PROJECT SUMMARY This project will provide Dr. Victor Lewis with postdoctoral research training in vertebrate developmental genetics and genomics to prepare for a career as an academic principal investigator. The applicant will follow a multi-faceted research and training plan at the University of Oregon under supervision of lead sponsor Dr. Kryn Stankunas with additional mentoring from co-sponsor Dr. John Postlethwait. A fundamental goal of developmental biology is to identify the cellular and molecular mechanisms synchronizing cell behaviors across lineages for complex organ regeneration. Zebrafish fins perfectly recapitulate their original form following resection, making them a striking example of and tractable research model for appendage regeneration. An injury-induced de-differentiated but organized mass of cells called the blastema produces replacement fin cells and directs regenerative outgrowth. Fin outgrowth then depends on spatially segregated proliferation and differentiation activities that progressively restore organized tissue, including ray bones integrated with stump tissue. The major signaling pathways, the signal-producing and responding cells, and their general roles (e.g. growth vs. differentiation) are becoming appreciated. However, how signals enable spatiotemporally coordinated actions across cell types remains largely mysterious. Empowered by new single cell transcriptomics, Dr. Lewis will test a novel hypothesis that a regulated mesenchymal to epithelial-like cell state transition forms the outgrowth-promoting population of the distal blastema. He will also test a hypothesis how systemic factors coordinate osteoblast differentiation with fin outgrowth. Finally, Dr. Lewis will map the cell-type specific regulatory landscape of fin regeneration by integrating sophisticated single cell technologies with chemical genetics providing acute pathway inhibition. Collectively, this proposal will explore and characterize innovative models and mechanisms for coordinated outgrowth during appendage regeneration. Broadly, these insights will impact design of regenerative medicine approaches and provide a framework to understand appendage injury repair. Dr. Lewis’s training plan will broaden single-cell sequencing and technical research skills, 2) improve writing and communication skills, 3) develop skills as a mentor and teacher, and 4) engage in professional development activities to build skills to succeed as a principal investigator. The UO, Institute of Molecular Biology, and UO’s internationally known zebrafish research community provide facilities, equipment, and intellectual environment ideally aligned with Dr Lewis’s postdoctoral research and training goals.

PROJECT NARRATIVE How vertebrate appendages are repaired following injury remains a fundamental mystery of developmental biology. The proposed research will reveal new concepts and mechanisms enabling zebrafish to robustly regenerate their fins. As such, the project will improve our understanding of congenital limb defects and how to enhance limb healing in humans.