1. A critical role for integrin  7 in muscle regeneration Isabelle Piec and Ulrike Mayer BMRC, UEA, Norwich Satellite cells are muscle-specific stem cells responsible for skeletal muscle regeneration. In normal conditions, these cells express Pax7 and rest quiescent between the sarcolemma and the basement membrane of the muscle fibre. Upon injury, satellite cells get activated, start to proliferate and express Myf5 and MyoD. Most cells then down-regulate Pax7, differentiate and express myogenin while others down-regulate MyoD and self renew. 0 10 20 30 40 cell velocity (  m/hr) B *** -/- +/+ 0 10 20 30 40 01234 number of divisions % of satellite cells A Model of satellite cell fate in muscle regeneration. Commitment of the satellite cell is determined by differential expression of myogenic factors (Pax7, Myf5, MyoD and myogenin) during activation, proliferation and differentiation. Compound deletion of α7 and dystrophin in mice results in loss of 50% of the muscle fibres in affected muscles. As mdx mice have normal muscle regeneration, this suggests that the loss of muscle fibres in the double-mutant mice is causally related to α7 deficiency and that α7 is required for efficient muscle regeneration. Integrin α7β1 is a prime candidate to efficiently regulate these muscle stem cells. Culturing myofibres in suspension models early events of muscle regeneration. We therefore used this ex vivo regeneration model as well as inducing regeneration in vivo to analyse in detail the defects that occur in satellite cell function in integrin  7 deficient mice. 15 days post injection 6 days post injection uninjected 1 st injection CTX2 nd injection CTX  7+/+  7-/-  7+/+  7-/- Haematoxylin and eosin staining of muscle sections before and 6 and 15 days after 1 and 2 injections of cardiotoxin in  7-/- and +/+ mice. Integrins are heterodimeric transmembrane glycoproteins of which at least 24 isoforms exist. Yet, integrin α7β1 is the predominant β1 integrin in adult skeletal muscle and mainly provides myotendinous junction (MTJ) stability and is expressed in quiescent and activated satellite cells.  7 +/+  -/- soleus myotendinous junction Myopathy in  7-/- muscles. Haematoxylin and eosin staining of cryosections prepared from adult wild- type and  7-/- muscles showing the dystrophic changes. Phase contrast images of cultured myofibres isolated from interosseus muscles show smooth-ended fibres in  7+/+ while MTJ are distorted in  7-/- fibres. Bars, 50 µm (B, C); 100 µm (D, E). Adapted from Nawrotzki et al., 2003. Integrin  7 deficiency induces a muscular dystrophy, primarily affecting the MTJ associated with mild dystrophic changes, mainly apparent in the soleus muscle. INTRODUCTION AIMS RESULTS CONCLUSIONS defective regeneration I- Integrin  7 deficiency delays muscle regeneration in vivo. Widespread muscle degeneration was induced by 2 consecutive injections of cardiotoxin (snake venom). Muscle sections were taken before and 6 and 15 days post-injury and stained with haematoxylin and eosin staining. II- Integrin  7 deficiency is associated with loss of satellite cells  Myofibres from α7-/- have 40% less quiescent Pax7 positive satellite cells.  This number decreases dramatically after activation.  The number of Myf5, MyoD and Mgn positive cells remained extremely low after several hours of culture.  Muscle regeneration is severely delayed in  7-/- muscle as compared to controls.  This effect is visible at day 6 and persist after full regeneration (15 days). This difference was further increased after the second injection of cardiotoxin. Fibres with their satellite cells from WT (grey) and α7-/- (dashed grey) mice were isolated and after 24, 48 and 72hrs of culture, were stained for indicated myogenic markers. * B % of non myogenic cells 0 5 10 15 20 25 +/+ -/- C fusion index 0 0.2 0.4 0.6 0.8 +/+ -/- * proliferation mediumdifferentiation 3 days 5 days 2 days -/- +/+ A IV- Integrin  7 deficiency is associated with a defect in satellite cell proliferation and migration. Satellite cells of both  7+/+ (grey) and  7-/- (dashed grey) were followed by time-lapse microscopy. This showed that:   Although  7-/- cells can divide four times, a higher number of  7-/- cells do not divide at all (A).   7-/- cells show an extremely reduced mobility on the fibres (B). Histograms showing the percentage of cell divisions (A) and velocity (B) of  7+/+ (grey) and  7-/- (dashed grey) satellite cells. III- Fusion of myogenic cells is not impaired in integrin  7 deficiency. Isolated muscle fibres were plated on matrigel and satellite cells allowed to outgrow and proliferate in proliferation medium. Differentiation was induced when the cells were subconfluent using low serum-containing medium. (A) Phase contrast images of outgrowing (3 and 5 days after plating) and differentiated satellite cells. Histograms showing the fusion index (B) and the percentage of non-myogenic cells (C) assessed after 2 days of differentiation in  7+/+ (grey) and  7-/- (dashed grey) satellite cells.  A lower number of cells outgrow from the  7-/- fibres (A)   7-/- cells fuse rapidly depleting the pool of myogenic cells (A)  The fusion index is therefore lower in  7-/- mice (B)  More cells dedifferentiate in  7-/- cultures (C). Using this model, we show that deficiency in integrin α7 leads to a loss of satellite cells after activation; impairing the self-renew of these cells. Time-lapse microscopy further demonstrates that integrin α7-/- satellite cells have a defect in migration and proliferation. Accordingly, we observed a defect in muscle regeneration after intramuscular injection of cardiotoxin. Integrin α7 is therefore crucial for satellite cell activation and muscle regeneration. Model of satellite cell activation in  7 deficiency.  This divisions occur at the same time points in  7-/- than  7+/+ cells (~every six hours after the first division occurred around 40hrs after isolation).