Figure 2. SC niche perturbation results in satellite cells activation.

(A) Schematic representation of the system used to generate Ckm-Tet-Off Hgf (MH) mice. In the absence of Doxycycline (-Dox), tTA binds to the Tetracycline Responsive Element (TRE), inducing the expression of HGF and eGFP in skeletal muscle. (B) Upper panel: fluorescent image of P10 mice. Middle panel: MH hindlimb under visible (Vis) and fluorescent (Fluo) light. Lower panel: fluorescent image of a muscle cross-section of a MH mouse. (C) Upper panel: semi-quantitative RT PCR of the indicated genes on Tibialis anterior muscles. Lower panel: fluorescent images of hindlimbs from a MH mouse with or without Dox treatment. (D) HGF-ELISA quantification in muscle and serum extracts from Dox-treated or control mice. (E) Distribution of Tibialis anterior cross-sectional areas showing a leftward shift in MH mice relative to their respective M controls. The mean value for MH mice was 585 ± 37 µm² (n=8); the mean value for control M mice was 844 ± 63 µm² (n=10). **p<0.01 (t test). (F) Representative H&E staining of Tibialis anterior cross sections. (Scale bar = 100 µm). (G) Quantification (mean ± SEM) of Pax7 positive cells/field in Tibialis anterior sections (M mice n=7; MH mice n=6). ***p<0.001 (t test). (H) Representative immunofluorescence of Pax7 (green), Laminin (red) and DAPI (blue) staining in G. Arrowheads indicate Pax7-positive cells. (Scale bar = 50 µm). (I) Quantification (mean ± SEM) of Pax7 positive cells/fiber after single fiber isolation (M mice n=13; MH mice n=12; MH +Dox n=2). *p<0.05; ***p<0.001 (t test). (J) Representative immunofluorescence of Pax7 (red) and DAPI (blue) staining in I. (K) Quantification (mean ± SEM) of MyoD-positive cells/fiber after single fiber isolation (M mice n=3; MH mice n=3; MH +Dox n=4). **p<0.01; ***p<0.001 (t test). (L) Representative immunofluorescence of MyoD (red) and DAPI (blue) staining in K. Dashed areas are shown at threefold magnification.

DOI: http://dx.doi.org/10.7554/eLife.12116.005

Figure 2—figure supplement 1. SC niche perturbation results in satellite cells activation.

(A) Body weight (mean ± SEM) of the indicated mice (M mice n=6; MH mice n=9). ^NSp>0.05 (t test). (B) Representative image of Tibialis anterior cross sections in the indicated mice. (C) Fiber size distribution (mean ± SEM) of Tibialis anterior showing a significant increase in the number of fibers with cross-sectional area of 300–800 μm² and a significant decrease in the number of fibers with cross-sectional area of 1000–1300 μm² in MH mice (n=8) respective to control M mice (n=10). *p<0.05; **p<0.01; ***p<0.001 (t test). (D) Quantification (mean ± SEM) of Pax7-positive cells/fiber after three days from single fiber isolation (M mice n=6; MH mice n=5). *p<0.05 (t test). (E) Quantification (mean ± SEM) of MyoD-positive cells/fiber after three days from single fiber isolation (M mice n=2; MH mice n=2). *p<0.05 (t test). (F) Quantification (mean ± SEM) of myonuclei/fiber after single fiber isolation (M mice n=5; MH mice n=7). **p<0.01(t test). (G) Representative immunofluorescence of MyoD (red), Laminin (green) and DAPI (blue) staining. Arrowheads indicate MyoD-positive cells. (Scale bar = 50 µm).

DOI: http://dx.doi.org/10.7554/eLife.12116.006