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Mechanisms coupling heart function and cardiac morphogenesis can be accessed in lower vertebrate embryos that can survive to swimming tadpole stages on diffused oxygen. Forward genetic screens in Xenopus tropicalis have identified more than 80 mutations affecting diverse developmental processes, including cardiac morphogenesis and function. In the first positional cloning of a mutation in X. tropicalis, we show that non-contractile hearts in muzak (muz) embryos are caused by a premature stop codon in the cardiac myosin heavy chain gene myh6. The mutation deletes the coiled-coil domain responsible for polymerization into thick filaments, severely disrupting the cardiomyocyte cytoskeleton. Despite the lack of contractile activity and absence of a major structural protein, early stages of cardiac morphogenesis including looping and chamber formation are grossly normal. Muz hearts subsequently develop dilated chambers with compressed endocardium and fail to form identifiable cardiac valves and trabeculae.
Fig. 2. muzak is encoded by myh6. WISH shows myh6 expression in wild type heart (A, black arrow) and jaw muscle (white arrow) is diminished in muz (B). (C, D) myh6.2 is expressed in jaw muscle (white arrow) but not heart (black arrow), and is unaffected by the mutation. (E) Schematic showing domain structure of wild type X. tropicalis myh6 and the truncated protein lacking the myosin coiled-coil tail encoded by the muz allele. (F) Western blot analysis does not detect sarcomeric MHC protein in extracts of muzheart; silver stained loading control below. (Movie S2 and G) myh6 morphant hearts do not beat and show strong depletion of sarcomeric MHC protein relative to control morpholino-injected tadpoles; silver stained loading control below.
Figure 2. muzak is encoded by myh6 WISH shows myh6 expression in wild type heart (A, black arrow) and jaw muscle (white arrow) is diminished in muz (B). (C, D) myh6.2 is expressed in jaw muscle (white arrow) but not heart (black arrow), and is unaffected by the mutation. (E)
Schematic showing domain structure of wild type X. tropicalis myh6 and the truncated protein lacking the myosin coiled-coil tail encoded by the muz allele. (F) Western blot analysis does not detect sarcomeric MHC protein in extracts of muzheart; silver stained loading control below. (Movie S2 and G) myh6 morphant hearts do not beat and show strong
depletion of sarcomeric MHC protein relative to control morpholino-injected tadpoles; silver stained loading control below.
Figure 3. MHC genes expressed in stage 40 wild type and muz
heartsRT-PCR from isolated stage 40 hearts shows lower levels of
myh6 in muz; myh7B
and myh8 are unaffected. (A) myh6.2 mRNA
is not detected in wild type or mutant tadpole hearts or wild type adult
heart, although it is amplified from whole-embryo mRNA;
myh15 is expressed in adult but not stage 40tadpoleheart(B).
Figure 4. Muz hearts lack myofibrils3D confocal projections of wild type (A) and muz (B) hearts immunostained with the pan-sarcomeric MHC A4.1025 antibody (green) and counterstained with phalloidin (red). In wild type hearts, MHC and actin colocalize to myofibrils, while muz hearts show very little A4.1025 immunostaining and no fibrillar structures.
Figure 5. Altered chamber morphology in muz heartsCoronal plastic sections of stage 40 wild type and muz
hearts (top rows) numbered from ventral side of cardiac cavity, and
indicated by white lines in 3D models (bottom rows). m= myocardium, e= inner
endocardial tube, v= ventricle, ot= outflow tract , a= atrium. No blood
cells are seen in the muz sections due to lack of
circulation, and myocardial layer appears thinner throughout the
muz heart compared to wild type. The
muz ventricle is wider than in wild type (sections 7
and 11), while outflow tract and atrium are dilated (sections 14, 23 and
41). Abnormal muz chamber morphology is highlighted in 3D
projections of outlines of myocardium (A, C, E, G, red=ventricle,
blue=outflow tract, green=atrium) and endocardium (B, D, F, H, orange),
including elongated ventricle, dilated outflow tract (black arrowhead in E)
and narrow cardiac tube at AVC level (black arrow in G).
muz endocardium is very compressed with drastically
reduced lumen (white arrows in 23, F and H)
Figure 6. Muz hearts become dilated and lack valves and
trabeculaeCoronal plastic sections of stage 42 wt and muz hearts (top
rows) numbered from ventral side of cardiac cavity, and indicated by white
lines in 3D models (middle rows). v= ventricle, ot= outflow tract , a=
atrium. Wild type hearts show a spiral valve in the outflow tract (sections
14, 23, black arrows), and thickening of endocardium preceding
atrioventricular valve formation (section 23, black asterisk). Valve
formation is not detected in muz hearts, and endocardial
lumen is drastically reduced in outflow tract and AVC regions (white
arrowheads sections 54, 58, also compare models B and F). Endocardial
cushion formation in AVC can also be seen in transverse sections of stage 42
wild type (I, white arrowhead) hearts but not in muz (J).
Trabeculation has initiated in the wild type ventricle (I, black arrowheads)
but is absent in muz (J). At this stage the ventricular
myocardium has a vacuolated appearance in both wt and mutant embryos (I, J
black arrows). Middle two rows: 3D projections of outlines of myocardium (A,
C, E, G) and endocardium (B, D, F, H) highlight abnormal
muz chamber morphology; red = ventricle, green =
atrium, blue = outflow tract, orange = endocardium. Muz
ventricles are elongated relative to wild type (E, G white arrows). A narrow
tube connects muzventricle and atrium (section 54 and G,
black arrowheads; compare to 23, C).
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