N‐cadherin is required for the differentiation and initial myofibrillogenesis of chick cardiomyocytes

To investigate initial stages of cardiac myofibrillogenesis, heart-forming mesoderm was excised from stage 6 chick embryos and explanted on fibronectin-coated coverglasses. The explants were fixed at various times and immunofluorescently stained with antibodies to N-cadherin, α-catenin, β-catenin, sarcomeric myosin, pan and sarcomeric α-actinins, or rhodamine phalloidin. After 7 hours in culture the cells appeared epithelial. N-cadherin, α- and β-catenin, pan α-actinin, and F-actin showed circumferential localization at cell borders. No cells in the explant were positive for sarcomeric α-actinin or sarcomeric myosin at this stage. Sarcomeric α-actinin and sarcomeric myosin were detected around 10 hours after plating. Sarcomeric α-actinin initially appeared as small beads along thin actin filaments. Mature Z-lines began to be organized at 20 hours, at the same time the cells started to contract. When the rat monoclonal antibody NCD-2, which inhibits N-cadherin function, was added to the culture at early time-points, cells lost cell–cell contacts, became spherical in shape, and contained tangled actin fibers. The expression of sarcomeric α-actinin and sarcomeric myosin was suppressed. These results indicate that 1) the precardiac mesoderm explant cells differentiate and form well-organized myofibrils in culture, 2) N-cadherin–mediated cell–cell interactions are necessary for early differentiation of cardiomyocytes and organization of myofibrils. Cell Motil. Cytoskeleton 39:52–62, 1998.