MYOBLAST DIFFERENTIATION DURING MAMMALIAN SOMITOGENESIS IS DEPENDENT UPON A COMMUNITY EFFECT

The differentiation potential of early mammalian myogenic cells was te sted under clonal culture conditions. Cells were isolated from paraxia l mesoderm and limb buds of transgenic mouse embryos at 9.5 days after conception and grown in culture at clonal density either on collagen- coated dishes or on various feeder cell layers. The transgene used con tained a reporter gene encoding beta-galactosidase with a nuclear loca lization signal under the control of regulatory sequences from the gen e for fast myosin light chain 3, so that beta-galactosidase staining i ndicated the presence of differentiated muscle cells. After 5 days in culture, the number and size of beta-galactosidase-positive (beta-gal( +)) clones were recorded. Cells isolated from somites I-V (the last fi ve somites to have formed) or from unsegmented paraxial mesoderm did n ot give rise to any beta-gal(+) clones. Cells isolated from somites VI -X or from the forelimb bud gave rise to beta-gal(+) clones, but only on feeder cells. Cells from somites XI or older gave rise to beta-gal( +) clones independently of the substrate. However, when cells isolated from unsegmented paraxial mesoderm or somites I-V were cultured with nontransgenic cells from the trunk (including neural tube and notochor d), differentiation occurred on condition that the cells were in a thr ee-dimensional aggregate, even though their specific position in the s omite had been lost. By culturing explants ranging in size from 1 to < 100 cells in the presence of an inhibitor of cell division, we determ ined that a minimal number of 30-40 cells is required for mesodermal c ells to differentiate.