Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

• Published in: Developmental biology Vol. 48; no. 2; pp. 284 - 307
• Main Authors: Turner, David C., Gmür, Rudolf, Lebherz, Herbert G., Siegrist, Marianne, Wallimann, Theo, Eppenberger, Hans M.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 1976
• Subjects: animal science; livestock; zoology
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/0012-1606(76)90091-9

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10 −5 M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.