Making connections: A three-dimensional visualization of musculoskeletal development

Researchers published their results in Development on March 26, 2025.

A key feature of this study is the use of a newly developed double-reporter mouse model combined with fluorescent imaging to investigate the complex processes required to form proper connections between cartilaginous and tendinous/ligamentous tissue that connect muscles and skeletal elements during embryogenesis. The double-reporter model enables the simultaneous tracking of two distinct reporter genes. In this study, mice expressing red and green fluorescent proteins in the expression domains of Scx and Sox9, respectively, were used to visualize the desired biological event of interest. The goal of this study is to elucidate how musculoskeletal components are integrated during organogenesis.

"Traditional methods using thin tissue sections have limitations in preserving structural integrity, which makes it difficult to study the 3D organization of these tissues. Our approach overcomes these challenges by combining tissue clearing of a newly established double fluorescent reporter mouse model with high-resolution fluorescence imaging," said Chisa Shukunami, a professor at Hiroshima University's Graduate School of Biomedical and Health Sciences.

The connection between muscle and cartilaginous primordia (early embryonic structures) via tendons, or between cartilaginous primordia via ligaments, must be precisely regulated in both spatial and temporal aspects to ensure the proper formation of a healthy and functional musculoskeletal system. To visualize this process, mouse models expressing fluorescent proteins are commonly used. Fluorescent proteins act as markers by emitting fluorescent light -- causing specific cell populations to glow -- and make it possible to observe where these cells are located and how they behave inside the body. This approach allows scientists to track these cells as they change position over time and to better understand how musculoskeletal tissues are assembled during development.

This study focuses on two molecules with tissue-specific expression patterns. One is Scleraxis (Scx), a basic helix-loop-helix transcription factor expressed during tendon and ligament formation. The other is SRY-box containing gene 9 (Sox9), a transcription factor essential for cartilage development. Mouse lines with ScxTomato and Sox9EGFP were crossed to obtain ScxTomato;Sox9EGFP mice. In the newly established double-reporter mouse model, red fluorescence highlights the Scx-expressing domains, corresponding to sites of tendon and ligament formation, while green fluorescence marks areas where Sox9 is active, indicating regions of cartilage formation.

"By combining double fluorescent reporter mice with Scx-deficient mice, we demonstrated that Scx not only regulates tendon and ligament maturation, but also plays an important role in controlling muscle morphology and its attachment to cartilaginous bone primordia. These findings provide new insights into how the musculoskeletal system is established," said Shukunami.

Additionally, it was found that the Scx⁺/Sox9⁺ cells exhibit heterogeneity in the expression levels of Scx and Sox9, meaning that there is some variability in the expression of these cells within the same cell population of cells in a given tissue. Such diversity in expression may be involved not only in developmental processes but also in tendon and ligament regeneration and the onset of related disorders, providing important implications for future research in musculoskeletal disorders.

Researchers hope to combine the double-fluorescent reporter mice with various genetically modified mice to investigate how the musculoskeletal system is established during embryonic development. While much of this research focuses on developing embryos, further investigation on postnatal mice -- using tissue clearing techniques and 3D fluorescent imaging -- may shift toward observing the postnatal maturation of tendons and ligaments. These efforts to uncover how tendons, ligaments and cartilage form connections could provide extremely valuable insights for the development of new diagnostic tools and treatment strategies for age or sport-related injuries related to these tissues.