<i>EHC</i> mutants display defects in cardiac development.

<p>A: Heterozygous littermate control at E11.5 showing developing heart in pericardial sac. B: <i>EHC</i> mutant has pericardial effusion and blood visible in pericardial sac (arrow). C: Transverse section of control littermate at E11.5. The boxed region indicates the segment shown in panel C’. C’: Note the absence of blood cells on the cardiac surface and in the pericardial space. D: Transverse section of <i>EHC</i> mutant at E11.5 showing enlarged atria. The boxed region indicates the segment shown in panel D’. D’: Blood cells are present on the surface of the <i>EHC</i> heart (black arrow) and in the pericardial space (orange arrow). E: In heterozygous littermates at E14.5 the ventricular chamber is surrounded by a thick compact myocardium, which is highly organised. F: At E14.5, <i>EHC</i> mutants show a reduced compact ventricular myocardium (indicated by asterisk), accompanied by a thinning of the ventricular septal tissue (arrow). Note the abnormal atrial morphology and different localisation of the atria with respect to the ventricle compared to control (E). G: Heterozygous heart at E16.5 shows expected ventricular vascular connections and also coronary vessels on the cardiac surface (arrow). H: <i>EHC</i> mutant heart shows double-outlet right ventricle (DORV) (asterisk) as well as an absence of surface coronary vessels (arrow). Note the vesicle-like structures present on the ventricular surface. I: <i>EHC</i>/<i>Myh10</i>∆ and J: <i>Myh10</i>∆/<i>Myh10</i>∆ hearts at E16.5 similarly show DORV (asterisks) and ventricular vesicles (arrows). Scale bar = 200 μm (E, F), 1mm (A-D, G-J). Abbreviations: Het: heterozygote; lb: limb bud; LA: left atria, LV: left ventricle, RA: right atria, RV: right ventricle, VS: ventricular septum, ∆: <i>Myh10</i>∆.</p

NMHC IIB ablated epicardial cells do not show migration defects <i>in vitro</i>.

<p>A: Cells cultured from E11.5 <i>Myh10</i>∆/+ heterozygote and B: <i>Myh10</i>∆ homozygous mutant heart explants form an epicardial monolayer. Epicardial status was confirmed by positive staining for the epithelial marker ZO-1 (left panels), and the epicardial marker, Wt1 (centre panels). Rhodamine-phalloidin staining of the actin cytoskeleton also revealed characteristic ‘cobblestone’ morphology, indicative of an epithelial cell population (right panels). C: Control and D: mutant epicardial monolayers were scratched at T0 (left panels) and imaged at 10-minute intervals for 20 hours. The migration of ten cells per field of view was tracked using ImageJ (right panels, coloured lines). E: Graph showing the subsequent comparison of mean cell migration speed (control = 0.3099μm/min, mutant = 0.3098μm/min, Mann-Whitney U-test p = 0.6717). F: Graph showing the comparison of cell migration directional persistence (control = 0.7342, mutant = 0.7411, Mann-Whitney U-test p = 0.2494). Total tracked cells = 240 control and 270 mutant. Cultures were generated from at least four hearts for each genotype. Control refers to data compiled from both wild type and ∆/+ genotypes. Scale bars: 250 μm. Abbreviations: ∆: <i>Myh10</i>∆.</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

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

The <i>l11Jus27</i> mouse line carries two embryonic lethal mutations, one of which is in <i>Myh10</i>.

