Synovial lining macrophages form during early postnatal life via fetal-derived CX3CR1+ cells and AQP1+ intermediates.

The synovial lining maintains joint integrity, produces lubricating fluid, and forms a sterile barrier disrupted in inflammatory joint disease. It consists of specialized fibroblasts and macrophages, but its developmental timing and mechanisms are not well understood. We used genetic mouse models, imaging, and single-cell transcriptomic profiling to delineate this process. We found that the lining is immature in fetal human and newborn mouse joints. In mice, fibroblasts provide full coverage at birth, whereas macrophages are scarce. The macrophage lining layer gradually forms during the first weeks of life in a colony-stimulating factor 1 (CSF1)-dependent process, largely monocyte-independent, involving fetal-derived Cx3cr1+ macrophages and Aqp1+ intermediates. Both lining macrophages and fibroblasts undergo substantial transcriptional changes postnatally, acquiring their specific identity, upregulating key genes such as Prg4 (fibroblasts) and Vsig4 (macrophages), and establishing active signaling units. Early postnatal life is therefore a critical window for synovial lining maturation with implications for joint health and disease.