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Although being of utmost importance for human health and mobility, stem cell identity and hierarchical organization of musculoskeletal progenitors remain largely unexplored. Here, cells from E10.5, E12.5, and E15.5 murine limbs are analyzed by high throughput single‐cell RNA sequencing to illustrate the cellular architecture during limb development. Single‐cell transcriptional profiling demonstrates the identity and differentiation architecture of musculoskeletal stem cells (MSSC), soft and hard tissue progenitors through expression pattern of musculoskeletal markers (scleraxis [Scx], Hoxd13, Sox9, and Col1a1). This is confirmed by genetic in vivo lineage tracing. Moreover, single‐cell analyses of Scx knockout mice tissues illustrates that Scx regulates MSSC self‐renewal and proliferation potential. A high‐throughput and low‐cost multi‐tissues RNA sequencing strategy further provides evidence that musculoskeletal system tissues, including muscle, bone, meniscus, and cartilage, are all abnormally developed in Scx knockout mice. These results establish the presence of an indispensable limb Scx+Hoxd13+ MSSC population and their differentiation into soft tissue progenitors (Scx+Col1a1+) and hard tissue progenitors (Scx+Sox9+). Collectively, this study paves the way for systematically decoding the complex molecular mechanisms and cellular programs of musculoskeletal tissues morphogenesis in limb development and regeneration., This study presents a comprehensive genomics resource that reveals the transcriptome dynamics of the developing limb. Single‐cell transcriptional profiling with lineage tracing data demonstrates the identity and differentiation trajectory of musculoskeletal stem cells into soft and hard tissues. These results emphasize an indispensable role of limb Scx+ musculoskeletal stem cell population and provide additional perspective on musculoskeletal biology.

In article number 2000938, Zi Yin, Junxin Lin, and co‐workers identify a novel musculoskeletal stem cell population by single‐cell transcriptional profiling with lineage tracing data during limb development. The differentiation potential and trajectory of musculoskeletal stem cells showed they could contribute to soft and hard connective tissues, as well as muscular tissue. These results suggest an indispensable role of limb Scx+ musculoskeletal stem cell population in musculoskeletal system morphogenesis. [Image: see text]