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, 15 (3), 316-27

Inhibition of Wnt Activity Induces Heart Formation From Posterior Mesoderm

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Inhibition of Wnt Activity Induces Heart Formation From Posterior Mesoderm

M J Marvin et al. Genes Dev.

Abstract

In the chick, heart mesoderm is induced by signals from the anterior endoderm. Although BMP-2 is expressed in the anterior endoderm, BMP activity is necessary but not sufficient for heart formation. Previous work from our lab has suggested that one or more additional factors from anterior endoderm are required. Crescent is a Frizzled-related protein that inhibits Wnt-8c and is expressed in anterior endoderm during gastrulation. At the same stages, expression of Wnt-3a and Wnt-8c is restricted to the primitive streak and posterior lateral plate, and is absent from the anterior region where crescent is expressed. Posterior lateral plate mesoderm normally forms blood, but coculture of this tissue with anterior endoderm or infection with RCAS-crescent induces formation of beating heart muscle and represses formation of blood. Dkk-1, a Wnt inhibitor of a different protein family, similarly induces heart-specific gene expression in posterior lateral plate mesoderm. Furthermore, we have found that ectopic Wnt signals can repress heart formation from anterior mesoderm in vitro and in vivo and that forced expression of either Wnt-3a or Wnt-8c can promote development of primitive erythrocytes from the precardiac region. We conclude that inhibition of Wnt signaling promotes heart formation in the anterior lateral mesoderm, whereas active Wnt signaling in the posterior lateral mesoderm promotes blood development. We propose a model in which two orthogonal gradients, one of Wnt activity along the anterior-posterior axis and the other of BMP signals along the dorsal-ventral axis, intersect in the heart-forming region to induce cardiogenesis in a region of high BMP and low Wnt activity.

