Fig. 1. Myogenesis in primary cultures derived from Drosophila embryos. (A-F) Fluorescence micrographs of freshly dissociated cells obtained from Drosophila gastrulating embryos carrying rp298-lacZ immediately following plating. Cells are stained using DAPI for nuclei (A, and blue in C), and antibodies targeting Dmef2 (B, and green in C,E), β-galactosidase (D, and red in E,F) and Lmd (green in F). (G-I) Primary cells derived from Dmef2-Gal4 embryos were mixed with those from UAS-2EGFP and allowed to develop for 48 hours at 18°C in culture. The GFP-positive myotube (G, and green in I) resulted from fusion of cells supplied by two genetically different embryos, and the GFP-negative one is most likely derived from the fusion of cells from two genetically identical embryos. Both myotubes expressed Mhc (H, and red in I). (J-L) Multinucleated myotubes are identified by staining for Mhc (J, and green in L) and for Dmef2 (K, and red in L). Note that not all Dmef2-positive nuclei are found in myotubes. The percentage of myotube nuclei among the total number of Dmef2-positive nuclei was used as an indication of the amount of fusion. (M) Time-course of myoblast fusion at 18°C and 25°C. Primary cell cultures were fixed and stained for Dmef2, Mhc or Actin at the times indicated. The number of Dmef2-positive nuclei was counted using Autoscope and Metamorph software. The number of nuclei in the myotubes was determined manually. The percentage of myotube nuclei was estimated by the number of myotube nuclei among the total Dmef2-positive nuclei, and used as an indication of the extent of myoblast fusion. Each point represents the average results of two or three trials. Arrows point to the time when fusion is nearly complete (pink for 25°C and blue for 18°C). (N) Fluorescence micrograph of a primary myotube from a 2-day culture at 18°C stained for Mhc. The white arrowhead points to the immature myofibril that formed along the side of the myotube. (O-R) Primary myotubes from 11-day cultures at 18°C, stained for Mhc (O), Actin (as detected using phalloidin) (P), Actn (Q) and Tropomyosin (R). The short arrow in O indicates the bundled myofibrils. Scale bars: 20 µm, in A for A-L and in N for N-Q.