<p>A: Two recombinant mice show linkage of the phenotype (lethality) with distinct sub-regions of the mouse chromosome 11 balancer interval. The blue line indicates the 129S5 non-mutagenised chromosome. The C57BL/6 region (red) in recombinant mouse 363 extends beyond the balancer chromosome endpoint at <i>Trp53</i>. B: <i>l11Jus27</i> heterozygous and homozygous mutant embryos at E10.5, E11.5, and E12.5. For this and all subsequent figures, images are representative findings from a minimum of n = 3 observations unless otherwise stated. C: Dissections of embryos from crosses of <i>l11Jus27</i> heterozygotes with recombinant 363 reveal that at E10.5 –E12.75 the <i>l11Jus27</i> phenotype is not apparent in homozygous C57BL/6 embryos. At E11.5 and E12.75 embryos homozygous for C57BL/6 DNA have prominent hydrocephalus (arrow), revealing a second embryonic lethal phenotype in the <i>l11Jus27</i> line. D: Sequencing of the <i>Myh10</i> gene reveals a ‘G’ to ‘T’ point mutation in the splice donor site of exon 18 (orange arrows). E: The <i>Myh10</i> point mutation causes exon 18 to be skipped in the <i>Myh10</i> mutant transcript. RT-PCR in three homozygous <i>EHC</i> mutant embryos (M) reveals a smaller transcript than in one wild type (WT) embryo. The reduction in size of the PCR product is consistent with skipping exon 18. F: Sequencing <i>Myh10</i> transcript from mutant embryos confirms that exon 18 is missing, causing exons 17 and 19 to be joined. G: A wild type transcript that contains exons 17, 18, and 19. The black and grey lines in (F) and (G) highlight the sequence from exon 17 and sequence from exon 19 respectively shown in both the mutant and wild type. Scale bars: 1mm. Abbreviations: L27: <i>l11Jus27</i>; L: molecular size ladder; Het: heterozygote; WT: wild type; M: mutant; neg: negative control.</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

<i>EHC</i> mutants lack mature coronary vessels.

<p>PECAM-1 immunohistochemistry reveals prominent coronary vessels (arrows) on the ventral (A) and dorsal (B) surface of <i>EHC</i> heterozygous hearts. Only clusters of PECAM-1 expressing cells (arrows) are apparent on <i>EHC</i> homozygous mutant ventral (C) and dorsal (D) heart surface, with no clear evidence of coronary vessels. E-F: Section immunofluorescence for PECAM-1 (green) on <i>Myh10</i>∆ heterozygote at E14.5, demonstrating cells in myocardial region surrounding a developing vessel (arrow). G-H: Section immunofluorescence for PECAM-1 on <i>Myh10</i>∆ homozygous mutant at E14.5, demonstrating staining of clusters of cells on cardiac surface (arrow), consistent with whole mount staining in panels C-D. Staining for the smooth muscle cell marker SM22α reveals few smooth muscle cells within the interventricular septum region of the heterozygote (I) and <i>Myh10</i>∆ homozygous mutant (L). SM22α expressing cells are localised around a developing coronary vessel (white dashed circle) in heterozygous control hearts (J-K, arrowheads), and nucleated blood cells within the vessel can be visualised with DAPI (yellow arrow). No such structures are present in <i>Myh10</i>∆ homozygous mutant hearts, and smooth muscle cells expressing SM22α are less clustered in mutant hearts (M-N, arrowheads). Cell nuclei were labelled with DAPI (blue). Scale bars: A-D = 1 mm, E-H = 100 μm, I and L = 50 μm, J-K and M-N = 25 μm. Abbreviations: CV: coronary vessel, epi: epicardium, IVS: interventricular septum, myo: myocardium, PECAM: platelet endothelial cell adhesion molecule-1, ∆: <i>Myh10</i>∆.</p

<i>EHC</i> mutant embryo morphology.

<p>A: Heterozygous littermate at E13.5. B: <i>EHC</i> mutant displays hydrocephalus in the mesencephalic vesicle (white arrow). C: Heterozygous littermate at E15.5. D: <i>EHC</i> mutant at E15.5 displays reduced hydrocephalus (arrow), although oedema in the spinal cord region is apparent (arrowhead). E: Heterozygous littermate at E17.5. F: <i>EHC</i> mutant has dome-shaped head (arrow), consistent with developmental defects arising from embryonic hydrocephalus. Scale bars: 1 mm. Abbreviations: MV: mesencephalic vesicle.</p

Apoptosis in <i>Myh10∆</i> mutant hearts.

