Single-cell atlas of mouse limb development reveals a complex spatiotemporal dynamics of skeleton formation

Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naïve limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb revealed three progenitor states: naïve, proximal and autopodial, as well as Msx1 as a marker for the naïve progenitors. In vivo lineage tracing confirmed this role and localized the naïve progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1 + naïve progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9 + chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest a new model for limb skeleton development.