Ectopic expression of wild-type Tbx20 rescues <i>whz</i> mutant embryos.

<p><b>(A, B)</b> Lateral view of <i>whz</i> mutant embryos control-injected with KCl <b>(A)</b> and wild-type zebrafish <i>tbx20</i> mRNA <b>(B)</b>, respectively. <b>(C)</b> Ectopic expression of wild-type zebrafish <i>tbx20</i> mRNA can rescue the heart phenotype of 73% of homozygous <i>whz</i> mutant embryos, whereas injection of KCl has no effect. <b>(D, E)</b> Dissected <i>whz</i> hearts injected with KCl <b>(D)</b> and wild-type <i>tbx20</i> mRNA <b>(E)</b> are stained against MEF-2 (red) after incorporation of 5-ethynyl-2'-deoxyuridine (EdU; green) to visualize cardiomyocyte proliferation. <b>(F)</b> <i>whz</i> mutant hearts injected with wild-type <i>tbx20</i> mRNA show significantly increased numbers of ventricular cardiomyocytes at 72 hpf <b>(E)</b> compared to control-injected <i>whz</i> mutants <b>(D)</b> (<i>whz+tbx20</i> mRNA: 142.5±10 SD, <i>whz</i> + KCl: 87.82±10 SD, n = 10; p = 0.0001). <b>(G)</b> Ventricular cardiomyocyte proliferation in <i>whz</i> mutant embryos injected with <i>tbx20</i> mRNA is significantly enhanced compared to control injected mutants at 72 hpf (<i>whz</i>+<i>tbx20</i> mRNA: 7±3% SD, <i>whz</i>+KCl: 1±2% SD, n = 10; p = 0.0001).</p

Overexpression of <i>tbx20</i> in wild-type zebrafish embryos results in reduced ventricular cardiomyocyte proliferation.

<p><b>(A, B)</b> Dissected wild-type hearts injected with KCl <b>(A)</b> and <i>tbx20</i> mRNA <b>(B)</b> at 72 hpf, stained against MEF-2 (red) after incorporation of 5-ethynyl-2'-deoxyuridine (EdU; green) to visualize the effect of <i>tbx20</i> overexpression on cardiomyocyte proliferation. <b>(C)</b> Wild-type zebrafish hearts injected with wild-type <i>tbx20</i> mRNA show significantly reduced numbers of ventricular cardiomyocytes at 72 hpf compared to control injected hearts (wt+KCl: 148.6±2.9; wt + <i>tbx20</i> mRNA: 64.0±2.017, n = 10, p< 0.0002). <b>(D)</b> Ventricular cardiomyocyte proliferation in wild-type embryos injected with <i>tbx20</i> mRNA is significantly reduced compared to control injected embryos at 72 hpf (wt+KCl: 8.9 ± 0.38; wt+<i>tbx20</i> mRNA: 1.1±0.28, n = 10, p = 0.0001).</p

<i>whz</i> encodes <i>Tbox transcription factor 20 (tbx20)</i>.

<p><b>(A)</b> Integrated genetic and physical map of the <i>whz</i> locus on zebrafish chromosome 16. The <i>whz</i> mutation interval is flanked by the microsatellite markers z1215 and z6240 and encodes 2 open reading frames, zebrafish <i>tbx20</i> and <i>herpud2</i>. <b>(B)</b> Missense mutation of Adenine to Cytosine of the stop codon of <i>tbx20</i> results in the loss of the original stop codon and the termination of <i>tbx20</i> transcription after 87 additional nucleotides. <b>(C)</b> Partial amino acid alignment of the C-terminus of zebrafish <i>whz</i> and wt as well as human and murine Tbx20. Tbx20 is highly conserved cross-species and the <i>whz</i> mutant Tbx20 protein is extended by 29 additional amino acids. <b>(D, E)</b> <i>Tbx20</i>-specific whole-mount antisense RNA <i>in situ</i> hybridization detects unaltered expression of zebrafish <i>tbx20</i> in <i>whz</i> mutant embryos compared to wild-types. <b>(F)</b> Quantitative RT-PCR analysis showing similar relative mRNA levels of <i>tbx20</i> in wt and <i>whz</i> embryos at 72 hpf (n = 4; p = 0.5957). <b>(G, H)</b> Tbx20 protein levels are significantly reduced in <i>whz</i> mutant embryos compared to wild-type littermates (n = 4; p = 0.0286).</p

Effects of the <i>whz</i> mutation on embryonic heart morphology and growth.

