Fibroblast diversification is an embryonic process dependent on muscle contraction.

Fibroblasts, the most common cell type found in connective tissues, play major roles in development, homeostasis, regeneration, and disease. Although specific fibroblast subpopulations have been associated with different biological processes, the mechanisms and unique activities underlying their diversity have not been thoroughly examined. Here, we set out to dissect the variation in skeletal-muscle-resident fibroblasts (mrFibroblasts) during development. Our results demonstrate that mrFibroblasts diversify following the transition from embryonic to fetal myogenesis prior to birth. We find that mrFibroblasts segregate into two major subpopulations occupying distinct niches, with interstitial fibroblasts residing between the muscle fibers and delineating fibroblasts sheathing the muscle. We further show that these subpopulations entail distinct cellular dynamics and transcriptomes. Notably, we find that mrFibroblast subpopulations exert distinct regulatory roles on myoblast proliferation and differentiation. Finally, we demonstrate that this diversification depends on muscle contraction. Altogether, these findings establish that mrFibroblasts diversify in a spatiotemporal embryonic process into distinct cell types, entailing different characteristics and roles.