Figure 3

Differentiated C2MIG myotubes, but not undifferentiated myoblasts, show (A) strong immunostaining (brown) for IGF-2, (Counterstain is Haematoxylin (blue)) and (B) bright green fluorescence (GFP). DAPI counterstained nuclei (blue) reveal that undifferentiated cells do not express the GFP transgene. Spontaneous differentiation into fast MyHC positive myotubes occurred (C) rarely in C2MG (B9) cells but more frequently (D) in C2MIG (H19) cells cultured under non-permissive conditions (DMF12 and 10% FCS) for 5 days. A majority of C2MG (B9) and a minority of C2MIG (H19) myotubes formed are fast MyHC antibody negative (C-D; white arrows). A minority of C2MG myotubes and a majority of C2MIG myotubes, are FMyHC positive (C-D; Black arrows). Total myotube numbers: MG, 16.4 ± 3.2; MIG 19.1 ± 3.5 ; Students t-test, p = 0.76. The average number of nuclei (per myotube) was 5.48 ± 1.17 (s.d.). (E) Immunoblotting shows a substantial increase in FMyHC in C2MIG compared to C2MG under these conditions. (F) After 3 days in differentiation permissive media there was a 50% increase (*, p = 0.003) in the proportion of FMyHC positive myotubes in C2MIG (H19) cells compared to C2MG (B9) and a concomitant 30% reduction (p = 0.05) in the proportion of SMyHC positive myotubes in C2MIG (H19) compared to C2MG (B9). Total myotube number was not altered (p = 0.47). Graph shows the mean and standard deviation of 3 separate experiments, each performed in duplicate. Immunoblot shows the increase in FMyHC in C2MIG compared to C2MG under the same regime. (G) E17.5 MIG embryos show a very strong generalised up-regulation of FMyHC in all skeletal muscles. (H) In MIG intercostals muscles almost all myotubes have intense FMyHC staining compared to (I) MG embryos at E17.5. (J) A probe recognising the intersection between the pmCK and Igf-2 was used to genotype MIG transgenic positive (track 4) and MIG transgene negative (track 3) mice. Track 1, MG and track 2, MIG plasmid PCR, demonstrate the specificity of the MIG PCR primers. M - 1Kb ladder. PCR to genotype MIG transgene positive (track 4) and MIG transgene negative (track 3) mice. Primer set mCKIgf-2 1. Data shown is of one MIG+ and one MG+ E17.5 embryo. The phenotype is representative of 3 other E17.5 MIG+ embryos immunostained for FMyHC. 2 MG+ E17.5 embryos and 4 E17.5 MIG- littermates showed an identical FMyHC phenotype to WT C57BL10 E17.5 embryos (Figure 3I and Figure 1).