Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation.

In whole mount preparations of the 9 somite stage chick embryonic hearts that were immunofluorescently double labeled for titin and alpha-actinin, presumptive myofibrils were recognized as rows of several periodically aligned titin spots. Within these titin spots, smaller alpha-actinin dots were observed. These periodical arrangements of titin spots and alpha-actinin dots were not found in the 7 somite stage hearts. In wide myofibrils in the 10 somite stage hearts, the alpha-actinin dots and titin spots simultaneously became 'lines.' To study the ultrastructural features of the titin-positive regions in the 6-9 somite stage hearts, the thoracic portions of the embryos were immunofluorescently labeled for titin and embedded in resin. Ultrathin sections were mounted on electron microscopic grids and examined in immunofluorescence optics. The titin-positive regions thus identified were then examined in the electron microscope. No readily discernable specific ultrastructural features were found in titin-positive regions of the 6 somite stage cardiac primodia. Examination of the sections of the 9 somite stage hearts, on the other hand, revealed the occasional presence of small dense bodies, Z bodies, in the titin-positive regions. These observations strongly suggest that these Z bodies are the ultrastructural counterparts of the alpha-actinin dots seen by immunofluorescence optics and that they are formed nearly at the time of the formation of the first myofibrils. In some of the nascent myofibrils the Z bodies were found to be considerably narrower than the myofibrils, implying that the Z bodies are required not for the assembly of myofibrils per se but for their stabilization. Immunofluorescent labeling for titin and alpha-actinin revealed that the length of the shortest sarcomeres in the first myofibrils is approximately 1.5 micron, approximately the width of the A bands of mature myofibrils. The possibility that the A bands might define the initial length of nascent sarcomeres was indicated.