PAX3-SIX2 muscle stem cell heterogeneity drives muscle-specific repair dynamics diversification

Skeletal muscle regeneration relies on muscle-specific stem cells (MuSCs) expressing PAX7. Its paralog, PAX3, a key regulator of embryonic myogenesis, is selectively expressed in a subset of adult MuSCs, with variable proportions across muscles. Here, using lineage tracing and muscle injury models, we show that PAX3-driven MuSC diversity underlies muscle-specific regeneration rates. PAX3⁺ MuSCs exhibit enhanced proliferation and differentiation, supporting rapid repair, while PAX3 loss leads to proliferation arrest and cell death. Single-cell RNA-sequencing revealed that PAX3 regulates SIX2 expression, and that SIX2 modulates MuSC proliferation, with its loss impairing, and ectopic expression promoting, repair dynamics. PAX3/SIX2 expression correlates with distinct proliferative and differentiation behaviors, influencing regeneration in a muscle-type-dependent manner. Our findings uncover an unrecognized layer of muscle-specific regulation in MuSCs-mediated repair and suggest that PAX3-SIX2 heterogeneity could be leveraged for targeted therapies in muscle-wasting diseases.