Approximately 8 million bone fractures occur annually and 10% of these are delayed and nonunion fractures which fail to heal despite therapeutic intervention. Current therapeutic strategies are severely hindered by the limited supplies of autograft bone and the poor mechanical properties of artificial materials. Zebrafish are masters of regeneration, and I have found that adult zebrafish can rapidly regenerate up to half of its lower jawbone after resection. Craniofacial bone development is highly conserved between zebrafish and humans, and hence understanding large-scale bone regeneration in zebrafish may lead to novel treatments in patients. A unique feature of zebrafish jawbone regeneration is the involvement of an unusual bone-producing chondrocyte population. In this proposal, I aim to identify the progenitor population that generates these ossifying chondrocytes during large-scale bone regeneration, as well as the role of macrophages in stimulating this progenitor population. Utilizing Cre-based transgenic lineage tracing strategies, I will test that a Runx2+/Sp7- population of pre-osteolasts in the periosteum generate the cartilage callus during regeneration. Using a genetic ablation strategy, I will then test that macrophages are required for the earliest events in bone repair, namely the shift of periosteal cells from making bone during homeostasis to making cartilage during repair. Lastly, I propose to utilize a RUNX2:GFP transgene to isolate these periosteal cells for deep sequencing of expressed mRNAs, which will help identify macrophage-dependent genes activated in periosteal cells after injury. Together, positive findings will illuminate the role of the immune system in shifting the fate of periosteal cells from bone to cartilage during bone repair, with knowledge gained in the zebrafish model being used in the future to develop new bone repair strategies in patients.