Zukunftssicherung durch Bewegung und Spiel im Kindesalter: Symposium, 13.3.2003 in Augsburg

<div><p><em>tbx5</em>, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt−Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an <em>in silico</em> screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on <em>miR-218-1</em> that, together with its host gene, <em>slit2</em>, is involved in heart development. We found correlated expression of <em>tbx5</em> and <em>miR-218</em> during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and <em>miR-218</em> dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of <em>miR-218</em> is able to rescue the heart defects generated by <em>tbx5</em> over-expression supporting the notion that <em>miR-218</em> is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.</p> </div

tbx5 over-expression causes eye, cardiac and fin defects.

<p>A, phenotypes generated by increasing doses of <i>tbx5a</i> mRNA. The percentage of embryos with the indicated defects was averaged across multiple independent experiments. The total number of embryos analyzed was as follows: mRNA-Tbx5a (35 pg) n = 48; mRNA-Tbx5a (100 pg) n = 199; mRNA-Tbx5a (200 pg) n = 131;. B, qRT-PCR analysis of <i>miR-218</i>a relative expression in 24 and 34 hpf embryos injected with 100 pg of <i>tbx5a</i> mRNA compared with embryos injected with 100 pg of GFP mRNA. C, phase-contrast and confocal images of representative transgenic <i>Tg(cmlc2:eGFP)</i> embryos at 72 hpf showing eye, heart and fin morphological defects induced by the injection of 100 pg (b,b′–c-c′) or of 200 pg (d,d′) of <i>tbx5a</i> mRNA. Arrowheads indicate eye alteration, arrows show fin absence. Labels: A, atrium, V, ventricle. Dotted lines encircle ventricle (white) and atrium (red). Scale bars: black 100 µm, red 25 µm.</p

miR-218 over-expression causes a delay in early heart field migration.

<p>A,B, images of <i>Tg(cmlc2:eGFP)</i> embryos injected with 260 pg of miR-Ct (A) or with 260 pg of <i>miR-218</i>a mimic (B) at different times of development. Dorsal view, anterior at the bottom. After confocal analysis, embryos were left to develop until 72 hpf when they were screened for the presence of edema. White scale bars: 150 µm. C, migration velocities of myocardial <i>Tg(cmlc2:eGFP)</i> cells as quantified from time-lapse images. Five embryos for each experiment were analyzed.</p

Down-regulation of miR-218 can rescue the defects generated by tbx5 over-expression.

<p>A, quantification of the phenotypes induced by the injection of 100 pg of <i>tbx5a</i> mRNA (n = 199), 8 ng of MO^D-218 (n = 182) or by the co-injection of 100 pg of <i>tbx5a</i> mRNA and 8 ng of MO^D-218 (n = 241). As control, non injected embryos were quantified. Each experimental point in the graph represents the mean ± SE of at least three independent experiments. Comparisons between groups were performed by one-way analysis of variance, followed by Bonferroni’s post-hoc for multiple comparisons. B, phase-contrast and confocal images of representative transgenic <i>Tg(cmlc2:eGFP)</i> embryos at 72 hpf comparing the phenotype of a control embryo (upper panels) to the rescued phenotype generated by the co-injection of 100 pg of <i>tbx5a</i> mRNA and 8 ng of MO^D-218 (lower panels). C, quantification of <i>tie-2</i> mis-expression in 72 hpf <i>Tg(tie-2:GFP)</i> embryos after the co-injection of <i>tbx5a</i> mRNA (100 pg) and MO-Ct (8 ng, n = 60) or of <i>tbx5a</i> mRNA (100 pg) and MO^D-218 (8 ng, n = 62). D, Confocal images of representative 72 hpf <i>Tg(tie-2:GFP)</i> embryos co-injected with <i>tbx5a</i> mRNA and MO-Ct (a-a′) or with <i>tbx5a</i> mRNA and MO^D-218 (b-b′). Labels: A, atrium, V, ventricle. Scale bars: black 100 µm, red 25 µm.</p

miR-218 over-expression affects cardiac development.

<p>A, <i>miR-218</i>a ISH of 72 hpf embryos. B, <i>slit2</i> and s<i>lit3</i> ISH of 48 hpf embryos. nt, neural tube; ht, heart. C,D, phenotypes induced at 72 hpf by increasing doses of <i>miR-218</i> mimic (C) or MO^M/MO^D-218 (D) injection. The percentage of embryos with the indicated defects was averaged across multiple independent experiments carried out in double blind. The total numbers of embryos analyzed were as follows: Ct miRNA (1 ng) n = 293; miR-214 mimic (1 ng) n = 104; miR-492 mimic (1 ng) n = 103; <i>miR-218</i> mimic (35 pg) n = 107; <i>miR-218</i> mimic (135 pg) n = 180; <i>miR-218</i> mimic (260 pg) n = 318; <i>miR-218</i> mimic (2 ng) n = 180; MO-Ct (8 ng) n = 207; MO^D-218 (12 ng) n = 323; MO^M-218 (2 ng) n = 112; MO^M-218 (4 ng) n = 165; MO^M-218 (8 ng) n = 182. E-H, phenotypic analysis of <i>miR-218</i>a misregulation in <i>Tg(cmlc2:eGFP)</i> embryos. Confocal images of representative transgenic embryos showing the presence or the absence of pericardial edema (e;top) and heart morphology (bottom). F,G examples of heart defects with different degrees of severity. a, atrium, v, ventricle, e, cardiac edema. Dotted lines encircle ventricle (white) or atrium (red). Red arrow in G (bottom panel) indicates a shrunken, elongated ventricle typical of the heartstring phenotype. Scale bars: white or black 100 µm, red 25 µm.</p

miR-218a over-expression leads to the expansion of tie-2 expression.

<p>A, confocal images of 72 hpf <i>Tg(tie-2:GFP)</i> embryos injected with 260 ng of control miRNA (a,a′), 260 ng of <i>miR-218</i>a mimic (b,b′), 2 ng of MO-Tbx5a (c,c′) or 100 pg of mRNA Tbx5a (d,d′). A, magnification of the control valve is shown in the inset in panel a′. Labels: A, atrium, V, ventricle. B, FACS analysis of cells dissociated from 72 hpf <i>Tg(tie-2:GFP-cmlc2:eRFP)</i> embryos injected as described in A. C, confocal images of 72 hpf <i>Tg(tie-2:GFP-cmlc2:eRFP)</i> embryos injected with 260 ng of miR-Ct (top) or with <i>miR-218</i>a mimic (bottom). The control valve is magnified in the inset in panel a′. White scale bars: 100 µm, red scale bars 25 µm.</p

tbx5 and miR-218 are co-expressed in cardiomyocyte differentiation of P19CL6 cells.

<p>A, qRT-PCR analysis of cardiac (α-MHC, Cx40, GATA4), muscle (Myosin), neural (Pax6, β-3-tubulin) and pluripotency (Oct4) markers in P19CL6 differentiating cells (8,10 days) compared to P19CL6 in growth medium (GM). B, qRT-PCR analysis of t<i>bx5</i>, <i>slit2</i>, <i>slit3</i> and <i>miR-218</i> relative expression in either expanding (GM) or differentiating (8,10,12 days) P19CL6 cells. (C-D), P19CL6 differentiating cells, 48 h after CMV-Tbx5 transfection compared to cells transfected with empty vector (C) or different times after Tbx5-siRNA transfection compared with cells transfected with siRNA-Ct (D). The timing course of the silencing experiment in (D) is described in the “Cell culture and transfection” section of methods. Results are standardized against GAPDH for genes, and against U6 for miRNAs.</p