The cellular mechanism by which the dermomyotome contributes to the second wave of myotome development

We have shown that a subset of early postmitotic progenitors that originates along the medial part of the epithelial somite gives rise to the primary myotome (Kahane, N., Cinnamon, Y. and Kalcheim, C. (1998). Mech. Dev. 74, 59-73). Because of its postmitotic nature, further myotome expansion must be achieved by cell addition from extrinsic sources. Here we investigate the mechanism whereby the dermomyotome contributes to this process. Using several different methods we found that cell addition occurs from both rostral and caudal edges of the dermomyotome, but not directly from its dorsomedial lip (DML). First, labeling of quail embryos with [3H]thymidine revealed a time-dependent entry of radiolabeled nuclei into the myotome from the entire rostral and caudal lips of the dermomyotome, but not from the DML. Second, fluorescent vital dyes were injected at specific sites in the dermomyotome lips and the fate of dye-labeled cells followed by confocal microscopy. Consistent with the nucleotide labeling experiments, dye-labeled myofibers directly emerged from injected epithelial cells from either rostral or caudal lips. In contrast, injected cells from the DML first translocated along the medial boundary, reached the rostral or caudal dermomyotome lips and only then elongated into the myotome. These growing myofibers had always one end attached to either lip from which they elongated in the opposite direction. Third, following establishment of the primary myotome, cells along the extreme dermomyotome edges, but not the DML, expressed QmyoD, supporting the notion that rostral and caudal boundaries generate myofibers. Fourth, ablation of the DML had only a limited effect on myotomal cell number. Thus, cells deriving from the extreme dermomyotome lips contribute to uniform myotome growth in the dorsoventral extent of the myotome. They also account for its expansion in the transverse plane and this is achieved by myoblast addition in a lateral to medial direction (from the dermal to the sclerotomal sides), restricting the pioneer myofibers to the dermal side of the myotome. Taken together, the data suggest that myotome formation is a multistage process. A first wave of pioneers establishes the primary structure. A second wave generated from specific dermomyotome lips contributes to its expansion. Because dermomyotome lip progenitors are mitotically active within the epithelia of origin but exit the cell cycle upon myotome colonization, they can only provide for limited myotome growth and subsequent waves must take over to ensure further muscle development.