PROJECT SUMMARY/ABSTRACT Healthy joints are important for everyday activities and the leading cause of disability is osteoarthritis. Ligament injury resolves with fibrous scar tissue that destabilizes the joint due to inferior scar tissue. There are no adequate treatments to address this problem due to the limited capacity of our ligamentocytes to regenerate native tissue. Current models used to study ligament repair experience fibrotic healing and are insufficient in addressing this challenge to regenerate. Our lab has developed a novel zebrafish model to study the basic biology underlying interopercular mandibular (IOM) craniofacial ligament regeneration. Our preliminary data shows that after IOM transection, zebrafish regenerate a scar-free ligament within a month. We show that ligamentocytes dedifferentiate and contribute to the regenerated ligament. This tightly regulated regenerative response is characterized by constant crosstalk between the dynamically changing extracellular matrix (ECM) and the different cell populations present in the injury microenvironment. For comparative analysis, we have developed a legumain (lgmn) mutant zebrafish model that experiences fibrotic healing to gain insight into the molecular and cellular regulation of regeneration vs. fibrosis. Lgmn is a cysteine protease involved in ECM remodeling and we show that lgmn mutants heal with a mis-patterned, scarred ligament after transection. In contrast to WT regeneration, lgmn mutant fibrotic healing is characterized by a defect in ligamentocyte dedifferentiation, failure to integrate new and old tissue, and disorganized collagen throughout the course of ligament healing. To investigate the role of Lgmn-mediated regeneration, this proposal will identify the subcellular localization and molecular mechanism of Lgmn in the context of craniofacial ligament regeneration. Additionally, this proposal will also characterize the proteomic profile of the zebrafish IOM ligament during homeostasis, regeneration, and fibrosis. This will yield new knowledge on differentially expressed proteins during these different states that can be used to identify potent molecular regulators of a pro-regenerative microenvironment. Our preliminary scRNAseq analysis shows a subset of macrophages at the injury site that express ECM remodeling factors including lgmn. Following IOM transection, lgmn mutants have less macrophages present at the injury site. Together, this indicates an important role of macrophage-derived lgmn in mediating a pro- regenerative microenvironment. To test this, I plan to use scRNAseq to characterize changes in macrophage subsets between WT and lgmn mutants. Further, I will functionally test lgmn mutant macrophages and use adoptive macrophage transfer to test if lgmn mutant scarred healing is rescued. Through these aims, I will uncover the role of Lgmn in mediating the injury microenvironment during ligament regeneration. Under the mentorship of Dr. Joanna Smeeton and Dr. Laura Johnston along with their combined expertise in zebrafish joint biology and genetics, I will be well prepared for my next role as a post-doctoral fellow. The training plan outlined will help me develop the necessary skills to succeed as an independent craniofacial research scientist.

PROJECT NARRATIVE Joints are used for everyday movements and ligament injury compromises joint health due to the fibrous scar tissue that forms and alters native joint movement. This proposed work will use the highly regenerative zebrafish as a model to study ligament regeneration to gain insight into the molecular factors promoting ligament regeneration vs. fibrosis after injury. By understanding the mechanisms driving scar-free ligament regeneration, we can better develop future therapies for acute ligament injury that will rebuild native tissue while also preventing fibrosis.