No abstract

We investigated the Thy-1-like immunoreactivity during the development of chicken skeletal muscle using a group of monoclonal antibodies raised and characterised against purified chicken brain Thy-1. The immunoreactivity attributable to bona fide Thy-1 in muscle was present in nerves, connective tissue associated with the intrafusal capsule and blood vessels, and the extracellular matrix of the muscle fibres. During development there was no change in the staining of nerves, blood vessels or intrafusal capsules. However, the extracellular staining of muscle first appeared around hatching and gradually increased in intensity reaching maximal levels in the adult. The intensity of staining varied within and between the muscles examined. One of the antibodies (SB1 20.11) recognised an additional molecule that is not Thy-1 and that was localised in the cytoplasm of slow muscle fibres. This immunoreactivity was first detectable at E10 in all myotubes that contained both alkali and acid stable myosin ATPase activity (presumptive slow), but not in those myotubes with only alkali-stable myosin ATPase activity (presumptive fast). Thereafter, the staining increased to a maximum in the newly hatched animal and then decreased until reactivity was undetectable in the adult (greater than 25 weeks). All positive fibres initially stained with a uniform intensity but the time of commencement and the rate of loss of staining was variable. Those fibres that contained both acid stable and acid labile myosin ATPase activity lost the antigen much faster than the fibres containing only acid-stable myosin ATPase activity, which also tended to increase in intensity for a longer period. These may represent, respectively, the slow tonic type III fibres and the slow twitch type I fibres classified by Barnard et al. (1982).

We have established that the monoclonal antibody (MAb) AA21, raised against a crude sarcolemmal fraction prepared from adult chicken anterior latissimus dorsi muscle, recognizes the slow twitch/cardiac isoform of calcium ATPase. This was done using a combination of immunohistochemistry at the light and electron microscopic level, the change in the cell distribution in skeletal muscle during development, the molecular weight of the principal protein recognized in Western transfers, and direct comparison with another MAb of known specificity. The antigen is initially expressed by all myotubes at E10 and with development is gradually lost from all presumptive fast fibers. In addition to its immunoreaction and slow extrafusal skeletal muscle fibers, AA21 displays a highly selective immunoreactivity with a number of other cell types in different tissues. The antibody stains a subset of intrafusal muscle fibers and intestinal and arterial smooth muscle, but not venous smooth muscle. In the nervous system, a subpopulation of neurons is intensely stained, most neurons are faintly stained, and glia are not stained at all.