Figures

Figure 1
Figure 1
Crescent is expressed anteriorly, whereas Wnt-8c and Wnt-3a are expressed posteriorly in gastrula stage chick embryos. In situ hybridization comparing crescent (AC), Wnt-8c (D–F), and Wnt-3a (G–I) expression patterns at the indicated gastrulation stages. (C,F,I) Sections of stage 6 embryos are at the levels indicated by the red lines in B, E, and H, respectively. Crescent expression is restricted to the germinal crescent, anterior endoderm, and prechordal plate. Wnt-8c is expressed in primitive streak and migrating lateral plate mesoderm. Wnt-3a is expressed in the epiblast of the primitive streak. Arrow in F shows expression of Wnt-8c in lateral plate mesoderm.
Figure 2
Figure 2
Crescent is an efficient Wnt-8 antagonist. (A) Crescent injection into one cell of a two-cell embryo enlarged anterior structures and inhibited posterior extension in injected Xenopus embryos (bottom series of embryos). Control embryos (top) were injected with globin mRNA. LacZ mRNA was included as a lineage tracer. (B) Crescent inhibited the induction of siamois by chick Wnt-8c in Xenopus animal caps. RT–PCR analysis of siamois and ornithine decarboxylase (ODC) expression in whole embryos (WE; lane 1) or animal caps from embryos injected with the following RNAs: globin RNA (lane 2), 1 ng of crescent RNA (lane 3), 200 pg of chick Wnt-8c RNA (lane 4), 200 pg of chick Wnt-8c and 1 ng of crescent RNA (lane 5), 10 pg of mWnt-3a (lane 6), or 10 pg of mWnt-3a and 1 ng of crescent (lane 7). All injected RNA was made up to 1.2 ng with globin RNA. The difference between the levels of siamois expression in lanes 6 and 7 was approximately threefold, when normalized to ODC levels.
Figure 2
Figure 2
Crescent is an efficient Wnt-8 antagonist. (A) Crescent injection into one cell of a two-cell embryo enlarged anterior structures and inhibited posterior extension in injected Xenopus embryos (bottom series of embryos). Control embryos (top) were injected with globin mRNA. LacZ mRNA was included as a lineage tracer. (B) Crescent inhibited the induction of siamois by chick Wnt-8c in Xenopus animal caps. RT–PCR analysis of siamois and ornithine decarboxylase (ODC) expression in whole embryos (WE; lane 1) or animal caps from embryos injected with the following RNAs: globin RNA (lane 2), 1 ng of crescent RNA (lane 3), 200 pg of chick Wnt-8c RNA (lane 4), 200 pg of chick Wnt-8c and 1 ng of crescent RNA (lane 5), 10 pg of mWnt-3a (lane 6), or 10 pg of mWnt-3a and 1 ng of crescent (lane 7). All injected RNA was made up to 1.2 ng with globin RNA. The difference between the levels of siamois expression in lanes 6 and 7 was approximately threefold, when normalized to ODC levels.
Figure 3
Figure 3
Stage 5 chick posterior lateral plate and posterior primitive streak express heart markers when cocultured with quail anterior endoderm. Stage 5 chick PLP mesoderm was explanted and cultured either alone (lane 1) or in the presence of quail anterior endoderm (lane 2). Stage 5 chick PPS was explanted and cultured either alone (lane 3) or in the presence of quail anterior endoderm (lane 4). Cultures were grown for 48 h and harvested for RNA. Gene expression for GAPDH, Nkx-2.5, vMHC, and aMHC were assayed for both quail (Q) and chick (C) tissue by RT–PCR. Restriction site polymorphisms were employed to distinguish quail and chick transcripts.
Figure 4
Figure 4
Wnt antagonists can induce cardiogenesis in PLP mesoderm but not in PPS explants. (A) Stage 5 posterior lateral plate (PLP) mesoderm (lanes 1,2) or posterior primitive streak (lanes 3,4) were infected with RCAS viruses encoding either alkaline phosphatase (AP; lanes 1,3) or crescent (lanes 2,4). Gene expression for the indicated genes was assayed by RT–PCR analysis. (B) Time course of Wnt-8c and Wnt-3a expression in stage 5 PLP and PPS mesoderm explants. PLP mesoderm (lanes 1–4) or PPS (lanes 5–8) were cultured for the indicated periods of time. At the end of the culture period, explants were harvested and transcript levels evaluated by RT–PCR. (C) Posterior tissues cocultured with COS cells expressing pCS2+–β-gal, pCMV–Dkk-1 (Xenopus), or pCS2+–crescent. PLP mesoderm (lanes 1,2,5,6) or PPS (lanes 3,4,7,8) were cultured with either control COS cells expressing CS2+–β-gal (lanes 1,3,5,7), COS cells expressing pCMV2–XDkk-1 (lanes 2,4), or COS cells expressing pCS2+-crescent (lanes 6,8). Transcript levels for the indicated genes were evaluated by RT–PCR.
Figure 5
Figure 5
Overexpression of Wnt genes blocks cardiogenesis in precardiac mesoderm. (A) Whole-mount in situ hybridization for Nkx-2.5 in chick embryos in which pellets of chick embryo dermal fibroblasts infected with either RCAS–mWnt-3a or control RCAS–AP were implanted into the precardiac region of embryos in New culture at stage 3+ to 4. Pellets of RCAS–Wnt-3a infected CEFs (red arrowheads) inhibit expression of Nkx-2.5 in a stage 9 embryo whereas pellets of control RCAS–AP-infected CEFs (open arrowheads) do not. (B) Red line in A indicates the level of this section. RCAS–Wnt3a-expressing cell pellets (red dotted circle) but not control cell pellets (black dotted circle) inhibit expression of cNkx-2.5 in the precardiac mesoderm and foregut endoderm but do not inhibit the accumulation of mesoderm lateral and ventral to the neural tube. (C) Ectopic Wnt expression suppresses cardiogenesis in anterior lateral plate mesoderm explants. Stage 5 anterior lateral plate mesoderm from the precardiac region was infected with either control virus (RCAS–GFP, lane 1; RCAS–AP, lane 3), or RCAS–Wnt-3a (lane 2) or RCAS–Wnt-8c (lane 4). Cultures were carried out in the presence of 200 ng/mL BMP-4 overnight followed by 48 h in 20 ng/mL BMP-4. Transcript levels were evaluated by RT–PCR.
Figure 5
Figure 5
Overexpression of Wnt genes blocks cardiogenesis in precardiac mesoderm. (A) Whole-mount in situ hybridization for Nkx-2.5 in chick embryos in which pellets of chick embryo dermal fibroblasts infected with either RCAS–mWnt-3a or control RCAS–AP were implanted into the precardiac region of embryos in New culture at stage 3+ to 4. Pellets of RCAS–Wnt-3a infected CEFs (red arrowheads) inhibit expression of Nkx-2.5 in a stage 9 embryo whereas pellets of control RCAS–AP-infected CEFs (open arrowheads) do not. (B) Red line in A indicates the level of this section. RCAS–Wnt3a-expressing cell pellets (red dotted circle) but not control cell pellets (black dotted circle) inhibit expression of cNkx-2.5 in the precardiac mesoderm and foregut endoderm but do not inhibit the accumulation of mesoderm lateral and ventral to the neural tube. (C) Ectopic Wnt expression suppresses cardiogenesis in anterior lateral plate mesoderm explants. Stage 5 anterior lateral plate mesoderm from the precardiac region was infected with either control virus (RCAS–GFP, lane 1; RCAS–AP, lane 3), or RCAS–Wnt-3a (lane 2) or RCAS–Wnt-8c (lane 4). Cultures were carried out in the presence of 200 ng/mL BMP-4 overnight followed by 48 h in 20 ng/mL BMP-4. Transcript levels were evaluated by RT–PCR.
Figure 6
Figure 6
Model of heart and blood inducing signals in early chick mesoderm. (A) BMP-2 and BMP-4 are expressed in the posterior primitive streak and in the lateral (future ventral) regions of the embryo. Wnt inhibitors such as crescent are expressed in the anterior of the embryo, whereas Wnt-3a and Wnt-8c are expressed in the posterior primitive streak and posterior lateral mesoderm. Nkx-2.5 expression appears at stage 5–6 in the region of the embryo where Wnt signals are blocked by crescent (and presumably other Wnt antagonists) and where BMP-2/BMP-4 are expressed. (B) Lateral plate mesoderm precursor cells are induced to become heart tissue by BMP signals that are transduced in the absence of Wnt-3a/Wnt-8c signaling. Conversely, lateral plate mesoderm precursors develop into primitive erythrocytes in the simultaneous presence of both BMP and Wnt-3a/Wnt-8c signals.

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