Abstract Mammalian bone has the capacity throughout life to regenerate in response to fracture injury. However, there is a ceiling for this regenerative potential, with hurdles to regeneration after a major trauma like limb amputation. This has a significant socioeconomic impact, as it is estimated that at least one in two Americans over age 50 is expected to have or be at risk of bone disease, and every year an estimated 1.5 million individuals suffer a fracture due to bone disease. Recently, we have developed imaging methods to study how osteoblasts drive bone regeneration in zebrafish, which display robust regeneration after major injury to bony structures like their fins, scales, and jaws. Our goal is to exploit this regenerative capacity, new imaging platforms we have created, and the molecular genetic approaches available in zebrafish to improve our ability to understand and manipulate the regenerative capacity of bone. The goal of this proposal is to generate an in toto map of the cellular and signaling events that regenerate patterned skeletal bone. Our experiments will test the hypothesis that correct patterning of regenerating bone requires dynamic signaling events that control osteoblast behaviors at individual and population levels. 1) We will use long-term live imaging, labeling with photo-convertible proteins, and computational analysis to generate a detailed map of how cell proliferation, hypertrophy and cellular flows, and interactions with neighboring tissues drive bone regeneration. 2) We will use cutting edge biosensors, live imaging, computational approaches, and mathematical modeling to dissect how traveling waves of chemical signals stimulate the growth of a regenerating osteoblast population. 3) We will use transcriptome profiling approaches to derive further insights on the dynamics of growth factor signaling, including single-cell sequencing-based approaches to link gene expression programs with osteoblast behaviors. These experiments will define a novel quantitative framework for understanding how osteoblast behaviors orchestrate bone regeneration.