Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation.

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.

Delta 1-activated notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis.

The myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. In this paper, we show that Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signalling is known to prevent differentiation in different systems and species, we sought to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. We conclude that activation of Notch signalling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stage.

Paraxis is expressed in myoblasts during their migration and proliferation in the chick limb bud.

The Paraxis gene encodes a basic helix-loop-helix (bHLH) transcription factor expressed in paraxial mesoderm and whose mutant displays an inability to form epithelial somites. We analyzed the later expression pattern of Paraxis transcripts in the chick limb. Paraxis transcripts are located in the Pax3-expressing myoblasts migrating from the somites and persist in proliferating myoblasts in the limb bud. Moreover, the QH1 antibody, which recognizes quail endothelial cells, reacts with a subset of the Paraxis/Pax3 expressing cells in the quail limb. Paraxis is then down-regulated during muscle differentiation.