IDENTIFICATION OF A NOVEL MARKER FOR PRIMORDIAL SMOOTH-MUSCLE AND ITSDIFFERENTIAL EXPRESSION PATTERN IN CONTRACTILE VS NONCONTRACTILE CELLS

The assembly of the vessel wall from its cellular and extracellular ma trix components is an essential event in embryogenesis. Recently, we u sed the de scending aorta of the embryonic quail to define the morphol ogical events that initiate the formation of a multilayered vessel wal l from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S, Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We gene rated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytologi cal and biochemical methods, The 1E12 antigen colocalizes with the act in cytoskeleton in smooth muscle cells grown on planar substrates in v itro; in contrast, embryonic vascular smooth muscle cells in situ cont ain 1E12 antigen that is distributed in threadlike filaments and in cy toplasmic rosette-like patterns, Initial biochemical analysis shows th at the 1E12 mAb recognizes a protein, M-r=100,000, in lysates of adult avian gizzard. An additional polypeptide band, M-r=40,000, is also re cognized in preparations of lysate, when stronger extraction condition s are used, We have identified the 100-kD polypeptide as smooth muscle oc-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we c onstructed a single chain antibody (sFv) using recombinant protein tec hnology. The sFv recognizes a single 100-kD protein in gizzard lysates . Additionally, the recombinant antibody recognizes purified smooth mu scle alpha-actinin. Our results suggest that the 1E12 antigen is a mem ber of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to t he smooth muscle lineage.