In contrast to humans, zebrafish robustly repair regenerate severely damaged bone. The long-term goal is to untangle such regenerative mechanisms at work in zebrafish and mimic these processes in human cells, thereby promoting new therapies for aged, damaged, or diseased bone. During zebrafish bone regeneration, coordinated Wnt and Bone Morphogenetic Protein (BMP) signaling directs pre-osteoblasts to self-renew and re-differentiate, respectively, to progressively re-form lost bone. While BMP-dependent osteoblast maturation is lineage intrinsic, the Wnt ligands needed to sustain pre-osteoblasts are produced by neighboring non- osteoblast cells, the Regenerating Fin Progenitor Niche (RPN). However, the cell lineage of RPN cells, and the transcriptional networks that define the RPN are unknown. The objectives of this proposal are to identify the cell lineage that contributes to the RPN and reveal transcriptional networks at work in these cells. The working hypothesis is that fin amputation induces cells of a unique lineage to dedifferentiate and form the RPN that resides at the distal tip of the regenerating fin. As regeneration completes, these unipotent RPN cells re- differentiate to their original state. The rationale for this work is that discovering how the RPN is formed and the transcription networks it uses will support novel therapies to generate and deploy human RPN-like cells at sites of bone damage, providing a pro-osteogenic microenvironment that enhances bone regeneration. This hypothesis will be tested by pursuing two aims: 1. Determine the origin and fate of RPN cells. To test our hypothesis that dedifferentiated intra-ray cells form the RPN, we will use transgenic fish to genetically label specific cell lineages in the fin and determine which comprises the RPN by virtue of expressing the RPN- specific marker Dach. 2. Establish transcriptional networks that define organization of RPN-lineage cells. To test the hypothesis that Dach+ RPN cells differentiate into Snail+ intra-ray mesenchymal cells, expression of these transcription factors (TFs) will be examined at high resolution over the course of bone regeneration to determine the spatial and temporal relationships among Dach+, Snail+, and Dach+/Snail+ populations. Further, determining the spatial and temporal expression dynamics of Dach/Eya1/Pax6/Six3 TFs will test the hypothesis that they represent a RPN-specific TF network. This approach is innovative because it utilizes a versatile model system, the zebrafish, which nature has endowed with extraordinary power to regenerate damaged bones. The proposed research is significant because it is expected to expand knowledge of how distinct cell lineages cooperate in an intact animal to achieve bone regeneration. Ultimately, this knowledge could promote development of novel therapies to repair and restore damaged bone.