Differential response of embryonic and fetal myoblasts to TGFβ: a possible regulatory mechanism of skeletal muscle histogenesis

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Abstract
Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGFβ. Under the conditions employed, TGFβ inhibited differentiation of 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 mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains 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 undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGFβ is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGFβ) can undergo differentiation, while fetal myoblasts are inhibited by TGFβ. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers. To test this model we developed an organ culture for limb buds which 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 were treated with TGFβ embryonic myotubes did form (as expected), but fetal myotubes never appeared. Conversely, when these cultures were treated with anti-TGFβ neutralizing antibodies, fetal myotubes developed earlier than in control cultures, suggesting that endogenously produced TGFβ may repress differentiation of fetal cells in vitro and, possibly, in vivo.