3-D observation of actin filaments during cardiac myofibrinogenesis in chick embryo using a confocal laser scanning microscope

Using a confocal laser scanning microscope (CLSM), we observed subcellular three-dimensional (3-D) arrangements of actin filaments stained with fluorescein-labeled phalloidin during myofibrinogenesis of chick embryonic heart (7- to 13-somite stages). Serial optical tomograms were obtained from whole-mounted heart tubes and reconstructed into stereoscopic images. Development of myofibrils in myocardial differentiation considerably differed in inner and outer myocardial cell layers. In the outer layer, initial myofibrils appeared along cell membranes at the 8-somite stage. They increased rapidly and constituted network structures with spatial extension over cell-cell junctions. In the inner layer, myofibrils appeared at the bottom, facing the cardiac jelly, at the 10-somite stage, and, when the straight heart tube began to bend, they were already aligned circumferentially in the direction of the heart tube. Double staining of fluorescein-phalloidin and DiI [1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate; DiI-C18-(3)] of the looped heart revealed that while myocytes in the outer layer were round, those of the inner layer were spindle-shaped, and their long axes coincided with the circumferential direction. These results suggest that the circumferentially arranged myofibrils at the bottom of the inner layer may play an important role in the looping of the heart tube.