miR-218 over-expression affects cardiac development.

A, miR-218a ISH of 72 hpf embryos. B, slit2 and slit3 ISH of 48 hpf embryos. nt, neural tube; ht, heart. C,D, phenotypes induced at 72 hpf by increasing doses of miR-218 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; miR-218 mimic (35 pg) n = 107; miR-218 mimic (135 pg) n = 180; miR-218 mimic (260 pg) n = 318; miR-218 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 miR-218a misregulation in Tg(cmlc2:eGFP) 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.

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

A, confocal images of 72 hpf Tg(tie-2:GFP) embryos injected with 260 ng of control miRNA (a,a′), 260 ng of miR-218a 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 Tg(tie-2:GFP-cmlc2:eRFP) embryos injected as described in A. C, confocal images of 72 hpf Tg(tie-2:GFP-cmlc2:eRFP) embryos injected with 260 ng of miR-Ct (top) or with miR-218a mimic (bottom). The control valve is magnified in the inset in panel a2. White scale bars: 100 μm, red scale bars 25 μm.

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

A,B, images of Tg(cmlc2:eGFP) embryos injected with 260 pg of miR-Ct (A) or with 260 pg of miR-218a 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 Tg(cmlc2:eGFP) cells as quantified from time-lapse images. Five embryos for each experiment were analyzed.

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

A, phenotypes generated by increasing doses of tbx5a 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 miR-218a relative expression in 24 and 34 hpf embryos injected with 100 pg of tbx5a mRNA compared with embryos injected with 100 pg of GFP mRNA. C, phase-contrast and confocal images of representative transgenic Tg(cmlc2:eGFP) 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 tbx5a 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.

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

A, quantification of the phenotypes induced by the injection of 100 pg of tbx5a mRNA (n = 199), 8 ng of MO^D-218 (n = 182) or by the co-injection of 100 pg of tbx5a 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 Tg(cmlc2:eGFP) 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 tbx5a mRNA and 8 ng of MO^D-218 (lower panels). C, quantification of tie-2 mis-expression in 72 hpf Tg(tie-2:GFP) embryos after the co-injection of tbx5a mRNA (100 pg) and MO-Ct (8 ng, n = 60) or of tbx5a mRNA (100 pg) and MO^D-218 (8 ng, n = 62). D, Confocal images of representative 72 hpf Tg(tie-2:GFP) embryos co-injected with tbx5a mRNA and MO-Ct (a-a2) or with tbx5a mRNA and MO^D-218 (b-b2). Labels: A, atrium, V, ventricle. Scale bars: black 100 μm, red 25 μm.

Rescue of cardiac defects induced by miR-218a over-expression was accomplished by co-injecting MO-218a. A, qRT-PCR analysis of miR-218a relative expression in 24 hpf embryos microinjected with 12 ng of control morpholino (MO-Ct) or MO^D-218a and with 260 pg of miR-Ct or miR-218a mimic. miR-218a relative expression was calculated as the ratio between the expression of injected and the expression of non injected embryos. B, representative transgenic Tg(cmlc2:eGFP) embryos at 72 hpf showing heart morphological defects induced by the injection of 260 pg of miR-218a mimic in the absence (a,b) or in the presence (c,d) of MO^D-218a (12 ng). Labels: a, atrium, v, ventricle. Black scale bars: 100 μm, red scale bars 25 μm.

miR-218 dysregulation does not affect vascular integrity. Confocal images of representative 72 hpf Tg(flk1:eGFP) embryos injected with 260 pg of miR-Ct (A), 260 pg of miR-218 mimic (B) or 8 ng MO^D-218 (C). Black and white scale bars: 100 μm.

tbx5 and miR-218a misexpression does not alter amhc and vmhc cardiac marker expression in zebrafish embryos. Ventral views of 48 hpf embryos injected with the indicated miRNA mimics (260 pg) or MOs (12 ng MO-Ct and MO^D-218a, 3 ng MO-Tbx5a) after mRNA in situ hybridization. Scale bar 100 μm.

miR-218 targets the 3′ UTR of robo1 in zebrafish embryos. Top: schematic representation of sensors and miRNAs used for in vivo sensor assay. Bottom: examples of 24 hpf embryos microinjected with 40 pg of RFP mRNA, 400 pg of 3′UTR robo 1 sensor and 160 pg of miR-Ct (a,c,e) or miR-218a (b,d,f). In figures C and D the percentage of the relative phenotypes were indicated. ~30 embryos for each thesis were injected. Scale bars 50 μm.

MO-Tbx5a and MO-Tbx5b effectively knockdown the two zebrafish tbx5 isoforms. A-D, 35 pg of pCS2 plasmid expressing GFP fused with MO-Tbx5a or MO-Tbx5b target sequences were injected in one-cell stage embryos in the absence (A and C) or in the presence (B and D) of 1,5 ng of the relative morpholino. Representative fluorescent images of 24 hpf embryos. ~20 embryos for each thesis were analysed. E-F, Tbx5 morphants analysis. Phenotypic analysis of Tbx5a (E) and Tbx5b (F) morphants: 2 ng of MO-Tbx5a, or 4 ng of MO-Tbx5b, were injected in Tg(cmlc2:eGFP) embryos. Phase-contrast images showing pericardial edema (arrowheads) and fin absence (brackets) or presence (arrows); in the bottom right corner of figures E and F, fluorescent images showing heart morphology. Quantification of Tbx5a (G) and Tbx5b (H) morphant phenotypes. The percentage of embryos with the indicate defects was averaged across multiple independent experiments. ~100 embryos for each thesis were analysed. Black scale bars: 100 μm, red scale bars 25 μm.

Injection of miR-218a in Tbx5a morphants increases the severity of heartstring phenotype. A, phenotypic analysis of Tbx5a morphants co-injected with 1 ng of MO-Tbx5a and either 130 pg of miR-218a mimic or 130 pg of miR-Ct. B, representative images of 48 hpf embryos showing the edema expansion caused by the co-injection of miR-218a mimic. C, phenotypic analysis of embryos co-injected with sub-phenotypic doses of both MO-Tbx5a(0.5 ng) and miR-218a mimic (35 pg). For comparison the same dose of MO-Tbx5a was co-injected with 35 pg of miR-Ct. (D-E) Representative confocal images showing heart morphology of transgenic Tg(cmlc2:eGFP) embryos injected with a sub-phenotypic dose of MO-Tbx5a and 35 pg of either miR-Ct (D) or miR-218a mimic (E). Embryo in D has normal looping while co-injected embryos in E show absence of looping, although with different degrees of heart defects. a, atrium, v, ventricle, e, cardiac edema. Black and white scale bars: 100 μm, red scale bars 25 μm.