Figure 3

Pw1 null satellite cells display altered self-renewal properties. (A) Quantification of PAX7 positive cells in Pw1^+/+ and Pw1^m−/p− 3 month TA muscle before and after two weeks of a single CTX injury (n = 4 for each genotype). NI = non injured; SI = single injured. Values represent the mean number of positive cells ± s.e.m. per 100 fibers. The number of PAX7 positive cells is higher in uninjured Pw1^m−/p− muscle as compared to Pw1^+/+. In contrast, the number of PAX7 positive cells in injured Pw1^m−/p− muscle is significantly lower as compared to Pw1^+/+ two weeks after injury. (B) Left Panel: Flow cytometric analyses of single cells from Pw1^+/+ and Pw1^m−/p− mice TA muscles five days after injury gated for α7-integrin⁺/CD34⁺/TER119⁻/CD45⁻/SCA1⁻. The gate used to identify CD34⁺/SCA⁻ quiescent satellite cells is shown in blue. Right Panel: Histograms showing percentages (left) and absolute cell numbers (right) of quiescent satellite cells CD34⁺/SCA⁻ fraction analyzed as shown in (left panel) (n = 3 for each genotype). Quiescent satellite cells CD34⁺/SCA⁻ population is reduced in Pw1^m−/p− injured muscle as compared to Pw1^+/+. (C) Left Panel: Representative cross-sections of Pw1^+/+ and Pw1^m−/p− TA muscles one week after two consecutive CTX injury immunostained for PAX7 (red), MYOD (green), and LAMININ (orange). Nuclei were visualized by DAPI. White and yellow arrows denote PAX7+/MYOD− (self-renewing) and PAX7+/MYOD+ (proliferating) myogenic cells respectively. Scale bar = 50 μm. Right Panel: Histograms showing proportions of myogenic cells PAX7+/MYOD− (red), PAX7+/MYOD+ (green) and PAX7−/MYOD+ (yellow). The proportion of self-renewing cells is decreased in Pw1^m−/p− compared Pw1^+/+, whereas the proliferating and committed myoblasts show a concomitant increase (n = 3 for each genotype). (D) Histograms showing proportions of PAX7+/KI67− cells (quiescent satellite cells, red), PAX7+/KI67+ cells (proliferating satellite cells, green) and PAX7−/KI67+ cells (proliferating myoblast, yellow) from Pw1^+/+ and Pw1^m−/p− TA muscles one week after two consecutive CTX injury (n = 3 for each genotype). The proportion of quiescent satellite cells is decreased in Pw1^m−/p− compared Pw1^+/+. (E) Histograms showing proportions of myogenic cells MYOD+/KI67− (committed cells, red), MYOD+/KI67+ (proliferating and committed cells, green) and MYOD−/KI67+ (proliferating cells, yellow) from Pw1^+/+ and Pw1^m−/p− TA muscles one week after two consecutive CTX injury (n = 3 for each genotype). The proportion of proliferating and committed cells is increased in Pw1^m−/p− compared Pw1^+/+. Values are expressed as mean ± s.e.m. Statistical analyses were performed using Student’s t-test *P < 0.05, **P < 0.01 and ***P < 0.001. In all the graphs Pw1^+/+ (+/+) and Pw1^m−/p− (m−/p−).