PROJECT SUMMARY/ABSTRACT: Skeletal tissues provide structure that allows movement and protects essential organs in the body from damage. Whereas bone displays some capacity for repair, non-healing bone injuries remain a major financial and medical burden. Understanding the potential for skeletal stem cells (SSCs) to improve bone repair therefore holds great promise. A challenge for the field of craniofacial bone repair is that these bones have a different developmental trajectory from the more studied limb bones and undergo direct ossification rather than cartilage-mediated repair in response to injury. Thus, it remains unclear the extent to which the repair of craniofacial intramembranous bones depends on the same suites of SSCs as those of the limbs. In this proposal, I investigate a hypothesis that there are two fundamentally distinct origins of SSCs in bones: one type of SSC derived from nave mesenchymal cells in the embryonic perichondrium and periosteum (PO- SSCs) and a second type derived from hypertrophic chondrocytes of the growth plate, which dedifferentiate and move into the marrow cavity (GP-SSCs). Using intersectional genetics, lineage tracing, conditional cell ablation, and assays of open chromatin, I will test that GP-SSCs are especially important for cartilage callus formation due to maintenance of accessible cartilage enhancers from their growth plate origin (i.e. epigenetic memory). In the craniofacial intramembranous bones, the lack of growth plates and hence GP-SSCs would result in direct ossification during repair. To test these models, I have developed innovative models of intramembranous (premaxilla) and endochondral (ceratohyal) bone regeneration in the adult zebrafish head. Using parallel Cre-Lox and Dre-Lox systems, I will be able to simultaneously trace PO-SSCs and GP-SSCs and assess their contributions to and requirements for the repair of intramembranous versus endochondral bone repair. Together, my studies should reveal mechanisms by which SSCs regenerate intramembranous bones differently than endochondral bones, which will inform approaches to specifically repair the intramembranous bones of the face and skull. My mentor, Dr. Gage Crump, has an exceptional training record and runs the Development, Stem Cells, and Regenerative Medicine program to which I belong. The Crump lab is located within the rapidly growing Broad Stem Cell Institute, which is a highly collaborative and dynamic environment for my scientific development. These interactions will help me in adapting emerging techniques such as scRNAseq and ATACseq to my novel zebrafish bone regeneration models. A training plan that incorporates acquisition of skillsets in zebrafish genetics and imaging, specialized coursework in genomics, the honing of presentation and writing skills, and career development will help me in achieving my goal of becoming a successful independent scientist in the field of craniofacial regenerative medicine.