PROJECT SUMMARY/ABSTRACT Ligament injuries are compounded by the drastically increased risks of reinjury and eventual osteoarthritis. These risks result from a failure of differentiation; torn mammalian ligaments reform with fibrous scar tissue rather than with true ligamentocytes, altering the biomechanical properties of the repaired ligament and destabilizing the nearby joint. While the current models (mice and rabbits) used to study ligament repair recapitulate ligament scarring, they are unlikely to yield novel insights into targets for regenerative therapeutics. To address this shortcoming, our lab has developed the highly regenerative zebrafish as a model for craniofacial ligament regeneration. Zebrafish have the remarkable capacity to regenerate their ligaments without scarring in under a month. Our preliminary data demonstrate that the jaw joint-supporting interopercle (IOP) ligament in zebrafish shows identical morphology to the uninjured ligament as soon as 28 days after transection. During this time, the injured ligament is replaced with a dense mesenchyme which deposits a complex extracellular matrix to remake a functional ligament. Additionally, preliminary lineage tracing experiments show that both the uninjured ligament and the regenerative mesenchyme are cranial neural crest-derived. This project aims to identify the source of the mesenchymal cells which reform the ligament, as well as the genetic and epigenetic changes which promote ligamentocyte fate. In Aim 1, I will use enhancer peaks with increased accessibility after ligament injury to firstly trace the lineage of the regenerative mesenchyme to the regenerated ligament, and secondly trace the lineage of the periosteum through regeneration. In Aim 2, I will analyze my single-nuclei multiomic (gene expression and chromatin accessibility for each cell) data from jaw joints through IOP ligament regeneration to generate a short list of candidate transcription factors using a selection funnel of motif accessibility, gene expression, and transcription factor binding. I will then assess in vivo if these transcription factors are expressed before ligamentocyte fate is adopted, and generate knockout zebrafish lines to assess if each transcription factor is necessary for ligamentocyte fate. Through these aims, we will unveil a novel population of cells capable of regenerating ligaments, as well as the transcription factors necessary for ligamentocyte differentiation. The training plan outlined through this proposal will develop the techniques, mentorship, and communication skills necessary to establish my future career as an independent scientist studying craniofacial regeneration. Through the mentorship of Dr. Joanna Smeeton (Sponsor), Dr. Stavros Thomopoulos (Co-sponsor), and my thesis advisory committee, I will be well prepared for my transition to a postdoctoral fellowship at a leading biomedical institution. Dr. Smeeton has extensive knowledge of the zebrafish as a model for craniofacial regeneration, and Dr. Thomopoulos is an expert in translational mouse tendon development with an outstanding mentorship record. Under their combined sponsorship, I will learn cutting-edge computational and in vivo genetics to develop a productive career in regenerative craniofacial biology.

PROJECT NARRATIVE Human and mouse ligaments reform with a fibrous scar after injury, leading to sharply increased risk of reinjury and osteoarthritis later in life. I plan to use the highly regenerative zebrafish as a novel model to study scar-free craniofacial ligament regeneration and identify which cells have the potential to reform the ligament as well as which genetic signals are necessary for ligament fate. In doing so, I will identify targets for future therapeutics to improve patient outcomes after acute ligament injury.