Tgfβ signaling hyperactivates glycolysis via positive feedback to promote interzone formation and joint tissue development

The role of bioenergetics in synovial joint development is unknown. With single-cell RNA sequencing (scRNA-seq) in the knee joint primordia, we discover that the Gdf5-expressing interzone cells are enriched for the expression of glycolysis genes. Cartilage anlagen were isolated from the hindlimbs of E13.5 or E14.5 embryos, and cleaned of soft tissues by rolling on Kimwipe paper. Presumptive knee joint regions were excised under a dissecting microscope (Leica) and digested in 4 mg/ml collagenase D (Sigma-Aldrich, Cat# 11088858001) on an orbital shaker at 120 rpm, 37℃ for 20 mins.