Fig. 4. (A) A single normal muscle fibre isolated from the flexor digitorum brevis muscle of a normal mouse. The fibre was maintained in culture for 48 h in the presence of bromodeoxyuridine (BrdU) to label DNA synthesis. Two satellite cells are adherent to the fibre; two additional cells (not identifiable with this staining technique) lie on the surface of the culture dish and have also incorporated BrdU during culture. Note that the post-mitotic nuclei within the muscle fibre have not incorporated BrdU. (Original micrograph x60.) (B) A single normal muscle fibre with BrdU-positive satellite cells beginning to migrate away from the fibre. Again, myonuclei inside the muscle fibre are not BrdU-positive. (Original magnification x140.) A and B after (Anderson and Pilipowicz, 2002). (C) A single normal muscle fibre prepared by immunostaining to demonstrate myogenin expression in activated satellite cells that are undergoing myogenesis on the fibre. The satellite cells have likely divided since the fibres were originally plated for culture, as they are present in pairs and quartets on the fibre surface. Note that the myofibre nuclei do not express myogenin, a muscle-specific regulatory gene expressed during the differentiation phase of myogenesis. (Original magnification x140.) (D) A selected field of myotubes in tissue culture, showing myogenin protein expression by immunostaining and fluorescence microscopy. Cultures are plated satellite cells isolated from skeletal muscle, and may contain `contaminating' cells of the fibroblast, adipocyte and endothelial lineages that are only partly distinguishable from myogenic cells in unstained cultures using structural and behavioural phenotypes. Myotubes form in culture as a result of cell fusion events, meaning that many myogenic cells were contained in a culture. Only those mononuclear cells expressing myogenin would be identified as myogenic. (Original magnification x140.) (E) A single dystrophic muscle fibre isolated from the flexor digitorum brevis muscle of an mdx dystrophic mouse, showing nuclei stained with DAPI (Z. Yablonka-Reuveni and J. E. Anderson, unpublished data). Nuclei are present within the fibre in one of three phenotypes. In the longitudinal regions or segments that have not undergone degeneration and regeneration, the nuclei are apparently `jumbled' and mainly appear out of the plane of focus. In other longitudinal segments of the fibre, the myonuclei are displayed as rows of centrally placed nuclei; these segments have undergone a cycle of degeneration and regeneration, and these central myonuclei originated from the fusion of satellite cell progeny into the muscle fibre remnant. The same regenerated segments are `decorated' with satellite cells, the nuclei of which appear at the periphery of the fibre. These cells lie outside the sarcolemma and within the external lamina that is present on these isolated fibres (Wozniak et al., 2003) and have contributed, through proliferative events, the myonuclei that lie within the adjacent regenerated segments. Note that this technique does not identify the possible heterogeneity between satellite cells regarding the number of cell cycles that have passed, and that the `parent' satellite cell may not lie in the same segment as the central myonucleus that was generated by the previous cell cycle. (Original magnification x140.)