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Fig. 2. 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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Fig. 5. Wnt signaling agonist treatment during organogenesis stages reduces heart marker gene expression, even in Wnt6 morphants. (A, B) Morphology of stage 38 embryos after control treatment with MeBIO (A) or treatment with the Wnt/β-catenin signaling agonist BIO at 12 μM, from embryonic stage 20 to control stage 32. Note multiple morphological defects in BIO-treated embryos in addition to absence of any discernable heart; including enlarged, elongated oval-shaped eyes, which extend toward the anterior; slightly reduced cement glands; reduced pigmentation; a shortened tail; and skin defects. (C–J) Analysis of marker gene expression at stage 32 with whole-mount RNA in situ hybridization in control embryos treated with 10 μM MBIO (C, E, G, I) or experimental embryos treated with 10 μM BIO (D, F, H, J) from stage 20 to 32. (K) Percentage bar chart of whole-mount RNA in situ hybridization analysis of marker gene expression in Wnt agonist-treated (BIO) and control-treated embryos (MBIO). Note that whole-mount RNA in situ hybridization analysis (C–K) shows that all of the marker genes associated with heart development are down-regulated to a various degree by treatment with BIO. (L) Bar chart of quantitative RT-PCR (qPCR) analysis of gene expression of heart development (GATA4, GATA6, Nkx2.3 and Nkx2.5) and heart muscle differentiation markers (TroponinIc and MLC2) in Wnt agonist- and control-treated embryos at stage 32. Note that the heart muscle differentiation markers TroponinIc and MLC2 are most affected along with Nkx2.5 at this stage of development. (M) Bar chart of quantitative RT-PCR (qPCR) analysis of marker gene expression at stage 32 in embryos injected with Wnt6 MO3 and subsequently treated with the Wnt/β-catenin signaling agonist BIO or MeBIO (10 μM from stage 20). Control embryos were injected with control MO and treated with MeBIO. Note that BIO-mediated activation of Wnt/β-catenin signaling during organogenesis stages rescues to a varying extent the deregulated expression of heart development and heart muscle marker genes in Wnt6 morphants. |