<p><b>(A-D)</b> Lateral view of wild-type (wt; <b>A, C</b>) and <i>whz</i> mutant (<b>B, D</b>) embryos at 72 hours post fertilization (hpf). <i>Whz</i> mutants show pericardial edema, blood congestion at the cardiac inflow tract and stretched heart chambers. <b>(E, F)</b> Hematoxylin and Eosin staining of sagittal histological sections of wt <b>(E)</b> and <i>whz</i> mutant <b>(F)</b> hearts at 72 hpf. Similar to wild-types, in <i>whz</i> atria and ventricles, myocardial (myo) and endocardial (endo) cell layers are clearly defined and separated by an atrio-ventricular canal (AVC). In contrast to wild-type hearts, <i>whz</i> mutant ventricles appear small and the myocardium monolayered. <b>(G-I)</b> Dissected wt <b>(G)</b> and <i>whz</i> mutant <b>(H)</b> hearts at 72 hpf, stained with a cardiomyocyte-specific MEF-2 antibody (nuclei; red) and co-stained with S46, exclusively marking atrial cardiomyocytes (green)<b>. (I)</b> <i>whz</i> mutant hearts show significantly reduced ventricular cardiomyocytes at 72 hpf (sib: 144.2±10 SD and <i>whz</i>: 94.9±10 SD, n = 10; p<0.0001), whereas cardiomyocyte numbers are comparable between wt and <i>whz</i> ventricles at 48 hpf (wt: 93.4±10 SD and <i>whz</i>: 88.2±10 SD, n = 10; p>0.05).</p

The <i>whz</i> mutation interferes with cardiomyocyte proliferation.

<p><b>(A, B)</b> TUNEL stainings of embryonic zebrafish hearts at 72 hpf show no difference in the number of apoptotic cardiomyocytes in wt <b>(A)</b> and <i>whz</i> <b>(B)</b> mutant embryos. TUNEL positive cells in the pericardium (peri) and ventricles are marked by arrows. <b>(C-F)</b> Dissected wt <b>(C)</b> and <i>whz</i> mutant <b>(D)</b> hearts at 72 hpf, stained against MEF-2 (red) after incorporation of 5-ethynyl-2'-deoxyuridine (EdU; green) to visualize cardiomyocyte proliferation. At 48 hpf, proliferation of ventricular cardiomyocytes appears unaltered between wt and <i>whz</i> mutant hearts (sib: 5±2% SD and <i>whz</i>: 4±2% SD, n = 10; p>0.05) <b>(E)</b>, whereas cardiomyocyte proliferation in <i>whz</i> mutant ventricles is significantly reduced compared to wt at 72 hpf (sib: 8±2% SD, <i>whz</i>: 2±2% SD, n = 10, p = 0.0001) <b>(F)</b>.</p

Knock-down of zebrafish <i>tbx20</i> phenocopies the <i>whz</i> mutant phenotype.

