ABSTRACT
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.
Footnotes
Author contributions
Conceptualization: C.S.; Data curation: X.Y., R.K., S.Y., H.W., S.M., G.K., R.S., M.I., T.I., C.S.; Formal analysis: X.Y., R.K., S.Y., M.I., C.S.; Funding acquisition: T.A., T.I., C.S.; Investigation: X.Y., R.K., S.Y., Y.Y., S.H., H.W., K.H., G.K., R.S., M.I., D.D., C.S.; Methodology: R.K., T.I., C.S.; Project administration: C.S.; Resources: T.S., H.A., M.I., C.S.; Supervision: C.S.; Validation: X.Y., R.K., S.Y., Y.Y., S.H.; Visualization: X.Y., R.K., M.I., C.S.; Writing – original draft: X.Y., R.K., C.S.; Writing – review & editing: X.Y., R.K., M.I., C.S.
Funding
This study was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (JP16H06280, JP18H02966, JP21H03107, JP21KK0161, JP22H04926 and JP23K23899), and the Cooperative Research Program of the Institute for Frontier Life and Medical Sciences, Kyoto University, Japan. This work was also supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), by the Frontier Development Program for Genome Editing funded by the Doctoral Program for World-leading Innovative and Smart Education, and by Japan Science and Technology Agency (JST), the establishment of university fellowships towards the creation of science technology innovation (JPMJFS2129 and JPMJSP2132).
Data availability
All relevant data can be found within the article and its supplementary information.
