Mouse limb muscle is determined in the absence of the earliest myogenic factor myf-5

myf-5 is the only member of the MyoD family of myogenic regulatory genes to be expressed in the mouse embryo prior to muscle cell differentiation. We have used the developing limb as a model in which to follow the formation of peripheral muscle, to address the question of whether myogenic precursor cells are already present in the limb bud before expression of myf-5. The lacZ reporter gene has been introduced into the myf-5 gene by homologous recombination so that its expression is under the control of the endogenous myf-5 locus. beta-Galactosidase (beta-gal) coloration provides a sensitive assay for myf-5+ cells. Embryos were generated from embryonic stem cells carrying this mutation, and the appearance of beta-gal+ (myf-5+) cells was followed during limb development in vivo. Limb buds, at a stage when they are beta-gal-, were cultured in vitro. After several days, beta-gal+ cells accumulated in the premuscle mass. We conclude that determined muscle precursor cells in the limb bud do not initially express any member of the MyoD family. Furthermore, myogenic precursor cells in the somite, which, according to the avian model, migrate from the ventral/lateral edge of the dermomyotome to form peripheral muscle masses, are also negative for these factors.