Synovial joint cavitation during limb skeletogenesis entails Na/K-ATPase ion pump expression and osmoregulatory activity

Limb synovial joints rely on a water/lubricant-filled cavity to sustain skeletal movement through life, but mechanisms generating the cavity within the primordial joint interzone during embryogenesis remain unclear. Because water accrual would be needed to create and inflate the cavity, its formation may require energy-consuming osmoregulatory mechanisms able to draw water and exert mechanical force. Our in situ hybridization and scRNAseq data reveal that interzone cells in developing mouse embryo joints strongly expressed the Na/K-ATPase ion pump genes Atp1a1, Atp1b1 and Atp1b3. There was also local and specific expression of water channel aquaporin 1 (Aqp1) and mechano-sensing genes. When pregnant mice were administered ouabain, which is a physiological glycoside that limits pump activity and osmoregulatory processes, joint cavitation in embryos was inhibited, as was lubricant gene expression. Joint development depends on signals from Indian hedgehog-expressing growth plate chondrocytes. Interference with hedgehog signaling coordinately inhibited pump, mechano-sensing and lubricant expression and cavitation. Our data provide a new understanding of joint cavitation as an energy-requiring osmoregulatory process that accrues a water-based fluid from interstitial and transcellular sources, and establishes a fluid-filled cavity in coordination with long bone development.