Connective tissue forms a three-dimensional network of fibroblasts and extracellular matrix that supports the musculoskeletal system and plays a decisive role during development, notably by providing positional information to muscle cells. In vertebrates, this tissue encompasses the muscle connective tissue—whose successive layers, the epimysium, perimysium and endomysium, surround the muscles, the fiber bundles and the individual fibers, respectively—as well as the tendon, itself wrapped in thin fibroblastic layers. Despite this importance, our understanding of fibroblast differentiation programs remains limited, largely because these cells are highly heterogeneous and poorly characterized. Unlike the muscle lineage, no master gene drives differentiation toward the dermis, the muscle connective tissue or the tendon, even though recognized markers exist for each, such as TWIST2 for the dermis, scleraxis (SCX) for the tendon and OSR1 for a population of fibro-adipogenic progenitors.
To explore this diversity, the authors combined several single-cell RNA sequencing approaches, including trajectory inference, with in situ hybridization analyses, applied to fetal limb development in the chicken. Trajectory inference was notably conducted using the STREAM pipeline, while in situ hybridizations—single or double, colorimetric or fluorescent—made it possible to localize marker expression on limb sections at embryonic stages E6 and E10, with immunostaining of sarcomeric myosin heavy chains to identify the muscles. The hybridization and immunohistochemistry experiments were performed on at least three limbs from different embryos.
This work shows that fibroblasts switch from a program based on positional information to a lineage diversification program at the onset of the fetal period. The muscle connective tissue and the tendon comprise several fibroblast populations that emerge asynchronously. Once the definitive muscle pattern is established, transcriptionally related populations occupy neighboring locations within the limb, foreshadowing the fibroblastic layers observed in the adult.
Based on these observations, the authors propose that limb connective tissue is organized into a continuum of "promiscuous" fibroblast identities—that is, weakly compartmentalized and related between neighboring populations. This organization would ensure a robust and efficient connection of muscle to bone and skin. The study thus provides a map of fibroblast diversity and of its developmental trajectories, in an area where these programs had until now been little described compared with those of the muscle lineage.