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

The human body contains around 640 distinct muscles, each capable of regeneration following injury through the action of muscle-specific stem cells (MuSCs), that express the transcription factor PAX7. Its paralog, PAX3, a master regulator of embryonic myogenesis, is selectively expressed in a subset of adult quiescent MuSCs. The proportion of PAX3-pos MuSCs varies across muscles. By combining lineage tracing and skeletal muscle injury, we demonstrate that PAX3 drives MuSC diversity and muscle-specific regeneration rates. PAX3-expressing MuSCs display enhanced proliferation and differentiation capacities, enabling a faster response following injury, whereas loss of PAX3 leads to proliferation arrest and cell death. Single-cell RNA-sequencing analyses revealed the specific expression of Six2 in PAX3-pos MuSCs. We show that PAX3 is required for SIX2 expression, and loss of SIX2 in MuSCs reduces proliferation and differentiation rates. Conversely, ectopic Six2 expression promotes proliferation of PAX3-neg MuSCs by directly activating cell cycle pathways. With this work, we establish that PAX3-SIX2 expression correlates with distinct MuSCs behavior, influencing regeneration rates in a muscle-type-dependent context. Our findings highlight a previously unrecognized layer of regulation in MuSC behaviour and muscle repair and suggest that PAX3-SIX2 heterogeneity could be leveraged for targeted therapeutic strategies in muscle-wasting diseases.