Figure 4. Increased ratio of MyoD-expressing myogenic cells and decreased cell number in overloaded HeyL-KO mice.

(A) Experimental scheme for analyzing Pax7⁺ cells on plantaris myofibers derived from control (Cont), Hey1-KO (1KO), or HeyL-KO (LKO) mice at 4 days after tenotomy (Ope). (B) Immunostaining of Pax7 (red), MyoD (green), and Ki67 (white) in MuSCs on freshly isolated myofibers at 4 days after tenotomy from Cont, 1KO, or LKO mice. Nuclei were counterstained with DAPI. Scale bar: 20 μm. (C) Frequency of MyoD⁻Ki67⁺ (left) or MyoD⁺Ki67⁺ (right) cells among Pax7⁺ cells on myofibers: sham (open circle) versus 4 days after tenotomy (closed circle), derived from Cont (light gray, n = 5), 1KO (black, n = 4), or LKO (blue, n = 5) mice. **p<0.01. N.S.: not significant. (D) Ratio of MyoD⁺ to MyoD⁻ cells among Ki67⁺Pax7⁺ cells on myofibers at 4 days after tenotomy, derived from Cont (light gray, n = 5), 1KO (black, n = 4), or LKO (blue, n = 5) mice. **p<0.01. N.S.: not significant. (E) Number of Pax7⁺ cells on myofibers: sham (open circle) versus 4 days after tenotomy (closed circle), derived from Cont (light gray, n = 5), 1KO (black, n = 4), or LKO (blue, n = 5) mice. **p<0.01. N.S.: not significant. Data represent the mean ± standard deviation. One-way ANOVA, followed by the Tukey–Kramer test, was used for statistical analyses.

Figure 4—figure supplement 1. Increased ratio of MyoD-expressing myogenic cells and decreased cell number in overloaded HeyL-KO mice at 7 days after tenotomy.

(A) Experimental scheme for analyzing Pax7⁺ cells on plantaris myofibers derived from control (Cont), Hey1-KO (1KO), or HeyL-KO (LKO) mice at 7 days after tenotomy (Ope) or derived from corresponding contralateral control muscles (Sham). (B) Frequency of MyoD⁻Ki67⁺ (left) or MyoD⁺Ki67⁺ (right) cells among Pax7⁺ cells on myofibers: sham (open circle) versus 7 days after tenotomy (closed circle) and derived from Cont (light gray, n = 3), 1KO (black, n = 4), or LKO (blue, n = 3) mice. **p<0.01. N.S.: not significant. (C) Ratio of MyoD⁺ to MyoD⁻ cells among Ki67⁺Pax7⁺ cells on myofibers at 7 days after tenotomy and derived from Cont (light gray, n = 3), 1KO (black, n = 4), or LKO (blue, n = 3) mice. **p<0.01. N.S.: not significant. (D) Number of Pax7⁺ cells on myofibers: sham (open circle) versus 7 days after tenotomy (closed circle) and derived from Cont (light gray, n = 3), 1KO (black, n = 4), or LKO (blue, n = 3) mice. N.S.: not significant. Data represent the mean ± standard deviation. One-way ANOVA, followed by the Tukey–Kramer test, was used for statistical analyses.

Figure 4—figure supplement 2. Normal muscle-regenerative ability in HeyL-KO mice.

(A) Immunostaining of M-cadherin (M-cad; red) and laminin α2 (LNα2; green) in sections of regenerating tibialis anterior muscle at 3 days after CTX injection (CTX-3d). Scale bar: 100 μm. Bar graph shows the frequency of M-cadherin⁺ cells per field in wild-type (Cont; n = 3) and HeyL-KO (LKO; n = 4) mice. N.S.: not significant. (B) Immunostaining of embryonic myosin heavy chain (eMyHC; red) in sections of regenerating tibialis anterior muscle at 4 days after CTX injection (CTX-4d). Scale bar: 200 μm. Bar graph shows percentages of eMyHC⁺ area in wild-type (Cont; n = 4) and LKO (n = 6) mice. N.S.: not significant. Data represent the mean ± standard deviation. A two-sided Student’s t test was performed.

Figure 4—figure supplement 3. Normal activation and proliferation ability of HeyL-KO MuSCs on cultured myofibers.

(A) Immunostaining of 2 day cultured myofibers from wild-type (Cont) and HeyL-KO (LKO) mice using Ki67 (white) and MyoD (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Bar graph showing the percentage of MyoD⁺ or MyoD⁻ cells among Ki67⁺ cells on 2 day cultured myofibers from wild-type (Cont) and LKO mice. The number of MyoD⁺ cells among Ki67⁺ cells is indicated in the numerator of each bar, and the number of Ki67⁺ cells is indicated in the denominator of each bar. (C) Bar graph showing the average number of Ki67⁺ cells per freshly isolated (0 d) or 2 day cultured (2 d) myofibers. The numbers in the graphs indicate the counted myofibers.