Craniofacial injuries require complex treatments to facilitate bone healing, however, current treatment methods for critical-sized bone loss fail to adequately promote complete healing. This is in part due to our limited understanding of how cell types in bone communicate via secreted factors to reconstruct native bone tissue. Specifically, peripheral nerves innervate bone to control bone formation during development and healing. In bone healing, nerves infiltrate into defects immediately after injury and retract as healing ensues. When nerves are inhibited prior to injury, bone formation is reduced. In non-healing defects, nerves fail to retract to baseline, suggesting that nerve retraction may be required for healing. Understanding the signaling interactions that control these phenomena could result in the identification of targets to facilitate improved bone formation. Our objective is to investigate the role of nerve infiltration and retraction on bone healing through identification of signaling interactions between nerves and osteoprogenitors in healing and non-healing defects. Nerves in the skull are challenging to visualize and require advanced 3D imaging. Further, it is difficult to identify secreted factors and signaling interactions that are specific to the distally located nerve cell bodies. Thus, I will combine advanced imaging and single cell transcriptomic techniques to reveal nerve 3D spatial associations and signaling interactions with osteoprogenitors in calvarial defect injuries. First, I will create sub critical- (1-mm) and critical- sized (4-mm) defects in the parietal bones of Baf53b-tdTomato mice, which constitutively express tdTomato in all peripheral nerves. I will use quantitative light sheet imaging (QLSM) to visualize tdTomato+ nerves and Osx+ osteoprogenitors at early and late timepoints following injury to reveal the spatial associations with and proliferation of EdU+ osteoprogenitors that occurs as nerves infiltrate into both defects and only fully retract in sub critical-sized defects. I will co-register QLSM images with μCT scans to investigate how neuroskeletal spatial associations correlate with regions of bone formation. To explore signaling interactions between nerves and osteoprogenitors, I will inject a tdTomato+ AAV retrograde tracer into the parietal bone to label nerve cell bodies in the trigeminal ganglia that innervate the defect region. I will harvest the trigeminal ganglia and defect region for single cell RNA-sequencing. Using differential gene expression and ligand-receptor analysis, I will uncover the signaling interactions between nerves and osteoprogenitors that mediate the osteoprogenitor proliferation, differentiation, and, ultimately, bone formation. I will next induce nerve retraction at early and late timepoints using Baf53b-tdTomato/TrkAF592A mice to establish the role of nerve retraction on osteoprogenitor proliferation and bone formation. Lastly, I will evaluate neural signaling factors id...