A novel GFP reporter mouse revealsMustn1expression in adult regenerating skeletal muscle, activated satellite cells and differentiating myoblasts

Abstract Aim Mustn1 has been implicated in myofusion as well as skeletal muscle growth and repair; however, the exact role and spatio‐temporal expression of M ustn1 have yet to be fully defined. Methods Transgenic mice were generated with a 1512‐bp sequence of the M ustn1 promoter directing the expression of GFP ( M ustn1 PRO ‐ GFP ). These mice were used to investigate the spatio‐temporal expression of M ustn1 PRO ‐ GFP during skeletal muscle development and adult skeletal muscle repair, as well as various phases of the satellite cell lifespan (i.e. quiescence, activation, proliferation, differentiation). Results Mustn1 PRO ‐ GFP expression was observed within somites at embryonic day 12 and developing skeletal muscles at embryonic day 15 and 18. While uninjured adult tibialis anterior muscle displayed no detectable M ustn1 PRO ‐ GFP expression, cardiotoxin injury robustly elevated M ustn1 PRO ‐ GFP expression at 3 days post‐injury with decreasing levels observed at 5 days and minimal, focal expression seen at 10 days. The expression of M ustn1 PRO ‐ GFP at 3 days post‐injury consistently overlaid with MyoD although the strongest expression of M ustn1 PRO ‐ GFP was noted in newly formed myotubes that were expressing minimal levels of MyoD. By 5 days post‐injury, M ustn1 PRO ‐ GFP overlaid in all myotubes expressing myogenin although cells were present expressing M ustn1 PRO ‐ GFP alone. The expression patterns of M ustn1 PRO ‐ GFP in regenerating muscle preceded the expression of desmin throughout the regenerative time course consistent with M ustn1 being upstream of this myogenic protein. Further, quiescent satellite cells located on freshly isolated, single myofibers rarely expressed M ustn1 PRO ‐ GFP , but within 24 h of isolation, all activated satellite cells expressed M ustn1 PRO ‐ GFP . Expression of M ustn1 PRO ‐ GFP in primary myoblasts diminished with prolonged time in proliferation media. However, in response to serum withdrawal, the expression of Mustn1 PRO ‐ GFP increased during myofusion (day 2) followed by declining expression thereafter. Conclusion Mustn1 PRO ‐ GFP is expressed in activated satellite cells and myoblasts but continued time in proliferation media diminished M ustn1 PRO ‐ GFP expression. However, myoblasts exposed to serum withdrawal increased M ustn1 PRO ‐ GFP expression consistent with its demonstrated role in myofusion. The in vivo expression pattern of M ustn1 observed in regenerating and developing skeletal muscle is consistent with its presence in satellite cells and its critical role in myofusion.