Synovial joints are critical for skeletal function, but we remain surprisingly ignorant about how they actually form during fetal life. In the limb, joint development starts with appearance of a mesenchymal interzone at future joint sites. Interzone progenitor cells are thought to give rise to tissues including articular cartilage and participate in joint morphogenesis, but it is not clear how the cells perform these critical tasks. The signaling proteins GDF-5 and Wnt-14 are strongly expressed by early interzone. GDF-5 was found to act as a growth and interzone determination factor, while Wnt-14 acted as an upstream regulator of interzone/early joint genes, including GDF-5 itself, Chordin and CD-44. In preliminary work we show for the first time that: Wnt-14 and GDF-5 are expressed in distinct patterns during interzone formation;peri-joint mesenchymal cells migrate into nascent interzone;GDF-5 expression becomes restricted to convex side during joint morphogenesis;and Wnt-14 signaling involves b-catenin. Our central hypotheses are: (i) interzone is made of progenitor cells derived from peri-joint sites;and (ii) GDF-5 and Wnt-14 have distinct, but interrelated roles during interzone and joint formation and morphogenesis. Our aims are to establish origin and fate maps of interzone cells, determine GDF-5 and Wnt-14 roles and mechanisms of action, and create conditionally Wnt-14-ablated mice and assess consequences. We will use cell tracing-tracking procedures, including ROSA reporter mice mated with available GDF-5-Cre mice;chimeric-microsurgical approaches;and Wnt-14 and GDF-5 gain- and loss-of-function approaches on chick and mouse autopods (paws), including those isolated from available GDF-5-null brachypodism mice and b-catenin-dependent axin-2 promoter reporter mice. The project will generate fundamental information on mechanisms of joint formation. The results will be invaluable in conceiving novel, directed and specific therapies to repair and restore malfunctioning joints common to aging individuals and otherwise affected patients.