The behaviour and commitment of myoblasts during mammalian skeletal muscle formation
During mammalian skeletal muscle development, muscle fibres form in a biphasic manner from the fusion of myoblasts. Primary fibres form first, which subsequently provide a surface for later secondary fibres to form on. The purpose of the present study was to successfully develop new and existing techniques and to employ them in order to study the commitment and behaviour of myoblasts during muscle development in mice. Following part 1; a general introduction into the development of skeletal muscle, the thesis is divided into two subsequent parts giving details of the investigations performed. In the main section (part 2) of this thesis, I investigated the commitment of myoblasts during the foetal development. It has been suggested, that separate populations of myoblasts are present, each committed to producing the different fibre types seen during development. The aim of this study was to see if different populations produced primary and secondary fibres, by seeing if clones of related cells were restricted to fusing with a single type of fibre. Following the injection of replication deficient retroviruses into the hindlimbs of Embryonic day (E)15 and El 7 foetal mice, cells became marked with the lac Z gene encoding for the enzyme [Special character omitted]-galactosidase. The infected cells, their descendants and the fibres they fused with could then be demonstrated histochemically. 83% of the clusters of marked fibres obtained following processing were found to contain both primary and secondary fibres as identified by electron microscopy. The clusters were assumed to be the result of the fusion of a single clone of cells. It was concluded that at these ages, a single population of cells contributes to primary and secondary fibres. Part 3 of the thesis describes a second, shorter study whereby the in vitro behaviour of El 7, El 9 and E21 myoblasts was investigated on artificial grooved substrata. Most cells on grooves with depths of 250nm-6um were found to align parallel with the direction of the grooves. Cells on the shallower grooves (40-140nm) either aligned parallel or perpendicular to the grooves. E21 cells however, orientated randomly on these groove sizes. It was generally concluded however, that myoblasts at the ages studied do align in grooves similar to those formed in vivo by adjacent primary and secondary fibres. It is suggested that grooves such as the ones mentioned may be a possible site for secondary myogenesis. The results of both my studies contribute to the current work being carried out on skeletal muscle development, and may also, in addition, provide useful information towards the development of myoblast transfer therapy, a possible treatment for Duchenne Muscular Dystrophy sufferers.