Tgfβ signaling stimulates glycolysis to promote the genesis of synovial joint interzone in developing mouse embryonic limbs

The initial interzone cells for synovial joints originate from chondrocytes, but such critical transition is minimally understood. With single-cell RNA Sequencing (scRNA-seq) of murine embryonic knee joint primordia, we discovered that heightened expression of glycolysis genes characterized developing interzone cells when compared to flanking chondrocytes. Conditional deletion of the glucose transporters GLUT1 and/or GLUT3, in either the incipient pre-skeletal mesenchyme with Prx1Cre or in chondrocytes with Col2Cre, disrupted interzone formation dose-dependently. In contrast, deletion of GLUT1/3 in established interzone cells with Gdf5Cre did not have similar severe disruption of joint development. scRNA-seq revealed that GLUT1/3 deletion by Prx1Cre impeded Tgfβ signaling in the developing interzone cells. Direct elimination of Tgfβ signaling with Prx1Cre partially phenocopied the deletion of GLUT1/3 in impairing interzone formation. Tgfβ stimulated glycolysis in chondrocytes via activation of mTOR and Hif1α in vitro. The data support that the essential conversion of chondrocytes to interzone cells requires a transient elevation of glycolysis partly dependent on Tgfβ signaling.