Comparison <i>Erbb2</i> null [22], [25] and <i>l11Jus8</i> phenotypes.

<p>Comparison <i>Erbb2</i> null <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107041#pone.0107041-Lee1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107041#pone.0107041-Erickson1" target="_blank">[25]</a> and <i>l11Jus8</i> phenotypes.</p

Numbers of distended hearts, hearts with atrioventricular block (AVb), atrial block (Ab), and atrial signal conduction gaps in optical mapping experiments.

<p>Numbers of distended hearts, hearts with atrioventricular block (AVb), atrial block (Ab), and atrial signal conduction gaps in optical mapping experiments.</p

Mapping of electrical signal propagation in E12.5 hearts.

<p>(<b>A</b>–<b>C</b>) Derivatives of the signal recorded in wt and homozygous <i>l11Jus8</i> E12.5 hearts. Black arrowheads point to the present peaks used for map generation, white arrowheads point to the missing peaks. Asterisk on (<b>C</b>) depicts the missing derivative for signal present on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107041#pone-0107041-g003" target="_blank"><b>Figure 3F</b></a>. The derivatives used in each map image are indicated on the electrical traces. (<b>D</b>–<b>F</b>) Optical maps showing spatio-temporal propagation of the electrical signal through the atria. Asterisks indicate gaps in normal electrical activity. White arrow shows the direction of the signal propagation in wild type and homozygous <i>l11Jus8</i> atria. (<b>G</b>–<b>I</b>) Optical maps showing electrical signal propagation through the ventricles. Maps are similar for all genotypes. Each colour (<b>D</b>–<b>I</b>) represents a one-millisecond interval with black as the initial time point and fuchsia as the final time point. RA, right atrium; LA, left atrium; LV, left ventricle; RV, right ventricle.</p

Analysis of DNA, protein, and morphological defects in the <i>l11Jus8</i> mutant mouse.

<p>(<b>A</b>) Three coding mutations found in the 35 Mb candidate intervals on mouse chromosome 11 are located in the <i>Med31</i>, <i>Nf1</i> and <i>Erbb2</i> genes. Only recombinant embryos with the C57BL/6 homozygous DNA fragment inherited from the mutagenised parent which contained the <i>Erbb2</i> mutation were lethal at weaning, whereas embryos with homozygous <i>Med31</i> and <i>Nf1</i> and heterozygous <i>Erbb2</i> mutations were viable. Straight and dotted lines depict homo- and heterozygous DNA, respectively. (<b>B</b>) Confirmation of the T98,433,986G base change in the <i>Erbb2</i> gene in <i>l11Jus8</i> mutants by Sanger sequencing. (<b>C</b>) Phenotype of wild type, homozygous <i>l11Jus8</i> and <i>Erbb2^M802R</i> mutant embryos and hearts. Scale bars: 2 mm for embryos, 500 µm for hearts. (<b>D</b>) Erbb2 protein structure: FLD - Furin-Like Domain, STKD - Serine/Threonine-Tyrosine Kinase Domain. Partial sequence of the STK domain (60 amino acids around the mutation point) is shown for mouse (M), human (H), chick (C) and zebrafish (Z) Erbb2 protein. Asterisks indicate conserved amino acids, colon and period indicate conservation between groups of strongly and weakly similar properties, respectively. Arrowhead demarcates the location of the amino acid change. (<b>E</b>) Predicted changes in the protein structure of the Erbb2^M802R conserved kinase catalytic domain based on the reported crystal structure of the Erbb2 protein. (<b>F</b>) Top panel: expression of the <i>Erbb2</i> mRNA in atria (A) and ventricles (V) of hetero- and homozygous <i>l11Jus8,</i> and embryonic erythrocyte (blood) samples. <i>Prpf8</i> mRNA expression is used as a positive control for cDNA isolation in blood sample. Bottom panel: <i>Erbb2</i> expression in the yolk sack (YS) of homozygous <i>l11Jus8</i> embryo, “-RT” control, genomic DNA (gen) control. A no template negative control (neg) is shown in both panels. (<b>G</b>) Erbb2 immunohistochemistry in heterozygote and <i>l11Jus8</i> atria (Atr) and ventricles (Ven). Embryonic erythrocytes within the cardiac chambers are indicated with an arrowhead. Scale bar  =  200 µm.</p

Effect of the homozygous <i>Erbb2^M802R</i> mutation on heart morphology in <i>l11Jus8</i> hearts.

