Project Summary Resident tissue-specific stem cells serve to repair and maintain many adult organs; however mammalian joint cartilage and ligaments have a very poor capacity for intrinsic repair. This lack of robust endogenous repair may be at the root of many degenerative diseases of joints, in particular osteoarthritis. Dr. Smeeton's postdoctoral work in Dr. Gage Crump's lab at USC uses the highly regenerative zebrafish to investigate the role of stem cells in rebuilding the cartilage and ligaments of the jaw joint. As in mammals, surgical injury to the ligament supporting the zebrafish jaw joint results in loss of articular cartilage. However, a remarkable difference from mammals is that both the ligament and joint cartilage subsequently regenerate in zebrafish. The aims outlined in this proposal will build on Dr. Smeeton's extensive preliminary data establishing this new surgical model and make use of novel transgenic and CRISPR/Cas9 knock-in lines to investigate the timeline and major cellular events of jaw joint regeneration (Aim 1), the respective roles of Sox9+ and Sox10+ stem cells in rebuilding ligaments and cartilage (Aim 2), and the molecular events driving regeneration (Aim 3). Aim 1 will be completed in the K99 phase. Aims 2 and 3 will be initiated during the K99 phase under the mentorship of Dr. Crump and her advisory committee and then completed during the R00 phase. In the long term, insights gained from studying the role of endogenous Sox9/10 stem cells in the zebrafish jaw joint could be harnessed to awaken analogous cell populations in mammalian joints to improve repair. The project and mentorship plan outlined in this proposal were designed to lay the groundwork for Dr. Smeeton's independent research program. Following her postdoctoral term, her goal is to obtain a tenure-track Assistant Professor position at a top-tier academic research institute. Using her novel surgical models and state-of-the-art genetics and imaging methods in zebrafish, her goal is use the zebrafish jaw joint to learn how to better rebuild cartilage and ligaments from resident stem cells ? a topic with a clear relevance for human health. During the K99 phase, both her scientific and career development will benefit from the expertise in zebrafish skeletal development and regeneration of her mentor, Dr. Crump. In addition, regular meetings with her advisory committee will allow her to acquire new expertise in joint biology (Denis Evseenko), ligaments (Amy Merrill), and RNAseq methodology (Andrew McMahon). Additional career development activities at USC, such as grant-writing, presentation and mentorship workshops will prepare Dr. Smeeton for her transition to a faculty position in the R00 phase. Dr. McMahon will be a particularly important advisor for the transition to independence given his extensive experience directing faculty searches at Harvard and USC. With its extensive network of craniofacial biologists and cartilage researchers, institutional support for career development, and outstanding technical resources, USC is a vibrant and exciting place to establish Dr. Smeeton's career in joint and craniofacial regeneration.