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

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

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

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