Cardiac differentiation in Xenopus laevis: Identification and expression analysis of a cardiac myosin heavy chain gene

To study Xenopus cardiogenesis at the molecular level, a cardiac myosin heavy chain cDNA, CMHC, was isolated and used as a molecular marker for cardiac differentiation. The expression of CMHC transcripts during development was examined using reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. The initial expression of CMHC occurs in the dorsal mesodermal mantle of early gastrula stage embryos. CMHC transcripts are detectable in the cardiac mesoderm at the early neurula stage and are expressed solely in the heart from mid-neurula to swimming tadpole stage. These results demonstrate that muscle-specific gene expression, and thus, myogenic differentiation, in the primitive myocardium has begun by the early neurula stage, approximately 30 hours before the heart beat begins. Xenopus, therefore, is similar to amniotes and mammals in that cardiac precursor cells begin to express muscle-specific markers soon after commitment to the cardiac myocyte lineage. Furthermore, the expression pattern of CMHC in the developing myocardium revealed that the cardiogenic differentiation program in Xenopus is biphasic. However, CMHC expression is not exclusive to the cardiac lineage; CMHC is expressed in the differentiating somite up to mid-neurula stage and in a subset of differentiating head muscles of the swimming tadpole.;CMHC expression is induced in blastula stage animal cap ectodermal cells injected with synthetic MyoD or Myf5 RNA suggesting that the CMHC gene can be regulated, either directly or indirectly, in non-cardiac cells by these skeletal-muscle specific transcription factors. To examine the ability of skeletal muscle transcription factors to regulate gene expression in the myocardium, Myf5 protein was ectopically expressed in the developing heart by microinjection of Myf5 RNA into a single blastomere of the eight-cell stage embryo. The ectopic expression of Myf5 induces skeletal-muscle specific gene expression in the myocardium and disrupts heart tube morphogenesis and function, demonstrating that cardiac precursor cells are sensitive to the effects of Myf5 expression.