Identification of a new population of Tnn+ progenitors to form tendon enthesis fibrocartilage

Elucidating the identity of enthesis-resident progenitors is critical for advancing regenerative strategies, particularly in the context of the long-standing question of how is fibrocartilage formed at tendon enthesis (bone-tendon interface) under mechanical loading. To address the question of cellular origins of entheseal fibrocartilage, we first employed spatial transcriptional and single cell sequencing to identify a novel population of Tnn ⁺ progenitor cells and delineate their lineage trajectories across developmental stages. Subsequently, we used a diphtheria toxin mediated ablation model targeting these Tnn ⁺ progenitors and demonstrated their functional importance, as ablation resulted in hypoplastic phenotypes characterized by impaired fibrocartilage maturation. Furthermore, comparative single-cell profiling between unloaded entheses and normal entheses revealed that tendon unloading significantly diminished both the abundance and chondrogenic potential of Tnn ⁺ progenitors. Collectively, these findings resolve fundamental questions regarding enthesis morphogenesis and provide mechanistic insights into how mechanical loading orchestrates this critical developmental process.