<p><b>(A-D)</b> Lateral view of wild-type embryos injected with zebrafish <i>tbx20</i>-specific control Morpholinos (MO-ctrl) <b>(A, C)</b> and <i>tbx20</i> start and splice Morpholinos (MO-<i>tbx20</i>) <b>(B, D)</b> at 72 hpf, respectively. Knock-down of <i>tbx20</i> phenocopies the <i>whz</i> mutant phenotype, whereas injection of the same amount of specific-control Morpholinos does not affect heart growth. <b>(E, F)</b> Hematoxylin and Eosin staining of sagittal histological sections of MO-ctrl <b>(E)</b> and MO-<i>tbx20</i> <b>(F)</b> injected hearts at 72 hpf. In contrast to control hearts, <i>tbx20</i> morphant ventricles appear small and the myocardium monolayered. <b>(G)</b> 78% (MO1-<i>tbx20</i>) and 76% (MO2-<i>tbx20</i>) of the injected embryos are indistinguishable from <i>whz</i> mutant embryos. <b>(H)</b> <i>Tbx20</i> morphant hearts show significantly reduced ventricular cardiomyocytes at 72 hpf (MO1-control: 130.7±10 SD, MO1-<i>tbx20</i>: 86.5±10 SD, n = 10; p = 0.0001). <b>(I-K)</b> Cardiomyocyte proliferation in <i>Tbx20</i> morphant ventricles is significantly reduced compared to controls at 72 hpf (MO1-control: 6±2% SD, MO1-<i>tbx20</i>: 1±2% SD, n = 10; p = 0.0001).</p

Targeted knock-down of Paxillin leads to heart failure in zebrafish.

<p><b>(A-C)</b> Co-immunostaining of Paxillin (A, red) and the known Z-disk protein Focal Adhesion Kinase (FAK) (B, green) on adult primary zebrafish cardiomyocytes revealed localization of Paxillin to sarcomeric Z-disks. Cell nuclei were counterstained with DAPI (C, blue). Scale bar 10 μm. <b>(D-G)</b> Lateral view of <i>paxillin</i> start MO (MO1-<i>paxillin</i>) (D) and <i>paxillin</i> 5bp-mismatch-MO (MO1-control) (E) injected embryos at 72 hpf. (F) 78% of embryos injected with 5.4 ng MO1-<i>paxillin</i> developed pericardial edema and blood congestion at the cardiac inflow tract (n = 3; *<i>P</i><0.0001), whereas control-injected embryos developed no pathological phenotype. (G) Fractional shortening (FS) measurements of Paxillin morphant ventricles at 48, 72 and 96 hpf. FS of Paxillin morphant ventricles was slightly reduced to 69.6% ± 5.07% compared to corresponding 5bp-mismatch-MO injected embryos (FS: 71.6% ± 3.96%) at 48 hpf. At 72 hpf, FS in Paxillin morphants was reduced to 49.29% ± 4.39% compared to control morphants (FS: 69.3% ± 4.36%), whereas ventricular chambers of Paxillin morphants became almost silent (FS: 4.23% ± 6.17%) compared to controls (FS: 74% ± 4.96%) at 96 hpf (n = 6–8 individuals per time point). <b>(H-K)</b> Lateral view of MO2-<i>paxillin</i> (H) and MO2-<i>paxillin</i>+<i>paxillin</i> mRNA (I) injected embryos at 72 hpf. (J) Co-injection of 4.5 ng of MO2-<i>paxillin</i> and 1 ng wild-type <i>paxillin</i> mRNA rescued the Paxillin morphant heart failure phenotype (n = 3; *P = 0.0073). (K) Quantification of ventricular FS revealed that ectopic expression of <i>paxillin</i> mRNA significantly improved contractile function in Paxillin morphants at 72 hpf (n = 8–9 individuals; *<i>P</i><0.0001). Error bars indicate s.d.</p

Knock-down of <i>fak1a</i> and <i>fak1b</i> phenocopies the Paxillin morphant phenotype.

