Fig. 1

Identification and characterization of Axin2⁺ cells in the resting zone after secondary ossification center formation. a–d Axin2⁺ cells in the proximal tibial growth plate in Axin2Cre^ERT2;R26R^ZsGreen mice. Axin2⁺ cells were labeled one day after three consecutive daily injections of tamoxifen (120 μg·g⁻¹ body weight) from P0 (n = 3) (a), P23 (n = 8) (b), P42 (n = 7) (c), and P84 (n = 6) (d). Bottom panels show magnified views of the resting zone of the growth plate. e Quantification of ZsGreen⁺ cells in the resting zone across different time points reported above in (a–d). Adjusted ****P =< 0.000 1. f–i Characterization of Axin2⁺ cells in the resting zone of the proximal tibial growth plate in Axin2Cre^ERT2;R26R^ZsGreen mice one day after three consecutive daily tamoxifen injections from P23. f Representative images of EdU labeling and Axin2⁺ cells and the percentage of EdU-negative cells among Axin2⁺ cells (n = 3). EdU (30 μg/g body weight) was injected daily from P23 to P26, and mice were euthanized 2 h after the last administration of EdU. g Representative images of in situ hybridization of Ki67 and Axin2⁺ cells and the percentage of Ki67+ cells among Axin2⁺ cells (n = 3). Arrow heads, Axin2⁺ cells expressing Ki67. h Representative images of in situ hybridization of Cd73 and Axin2⁺ cells and the percentage of Cd73⁺ cells among Axin2⁺ cells (n = 3). Arrow heads, Axin2⁺ cells expressing Cd73. i Representative images of in situ hybridization of Pthrp and Axin2⁺ cells and the percentage of Pthrp⁺ cells among Axin2⁺ cells (n = 3). Arrow heads, Axin2⁺ cells expressing Pthrp. Tx tamoxifen, SOC secondary ossification center. The white dashed lines demarcate the growth plate from the surrounding tissues. Scale bars: 200 μm (a–d [upper panels], f [left]), 40 μm (a–d [bottom panels]), 20 μm (f [right], g–i). All data are presented as the mean ± SD. Statistical significance was determined by one-way analysis of variance and Tukey’s multiple comparison test