Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. I. Presence of immunofluorescent titin spots in premyofibril stages.

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Abstract
Our initial attempts to immunolabel intact myocardial walls of 4-12 somite stage chick embryos were hindered by the presence of the cardiac jelly that covers the inner myocardial wall surface and prevents the access of antibodies to that surface. We overcame this difficulty by treating the specimens with hyaluronidase, which made the cardiac jelly permeable to the antibodies. An additional nonionic detergent treatment made the two more cell layers of the myocardial wall accessible to the antibodies from both surfaces of the wall. Specimens treated in this manner were fluorescently labeled with antibodies to titin, myosin, or actin or with NBD-phallacidin for F-actin and examined as whole mount preparations or cut into semithin sections after resin embedding. These preparations and sections revealed that titin, a putative scaffolding protein of sarcomeres, is present in a punctate state and also in a diffuse form throughout the cytoplasm of cardiac myocytes in the premyofibril stages (4-7 somite stages) as well as in the early stages of myofibril formation. We interpreted the punctate and diffuse states to represent an aggregated state of several titin molecules, respectively. In the 4-7 somite cardiac primordia, myosin and actin show only a uniform labeling throughout the cytoplasm of the myocytes. These observations are in contrast to a previous report that titin and myosin are tightly linked during in vitro skeletal myofibrillogenesis (Hill, C. S., S. Duran, Z. Ling, K. Weber, and H. Holtzer, 1986, K. Cell Biol., 103:2185-2196). In the 8-II somite stage hearts, the number of individual titin spots rapidly reduces, while the number of myofibrils with periodically aligned titin spots increases, which strongly suggests that the titin spots are incoporated into the newly arising myofibrils. Titin spots were been as doublets only after titin spots were incorporated into the first myofibrils. However, the fact that the distance between the components of the narrowest doublet was close to the resolution limit of the light microscope left open the possibility that undiscernible doublets of sub-microscopic separations might exist in the premyofibril stages. The myosin labeling revealed the sarcomeric periodicity in an earlier stage of myofibril development than the F-actin labeling. In addition, we made two morphogenic observations. One was that immunolabeling of titin and ventricle-specific myosin always occurred for a short distance beyond the physical boundaries of the 8-9 somite stage heart troughs, indicating that myogenic cells occur in a wider area than that which eventually develops into the heart wall. Another observation was that in the 5-6 somite stage, the endothelial cells of the endocardium were positively labeled for ventricle-specific myosin, which implies that the endocardium is more closely related to the myocardium in origin than hitherto considered.