A human embryonic limb cell atlas resolved in space and time

Human limbs emerge during the fourth post-conception week as mesenchymal buds which develop into fully-formed limbs over the subsequent months. Limb development is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common. Decades of work with model organisms has defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterisation of this process in humans has yet to be performed. Here, we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells, progressing from few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterised by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using our VisiumStitcher, we map cell clusters across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of mesenchyme in the autopod. Finally, we perform scRNA-seq on murine embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.