Three-dimensional observation with a confocal scanning laser microscope of fibronectin immunolabeling during cardiac looping in the chick embryo

From the beginning of cardiac myofibrillogenesis in the chick embryo, developing myofibrils at the bottom of the inner myocardial cell layer facing the cardiac jelly are already aligned circumferentially in the direction of the heart tube. To elucidate the mechanism of this alignment, we investigated the temporal and spatial expression of fibronectin and its relationship to actin filaments before and during looping (4- to 13-somite stages) by using a confocal scanning laser microscope. Serial optical tomograms were obtained from whole-mounted heart tubes stained with fluorescein-conjugated antibody against cellular fibronectin and rhodamine-conjugated phalloidin. Before looping (4- to 7-somite stages), particulate and speckled fibronectin formed loose networks. At the onset of looping (8- to 9-somite stages), fine fibrils of fibronectin appeared. They became dense and were arranged circumferentially in the direction of the heart tube. They were aligned parallel with the thick actin bundles that appeared as an initial stage of developing myofibrils. During looping, (10- to 13-somite stages), fibronectin fibrils were fragmented and showed a speckled pattern, while the number of circumferentially aligned mature striated myofibrils increased. These observations suggest that the temporal arrangement of fibronectin fibrils at the beginning of looping plays a role in the circumferential alignment of developing myofibrils.