<p>(<b>A</b>–<b>L</b>) Representative H and E stained coronal sections of E12.5 hearts. (<b>A</b>–<b>B</b>) Atrioventricular cushions, (<b>C</b>–<b>D</b>) dorsal outflow tract, (<b>E</b>–<b>F</b>) ventricles, (<b>G</b>–<b>H</b>) atria. Arrows in (<b>G</b>–<b>H</b>) point to the atrial wall. AVC, atrioventricular cushion; OFT, outflow tract; Ven, ventricle; Atr, atrium; Ep, epicardium; En, endocardium; My, myocardium. (<b>I</b>–<b>J</b>) Longitudial atrial and (<b>K</b>–<b>L</b>) ventricular sections; homozygous <i>l11Jus8</i> heart had distended atria. Embryos in <b>I</b>–<b>L</b> are littermates. Magnified areas in (<b>I</b>–<b>J</b>) show the developing pectinate muscles (arrows). (<b>M</b>–<b>N</b>) Activated Caspase 3 (Cas3) staining of atrial wall at E11.5. Inset shows positive activated Caspase3 staining from trigeminal ganglion on same embryo section. (<b>O</b>–<b>P</b>) Propidium Iodide (PI) labelling of the necrotic cells in E12.5 hearts. RA, right atrium; LA, left atrium. Arrowheads point to the unspecific PI staining resulting from cells of the OFT damaged during dissection. Scale bars: 200 µm in <b>A</b>–<b>H</b> and <b>M</b>–<b>N</b>, 50 µm in <b>I</b>–<b>L</b>.</p

Mapping of electrical signal propagation in E11.5 hearts.

<p>(<b>A</b>) Pattern of wild type electrical signal propagation in the E11.5-12.5 heart. Different colours encode the time scale of the signal conduction; each colour represents a one-millisecond interval with black as the initial time point and fuchsia as the final time point. (<b>B</b>–<b>C</b>) Derivatives of the signal recorded in wt and homozygous mutant <i>l11Jus8</i> E11.5 hearts. Black arrowheads point to the peaks used for map generation. (<b>D</b>–<b>F</b>) Optical maps showing spatio-temporal propagation of the electrical signal through the atria. Asterisk on (<b>F</b>) points to the gap in normal electrical activity in homozygous mutant <i>l11Jus8</i> atria. (<b>G</b>–<b>I</b>) Optical maps showing electrical signal propagation through the ventricles. Maps are similar for all genotypes. SAN, sinoatrial node; RA, right atrium; LA, left atrium; LV, left ventricle; RV, right ventricle; AVC, atrioventricular canal; IVG, interventricular groove.</p

Optical mapping of the cardiac electrical activity obtained with di-4-ANEPPS voltage sensitive dye.

<p>(<b>A</b>–<b>B</b>) and (<b>C</b>–<b>F</b>) show reading of E11.5 and E12.5 hearts, respectively. The location from which the electrical reading was taken is shown with lines onto the cardiac dissection image. Black arrowheads point to matched atrial and ventricular signals. (<b>D</b>) White arrowhead indicates absent signal in left ventricle corresponding to a present signal in left atria (black arrowhead), indicative of AV block. (<b>E</b>–<b>F</b>) White arrowheads indicate absent atrial signals corresponding to present ventricular signals (A block), a phenotype only present in mutant samples. Asterisks on RA and LA traces in (<b>F</b>) indicate signal described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107041#pone-0107041-g005" target="_blank"><b>Figure 5C</b></a>. RA, right atrium; LA, left atrium; LV, left ventricle; RV, right ventricle.</p

Examination of ECM composition in <i>Myh10</i>∆ control and mutant embryos.

