Résumé :
|
Communication n° 702. We previously demonstrated that the fetal vascular endothelial cells (FVECs) isolated from mouse embryonic muscles can be a source of myogenic progenitors (Le Grand et al, 2004). Indeed, these cells primarily expressing the endothelial markers CD34 and Flk1, are able to differentiate spontaneously and predominantly into skeletal myofibres in vitro. When transplanted into a dystrophic adult muscle, the FEVCs displayed a particularly high propensity to fuse among themselves or with the host myofibres. With the aim to unravel the mechanisms of myogenic fusion between FVECs, we are presently investigating their in vitro course of myogenic commitment and differentiation. FEVCs are purified from the limb muscles of 17-dpc mouse fetuses by immunomagnetic separation on the basis of CD34 and Flk1 expression. Myogenesis of cultured FVECs was monitored by RT-PCR, immunocytostaining and/or Western blotting techniques and compared with cultured fetal myoblasts. We designed an in vitro cell culture system in which the myogenic commitment is delayed. In that way, expression of the myogenic regulatory factors and vascular endothelial specific genes remains stable. By contrast, expression of the myogenic stem cell transcription factor, Pax7, diminishes along with the up-regulation of Wnt signalling. Fusion takes place between days 3 and 4, and differentiated myotubes appear after 5 days. FVECs express the muscle-specific intermediate filament protein Desmin before contacting each others. Then the adhesion protein M-Cadherin is immunolocalised in focal adhesion clusters. Myosin Heavy Chains Isoforms and Dystrophin proteins are visualised as indicators of a mature phenotype in multinucleated myotubes. The hypothesis that ionic currents are involved in the fusion process of FVECs is also investigated. In conclusion, we propose that vessels-associated foetal progenitors commit in the myogenic lineage using a very special pathway, resembling in some ways skeletal muscle regeneration.
|