<p><b>(A)</b> Western blot analysis of FAK protein levels in Paxillin-deficient zebrafish embryos compared to control-injected embryos. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. The figure shows one representative western blot out of three independent experiments. <b>(B, C)</b> Quantitative real time PCR of Paxillin-depleted and control-injected embryos showed no significant (ns) alteration of <i>fak1a</i> (n = 3; *<i>P</i> = 0.9017) (B) and <i>fak1b</i> (n = 3; *<i>P</i> = 0.8878) (C) mRNA expression. For statistical analysis student’s <i>t</i>-test was performed. <b>(D-F)</b> Lateral view of MO1-<i>fak1a</i>/<i>fak1b</i> (D) and 5bp-mismatch-MOs (MO1-control) (E) injected embryos at 72 hpf. Embryos co-injected with 2.7 ng of MO1-<i>fak1a</i> and 2.15 ng of MO1-<i>fak1b</i> showed a severe heart failure phenotype whereas injection of the same amount of <i>fak1a</i> and <i>fak1b</i> 5bp-mismatch MOs (MO1-control) caused no significant pathological cardiac phenotype (F) (n = 3; *<i>P</i><0.0001). <b>(G)</b> FS measurements of FAK morphant ventricles at 36, 48 and 72 hpf. FS of FAK double-knock-down morphant ventricles was slightly reduced to 56.38% ± 5.85% compared to corresponding 5bp-mismatch-MO injected embryos (FS: 62.4% ± 5.77%) at 36 hpf. At 48 hpf, FS in FAK morphants was reduced to 63.57% ± 9.18% compared to control morphants (70.33% ± 3.72%), whereas ventricular chambers of FAK-deficient embryos became almost silent (FS: 2.6% ± 3.71%) compared to controls (FS: 69.33% ± 4.36%) at 72 hpf (n = 5–8 individuals per time point). <b>(H)</b> Western blot analysis of Paxillin levels in control-, MO1-<i>fak1a</i>/<i>fak1b-</i> and MO1-<i>paxillin</i>-injected embryos. Paxillin protein levels are severely reduced after targeted knock-down of FAK and Paxillin, respectively. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. The figure shows one representative western blot out of three independent experiments. <b>(I)</b> Quantitative real time PCR of FAK-depleted and control-injected embryos showed no significant (ns) alteration of <i>paxillin</i> mRNA expression (n = 3; *<i>P</i> = 0.0937). For statistical analysis student’s <i>t</i>-test was performed. <b>(J)</b> Western blot analysis of embryos co-injected with zebrafish <i>paxillin</i> mRNA and MO2-<i>paxillin</i> compared with embryos injected with MO2-<i>paxillin</i> or <i>paxillin</i> mRNA alone. Ectopic expression of zebrafish <i>paxillin</i> mRNA was able to restore FAK protein expression. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. Error bars indicate s.d.</p

Vinculin does not localize to focal adhesion sites in Paxillin- and FAK-deficient zebrafish embryos.

<p><b>(A-C)</b> Co-immunostaining of dissected embryonic (72 hpf) zebrafish hearts with antibodies against β-Catenin (red) and Vinculin (green) of control- (A), MO1-<i>fak1a/fak1b-</i> (B) and MO1-<i>paxillin</i>- (C) injected hearts. Arrow heads indicate focal adhesion sites. Scale bar 5μM.</p

Vinculin protein levels are severely decreased in Paxillin- and FAK-deficient zebrafish embryos.

<p><b>(A)</b> Western blot analysis of MO-mediated Paxillin inactivation (MO1-<i>paxillin)</i> led to severely reduced Vinculin protein levels <i>in vivo</i>. Similar to loss of Paxillin, targeted ablation of FAK (MO1-<i>fak1a</i>/<i>fak1b)</i> also resulted in degradation of Vinculin, whereas Vinculin levels in control-injected embryos were completely unaffected. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. The figure shows one representative western blot out of three independent experiments. <b>(B)</b> Western blot analysis of embryos co-injected with zebrafish <i>paxillin</i> mRNA and MO2-<i>paxillin</i> compared with embryos injected with MO-<i>paxillin</i> or <i>paxillin</i> mRNA alone. Ectopic expression of zebrafish <i>paxillin</i> mRNA was able to restore Vinculin protein levels. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. <b>(C, D)</b> Bar graphs compare average of <i>vinculin</i> mRNA expression in Paxillin- (n = 3; *<i>P</i> = 0.1179) (C) and FAK-depleted (n = 3; *<i>P</i> = 0.8427) (D) compared to control-injected embryos. For statistical analysis student’s <i>t</i>-test was performed.</p