M-twist expression inhibits mouse embryonic stem cell-derived myogenic differentiation in vitro

The mouse M-twist gene codes for a basichelix-loop-helix protein which was shown to be inhibitory for differentiation of myogenic cells inculture. Mouse embryonic stem (ES) cells of line BLC6 efficiently differentiating into skeletal muscle cells when cultivated as embryo-like aggregates (embryoid bodies) were stably transfected with the plasmid pME18s-twist containing the M-twistgene under the control of the modified SV40 earlypromoter SPα. Two pME18s-twist-expressing clones showed delayed and reduced skeletal musclecell differentiation depending on the level of exogenous M-twist expression compared to control cells. By morphological analysis using phase contrast microscopy and hematoxylin-eosin staining, thedevelopment of first myocytes and formation of myotubes in embryoid body outgrowths of these clones were found to be delayed for about 3 days in comparison to control cells. Immunofluorescence studies with a monoclonal antibody against sarcomeric myosin heavy chain revealed that myogenic cellsappeared in so-called myogenic centers showing a reduced number of myocytes and myotubes in the M-twist-expressing clones. Using RT-PCR analysis the expression of the skeletal muscle determinationgenes myf5, myogenin, and MyoD as well as muscle-specific genes coding for the γ-subunit ofthe nicotinic acetylcholine receptor and the celladhesion molecule M-cadherin were found to appearwith a delay of at least 1 to 4 days in the pME18s-twist-transfected cells during the development of embryoid bodies. We conclude that the constitutive expression of the mouse M-twist gene during ES-cell-derived differentiation has an inhibitoryeffect on skeletal muscle cell development depending on the level of exogenous M-twist expression.