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Fig. 4. Overexpression of β-catenin during organogenesis stages inhibits heart muscle development. (A) Schematic representation of transgene for concomitant overexpression of a stabilized form of β-catenin and GFP in transgenic Xenopus embryos. (B–E) Identification of non-transgenic control (B,D) versus transgenic embryos (C, E) induced at stage 22 by heat shock treatment to overexpress concomitantly GFP and β-catenin, viewed at stage 28 under UV light (D, E, compare with the same embryos viewed under visible light in B and C, respectively). Note strong fluorescence due to GFP expression in transgenic embryo (E). (F, G) External morphology and TroponinIc expression of non-transgenic control (F) and β-catenin-overexpressing transgenic (G) embryo at stage 36. Note abnormal morphology in β-catenin-overexpressing embryo, particularly the shortened axis and tail, as well as, much reduced TroponinIc expression. (H–Q) Analysis of heart marker gene expression with whole-mount RNA in situ hybridization in non-transgenic control (H, J, L, N) and β-catenin-overexpressing transgenic embryos (I, K, M, O) at stage 32. Note much reduced and restricted GATA4 expression (I), absence of detectable Nkx2.5 (K) and TroponinIc (TnIc) (M) expression and restricted domain of MLC2 expression (O) in β-catenin-overexpressing embryos. (P) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in non-transgenic (NT) control embryos, in transgenic embryos with weak GFP and therefore presumably weak β-catenin expression (weak expr.) and in transgenic embryos with strong GFP and therefore presumably strong β-catenin expression (strong expr.). Note reduction of GATA4, Nkx2.5, TnIc and MLC2 expression in embryos overexpressing β-catenin. (Q) Bar chart of qPCR analysis of heart marker gene expression in stage 32 embryos. Note reduced cardiogenic gene expression in β-catenin-overexpressing transgenic embryos. |
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Fig. 6. Stage-specific activation of GATA4 during organogenesis stages rescues reduction of cardiomyogenic genes caused by activation of Wnt/β-catenin signaling. (A–X) Analysis of Nkx2.5 (A–H), MLC2 (I–P) and TroponinIc (Q–X) expression with whole-mount RNA in situ hybridization at stage 32 in uninjected (A–D, I–L, Q–T) and GATA4GR-injected (E–H, M–P, U–X) embryos treated from stage 20 with DMSO (A, E, I, M, Q, U); Dexamethasone alone (B, F, J, N, R, V); BIO alone (C, G, K, O, S, W); and BIO and Dexamethasone together (D, H, L, P, T, X). Note in embryos with activated GATA4GR increased Nkx2.5 (F), MLC2 (N) and TroponinIc expression (V); in embryos with BIO-mediated activated Wnt/β-catenin signaling clearly reduced Nkx2.5 (C, D, G), MLC2 (K, L, O) and TroponinIc expression (S, T, W); but note restored Nkx2.5 (H) MLC2 (P) and TroponinIc (X) expression in embryos where BIO-mediated activated Wnt/β-catenin signaling is combined with activated GATA4GR. (Y-AA) Bar charts showing the percentage of embryos with high, normal, or low Nkx2.5 (Y), MLC2 (Z) and TroponinIc (AA) expression in the experiments illustrated in panels A–X (n = number of embryos assayed for each treatment). |
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Fig. 7. Stage-specific activation of GATA6 during organogenesis stages rescues reduction of cardiomyogenic genes caused by activation of Wnt/β-catenin signaling. (A–X) Analysis of Nkx2.5 (A–H), MLC2 (I–P) and TroponinIc (Q–X) expression with whole-mount RNA in situ hybridization at stage 32 in uninjected (A–D, I–L, Q–T) and GATA6GR-injected (E–H, M–P, U–X) embryos treated from stage 20 with DMSO (A, E, I, M, Q, U); Dexamethasone alone (B, F, J, N, R, V); BIO alone (C, G, K, O, S, W); and BIO and Dexamethasone together (D, H, L, P, T, X). Note in embryos with activated GATA6GR only slightly increased Nkx2.5 (F), MLC2 (N) and TroponinIc expression (V); in embryos with BIO-mediated activated Wnt/β-catenin signaling clearly reduced Nkx2.5 (C, D, G), MLC2 (K, L, O) and TroponinIc expression (S, T, W); but note restored Nkx2.5 (H) MLC2 (P) and TroponinIc (X) expression in embryos where BIO-mediated activated Wnt/β-catenin signaling is combined with activated GATA6GR. (Y-AA) Bar charts showing the percentage of embryos with high, normal, or low Nkx2.5 (Y), MLC2 (Z) and TroponinIc (AA) expression in the experiments illustrated in panels A–X (n = number of embryos assayed for each treatment). |
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Fig. 3. Overexpression of Wnt6 during organogenesis stages inhibits heart muscle development. (A) Schematic representation of transgene for concomitant overexpression of xWnt6 and GFP in transgenic Xenopus embryos. (B-E) Identification of a non-transgenic control embryo (B, D) versus a transgenic embryo (C, E) induced at stage 22 by heat shock treatment to overexpress concomitantly GFP and xWnt6 viewed at stage 28 under UV light (D, E; compare with the same embryos viewed under visible light in panels B and C, respectively). Note only faint background fluorescence (mainly from yolk) in non-transgenic embryo (D), but strong fluorescence due to GFP expression in transgenic embryo (E). External morphology of whole embryos (F, G) and of the heart forming regions of the same embryos (H, J) at stage 40; TroponinT immunohistochemisty analysis of sections through the heart forming region at stage 42 (I, K); and analysis of marker gene expression with whole-mount RNA in situ hybridization at stage 32 (L–U) in non-transgenic control (F, H, I, L, N, P, R, T) and xWnt6-overexpressing transgenic embryos (G, J, K, M, O, Q, S, U). Note abnormal morphology in xWnt6-overexpressing embryos, particularly in the eye (G) and the heart-forming region (G, J). Note much reduced TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (panel K, see also panel V). Note significantly reduced and restricted GATA4 (M) and GATA6 (O) expression, dramatic reduction of Nkx2.5 expression (panel Q, but see panel W and panel X) and restricted domains of TroponinIc (TnIc) (S) and Myosin Light Chain 2 (MLC2) (U) expression in xWnt6-overexpressing embryos. (V) Percentage bar chart of TroponinT immunohistochemistry analysis of relative size of myocardial tissue in non-transgenic control and xWnt6-overexpressing transgenic embryos; note much smaller TroponinT-expressing myocardial tissue in xWnt6-overexpressing embryos (see also panels I, K). (W) Percentage bar chart of whole-mount RNA in situ hybridization analysis of heart marker expression in non-transgenic control (NT) and weak or strong xWnt6 overexpression in transgenic embryos. Note reduction of GATA4, GATA6, TroponinIc (TnIc), myosin light chain 2 (MLC2) and generally Nkx2.5 expression in embryos with xWnt6 overexpression. (X) Bar chart of quantitative RT-PCR (qPCR) analysis of heart development marker gene expression in stage 32 embryos. Note reduced expression in xWnt6-overexpressing embryos, apart from Nkx2.3 and Nkx2.5. |