<p>A: Laminin immunofluorescence (green) is present at E11.5 and E14.5 in the <i>Myh10∆</i> heterozygous ventricular myocardium and is continuously distributed in the epicardium (arrow). <i>Myh10∆</i> homozygous mutants show reduced laminin immunoreactivity in the epicardium (arrow) at both E11.5 and E14.5. Nuclei are marked with DAPI (blue). B: Fibronectin immunofluorescence (green) is present at E11.5 and E14.5 in the <i>Myh10∆</i> heterozygous ventricular myocardium and is continuously distributed in the epicardium (arrow). <i>Myh10∆</i> homozygous mutants appear to have reduced signal, especially in the epicardium (arrow) at E11.5 and E14.5. Nuclei are marked with DAPI (blue). C: Collagen 1 immunofluorescence (green) is present at E11.5 and E14.5 in the <i>Myh10∆</i> heterozygous ventricular myocardium and is continuously distributed throughout the epicardium (arrow). <i>Myh10∆</i> homozygous mutants have reduced collagen 1 signal in the epicardium (arrow) at E11.5 and E14.5. Nuclei are marked with DAPI (blue). Genotypes and developmental stages are labeled on the image. D: Quantification of the ratios of expression levels of epicardial immunofluorescence intensity to myocardial immunofluorescence intensity for each protein. Three embryos of each genotype were analysed, with measurements taken from five different areas of three different sections from each embryo. All markers and time points showed a statistically significant reduction (*) in immunofluorescence intensity of the epicardium relative to the myocardium in <i>Myh10∆</i> homozygous mutants as compared to controls (two-tailed t-test, p<0.05). Ratios of expression for each genotype were compared to each other for each marker and developmental stage. Error bars represent standard deviation. Genotypes, markers and stages are labeled on the graphs. Scale bars: 15 μm. Abbreviations: Lam: laminin, FN: fibronectin, Col1: Collagen I.</p

<i>EHC</i> epicardial-derived cells show defective migration <i>in vivo</i>.

<p>A: Representative images of Wt1 immuno-stained coronal cryo-sections of <i>EHC</i> heterozygous and B: homozygous mutant E14.5 hearts. The dashed box indicates the area of ventricular wall used for measurement analysis in (C) and (D). C: Wt1 positive epicardial-derived cells were marked with yellow crosshairs in heterozygous and D: mutant hearts. The distance between these cells and the cardiac surface (C-G: dashed line) was manually measured using ImageJ. E: Wt1 positive cells (white) in the heterozygous heart can be seen at the cardiac surface (dashed line) indicating localization in the epicardium, as well as in deeper cardiac tissue below the dashed line. F: <i>EHC</i> mutants have Wt1 positive cells (white) primarily at the cardiac surface (dashed line), but these cells are organised into abnormal clusters (arrowheads), with few cells at deeper positions in the underlying myocardium. G: Wt1 positive cells surround the ventricular surface blisters (arrows) present in <i>EHC</i> mutant hearts. H: Graph comparing the mean migration distance of Wt1 positive cells: Het = 33.37 μm (+/-1.197 μm SEM, n = 975), mutant = 20.14 μM (+/-0.8136 μm SEM, n = 1180), Mann-Whitney U-test p = <0.0001). I: Histogram showing the relative frequency of Wt1 positive cells at increasing depths within the myocardial wall. A higher proportion of mutant Wt1 positive cells reside in the subepicardial region (<50 μm from the apical epicardial boundary) compared to controls. J. Comparison of the mean number of Raldh2 expressing cells found on the cardiac surface of hearts per unit of epicardial length for each genotype at E11.5 and E14.5. Mean number of epicardial cells/100μm in E11.5 controls = 7.975 (+/- 0.4008, n = 4) and mutants = 11.93 (+/- 1.145, n = 7); Mean number of epicardial cells/100μm in E14.5 controls = 7.3 (+/- 0.4243, n = 4) and mutants = 11.27 (+/- 0.9502, n = 10). A significant difference is detected between genotypes at E11.5 (2-tailed unpaired t test, p = 0.0335) and E14.5 (2-tailed unpaired t test, p = 0.0257). Error bars represent standard error of the mean. K: Raldh2 protein localisation (red) in <i>Myh10∆</i> heterozygous control and L-M: <i>Myh10∆</i> homozygous mutant heart at E11.5. Sections from two different embryos are shown. N: Raldh2 protein localisation (red) in <i>Myh10∆</i> control and O-P: <i>Myh10∆</i> homozygous mutant heart at E14.5. Sections from one embryo at different cardiac depths are shown (O-P). Q: Vimentin immunofluorescence in E14.5 control heart. Spindle-shaped mesenchymal cells are present (arrow). R: Merged image showing nuclei (blue). S: Vimentin immunofluorescence in E14.5 <i>EHC</i> mutant heart. Spindle-shaped mesenchymal cells are present (arrow). T: Merged image showing nuclei (blue). The epicardial boundary with the myocardium is labeled with a dashed line. Scale bars: A and B = 250 μm, C, D, Q-T = 100 μm, E-G = 50 μm, K and N = 25 μm. Abbreviations: epi: epicardium, myo: myocardium.</p

