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Figure 3 | BMC Developmental Biology

Figure 3

From: Hedgehog can drive terminal differentiation of amniote slow skeletal muscle

Figure 3

Shh promotes slow differentiation. A. Mouse C2X myoblasts were treated with control QT6 or ShhQT6 conditioned medium for two days growth and three days differentiation. Dual immunofluorescent analysis with pan MyHC (A4.1025, upper panels) and slow MyHC (A4.840, lower panels). Insets: anti-slow MyHC monoclonal antibodies A4.840 and BA-D5 confirm the induction of slow MyHC [61]. B, C. The number of differentiated myocytes, i.e. nuclei in MyHC-positive cytoplasm (B) and the proportion of differentiated myocytes expressing slow MyHC (C) was determined under various treatment regimes. Shh conditioned medium significantly increased (p < 0.001, t-test, n = 18 replicate wells in 3 experiments) both differentiation and the proportion of myocytes expressing slow MyHC compared to either untreated cells or control conditioned medium. This effect was blocked by addition of the 5E1 (1:300 diluted from 1.9 mg/ml) functionally-blocking anti-Shh monoclonal antibody, although basal differentiation and slow MyHC expression in control myocytes was unaffected. Purified preparations of mouse or zebrafish Shh N-terminal fragment also significantly induce differentiation and slow MyHC (p < 0.01, t test, n = 11 replicate wells in 2 experiments). D. Shh enhances differentiation and fusion of C2X myoblasts (left panel). Shh enhances slow MyHC accumulation in all classes of myotubes (right panel). Note that although the number of mononucleate myocytes is unaltered by Shh exposure, mononucleate myocytes are as efficiently induced to express slow MyHC as more mature multinucleate myotubes.

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