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

The developmental biology of limb synovial joints and their water/lubricant-filled cavity remains unclear. Joint formation is known to start with emergence at each prescribed anatomical site of a compacted Gdf5-expressing mesenchymal interzone flanked by cartilaginous skeletal primordia. We and others previously showed that the interzone cells serve as progenitors and generate most joint tissues over time, including articular cartilage and ligaments. But how is the cavity formed within the compacted interzone? Because water accrual would be needed to create and inflate the cavity and in turn distance the flanking skeletal elements, cavity formation may require energy-consuming osmoregulatory mechanisms able to draw water and exert mechanical force. Our data now reveal that interzone cells in cavitating knee and digit mouse embryo joints strongly express the Na/K-ATPase pumps Atp1a1, Atp1b1 and Atp1b3, members of a key cell surface osmoregulatory protein family that regulates fluid tonicity and drawing of water. There was also local and specific expression of the water channel Aquaporin 1 (Aqp1). Single cell RNA seq (scRNAseq) showed that Gdf5+ interzone cells expressing those genes culminated in number at cavitation and were characterized by other osmoregulatory and mechano-sensing genes also. Indeed, when pregnant mice were administered ouabain -a physiological glycoside that normally limits Na/K-ATPase pump activity and osmoregulatory processes-, limb joint cavitation in the embryos was inhibited as was lubricant gene expression. Joint development is known to depend on signals from Indian hedgehog-expressing growth plate chondrocytes in flanking skeletal primordia. Interference with hedgehog signaling did coordinately inhibit expression of pump, mechano-sensing and lubricant genes as well as cavitation. Together, our data suggest a novel understanding of joint cavitation as an active energy-requiring osmoregulatory process able to accrue a water-based fluid, distance the flanking articulating surfaces and establish a synovial cavity in close coordination with long bone development and growth. cell atlas of mouse knee joint cells at E16.5 timepoint肢体滑膜关节及其充满水/润滑剂的关节腔的发育生物学机制仍未阐明。已知关节形成起始于：在每个预设解剖位点，由软骨性骨骼原基侧翼包裹的、表达生长分化因子5（Gdf5）的致密间充质交界区出现。我们与其他研究团队此前已证实，交界区细胞作为祖细胞，随发育进程可生成绝大多数关节组织，包括关节软骨与韧带。但致密交界区内的关节腔隙究竟是如何形成的？由于腔隙的形成与扩张需要蓄积水分，进而使两侧的骨骼原基相互分离，因此腔隙形成可能依赖消耗能量的渗透调节机制，以摄取水分并产生机械力。我们的最新研究数据显示，正在发生腔隙形成的小鼠胚胎膝关节与指关节的交界区细胞，会高表达钠钾ATP酶泵Atp1a1、Atp1b1与Atp1b3——这类细胞表面关键的渗透调节蛋白家族成员，可调控体液张力与水分摄取。同时还存在水通道蛋白1（Aquaporin 1，Aqp1）的局部特异性表达。单细胞RNA测序（single cell RNA sequencing，scRNAseq）结果表明，表达上述基因的Gdf5+交界区细胞的数量在腔隙形成阶段达到峰值，且同时还具备其他渗透调节与机械感应相关基因的表达特征。事实上，当对孕鼠施加哇巴因——一种通常会抑制钠钾ATP酶泵活性与渗透调节过程的生理性糖苷时，胚胎的肢体关节腔隙形成过程受到显著抑制，润滑剂相关基因的表达也同样被阻断。已知关节发育依赖于侧翼骨骼原基中表达印度刺猬因子（Indian Hedgehog，Ihh）的生长板软骨细胞所传递的信号。干扰刺猬信号通路也会协同抑制钠钾泵、机械感应与润滑剂相关基因的表达，并阻碍关节腔隙形成。综上，我们的数据表明，关节腔隙形成是一种全新的、需要消耗能量的渗透调节过程：该过程可蓄积水性体液，使两侧的关节面相互分离，并与长骨的发育及生长紧密协同，最终形成滑膜关节腔。本数据集为E16.5时间点的小鼠膝关节细胞图谱。