The importance of timing differentiation during limb muscle development

Background: Skeletal muscle of trunk, limbs and tongue develops from a small population of cells that originates from somites. Although promoters and inhibitors of muscle differentiation have been isolated, nothing is known about how the amplification of the muscle precursor pool is regulated; this amplification provides muscle mass during development. Furthermore, little is known about how cells accumulate in the pre-muscle masses in the limbs. We investigated the role of bone morphogenetic proteins (BMPs) and Sonic hedgehog (Shh) during proliferation, differentiation and positioning of muscle. Results: The proliferation of muscle precursors in limbs was linked to Pax-3 expression. Ectoderm removal downregulated Pax-3 expression, arrested proliferation and prematurely initiated muscle differentiation which exhausted the muscle precursor pool and prevented further muscle growth. BMP-2, BMP-4 and BMP-7 had a dose-dependent effect on pre-myogenic cells: low concentrations maintained a Pax-3-expressing proliferative population, substituting for ectoderm-derived proliferative signals and delaying differentiation, whereas high concentrations prevented muscle development, probably by inducing apoptosis. In the limb, Shh upregulated Bmp-2 and Bmp-7 expression which delayed muscle differentiation, upregulated Pax-3, amplified the muscle precursor population and stimulated excessive muscle growth. Conclusions: These data indicate that embryonic muscle growth requires muscle differentiation to be delayed. Muscle differentiation may occur through a default pathway after cells escape proliferative signals. Positioning of muscle is regulated by high concentrations of BMPs, thus a single type of signalling molecule can determine crucial steps in muscle development: when and where to proliferate, and when and where to differentiate.