Figure 1.

Ezh2 is expressed early in the myotomal compartment of developing somites and in undifferentiated skeletal myoblasts. (A) Myogenin (left panels) and Ezh2 (right panels) transcripts were analyzed by in situ RNA hybridization on sections from E9.5, E11.5, and E15.5 mouse embryos. In a sagittal section at E9.5, myogenin mRNA was detected in myotomes of the developing somites (s). (nt) Neural tube; (t) tail; (ov) otic vesicle; (ba) branchial arch; (h) heart. Ezh2 mRNA was detected in all structures at a high level. In transverse sections at E11.5, myogenin was detected in developing myotomes (myo; arrows) and at a low level in limb buds (lb). (li) Liver; (h) heart; (nt) neural tube. At E11.5, Ezh2 mRNA levels have decreased significantly from E9.5. Ezh2 was detected in neural tube, liver, limb buds, and paraxial mesoderm. In a transverse section at the level of the jaw (j) at E15.5, myogenin mRNA was detected in tongue (to) muscle, shoulder muscle (shm), pectoral muscle (pm), and all other skeletal muscle. (sg) Salivary gland. At E15.5, Ezh2 mRNAs were only detected in thymus (th). (B) Mouse primary muscle satellite cells were cultured in either growth conditions (P, proliferating) or, once confluent, induced to differentiate for 0, 24, and 48 h. The RNA was isolated and RT-PCR was performed with specific primers for Ezh2, MCK, and GAPDH. (C) Immunoblot of muscle satellite cell extracts with Pax7, MHC, and tubulin antibodies at different differentiation stages. (D) C2C12 proliferating myoblasts (MB) and differentiated myotubes (MT) were immunostained for Ezh2 and nuclei visualized with DAPI. (E) Nuclear and cytoplasmic extracts of proliferating myoblasts (MB) and myoblasts at different stages of differentiation (12, 36, and 48 h in differentiation medium) were fractionated and immunoblotted for Ezh2 and tubulin.