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Published December 6, 2023 | in press
Journal Article Open

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 months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization 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 from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized 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 VisiumStitcher, we map cells 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 the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.


We thank M. Thomson’s laboratory for help with mouse 10x loading; members of the Teichmann laboratory, Zhang laboratory, Marioni laboraory, Haniffa laboratory and Behjati laboratory for discussion and feedback; O. Pourquié for feedback on muscle development; A. Pyle, H. Xi and J. Langerman for sharing their raw data with us; and K. To for proofreading the manuscript. This work was supported by the Wellcome Trust grants 206194, 108413/A/15/D and 211276/Z/18/Z, as well as the National Key Research and Development Program (grants 2019YFA0801703 and 2022YFA1104904), the National Natural Science Foundation of China (grants 31871370 and 82203952), the Science and Technology Program of Guangzhou (grant 202002030429) (to H.Z.); and the China Postdoctoral Science Foundation (grant 2021M700936) and the Natural Science Foundation of Guangdong (grant 2019A1515011342) (to S.W.). P.H. holds a non-stipendiary research fellowship at St Edmund’s College, University of Cambridge. J.E.G.L. is funded by the Wellcome Trust under the clinical PhD programme (grant 222902/Z/21/Z) and supported by Darwin College, Cambridge through a Geoffrey Fisk Studentship. A.C. is funded by the INSERM cross-cutting programme HuDeCA 2018. R.B. was a recipient of a fellowship from the ‘Fondation pour la Recherche Médicale’ (FRM). This publication is part of the Human Cell Atlas (www.humancellatlas.org/publications/#).


These authors contributed equally: Bao Zhang, Peng He, John E. G. Lawrence, Shuaiyu Wang. 

S.A.T. and H.Z. supervised the project. S.A.T. initiated and designed the project. X.H. and Y.F. carried out human tissue collection. B.A.W. carried out mouse tissue collection. B.A.W., L.M., L.B., R.E. and E.S.F. performed scRNA-seq. E.T. performed the Visium spatial experiments. S.W., K.R. and E.T. carried out the in situ staining and functional experiments. R.B. performed the light-sheet fluorescence microscopy. H.Y., C.L. and H.Z. provided experimental support. B.Z., P.H. and J.E.G.L. analysed the sequencing data and generated figures. V.K., K.P., M.P., N.Y. and T.L. provided computational support. D.R.F., H.F., A.D., M.A.S. and B.J.W. contributed to interpretation of the results. B.Z., P.H., J.E.G.L., S.W., H.Z. and S.A.T. wrote the manuscript. A.C., O.A.B., J.C.M., R.A.B., H.Z. and S.A.T. supervised team members. All authors contributed to the discussion and editing of the manuscript.

Data Availability

All of our newly generated raw data are publicly available on ArrayExpress (mouse scRNA-seq, E-MTAB-10514; human Visium, E-MTAB-10367; and human scRNA-seq, E-MTAB-8813). Previously published raw data can be found from the ENCODE portal (ENCSR713GIS) and the Gene Expression Omnibus (GSE137335 and GSE142425). Processed data can be downloaded and visualized at our data portal (https://limb-dev.cellgeni.sanger.ac.uk/). The data deposited and made public are compliant with the regulations of Ministry of Science and Technology of the People’s Republic of China. Source data are provided with this paper.

Code Availability

All in-house code can be found on GitHub (https://github.com/Teichlab/limbcellatlas/), which is central to our conclusions.

Conflict of Interest

In the past 3 years, S.A.T. has consulted for or been a member of scientific advisory boards at Qiagen, Sanofi, GlaxoSmithKline and ForeSite Labs. She is a consultant and equity holder for TransitionBio and EnsoCell. J.C.M has been an employee of Genentech, Inc. since September 2022. The remaining authors declare no competing interests.

Copyright and License

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.


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Additional details

February 21, 2024
February 21, 2024