A spatial and single-cell transcriptomic atlas of the embryonic synovial joint and its explant model

Synovial joint formation requires the coordinated differentiation of multiple cell types while balancing opposing migratory cues that recruit joint progenitors and exclude other populations. More specifically, a subset of limb bud mesenchyme is recruited into the interzone while neighboring chondrocytes and endothelial cells are excluded. Existing single-cell references sample this process sparsely, without position, or without the surrounding mesenchyme. Here, we present an atlas of the mouse hindlimb from E11.5 to E15.5 that pairs 228,703 single-cell transcriptomes with 47 spatial transcriptomic sections, annotates 56 cell types by their location, and extends the same profiling to limb explant culture. Explants preserve the lineages and axial patterning of the limb for three days, with delayed chondrocyte hypertrophy. Using the atlas, we found that Sema3c is co-expressed with Sox9 and Gdf5 during condensation and is required for chondrogenesis and joint formation in culture; that interzone cells carry a chondrogenic signature early and a tenogenic signature late; and that a core interzone signature includes eleven previously unreported markers, one of which, Igfbp5, is required for knee formation in explants.