TGFβ activation and inhibition of Wnt and MAPK signaling pathways promote the specification of joint lineage cells in vitro and ex vivo

The developmental processes of synovial joint initiation and subsequent differentiation of progenitor cells towards anatomically and functionally distinct joint tissues are not well understood. Here we quantified the induction of cells expressing growth and differentiation factor 5 (Gdf5), an early marker of joint formation, and Prg4, a proteoglycan found in the superficial zone of articular cartilage and other joint tissues, utilizing a dual reporter mESC line and directed differentiation methodologies. We found TGF signaling to be necessary and sufficient for the induction of Gdf5-RFP and Prg4-GFP. Inhibition of either canonical Wnt signaling or MAPK signaling significantly increased the induction of Gdf5-RFP and chondrogenesis, while activation of either pathway prohibited this. Activation of MAPK significantly promoted the emergence of Prg4-GFP-expressing cells and elevated levels of tendon-ligament fibroblast genes such as Tnmd. We used single cell transcriptomics to determine the cellular composition of these cultures and determined that Gdf5+ cells are chondrogenic in both in vitro cultures and in mouse E12.5 and E14.5 limb bud cells. We validated the joint cell- promoting role of Wnt and MAPK inhibition in both systems. In summary, we used a novel ESC model to elucidate the roles of TGF, Wnt and MAPK signaling in the specification and differentiation of joint lineage cells.