NMHC IIB is not required in cardiomyocytes for coronary vessel formation.

<p>A: Genomic PCR on tissues isolated from mice carrying the α<i>MHC-Cre</i> transgene and <i>Myh10 flox/+</i> alleles shows a PCR product of 600 bp in cardiac tissue, consistent with deletion of <i>Myh10</i> exon 2 from the genome (arrow). This product is not seen in tail, brain, or liver tissue. PCR results from a <i>Myh10∆/+</i> heterozygous animal are shown for the corresponding tissues, with the 600 bp product present in all tissues. The 1 Kb product represents the <i>Myh10</i> allele lacking exon 2 deletion. Products were sequenced to confirm specificity of PCR reactions. B: NMHC IIB protein (green) and C: actin (red; phalloidin stain) localisation in fibroblasts cultured from α<i>MHC-Cre; flox/flox</i> hearts. D: Merged image shows DAPI labeling of nuclei (blue). E: NMHC IIB protein (green) and F: actin (red; phalloidin stain) localisation in cardiomyocytes cultured from α<i>MHC-Cre; flox/flox</i> hearts. G: Merged image shows DAPI labelling of nuclei (blue). H: Expression patterns of NMHC IIB (white) and cardiac Troponin T (red), and nuclei stained with DAPI (blue) in α<i>MHC-Cre; Myh10+/+</i> control embryo with presence of coronary vessel (orange arrow). NMHC IIB expression is seen throughout heart including valve tissue (white arrow). Co-expression of NMHC IIB and cTnT appears pink. I: Higher magnification image of cardiac tissue from α<i>MHC-Cre; Myh10+/+</i> control embryo. J: Expression patterns of NMHC IIB (white) and cardiac Troponin T (red), and nuclei stained with DAPI (blue) in α<i>MHC-Cre; Myh10 flox/flox</i> control embryo with presence of coronary vessel (orange arrow). NMIIB expression is seen in valve tissue (white arrow) but reduced in myocardial region of heart, and is absent from cTnT positive cells, so that the pink staining indicating NMHC IIB and cTnT co-expression is reduced in the myocardium. K: Higher magnification image of cardiac tissue from α<i>MHC-Cre; Myh10 flox/flox</i> control embryo. Epicardial expression of NMHC IIB persists (white arrows). L: Ventral view of cardiac surface stained with DAB to identify blood cells in α<i>MHC-Cre; Myh10+/+</i> heart (arrow). M: Dorsal view of cardiac surface stained with DAB to identify blood cells in α<i>MHC-Cre; Myh10+/+</i> heart (arrows). N: Ventral view of cardiac surface stained with DAB to identify blood cells in α<i>MHC-Cre; Myh10 flox/∆</i> heart (arrows). O: Dorsal view of cardiac surface stained with DAB to detect endogenous peroxidase activity from blood cells in α<i>MHC-Cre; Myh10+ flox/∆</i> heart (arrows). Blood cells within vessels are present on the cardiac surface of cardiomyocyte-specific <i>Myh10</i> mutant hearts. Similar results were seen for α<i>MHC-Cre; Myh10 flox/flox</i> hearts. Scale bars: B-G = 50 μm, H and J = 100 μm, I and K = 15 μm, L-O = 400 μm. Abbreviations: α<i>MHC-Cre</i>: <i>Tg(Myh6-cre)</i>^{<i>2182Mds/J</i>}, CM: cardiomyocyte, cTnT: cardiac troponin T, Fibro: fibroblast, NMIIB: NMHC IIB, ∆: <i>Myh10</i>∆.</p