Abstract The meniscus plays a vital role in knee function, but injury is common and healing is limited in adults. Development of effective regenerative solutions is challenged by our limited understanding of the cellular mechanisms that regulate meniscus formation, maturation, and maintenance. In the previous funding cycle, we queried the origins of cell fate and biosynthetic function in the developing meniscus. We found that the embryonic-to-early postnatal growth period is the most active time frame of meniscus cell specification, patterning, and regional specialization. We also demonstrated a central role of external mechanical loading and internal cell mechanosensing in regulating meniscus patterning, growth and matrix organization during this early developmental phase. Despite these findings, the timing and mechano-epigenetic mechanisms controlling the early development of meniscus remain unresolved. To address this, in this renewal, we aim to elucidate the roles of cellular force generation and mechanosensing machinery in the formation of the meniscus during early development, as well as its maintenance in the adult. Our central hypothesis is that cell generated tension is essential for meniscus cell inner-to-outer specification and maintenance across the meniscus lifecycle. To test this hypothesis, we will induce timed ablation of cellular force generating machinery, the non-muscle myosin (NMII) genes Myh9 and Myh10 (Myh9/10) in meniscus progenitors at key developmental time points, as well as in the adult meniscus. We will test if this ablation results in aberrant patterning and matrix formation during early development and if it results in loss of cell phenotype and matrix degeneration in adults. Specifically, Aim 1 will determine the role of acto-myosin contractility in the specification and development of the embryonic meniscus. We will test if early ablation of Myh9/10 in the murine embryonic meniscus results in aberrant patterning, and if later ablation leads to insufficient matrix elaboration and impaired maturation. Aim 2 will establish the mechano- epigenetic basis of contractility-mediated regional specification within the embryonic meniscus. Single cell RNA- seq will be applied to evaluate phenotypic heterogeneity at key time points. We will also apply ATAC-seq to determine if ablation of contractility and/or Myh9/10 decreases the accessibility at fibrous matrix genomic loci and increases the accessibility at chondrogenic/remodeling loci in porcine meniscus progenitors cultured in vitro. Aim 3 will determine if loss of cellular force generation and mechanosensing machinery in the adult instigates meniscus degeneration. We will test the effect of Myh9/10 ablation in adult mice by evaluating meniscus cell mechanosensing, fate, matrix production and mechanical properties, and assess how adult porcine and human meniscus cells shift their phenotype upon the loss of tension in vitro. We expect the outcomes to generate novel data defin...