Schneider VA and Mercola M (2001), Wnt antagonism initiates cardiogenesis in Xenop...

XB-ART-9675

Genes Dev 2001 Feb 01;153:304-15. doi: 10.1101/gad.855601.

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Wnt antagonism initiates cardiogenesis in Xenopus laevis.

Schneider VA , Mercola M .

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Heart induction in Xenopus occurs in paired regions of the dorsoanterior mesoderm in response to signals from the Spemann organizer and underlying dorsoanterior endoderm. These tissues together are sufficient to induce heart formation in noncardiogenic ventral marginal zone mesoderm. Similarly, in avians the underlying definitive endoderm induces cardiogenesis in precardiac mesoderm. Heart-inducing factors in amphibians are not known, and although certain BMPs and FGFs can mimic aspects of cardiogenesis in avians, neither can induce the full range of activities elicited by the inducing tissues. Here we report that the Wnt antagonists Dkk-1 and Crescent can induce heart formation in explants of ventral marginal zone mesoderm. Other Wnt antagonists, including the frizzled domain-containing proteins Frzb and Szl, lacked this activity. Unlike Wnt antagonism, inhibition of BMP signaling did not promote cardiogenesis. Ectopic expression of GSK3beta, which inhibits beta-catenin-mediated Wnt signaling, also induced cardiogenesis in ventral mesoderm. Analysis of Wnt proteins expressed during gastrulation revealed that Wnt3A and Wnt8, but not Wnt5A or Wnt11, inhibited endogenous heart induction. These results indicate that diffusion of Dkk-1 and Crescent from the organizer initiate cardiogenesis in adjacent mesoderm by establishing a zone of low Wnt3A and Wnt8 activity.

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Species referenced: Xenopus laevis
Genes referenced: actl6a chrd dkk1 frzb frzb2 gsk3b myh4 myh6 nog sia1 szl tbx5 tnni3 wnt11 wnt3a wnt5a wnt8a

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	Figure 1. dkk-1 and crescent, but not frzb, induce cardiac specific gene expression in noncardiogenic tissue. (A) mRNAs encoding various Wnt and BMP antagonists were injected equatorially into ven- tral blastomeres at the four-cell stage. Ventral marginal zone (VMZ) tissue was then explanted at stage 10 and cultured until analyzed by RTCR for gene expres- sion at stage 30 (see Materials and Meth- ods). (B) Injection of dkk-1 or crescent induced both markers of cardiac mesoderm (Tbx5 and Nkx2.5) and heart musclepecific genes (cardiac isoform of troponin-I, TnIc, and myosin heavy chain-alpha, MHC-alpha) in VMZ tissue. frzb, in contrast, induced muscle actin (m. actin), which is primarily a skeletal muscle marker, but not cardiac specific gene expression. Induced genes were expressed at levels comparable to endogenous expression in control dorsal marginal zone (DMZ) explants. (C) TnIc transcripts induced by injection of 1.5 ng of dkk-1 or crescent mRNAs were highly localized, similar to endogenous expression (cf. with control DMZ shown in Figs. 3Cﲒ and 5G), whereas injection of frzb mRNA does not induce TnIc. (F,G) dkk-1, crescent, and frzb block Wnt8 induction of Siamois in animal cap tissue. Wnt8 and Wnt antagonist mRNAs were injected into the animal region of two-cell-stage embryos and caps were isolated at stage 9, cultured, and processed for RTCR at stage 10.5 (F). Antagonism of Wnt8 signaling indicates that functional protein is translated from the injected mRNAs in each case (G). EF1-alpha expression is shown as a control for the RT reaction in all cases.
	Figure 2. Injection of the Wnt antagonists dkk-1 and crescent resulted in the formation of beating hearts in VMZ tissue. Embryos were injected ventrally with 900pg dkk-1, 1.5 ng crescent, or 1.5 ng frzb mRNA at the four-cell stage, and VMZ explants isolated and cultured as above. (A) The explants were scored for rhythmic beating when sibling controls reached stage 41. Uninjected VMZ and DMZ explants were analyzed as negative and positive controls, respectively. (B) Control DMZ explants formed an embryoid-like structure having a well-developed anteroposterior body axis (B). The heart tube contained a myocardial layer that stained with CT-3, which recognizes the cardiac isoform of troponin-T (C), lined by a thin layer of CT-3 negative endothelial cells visualized with DAPI (arrow in D). (E) dkk-1 injected VMZ explants formed simple structures resembling a small epithelial sac encapsulating a CT-3 positive myocardial tube (F) also lined by endothelial cells (G). (H) crescent-in- jected VMZs formed similar structures. Pigmented melanocytes were seen scattered on the surface of the dkk-1- and crescent- injected VMZ explants, and cement gland tissue was often ob- served (cluster of pigmented cells on surface of tissue in E). Line represents 25 μm.
	Figure 3.Induction of cardiogenesis in the VMZ assay is specific to certain Wnt antagonists. (A) The BMP antagonists Noggin and Chordin did not induce specific markers of cardiogenesis (TnIc or MHC-alpha) despite induction of m. actin and elongation of the explants (not shown). Noggin did not induce Tbx5, Nkx2.5, or Nkx2.10, whereas chordin weakly induced these genes. Note that Tbx5 and Nkx2.5 are expressed in tissues other than cardiac mesoderm and that induction of these genes (in the absence of other markers) does not necessarily indicate heart field specification (see text). (B) Wnt antagonists not normally present in gastrula-stage embryos induced weak expression of Tbx5, Nkx2.5, and Nkx2.10 but did not induce the more specific cardiac markers TnIc or MHC-alpha. In situ hybridization for expression of Nkx2.5 (C) and TnIc (C'') indicated that only WIF-1 induced detectable levels of Nkx2.5 expression (arrow in H; 4 of 24 explants showed expression) and that none of these mRNAs induced TnIc. Arrowheads in F and F' show pigmented cement glands that formed in explants injected with chordin mRNA
	Figure 5. Overexpression of Wnt3A and Wnt8, but not Wnt5A and Wnt11, blocks endogenous expression of Nkx2.5 and TnIc in DMZ tissue. (A) Expression was targeted to the heart-forming region by injection of a plasmid encoding Wnt cDNA into dorsal blastomeres at the four-cell stage. DMZ explants were dissected at stage 10 and analyzed when sibling controls reached stage 23 (Nkx2.5) or stage 30 (TnIc). (B) Percentage of explants expressing Nkx2.5 and TnIc as determined by in situ hybridization. (C) Examples of TnIc in situ hybridization patterns in DMZ explants overexpressing Wnt cDNAs. Note that nearly all control DMZ explants expressed both markers (G), as did DMZ explants overexpressing Wnt5A and Wnt11 (E,F). In contrast, Wnt3A and Wnt8 reduced the incidence of Nkx2.5 and TnIc expression (B). Whereas Nkx2.5 expression was lost entirely in affected explants, TnIc expression was either absent (C,D) or greatly reduced in area (C',D')

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