Expression of several muscle-specific genes during differentiation of cultured quail pineal body cells under artificial (high NaCl) conditions.

A change in cell differentiation of pineal body cells to cells with myogenic features can be induced when such cells are cultured with stepwise increases in the NaCl concentration up to a 125 mM NaCl excess. Further differentiation can be achieved by changing the culture medium to Cosmedium (serum-free) with the same high NaCl concentration. This system is a good candidate as an in vitro model for the commitment of pre-myoblastic cells to myogenic cells, and for studies on early phases of myogenic differentiation, since the myogenic fate is not selected under conventional isotonic culture conditions. Our first step was to analyze the expression schedule of an intermediate filament protein, desmin, as an early stage marker of muscle differentiation. Desmin expression was detected at a low level in mononucleated cells during early differentiation stages (two days in medium with a 50 mM excess of NaCl), and a higher order of expression was observed in later stages in 20-40% of cells corresponding to myotubes. Expression of qmf1 and qmf2 mRNA, quail counterparts of MyoD and myogenin, respectively, was detected in two different stages: in the pineal body of 9-day-old embryos and in myogenically differentiated pineal cells, by Northern blotting and RT-PCR analysis. When pineal cells were transferred to the culture system, qmf1 and qmf2 expression was not present in isotonic medium and no myogenic differentiation occurred under these conditions. However, they reappeared and increased during the final stages of myogenic differentiation in the high NaCl medium. Furthermore, by use of muscle-specific domain (MSD)-specific antibodies we found the expression of a muscle-specific neural cell adhesion molecule (NCAM) species at the late stages of differentiation. These results show that the gene expression and mRNA splicing patterns of the cells changed from pineal body-specific to muscle-specific. On the basis of these results, we conclude that this system is appropriate for studying the differentiation of non-muscle cells to muscle cells.