M-TWIST EXPRESSION INHIBITS MOUSE EMBRYONIC STEM CELL-DERIVED MYOGENIC DIFFERENTIATION IN-VITRO

The mouse M-twist gene codes for a basic helix-loop-helix protein whic h was shown to be inhibitory for differentiation of myogenic cells in culture. Mouse embryonic stem (ES) cells of line BLC6 efficiently diff erentiating into skeletal muscle cells when cultivated as embryo-like aggregates (embryoid bodies) were stably transfected with the plasmid pME18s-twist containing the M-twist gene under the control of the modi fied SV40 early promoter SR alpha. Two pME18s-twist-expressing clones showed delayed and reduced skeletal muscle cell differentiation depend ing on the level of exogenous M-twist expression compared to control c ells. By morphological analysis using phase contrast microscopy and he matoxylin-eosin staining, the development of first myocytes and format ion of myotubes in embryoid body outgrowths of these clones were found to be delayed for about 3 days in comparison to control cells, Immuno fluorescence studies with a monoclonal antibody against sarcomeric myo sin heavy chain revealed that myogenic cells appeared in so-called myo genic centers showing a reduced number of myocytes and myotubes in the M-twist-expressing clones. Using RT-PCR analysis the expression of th e skeletal muscle determination genes myf5, myogenin, and MyoD as well as muscle-specific genes coding for the gamma-subunit of the nicotini c acetylcholine receptor and the cell adhesion molecule M-cadherin wer e found to appear with a delay of at least 1 to 4 days in the pME18s-t wist-transfected cells during the development of embryoid bodies. We c onclude that the constitutive expression of the mouse M-twist gene dur ing ES-cell-derived differentiation has an inhibitory effect on skelet al muscle cell development depending on the level of exogenous M-twist expression. (C) 1995 Academic Press, Inc.