|
Fig. 1. Wnt/β-catenin-mediated inhibition of cardiogenesis in Xenopus is via GATA4/6 function. Gene expression analysis at stage 32 by RT-PCR of animal cap explants, injected with RNA encoding Activin (Act, 50 fg) to induce cardiogenesis (A), dorsal marginal zone (DMZ) explants (B) and whole embryos (C), which were injected with β-cateninGR (βGR, 50 pg), GATA4GR (G4GR, 200 pg) or GATA6GR (G6GR, 200 pg), as indicated, and were cultured with dexamethasone from stages 8, 10 or 18 where indicated. Activation of β-catenin abolishes activin-induced (A) and endogenous (B) expression of GATA4 and GATA6, as well as cardiogenesis, as monitored by marker gene expression. (C) In whole embryo analysis, activation of β-catenin abolishes heart muscle-specific differentiation markers (MLC2, TnIc), but causes only a relatively weak reduction of markers that are not exclusively heart specific (GATA4, GATA6, Wnt11, Nkx2-5). However, β-catenin-mediated inhibition of cardiogenic marker gene expression is rescued by concomitant activation of GATA4GR or GATA6GR in all three assays. N/D, not determined; VMZ, ventral marginal zone explant.
|
|
Fig. 2. GATA4 and GATA6 are required for Wnt11 expression and heart muscle differentiation in Xenopus. RT-PCR analysis of Nkx2-5, Wnt11, MLC2 and TnIc gene expression in embryonic explants and whole embryos at control stages 24 and 32, as indicated. (A) Animal cap explants injected with RNA encoding Activin (Act, 50 fg) to induce cardiogenesis and in combination with morpholinos targeting GATA4 or GATA6 (G4MO, G6MO), as indicated. (B) Ventral marginal zone (VMZ) explants injected with Dickkopf-1 mRNA (Dkk-1, 800 pg) to induce ectopic cardiogenesis and together with G4MO, or G6MO as indicated. (C) Analysis of gene expression in whole embryos injected with G4MO or G6MO as indicated. Note that morpholino-mediated inhibition of either GATA4 or GATA6 reduces Wnt11 and cardiogenic marker gene expression, but that inhibition of both GATA4 and 6 abolishes them in all three assays.
|
|
Fig. 3. Wnt11 and Nkx2-5, but not MHC are direct targets of regulation by GATA4 and GATA6. RT-PCR analysis of Nkx2-5, Wnt11 and MHC gene expression in Xenopus animal cap explants that were injected with GATA4GR or GATA6GR RNA and cultured in the presence or absence of cycloheximide (CHX) and dexamethasone (Dex), as indicated. In A, 100 pg or 200 pg GATA4GR or GATA6GR mRNA were injected, as indicated; in B, 100 pg. (A) CHX followed by Dex treatment from stage 8 and analysis of gene expression at control stage 11. (B) CHX followed by Dex treatment from control stage 18 and analysis at stage 20. Activated GATA transcription factors induce Nkx2-5 and Wnt11, but not MHC gene expression in the presence of the protein synthesis inhibitor (CHX) at different stages, thereby identifying them as direct target genes of GATA transcription factors.
|
|
Fig. 4. Wnt11 is required for induction of cardiomyogenesis by GATA4 and GATA6. RT-PCR analysis of cardiomyogenic gene expression (MLC2, TnIc) at control stage 32 (A,C,E) and analysis of cardiomyocyte differentiation (detectable rhythmic beating) at control stage 45 (B,D,F). (A) Xenopus animal cap explants that were injected with GATA4GR or GATA6GR mRNA (G4GR, G6GR, 100 pg), alone or in combination with Wnt11 morpholino (11MO) as indicated, and cultured in the absence or in the presence of dexamethasone from stage 8. Induction of cardiomyogenic gene expression by G4GR and G6GR is abolished by inhibition of Wnt11. (B) Numerical analysis of spontaneous rhythmic beating in animal cap explants injected with G4GR or G6GR mRNA (500 pg or 1000 pg and activated with dexamethasone at stage 8, as indicated). Only G4GR mRNA is capable of inducing rhythmic beating in animal cap explants. (C) Analysis of cardiomyogenic gene expression in sibling animal cap explants of those used for cardiomyocyte differentiation in D, injected with G4GR (500 pg, activated at stage 8) with 11MO as indicated. (D) Numerical analysis of spontaneous rhythmic beating in sibling animal cap explants. Under these conditions (G4GR mRNA, 500 pg), inhibition of Wnt11 reduces, but does not abolish, cardiomyogenic gene expression, consistent with reduced cardiomyocyte differentiation in explants. (E) Cardiomyogenic gene expression in sibling dorsal marginal zone explants (DMZ) or in sibling whole embryos (W/E) of those used for phenotypic analysis in F, which were injected with 11MO as indicated. (F) Numerical analysis of rhythmic beating in DMZ explants and whole embryos, as in E. Morpholino-mediated inhibition of Wnt11 causes strong but not complete reduction of cardiomyogenic gene expression, consistent with the decreased cardiomyogenesis in both DMZ explants and whole embryos. In B,D, the results of three different experiments are combined but in F only the result of one experiment is presented with n representing the number of explants or embryos scored. VMZ, ventral marginal zone.
|
