The Role of Sox9 in Regulating Joint Development through a Gdf5 Enhancer

Abstract

Sox transcription factors are involved in a number of developmental processes including joint formation. Sox9, Sox5, and Sox6 (the Sox‐trio) form a complex that promotes chondrogenesis. In joint‐associated cells, Sox9 may give rise to a subpopulation of cells expressing growth differentiation factor 5 (Gdf5). Gdf5, a secreted morphogen critical to joint development, regulates cartilage growth and tissue differentiation. Osteoarthritis (OA) is condition characterized by articular cartilage degradation and is a leading cause of disability worldwide. Gdf5 dysregulation has been identified as a risk factor for OA, and in the joints of OA patients, the Sox‐trio are downregulated. The link between Sox, Gdf5, and joint development is not fully understood. We previously identified an enhancer for Gdf5, known as Gdf5‐Associated Regulatory Region (GARR), and have identified possible Sox9 and 5/6 binding sites within GARR by in silico analysis. Thus, we hypothesized that Sox9 is required for Gdf5 upregulation through GARR, while other Sox transcription factors (Sox5/6) enhance or stabilize this interaction. To test this hypothesis, we analyzed publicly available single cell RNA sequencing (scRNA‐seq) data from mouse embryo (E12‐5‐E15.5) knee joints. Analysis revealed a strong correlation between Gdf5 and Sox9. Sox9 was upregulated in Gdf5 positive cells. Sox5 and 6 were upregulated in Gdf5 positive cells but had a lower correlation to Gdf5 expression indicating a weaker or indirect/secondary role in Gdf5 regulation. We also compared the activity of the wildtype GARR enhancer‐reporter construct to constructs with mutated Sox binding sites in developing chicken limbs to determine the role of these sites on enhancer activity. We found that in developing chicken embryo limbs, constructs with mutated Sox9 binding sites had a marked decrease in enhancer activity in the elbow but not in the autopod. Mutation of predicted Sox5/6 binding sites did not seem to affect activity relative to the non‐mutated control in the forelimb joints. Our findings suggest that Sox9 is a key regulator of elbow development through GARR‐mediated expression of Gdf5. The correlations identified in the transcriptomic data may be relevant only to the elbow and knee joints. Alternatively, the Sox transcription factors may be acting though other Gdf5‐associated enhancers in the autopod joints. Further investigation of enhancer activity in the presence of various combinations of Sox5/6/9 will throw more light on how they collectively affect Gdf5 expression as well as joint development and OA progression.