<p>A: TUNEL analysis (green) of <i>Myh10∆</i> heterozygous heart at E14.5 shown with nuclei stained with DAPI (blue). B: Single channel of TUNEL staining from (A). Apoptotic cells (arrowhead) are present along with autofluorescent erythrocytes (asterisk). C: Autofluorescence of erythrocytes (red) from (A). D: TUNEL analysis (green) of <i>Myh10∆</i> homozygous mutant heart at E14.5 shown with nuclei stained for DAPI (blue). E: Single channel of TUNEL staining from (D). Apoptotic cells (arrowhead) are present along with clusters of erythrocytes (asterisks). F: Autofluorescence of erythrocytes (red) from (D). G: Graph comparing mean number of TUNEL positive cells as a proportion of total cells in either the epicardium, or myocardial wall. Mean percentage of apoptosis in control = 0.4484% (+/- 0.3082%), and mutant epicardium = 1.762% (+/- 0.662%) (unpaired 2-tailed Mann-Whitney U-test, p = 0.0744). Mean percentage of apoptosis in control = 0.4532% (+/- 0.204%), and mutant myocardial wall = 1.097% (+/- 0.2132%) (unpaired 2-tailed Mann-Whitney U-test, p = 0.0053). Three hearts per genotype and six non-consecutive sections per heart were analysed. H: Sagittal section of control and I: <i>Myh10∆</i> homozygous mutant hearts at E9.5 showing immunofluorescence for activated Caspase-3 (turquoise, arrows), with nuclei visualised with DAPI (blue). J: Graph comparing mean number of activated caspase-3 positive cells as a percentage of total cells in cardiac sections of control and mutant E9.5 hearts. Mean percentage of apoptosis in control = 0.435% (+/- 0.0.1579%), and mutant hearts = 0.4885% (+/- 0.1722%). Three hearts were examined per genotype, and three sections were analysed for each heart. There is no significant difference in apoptosis between the two genotypes (unpaired 2-tailed Mann-Whitney U-test, p = 0.9292). Error bars in G and J represent standard error of the mean. Scale bars: A-F = 25 μm, H-I = 50 μm.</p

NMHC IIB is the predominant form of NMII in the embryonic heart and epicardium.

<p>A-C: Immunohistochemical comparison of the three different NMHC II isoforms (green) in the <i>Myh10</i>∆/+ E14.5 heart. NMHC IIB is the predominant isoform, and shows intense staining in the epicardium (B, arrows). D-F: E14.5 embryonic lung tissue was used as a control for the NMII isoform immunohistochemical analysis (arrows). Cell nuclei are stained with DAPI (blue). G-I: Western blot analysis of NMII isoforms in E11.5 <i>Myh10</i>∆ heterozygous control and homozygous mutant whole heart protein extracts. Protein extract from adult mouse lung was run as a positive (+ve) control. NMIIA and IIB are found in relative abundance at this developmental stage in heterozygous hearts (G and H). As expected, NMIIB is absent from mutant samples (H, lane 3), which also show a reduction in NMIIA (G, lane 3). NMIIC is not detectable in either control or mutant samples (I). Protein extracts pooled from at least n = 3 hearts for each genotype. J-O: Immunocytochemistry for the predominant IIA and IIB isoforms in <i>Myh10</i>∆ heterozygote (J, K, M and N) and mutant (L, O) epicardial cell cultures from E11.5 heart explants. Note localisation of NMIIA to the periphery of epicardial cells (J, K, arrows), whilst NMIIB is localised throughout the whole cell body (M, N, asterisks), and is frequently associated with actin stress fibres (M, N, arrow). Consistent with western blot analysis (H), NMHC IIB is not detectable in mutant samples (O). Images representative of cell cultures obtained from four hearts per genotype. P: NMIIB immunofluorescence (green) in E11.5 control sample. NMIIB is expressed in the epicardium (arrow). Nuclei are labeled with DAPI (blue). Q: NMIIB expression in <i>Myh10</i>∆ homozygous mutant heart at E11.5. R: NMIIB immunofluorescence (green) at E14.5 in control sample. NMIIB is highly expressed in the epicardium (arrow). Nuclei are labeled with DAPI (blue). S: NMIIB expression in <i>Myh10</i>∆ homozygous mutant heart at E14.5. NMIIB is not detectable in <i>Myh10</i>∆ homozygous mutant samples, consistent with western blot (H) and cell culture results (O). Scale bars: A-F, J, L, M and O = 100 μm, K, N, P-S = 50 μm. Images in A-C and D-F obtained at same magnification. Abbreviations: ∆: <i>Myh10</i>∆.</p