Abstract
The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.
Highlights
There is currently a significant focus on strategies to promote regeneration in adult mammals and a renewed interest in the mechanisms that underlie regeneration in urodele amphibians
The myotubes were effectively converted to mononucleate cells that proliferated in the blastema, and this process occurred in cells that were blocked from cell cycle reentry (Velloso et al 2000), showing that the two aspects of plasticity are not linked mechanistically
The striated myofibers readily attached to the culture dish (Figure 1; Figure S1) and were found to express myosin heavy chain (MHC) and titin after antibody staining
Summary
There is currently a significant focus on strategies to promote regeneration in adult mammals and a renewed interest in the mechanisms that underlie regeneration in urodele amphibians. A second aspect of plasticity is the ability of multinucleate skeletal muscle cells to fragment into viable mononucleate cells that contribute to the regenerate This process, sometimes referred to as cellularisation, was described in classical studies of limb regeneration (Thornton 1938; Hay 1959), but was first analysed with marked cells by implantation of cultured newt myotubes labelled by microinjection with a lineage tracer (Lo et al 1993) or by an integrated retrovirus (Kumar et al 2000). After amputation of the tail, the myofibers fragmented into viable mononucleate cells, establishing that cellularisation occurs during regeneration and contributes to the proliferative zone or blastema
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