Yu Xinyi, Kawakami Ryosuke, Yambe Shinsei, Yoshimoto Yuki, Sasaki Takako, Higuchi Shinnosuke, Watanabe Hitomi, Akiyama Haruhiko, Miura Shigenori, Hu Kadi, Kondoh Gen, Sagasaki Ramu, Inui Masafumi, Adachi Taiji, Docheva Denitsa, Imamura Takeshi, Shukunami Chisa
Department of Molecular Biology and Biochemistry, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Ehime 791-0295, Japan.
Development. 2025 Mar 15;152(6). doi: 10.1242/dev.204512. Epub 2025 Mar 26.
Proper connections between cartilaginous and muscular primordia through tendinous/ligamentous primordia are essential for musculoskeletal integration. Herein, we report a novel double-reporter mouse model for investigating this process via fluorescently visualising scleraxis (Scx) and SRY-box containing gene 9 (Sox9) expression. We generated ScxTomato transgenic mice and crossed them with Sox9EGFP knock-in mice to obtain ScxTomato;Sox9EGFP mice. Deep imaging of optically cleared double-reporter embryos at E13.5 and E16.5 revealed previously unknown differences in the dynamic interactions between cartilaginous and tendinous/ligamentous primordia in control and Scx-deficient mice. Tendon/ligament maturation was evaluated through simultaneous detection of fluorescence and visualisation of collagen fibre formation using second harmonic generation imaging. Lack of deltoid tuberosity in Scx-deficient mice caused misdirected muscle attachment with morphological changes. Loss of Scx also dysregulated progenitor cell fate determination in the chondrotendinous junction, resulting in the formation of a rounded enthesis rather than the protruding enthesis observed in the control. Hence, our double-reporter mouse system, in combination with loss- or gain-of-function approaches, is a unique and powerful tool that could be used to gain a comprehensive understanding of musculoskeletal integration.
通过腱/韧带原基实现软骨原基与肌肉原基之间的正确连接对于肌肉骨骼整合至关重要。在此，我们报告一种新型双报告基因小鼠模型，用于通过荧光可视化硬骨素（Scx）和含SRY盒基因9（Sox9）的表达来研究这一过程。我们构建了ScxTomato转基因小鼠，并将它们与Sox9EGFP基因敲入小鼠杂交，以获得ScxTomato;Sox9EGFP小鼠。对E13.5和E16.5期光学透明的双报告基因胚胎进行深度成像，揭示了对照小鼠和Scx缺陷小鼠中软骨原基与腱/韧带原基之间动态相互作用的先前未知差异。通过同时检测荧光和使用二次谐波生成成像观察胶原纤维形成来评估肌腱/韧带成熟度。Scx缺陷小鼠中三角肌粗隆的缺失导致肌肉附着方向错误并伴有形态学改变。Scx的缺失还会使软骨-肌腱连接处祖细胞命运决定失调，导致形成圆形的附着点，而不是对照中观察到的突出附着点。因此，我们的双报告基因小鼠系统与功能丧失或功能获得方法相结合，是一种独特而强大的工具，可用于全面了解肌肉骨骼整合。
