DIFFERENTIAL RESPONSE OF EMBRYONIC AND FETAL MYOBLASTS TO TGF-BETA - A POSSIBLE REGULATORY MECHANISM OF SKELETAL-MUSCLE HISTOGENESIS

Embryonic and fetal skeletal myoblasts were grown in culture in the pr esence of TGF beta. Under the conditions employed, TGF beta inhibited differentiationof fetal but not of embryonic myoblasts, To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesoder mal cells in the proximal region of the limb bud at the time of primar y fiber formation. BrdU labeling and immunostaining for myosin heavy c hains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers fist appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers , suggesting that the latter may be a source of mitogens for undiffere ntiated myoblasts. Coculture experiments supported this hypothesis, sh owing that medium conditioned by fiber-containing explants can stimula te myoblast proliferation.Taken together these data suggested a possib le mechanism for the regulation of muscle fiber formation. The model a ssumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryoni c myoblasts (which do not respond to TGF beta) can undergo differentia tion, while fetal myoblasts are inhibited by TGF beta. Once formed, pr imary fibers may stimulate a new wave of proliferation in fetal myobla sts, in order to expand the pool of cells needed to form secondary fib ers. o test this model we developed an organ culture for limb buds whi ch resulted in the production of myotubes with a phenotype similar to embryonic (primary) and fetal (secondary) fibers, roughly at the time when primary and secondary fibers form in vivo. When these cultures we re treated with TGF beta, embryonic myotubes did form (as expected), b ut fetal myotubes never appeared. Conversely, when these cultures were treated with anti-TGF beta neutralizing antibodies,fetal myotubes dev eloped earlier than in control cultures, suggesting that endogenously produced TGF beta may repress differentiation of fetal cells in vitro and, possibly, in vivo.