Figure 6. The actin::MuSK-L transgene is sufficient to initiate but not maintain muscle prepatterning.
Whole mounts of diaphragm muscles from E13.5 (A–C) or E18.5 (D–F) mice that lack (A, D) motor neurons (HB9cre; Isl2DTA), (B, E) motor neurons and endogenous MuSK (HB9cre; Isl2DTA; MuSK−/−) or (C, F) motor neurons and endogenous MuSK but carry the MuSK-L transgene (HB9cre; Isl2DTA; MuSK−/−; HSA::MuSK-L) were stained with Alexa594-α-BGT. AChRs are clustered in the central region of muscle from mice that lack motor axons (A, D) but not in muscle from mice that lack both motor axons and endogenous MuSK (B, E). (C) Expression of the MuSK-L transgene restores AChR clustering in the central region of muscle from E13.5 mice that lack motor neurons and endogenous MuSK, and the width of this zone is indistinguishable from control mice (G) (n = 3 HB9cre; Isl2DTA and 2 HB9cre; Isl2DTA; MuSK−/−; HSA::MuSK-L mice). By E18.5, however, this restricted pattern of AChR expression is lost, since AChR clusters are found throughout the muscle of mice that carry the MuSK-L transgene and lack motor neurons and endogenous MuSK. (H, I) A model for initiating and maintaining muscle prepatterning. (H) Muscles grow in length by the fusion of myoblasts at the ends of developing myotubes. The MuSK gene is activated early during myotube formation. Stochastic dimerization of MuSK protein in small myotubes leads to tyrosine phosphorylation and activation of MuSK. Activated MuSK stimulates two positive feedback loops, a post-translational feedback loop, which clusters MuSK protein, and a transcriptional feedback loop, which stimulates MuSK expression. Because MuSK activation occurs early during myotube formation and because muscles grow extensively from their ends, these feedback loops specialize the central region of the muscle and initiate and maintain muscle prepatterning. (I) The actin gene is likewise activated early during myotube formation, but the actin gene is uniformly expressed throughout muscle. Thus, in mice that lack endogenous MuSK and carry the actin::MuSK-L transgene, expression of MuSK protein is initiated early, leading to stimulation of the post-translational feedback loop, but MuSK RNA expression is not elevated in the central region of the muscle, since the actin promoter is not responsive to the transcriptional feedback loop. Thus, in these mice, muscle prepatterning is initiated but not maintained. Scale bar = 100 μm for (A–C) and 200 μm for (D–F).