, we demonstrate that all three MOs inhibit xWnt6 translation, with Wnt6MO2 and Wnt6MO3 being the most effective (Fig. 1E). We next tested Wnt6MO2 and MO3 in vivo and found that they both inhibited translation of endogenous xWnt6 as detected by Western blot using a Wnt6 antibody raised against chick Wnt6 (Fig. 1F, for details see Material and methods). Injection of Wnt6MO2 or Wnt6MO3 into Xenopus embryos induced almost identical phenotypes (Figs. 2H, I). Both Wnt6 morpholinos cause morphological defects in the heart forming region (see below), reduced (or in some cases missing) eyes, enlarged fluid filled fins; many suffered from edema; and some had a bent and shortened body axis, as well as defects in gut looping and cloaca formation. This is not seen in embryos injected with Control MO (Fig. 2G). Additionally, we noticed that Wnt6 MO2- or Wnt6 MO3-injected embryos do not swim or respond to physical stimuli (see Movies 1–3 in Supplementary data).


Wnt6 signaling regulates heart muscle development during organogenesis.

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

xWnt6 is required for regulating heart muscle development. Morphology of the head and heart forming region at stage 42 (A, B), TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (C, D) and whole-mount RNA in situ hybridization analysis of marker gene expression at stage 32 (E–N) or at stage 40 (O, P) in embryos that were injected into the marginal zone of each blastomere at the two-cell stage with 20 ng of either the Control MO (A, C, E, G, I, K, M, O) or Wnt6MO3 (B, D, F, H, J, L, N, P). Note enlarged tissue mass in Wnt6 morphants in the heart forming region (panel B, compare to panel A) and more TroponinT-expressing myocardial tissue (panel D, compare to panel C, scale bar indicates 100 μm, see also panel Q). Also note slightly stronger and extended expression of GATA4 (panel F, compare to panel E) and GATA6 (panel H, compare to panel G), and much stronger and extended expression of Nkx2.3 (panel J, compare to panel I), Nkx2.5 (panel L, compare to panel K), TnIc (panel N, compare to panel M) and MLC2 (panel P, compare to panel O) (see also panel R). (Q) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in Wnt6 MO- and control MO-injected embryos at stage 42 (see also panels C, D). Note larger TroponinT-expressing tissue in Wnt6 MO-injected embryos. (R) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in xWnt6 MO3- and Control MO-injected embryos at stage 32 (see also panels E–P). Note slightly increased expression of GATA4, and GATA6; and strongly increased expression of Nkx2.3, Nkx2.5, TroponinIc (TnIc) and myosin light chain 2 (MLC2) in xWnt6 MO3-injected embryos compared to controls. (S) Bar chart of qPCR analysis of cardiogenic transcription factor gene expression and heart muscle differentiation markers in xWnt6 MO3- and Control MO-injected embryos at stage 20 and stage 32. Note increased expression of GATA4 in particular at stage 20 and increased expression of heart muscle differentiation markers (MLC2, TnIc) in particular at stage 32 of development.
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fig2: xWnt6 is required for regulating heart muscle development. Morphology of the head and heart forming region at stage 42 (A, B), TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (C, D) and whole-mount RNA in situ hybridization analysis of marker gene expression at stage 32 (E–N) or at stage 40 (O, P) in embryos that were injected into the marginal zone of each blastomere at the two-cell stage with 20 ng of either the Control MO (A, C, E, G, I, K, M, O) or Wnt6MO3 (B, D, F, H, J, L, N, P). Note enlarged tissue mass in Wnt6 morphants in the heart forming region (panel B, compare to panel A) and more TroponinT-expressing myocardial tissue (panel D, compare to panel C, scale bar indicates 100 μm, see also panel Q). Also note slightly stronger and extended expression of GATA4 (panel F, compare to panel E) and GATA6 (panel H, compare to panel G), and much stronger and extended expression of Nkx2.3 (panel J, compare to panel I), Nkx2.5 (panel L, compare to panel K), TnIc (panel N, compare to panel M) and MLC2 (panel P, compare to panel O) (see also panel R). (Q) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in Wnt6 MO- and control MO-injected embryos at stage 42 (see also panels C, D). Note larger TroponinT-expressing tissue in Wnt6 MO-injected embryos. (R) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in xWnt6 MO3- and Control MO-injected embryos at stage 32 (see also panels E–P). Note slightly increased expression of GATA4, and GATA6; and strongly increased expression of Nkx2.3, Nkx2.5, TroponinIc (TnIc) and myosin light chain 2 (MLC2) in xWnt6 MO3-injected embryos compared to controls. (S) Bar chart of qPCR analysis of cardiogenic transcription factor gene expression and heart muscle differentiation markers in xWnt6 MO3- and Control MO-injected embryos at stage 20 and stage 32. Note increased expression of GATA4 in particular at stage 20 and increased expression of heart muscle differentiation markers (MLC2, TnIc) in particular at stage 32 of development.
Mentions: In order to understand what function xWnt6 may normally play in development; we performed loss of function studies. A knockdown strategy was developed involving anti-sense morpholinos (MO) (see Materials and methods). Three different MOs were tested to examine which would be the most effective and specific. Using an in vitro transcription/translation assay, we demonstrate that all three MOs inhibit xWnt6 translation, with Wnt6MO2 and Wnt6MO3 being the most effective (Fig. 1E). We next tested Wnt6MO2 and MO3 in vivo and found that they both inhibited translation of endogenous xWnt6 as detected by Western blot using a Wnt6 antibody raised against chick Wnt6 (Fig. 1F, for details see Material and methods). Injection of Wnt6MO2 or Wnt6MO3 into Xenopus embryos induced almost identical phenotypes (Figs. 2H, I). Both Wnt6 morpholinos cause morphological defects in the heart forming region (see below), reduced (or in some cases missing) eyes, enlarged fluid filled fins; many suffered from edema; and some had a bent and shortened body axis, as well as defects in gut looping and cloaca formation. This is not seen in embryos injected with Control MO (Fig. 2G). Additionally, we noticed that Wnt6 MO2- or Wnt6 MO3-injected embryos do not swim or respond to physical stimuli (see Movies 1–3 in Supplementary data).

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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