Figure 3

Gene expression analysis using quantitative RT-PCR, along with TUNEL immunostaining, show an increase in the myogenic program and cell death. (A) Terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labeling (TUNEL) staining shows an increased number of apoptotic nuclei in Lama2^DyW (DyW) muscles at each of the four time points. Desmin costaining was used to delineate differentiated myogenic cells. Arrows point to those cells that are both desmin-positive (red) and TUNEL-positive (green). (DyW is also referred to as KO, Knockout.) (B) Quantitative analysis of the TUNEL staining shows the highest incidence of apoptosis at postnatal week 1, which decreased by 50% at week 3. (C) Gene analysis of MyoD shows the highest expression for both groups early in life, followed by decreases over time. DyW mice had significantly increased expression compared to age-matched wild-type (WT) mice at all time points (1 week: P < 0.05, 2 weeks: P < 0.001, 3 weeks: P < 0.0001 and 4 weeks: P < 0.001, all by two-way analysis of variance (ANOVA) (n = 5 for all groups). (D) Myogenin expression shows a similar trend of having the highest expression early on, and DyW levels were also increased compared to all age-matched WT groups (1 week: P < 0.01, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5 for all groups). (E) Embryonic myosin heavy chain (eMYHC) expression was significantly elevated in the DyW mice at each of the four time points (1 week: P < 0.0005, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5). Asterisks indicate statistical significance between age-matched DyW and WT animals, hash tags indicate significant changes within the DyW group and Deltas indicate significant changes within the WT group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. These statistical representations also apply other symbols (Δ, #). Bar = 100 μm.