Project Summary. The long-term objective of this study is to understand signals normally involved in development of the axial skeleton so that therapies that mimic normal development can be applied to the spine. Members of the Tgfb superfamily are secreted signaling proteins that regulate many aspects of skeletal development and maintenance. Polymorphisms and mutations in genes that regulate Tgfb activity have been associated with pathology in the spine. Previously, we and others showed using genetically engineered mice and cell culture models that Tgfbr2 is required for development and maintenance of the fibrous tissues in the spine: the annulus fibrosus of the intervertebral disc, ligaments, and tendon. Results obtained during the previous funding periods indicate that Tgfb regulates cell fate decisions in the sclerotome, the progenitor of the connective tissues in the spine. Based on published literature and preliminary data we proposed that Tgfb favors the formation of fibrous cell types while BMP favors chondrogenesis and formation of the vertebral bodies. In this application we propose to address the instructive mechanisms whereby Tgfb regulates formation and maintenance of fibrous tissues in the spine. In addition, we propose to address the problem of sclerotome resegmentation during development of the axial skeleton. Resegmentation is a process that creates the spatial organization of tissues in the spine that allow motion. Alterations in resegmentation would be expected to alter the context in which cells differentiate, affecting permissive signals and competence to respond to instructive signals that govern pair-wise cell fate decisions. Finally, using a mouse model and TGF-ß loaded scaffolds, both developed in the last funding period, we will determine the role and effects of TGF-ß on mechanically induced disc degeneration. Specific hypotheses based on published results and preliminary data will be tested: 1) Tgfb acts through a non-canonical signaling pathway involving Erk to regulate Scx mRNA expression and then through cooperation of Scx and Smad3 proteins to regulate fibrous differentiation in the spine;; 2) Tgfb regulates resegmentation of sclerotome;; and 3) TGF-ß protects and/or repairs fibrous tissues subjected to mechanically induced degeneration. The experiments described here will address questions about development in the axial skeleton and provide information necessary for future efforts to engineer therapies for the spine.