Dcx expression defines a subpopulation of Gdf5 ⁺ cells with chondrogenic potentials in E12.5 mouse embryonic limbs

Growth differentiation factor 5 (Gdf5) and doublecortin (Dcx) genes are both expressed in joint interzone cells during synovial joint development. In this study, we re-analyzed the single cell RNA-sequencing data (Gene Expression Omnibus GSE151985) generated from Gdf5 ⁺ cells of mouse knee joints at embryonic stages of E12.5, E13.5, E14.5, and E15.5, with a new focus on Dcx. We found that Dcx expression was enriched in clusters of Gdf5 ⁺ cells, with high expression levels of pro-chondrogenic genes including sex determining region Y-box transcription factor 5 (Sox5), Sox6, Sox9, Gdf5, versican, matrilin 4, collagen type II α 1 chain (Col2a1), Col9a1, Col9a2, and Col9a3 at E12.5. Dcx ⁺ and Dcx ^- cells had differential gene expression profiles. The up-regulated genes in Dcx ⁺ vs. Dcx ^- cells at E12.5 and E13.5 were enriched in chondrocyte differentiation and cartilage development, whereas those genes up-regulated at E14.5 and E15.5 were enriched in RNA splicing, protein stability, cell proliferation, and cell growth. Gene expression profiles in Dcx ⁺ cells showed rapid daily changes from E12.5 to E15.5, with limited number of genes shared across the time period. Expression of Gdf5, Sox5, Sox6, melanoma inhibitory activity, noggin, odd-skipped related transcription factor 2, matrilin 4, and versican was positively correlated with Dcx expression. Our results demonstrate that Dcx expression defines a subpopulation of Gdf5 ⁺ cells with chondrogenic potentials in E12.5 mouse embryonic limbs.