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Figure 2. Depletion of either mef2c or mef2d leads to severe cardiac defects.A, C. Mef2c- or Mef2d MO-injected embryos developed cardiac edema and morphological heart defects at stage 42, as compared with Control MO-injected sibling embryos. The amount of MO injected in total (two blastomeres) is given in ng. In addition, cardiac beating was reduced upon Mef2c or Mef2d MO depletion. The dotted dark gray lines indicate the heart. a: atria; v: ventricle; oft: outflow tract. B, D. Quantitative presentations are shown. N: number of examined embryos; n: number of independent experiments.
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Figure 3. Loss of Mef2c or Mef2d affects the cardiac progenitor cell population.Mef2c or Mef2d MO (10 ng) was unilaterally injected and expression of cardiac marker genes was monitored at stage 20. A.
tbx20 expression was down-regulated in Mef2c MO- or Mef2d MO-injected embryos (arrowheads). In addition, Mef2d- but not Mef2c-depleted embryos showed reduced tbx1 expression (arrowhead). Expression of isl1 as well as bmp4 remained unchanged upon loss of Mef2c or Mef2d. Anterior views of embryos are shown. B. Quantitative presentation of observed phenotypes in A is given. N: number of examined embryos; n: number of independent experiments; *, pâ¤0.05.
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Figure 4. Cardiac differentiation is affected upon loss of Mef2c or Mef2d.A. At stage 28, depletion of Mef2c or Mef2d led to a reduced expression of cardiac markers including isl1, bmp4, nkx2.5, tbx1, tbx20, gata6b, myh6, actc1, and tnni3 (arrowheads). Ventral views of embryos are shown. B. Quantitative presentation of the observed phenotype in A is given. Nâ=ânumber of examined embryos; nâ=ânumber of independent experiments; *, pâ¤0.05; **, pâ¤0.01.
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Figure 5. Specificity of phenotype observed upon knock down of Mef2c.A.
mef2cγ- and mef2cγ+ isoforms are expressed on RNA level in heart tissue enriched explants at stages 24, 28, and 32 as revealed by qPCR. Expression is shown relative to gapdh. B. mMef2cγ- and mMef2cγ+ are expressed on protein level upon RNA injection into Xenopus embryo at comparable levels. β-Tubulin served as loading control. Note that the Mef2c antibody used does not recognize endogenous Xenopus Mef2c protein. The asterisk indicates unspecific background. CâF. Mef2c MO was unilaterally injected together with GFP, mMef2cγ+ or mMef2γ- RNA as indicated. C, E Expression of the cardiac marker genes myh6 and tnni3 was monitored at stage 20 or stage 28. Black arrowheads indicate reduced marker gene expression, white arrowheads highlight the rescued situation. Ventral views of embryos are shown. D, F. Quantitative presentations are shown. N: number of examined embryos; n: number of independent experiments; st: stage; **, pâ¤0.01.
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Figure 6. Specificity of phenotype observed upon knock down of Mef2d.A.
mef2d isexpressed on RNA level in heart tissue enriched explants at stages 24, 28, and 32 as revealed by qPCR. Expression is shown relative to gapdh. B. hMEF2D is expressed on protein level upon RNA injection into Xenopus embryo. β-tubulin served as loading control. Note the Mef2d antibody used does not recognize endogenous Xenopus Mef2d protein. C. Mef2d MO was unilaterally injected together with GFP or hMEF2D as indicated. Expression of cardiac marker genes myh6 and tnni3 was monitored at 28. Black arrowheads indicate reduced marker gene expression, white arrowheads highlight the rescued situation. D. A quantitative presentation of results is given. N: number of examined embryos; n: number of independent experiments; **, pâ¤0.01.
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Figure 7. Cooperation of Mef2c and Mef2d in Xenopus.A, B. The injection of 7(in both cases 3.5 ng per blastomere) did not result in cardiac defects. The co-injection of 7 ng Mef2c together with 7 ng Mef2d MO led to a significant increase of the phenotype. The dotted black lines indicate the heart. C, D. Mef2c MO was unilaterally injected along with RNA coding for hMEF2D. Expression of cardiac marker genes was monitored at stages 20 (tbx20, bmp4; anterior views) or stage 28 (myh6, tnni3; ventral views), respectively. E, F. Mef2d MO was unilaterally injected along with mMef2cγ+ RNA. Expression of cardiac marker genes was monitored at stages 20 (tbx1, tbx20; anterior views) or stage 28 (bmp4, myh6, tnni3; ventral views), respectively. G, H. Mef2d MO was unilaterally injected along with mMef2cγ Ë RNA. Expression of cardiac marker genes was monitored at stage 28 (ventral view). In all cases, black arrowheads indicate reduced marker gene expression, white arrowheads highlight the rescued expression. B, D, F, H. Quantitative presentations of the experiments shown in C, E, G are shown. N: number of examined embryos; n: number of independent experiments; *, pâ¤0.05; **, pâ¤0.01.
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Figure 8. Gain of function analyzes reveals an earlier onset of cardiac differentiation.A, C. RNA coding for mMef2cγ+, mMef2cγ- or hMEF2D was injected unilaterally into the dorsal-vegetal blastomere at eight cell stage and tnni3 expression was monitored at stages 24 (A) or 28 (C). Ventral views of embryos are shown. B, D. The percentage of embryos with enhanced expression of tnni3 on the injected side is given. N: number of examined embryos; n: number of independent experiments; **, pâ¤0.01.
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Figure 1. Synteny analyses of Mef2a, b, c, and d.
A. Synteny analysis of mef2a. Schematic overview comparing the mef2a gene in Homo sapiens (chromosome 15), Mus musculus (chromosome 7) and Xenopus tropicalis (scaffold_3, Xenbase G-Browse). B. Synteny analysis of mef2b. Schematic overview comparing the mef2b gene in Homo sapiens (chromosome 19), Mus musculus (chromosome 8) and the mef2b neighboring genes in Xenopus tropicalis (scaffold_1, Xenbase G-Browse). C. Synteny analysis of mef2c. Schematic overview comparing the mef2c gene in Homo sapiens (chromosome 5), Mus musculus (chromosome 13) and Xenopus tropicalis (scaffold_1, Xenbase G-Browse). D. Synteny analysis of mef2d. Schematic overview comparing the mef2d gene in Homo sapiens (chromosome 1), Mus musculus (chromosome 3) and Xenopus tropicalis (scaffold_27, Xenbase G-Browse). In all panels conserved genes are indicated by a color code. The orientation of the open reading frames of some genes is depicted by arrowheads. Gene length or distances between genes are not drawn to scale. A list of gene abbreviations used here is given in Table S1.
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