The synovial joints are critical for skeletal motion and function, and their structure, organization, distinct tissues and susceptibility to diseases, including osteoarthritis, are well understood. However, basic aspects of their developmental biology remain unclear. If available, such information and insights could be used to create new joint tissue repair strategies. In early fetal limbs, the skeletal primordia are initially continuous and uninterrupted by joints. Joint development starts with emergence at each prescribed anatomical site of mesenchymal cells, called the interzone, that are dense and compacted and express growth-and- differentiation-factor 5 (Gdf5). Cell lineage tracing and tracking in transgenic mice showed that the Gdf5 expressing cells and progenies represent unique stem cells and produce most, if not all, joint tissues over time, including articular cartilage, ligaments and synovial lining. Though new and broadly relevant, these and other studies left much unclear. Notably, one of the least understood processes in joint formation is how the synovial cavity forms, how it can be created within the compacted interzone and what mechanisms attract and accrue its fluid, surprising gaps in knowledge given the cavity’s critical nature and essential roles. Previous studies showed that joint cavitation is associated with local hyaluronate production, protease expression and muscle motion, all processes contributing to interzone cell-cell contact relaxation. But how does the cavity itself form and enlarge? In preliminary studies, we have found that cavity enlargement requires active and energy- requiring mechanisms able to attract fluid and distance the opposing articulating surfaces from each other, eliciting a synovial cavity space. Using pharmacological approaches, we have found that these mechanisms and their activities are in fact required for cavity formation and growth. These and other preliminary data lead to the central hypothesis that cavitation is a stepwise process brought about by convergence and coordination of distinct regulatory mechanisms. We propose two interrelated Aims in which we will carry out single cell analyses to delineate genes involved and upstream regulatory mechanisms (Aim 1) and will test the roles of these mechanisms in postnatal joint maintenance, endurance and structural and functional capacities (Aim 2). We will make use of diverse analytical approaches including molecular genetics; histomorphometry; microCT; single cell RNAseq; in situ hybridization; tissue isolation; cell cultures and cell fractionation; and imaging quantification and reconstruction. This high risk-high return R21 project is expected to provide wholly novel and previously unsuspected data and insights into joint cavitation and function. Limb joints are affected by various diseases for which current treatments are only partially effective and not long-lasting. With its novel concepts and data, the present project promises to pave the...