A point mutation in Myh10 causes major defects in heart development and body wall closure

Abstract

Background: The 3 isoforms of nonmuscle myosin (NM) II (NMII-A, NMII-B, and NMII-C) play various roles during mouse embryonic development. Previous work, using knockout and hypomorphic mice, showed that Myh10 encoding myosin heavy chain II-B is critical for cardiac and brain development. Ablating or decreasing NMII-B by 80% results in cardiac (ventricular septal defect, double outlet of the right ventricle) and brain defects but not midline fusion defects. Neither NMII-A nor II-C seems to play roles in early myocardial development.

Methods and results: We had previously generated point mutant knock-in mice and now report novel findings as a result of expressing motor-deficient NMII-B at wild-type levels. Homozygous mice die at embryonic day 14.5 in cardiac failure, exhibiting abnormalities not seen in NMII-B null and hypomorphic mice: a failure in midline fusion resulting in a cleft palate, ectopia cordis, and a large omphalocele. Fusion of the sternum and endocardial cushions is impaired in the mutant mice associated with a failure in apoptosis of the mesenchymal cells. Failure to disassemble myocyte cell-cell adhesions during cardiac outflow tract development contributes to impaired outflow tract myocardialization and displacement of the aorta to the right ventricle.

Conclusions: Expression of motor-impaired NMII-B disrupts normal ventral body wall closure because of a dominant-negative effect. This is not because of the loss of NMII-B function but rather a gain-of-function resulting from prolonged cross-linking of NMII-B to actin filaments, thereby interfering with the dynamics of actomyosin cytoskeletal structure. Furthermore, impaired NMII-B motor activity inhibits outflow tract myocardialization, leading to mislocalization of the aorta.

Keywords: apoptosis; cell adhesion; double outlet right ventricle; ectopia cordis; hernia, diaphragmatic; nonmuscle myosin type IIA.

© 2014 American Heart Association, Inc.

Figures

Figure 1

Congestive Heart Failure and Midline Fusion Defects in BR709C/BR709C Mice. a,b. Representative images of wild-type (B+/B+, a) and BR709C/BR709C (b) mice at E14.5 showing generalized edema (white arrow) and an umbilical hernia (orange arrow) in a BR709C/BR709C mouse. c,d. H&E stained sagittal sections of E13.5 embryos show a herniated liver in B+/BR709C (c, arrow) and BR709C/BR709C (d, arrow) mice. e,f. H&E stained cross sections of E14.5 embryos show ectopia cordis in a BR709C/BR709C mouse (f, black arrow). A similar section from a B+/B+ mouse is shown in panel e. In 50% of BR709C/BR709C mice, the two halves of the lower sternum are widely separated (f, green arrows; compare to e, green arrow) allowing the heart to protrude outside the thoracic chamber. g,h. H&E stained cross sections of E14.5 embryos show a cleft palate in a BR709C/BR709C mouse (h, arrows). In the B+/B+ section (g) the two palatal shelves contact each other (arrow). Scale bars: a-f, 1 mm; g and h, 500 μm.

Figure 2

Defects in Diaphragm Development in B+/BR709C and BR709C/BR709C embryos. a-i. Immunofluorescence confocal images of E13.5 mouse sagittal sections near the middle of the torso stained for NMHCII-A (green) and striated muscle myosin (MF20, red) show loss of skeletal muscle cells in the lateral-most region of the diaphragm in B+/BR709C and BR709C/BR709C embryos (b,c, yellow boxes; enlarged in e,f). In the B+/B+ embryo skeletal muscle cells are numerous in this region (a, yellow box; enlarged in d). Skeletal muscle cells accumulated near the midline of the B+/BR709C and BR709C/BR709C diaphragm (b,c, white boxes; enlarged in h,i) compared to the B+/B+ diaphragm (a, white box; enlarged in g). DAPI (blue) stains nuclei. Scale bars: a-c, 200 μm; d-i, 50 μm.

