Project Summary Injuries to the tendon-bone attachment, or enthesis, are common and challenging to treat clinically, with re-rupture rates of up to 94% in patients. The enthesis is composed of a special cellular and matrix composition which is uniquely tailored to transmit force from tendon to bone, enabling movement. While the mammalian enthesis can heal, its distinctive structure cannot be restored after injury. Even with existing treatments, healing forms disorganized scar tissue with compromised function, leading to a higher likelihood of both re-injury and developing joint degenerative diseases, ultimately impacting patient quality of life. As current treatment options are limited and highly variable in efficacy, better clinical strategies are needed. Unlike mammals, zebrafish can fully regenerate their tendon-bone attachments. Notably, following injury, preliminary data indicate that endogenous scleraxis a (scxa)-expressing tendon cells in zebrafish proliferate and migrate to regenerate the enthesis, while their mammalian Scx+ counterparts remain quiescent. The mechanisms underlying these divergent scxa/Scx+ tendon cell responses after enthesis injury remain unknown yet may be key to redirecting fibrotic responses into regenerative outcomes in mammals. The goal of this proposal is to identify molecular and cellular mechanisms which control cell activation after injury by employing a combination of multiphoton imaging, chemical, genetic, and cross-species comparative genomic approaches in both zebrafish and mice. During the mentored K99 phase, mechanisms that are required and sufficient for tendon cell proliferation and recruitment after enthesis injury will be identified (Aims 1 and 2). In the independent R00 phase, Dr. Tsai will investigate the role of surrounding cell populations in enthesis regeneration and elucidate how their injury-responsive signaling interactions with tendon cells diverge between zebrafish and mammals to drive regeneration or scarring (Aim 3). These findings will not only uncover instructive cues directing regenerative and fibrotic responses which may be leveraged to inspire novel strategies to treat enthesis injuries, but also expose basic principles underlying scarring which may extend to other tissues. The extensive training plan in the K99 phase will be conducted under the mentorship of Dr. Jenna Galloway and co-mentor Dr. Jayaraj Rajagopal at the Center for Regenerative Medicine at MGH, a collaborative and intellectually vibrant research community with ample opportunities for scientific and professional skill development. In addition to boosting grant writing and leadership skills, Dr. Tsai will also acquire scientific expertise in new areas with the aid of her advisory committee and collaborators. These include enthesis healing and biomechanics (Dr. Elazar Zelzer, Dr. Stavros Thomopoulos), single cell ATAC-sequencing (Dr. Mansi Shrivastava, Dr. Rajagopal), and cross-species comparative genomics (Dr. Bo Wang)...