Pax7 creert2 mice

C57BL/6J mice were purchased from Charles River Laboratories (Yokohama, Kanagawa, Japan). Hey1^−/− mice and Heyl^−/− mice were described previously (Fukada et al., 2011 (link); Kokubo et al., 2005 (link)). Pax7^CreERT2+ mice (stock No: 012476) were obtained from Jackson Laboratories (Bar Harbor, ME, USA; Farmington, CT, USA; or Sacramento, CA, USA). Relocation of Rosa26^EYFP/+ mice (stock No: 006148) from the National Center of Neurology and Psychiatry to Osaka University was approved by Jackson Laboratories. Pax7^CreERT2+::Rosa26^EYFP/+ mice were injected intraperitoneally twice (24 hr apart) with 200 μl to 300 μl tamoxifen (20 mg/ml; #T5648; Sigma-Aldrich, St. Louis, MO, USA) dissolved in sunflower seed oil (#S5007; Sigma-Aldrich) and 5% ethanol, and the mice were maintained in a controlled environment (temperature, 24 ± 2°C; humidity, 50 ± 10%) under a 12/12 hr light/dark cycle. The mice received sterilized standard chow (DC-8; Nihon Clea, Tokyo, Japan) and water ad libitum. All procedures used for the experimental animals were approved by the Experimental Animal Care and Use Committee of Osaka University (approval number: 25-9-3, 30–15).

All experiments were conducted in female mice maintained in specific pathogen-free animal facility in individually ventilated cages with controlled temperature (22 ± 1°C) and light (12 h light/dark cycle). Animal care and use were in accordance with the guidelines of the institution. The study protocol has been approved by ethics committee of local institution (Approval No. XHEC-F-2021-012). Pax7-CreERT2 mice were purchased from Jackson Laboratory, and Esr1^f/f mice were purchased from Gem Pharmatech. Muscle stem cell specific gene knockout was induced by i.p. injection of 100 μL of 10 mg/mL tamoxifen (ABCONE, cat#T56488) every 2 days for a week.
Bipedal mice were established to simulate upright posture as described previously^{51 (link)}. Briefly, forelimb and tail of mice were firstly removed at 3 weeks old. With progressively raising water and food, these bipedal mice will gradually maintain standing posture. Unilateral intramuscular injection of 100 μL 100 nM MPP (Tocris, cat#1991) at the left side of the para-spinal muscle was performed in 3-week-old mice twice every week for 3 weeks.
In the scoliosis mouse model, intramuscular injections of 10 nM Raloxifene (Tocris, cat#2280) were performed at the concave para-spinal muscle once every 3.5 days for 2 weeks. The spinal alignment and para-spinal muscle size were then analyzed at 2 weeks after Raloxifene injection.

Dnmt3a-flox mice were kindly provided by Dr. M. Okano. Dnmt3a-floxed allele was previously described [66 (link)]. Pax3-Cre mice and Pax7-CreERT2 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Pax3-Cre allele and Pax7-CreERT2 allele were previously described [67 (link),68 (link)]. Genomic DNA was isolated from muscle tissues using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Gene deletion efficiency was calculated by genomic DNA qPCR. Relative genomic DNA level was determined by the standard curve method. All primer sequences are listed in S1 Table.