Figure 3

Impaired Apoptosis in the Fusing Lower Sternum of BR709C/BR709C Embryos. a,b. H&E stained mesenchymal cells in the middle of the E14.5 fusing sternum show an extensive accumulation of apoptotic cells with condensed and/or fragmented chromosomes in B+/B+ mice (a, green arrows). Very few apoptotic cells are seen in BR709C/BR709C mice (b). c,d. Confocal images of TUNEL assays show apoptotic cells near the midline in the fusing sternum of B+/B+ mice (c, green) which are not seen in BR709C/BR709C mice (d). The insets (H&E images) indicate areas shown in panels c and d. e,f. Confocal images of the sternal area stained with antibodies for activated-caspase-3 (red) and p53 (green) from E14.5 mouse embryos show a decrease in caspase-3 positive cells in BR709C/BR709C mice (f, red) compared to B+/B+ mice (e, red). No difference in p53 staining was seen between B+/B+ and BR709C/BR709C mice (e,f, green).g-i. Confocal images of E14.5 mouse embryos stained with antibodies for NMHCII-A (g, red), II-B (h, red) and II-C (i) show that both NMHCII-A and II-B, but not II-C, are expressed in the fusing sternum. DAPI (blue) stains nuclei. Scale bars: a and b, 25 μm; c-i, 50 μm.

Figure 4

Defects in Fusion and Remodeling of the Atrioventricular Cushions in BR709C/BR709C Mouse Hearts. a-i. H&E stained heart sections of B+/B+, BR709C/BR709C, and B−/B− embryos show developmental progression of atrioventricular (AV) cushions from E11.5 to E14.5. E11.5 AV cushions show no differences in size, morphology and positioning between B+/B+ (a), BR709C/BR709C (b) and B−/B− (c) hearts. B+/B+ AV cushions fuse and start to elongate at E12.5 (d), and acquire mature mitral (MV) and tricuspid (TV) valve leaflets by E14.5 (g). BR709C/BR709C cushions remain unfused and show no sign of maturation at E12.5 (e) and E14.5 (h). The fusion of AV cushions in B−/B− hearts appears normal at E12.5 (f), however further maturation into cardiac valves is delayed at E14.5 (i) compared to the B+/B+ mouse (e). IC, inferior AV cushion; SC, superior AV cushion. j,k. TUNEL assay shows defective apoptosis in developing BR709C/BR709C cushions. Apoptotic cells are readily seen in B+/B+ cushions (j, green), but very few apoptotic cells are found in BR709C/BR709C cushions (k). DAPI (blue) stains nuclei. Scale bar: a-i, 40 μm; j,k, 25μM.

Figure 5

Defective Myocardialization of Developing Outflow Tract in BR709C/BR709C Mouse Hearts. a-c. Serial H&E stained heart sections from an E14.5 BR709C/BR709C embryo show abnormal configuration of the great arteries with DORV. AO, aorta; PA, pulmonary artery; RV, right ventricle. d,e. H&E stained sections of E11.5 mouse hearts show that the cardiac myocytes in the developing OFT are invading the underlying cardiac cushions (CC) in the B+/B+ mouse heart (d, arrow) but not in the BR709C/BR709C heart (e, arrow). f,g. Immunofluorescence confocal microscope images of the OFT from E11.5 mouse hearts stained with antibodies for NMHCII-B (green) and MF20 (red, marker for sarcomeric myosin indicating cardiac myocytes) show that the B+/B+ cardiac myocytes are invading the cardiac cushion (f, small arrows), but the BR709C/BR709C myocytes are not (g). h,i. Immunofluorescence images of the developing OFT from E11.5 mouse hearts stained with antibodies for N-cadherin (green) show that in the B+/B+ OFT there is no obvious enrichment of N-cadherin at the boundaries between cardiac myocytes (h). In contrast, in the BR709C/BR709C myocytes, N-cadherin is enriched at the cell-cell boundaries (i, arrows). DAPI (blue) stains nuclei. Scale bars: a-e, 200 μm; f and g, 50 μm; h and i, 10 μm.

Figure 6

Defective Myocardialization of the Developing Outflow Tract in B−/B− Mouse Hearts. a-d. Immunofluorescence confocal microscope images of E11.5 mouse cardiac outflow tracts stained with antibodies for desmin (a,c red, a marker for cardiac myocytes) or N-cadherin (a-d, green). N-cadherin localization shows that the cardiac myocytes are invading the underlying cardiac cushions in the B+/B+ mouse heart (a, red) but not in the B−/B− heart (c, red) causing a defect in OFT myocardialization in B−/B− mouse hearts. Staining of the cardiac intercellular adhesion molecule N-cadherin shows that in the B+/B+ OFT there is no obvious localization of N-cadherin at the boundaries between cardiac myocytes (a,b, green). In B−/B− OFT, N-cadherin is localized at the cell-cell boundaries (c,d, green) indicating a failure in disassembly of cardiac myocyte cell-cell adhesions. Scale bars: 10 μm.