Three‐dimensional observation of the muscle–tendon integration process in mouse embryos

BACKGROUND: Muscle-tendon integration is a crucial step in the morphogenesis of the vertebrate locomotion system. As the limb muscle and tendon progenitor cells derive from distinct embryonic origin, their integration requires precise mutual positioning. This process has been described mainly by tissue sectioning and in situ hybridization, which are not well suited for understanding the three-dimensional (3D) organization. To address these limitations, this study applied tissue clearing and light-sheet microscopy to visualize the muscle-tendon integration process in 3D in mouse embryonic limbs. RESULTS: By combining CUBIC reagents, immunofluorescence, and fluorescent reporter mice, we were able to resolve the 3D arrangement of forelimb muscles and tendons in mouse embryos from E11.5 to E15.0. Interestingly, we found that a subpopulation of Scleraxis+ tendon lineage cells remains within the interstitial space of myofibers even after muscle-tendon integration is established, implying the existence of previously undescribed cellular heterogeneity within the Scleraxis+ cell population at this stage. Moreover, our approach successfully captured the muscle-tendon morphologies altered by genetic perturbations, such as Scleraxis-lineage ablation and Myomaker knockout. CONCLUSIONS: In summary, this tissue clearing and light-sheet microscopy approach provides a versatile method for analyzing dynamically changing embryonic muscle-tendon morphologies and will facilitate deeper understanding of the muscle-tendon integration process.