Figure 1

Immunostaining pattern of fast myosin and Igf-2 in the wild type (C57BL10) mouse embryo. (A, E) Pan-myosin (MF20) staining and (B, F) Pax 7 respectively, show the location of differentiated myotubes and undifferentiated myoblasts at E11.5 in intercostal (ic), limb (l), trunk muscles (t) and upper proximal limb muscles (pr); (C, G) Igf-2 is absent from E11.5 embryonic myotubes, (D, H) a small number of secondary myotubes stain with My32 (fast MyHC). (I) Immunoblot of whole embryo protein (E11.5 to E17.5) showing fast MyHC (My32), Igf-2 and tubulin. FMyHC increases with stage. Igf-2 peaks at E15.5 and is downregulated in muscle at E17.5. Igf-2 is also present in non-muscle tissues (including liver, kidney, heart, gut) between E11.5 and E17.5. (J-M, O-Z) Imunostaining pattern of Igf-2 and FMyHC in upper proximal limb muscles. (J-L) E13.5 embryos show an overlapping pattern of Igf-2 (J, L) and fast myosin (K, M) in secondary myotubes. Enlarged areas (L, M) show secondary myotubes containing both Igf-2 and fast myosin (indicated by white arrows). (N-R) At E15.5 Igf-2 and fast Myosin are localised to ~half of myotubes and show a strikingly similar pattern. (N-O) White arrows, double positive myotubes. Black arrows FMyHC positive, Igf-2 negative muscle group. (Q-R) most Igf-2 positive myotubes also stain for FMyHC (Black arrows, double positive myotubes). (N) Igf-2 mRNA is expressed predominantly in secondary myotubes broadly coincident (O) with the Igf-2 and (P) fast Myosin protein localisation patterns. (S-V) At E17.5 Igf-2 staining (S, U) is almost undetectable. Fast Myosin (T, V) staining shows increased heterogeneity compared to earlier stages (black arrows, reduced FMyHC staining). Skeletal muscles of day 1 pups (P1) contain (W, Y) no Igf-2 and (X, Z) have reduced and heterogenous fast Myosin staining (X, Z). (S-T, W-X) intercostals (ic); U-V, Y-Z upper proximal (del, deltoid) muscles. Images shown in A-D (E-H); J-K (L-M); O-P (Q-R); S, T and U, V; W, X and Y-Z are adjacent sections. Immunostaining patterns were confirmed by at least three different runs on separate embryos (many more for the Igf-2 pattern) per stage. Images shown are fully representative of each run. Comparison of FMyHC, Igf-2, Pax 7 and MF20 was achieved by staining separate adjacent sections for each antibody in the same run. Negative second antibody controls were included in each run. Peptide absorption was carried out at each stage for the Igf-2 pattern. Magnification bars indicate 10 μC on all images.