PROJECT SUMMARY / ABSTRACT Intervertebral disc (IVD) injury such as herniation and degeneration can result in low back pain which is a leading cause of global disability. Tissue engineering strategies which successfully recapitulate IVD structure could restore function and reduce the burden of IVD injury. However, intervertebral disc development is not well understood, hindering the therapeutic targeting of key molecular factors. Initially, the IVD center, the nucleus pulposus, arises from the segmentation of the continuous notochord through the movement of notochordal cells out of the future vertebra body and into the intervertebral disc. The IVD fibrous exterior, the annulus fibrosus, arises from sclerotomal cells which surround the notochord and differentiatiate through the deposition of aligned collagen fibers production of extracellular matrix. Molecularly, the notochord is a source of the secreted morphogen Sonic Hedghehog (Shh), and annulus fibrosus expresses the growth factor Tgfβ. This grant will test the necessity of the notochord in two different stages of IVD development: proliferation, and actin fiber formation in the AF. The second aim will determine the role of the mechanical force generation, myosin, actin filament formation and Rho pathway activation on AF formation. The last aim determines the quantitiative expression and necessity of Tgfβ signaling in AF differentiation including cell alignment and extracellular matrix deposition. This project utilizes mouse genetics to manipulate molecular cues, and assays the molecular and phenotypic effects with in situ hybridization, immunohistochemistry and RNA sequencing. The successful completion of the project will provide insights on molecular and mechanical drivers of intervertebral disc formation and yield insights which can be used for developing repairs for painful IVD injury.