Tgfß 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. Overall design: 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.