Project Summary Skeletal muscle tissue is essential for life, requiring continuous maintenance and repair by a resident population of skeletal muscle stem cells or satellite cells (SCs). Maintenance and repair of skeletal muscle re- quires fusion of SCs into existing myofibers or fusion of SCs with each other to replace a damaged myofiber, as inhibiting SC fusion prevents hypertrophy of myofibers and elimination of SCs prevents skeletal muscle repair. Inactive SCs are mitotically quiescent, residing between the myofiber plasma membrane and the basement membrane, an inherently asymmetric environment. Following a muscle injury, quiescent SCs activate, exiting quiescence and proliferate, eventually terminally differentiating with daughter cells fusing or re-acquiring qui- escence and re-occupying their niche to replenish the SC population. Replenishment of SCs occurs via symmet- ric or asymmetric division, likely balanced by the extent of muscle repair or maintenance. Understanding the mechanisms regulating SC division will provide knowledge for development of therapeutics targeting SCs for improving muscle repair to combat aging and muscle diseases. Single cell sequencing and single nuclear se- quencing of skeletal muscle has revealed unexpected heterogeneity in skeletal muscle myonuclei as well as the unexpected contribution of non-SC progenitors to myonuclei. Because skeletal muscle cells are syncytial, de- rived from fusion of many hundreds of progenitors or SCs, lineage tracing the parental cells producing myonu- clei has proven highly technically challenging, broadening a knowledge gap that does not exist in tissues popu- lated by mononuclear cells. To fill this knowledge gap, we have developed a novel approach to lineage trace my- onuclear parents that is a flexible, quantitative method that can be applied to SCs and non-myogenic myonu- clear progenitors. Moreover, our method allows quantification of the cell divisions a myonucleus underwent prior to terminal differentiation, which has not previously been attempted in most lineage tracing experiments. We plan employ the methodology we have developed to aid in answering: (i) Are clonally diverse myonuclei derived from distinct progenitors?, (ii) Are heterogeneous populations of SCs responsible for producing myo- nuclei and SCs, respectively?, and, (iii) What environmental signals affect SC behavior during production of centrally located myonuclei vs. production of peripheral nuclei? to fulfill broad knowledge gaps in understand- ing how myonuclear diversity ensures that skeletal muscle is functional and healthy.