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 1 . This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common 2 . 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.


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Population characteristics
We used first trimister embyros (age, post conception weeks 5-9) from voluntary medical abortion.Tissue samples used for human scRNA-seq/Visum and validation experiments were obtained from donors of British and Chinese, respectively.No developmental abnormalities were visible or known in any of the embryos collected.

Recruitment
Medical aborted embryos were collected with the agreement of the pregnant female.The termination time point were decided by the female and doctor.

Ethics oversight
First trimester human embryonic tissue was collected from elective termination of pregnancy procedures at Addenbrookes Hospital, Cambridge, UK under full ethical approval (REC-96/085; for scRNA-seq and Visium), at Guangzhou Women and Children's Medical Center, China under approval of the Research Ethics Committee of Sun Yat-sen University (ZSSOM-2019-075) and Guangzhou Women and Children's Medical Centre (2022-050A01, for In-situ hybridisation and immunohistochemistry).Experiments were also followed the 2021 International Society for Stem Cell Research (ISSCR) guidelines in working on human embryos.Informed written consent was obtained from all donors before abortion and tissue collection.No developmental abnormalities were visible or known in any of the embryos collected.All human data generated from China was registered at China National Center for Bioinformation (PRJCA012474) and has been approved by the Chinese Ministry of Science and Technology for the Review and the Approval of Human Genetic Resources (2023BAT0445).For lightsheet fluorescence microscopy, tissues were obtained through INSERM's HuDeCA Biobank and made available in accordance with the French bylaw.Permission to use human tissues was obtained from the French agency for biomedical research (Agence de la Biomédecine, Saint-Denis La Plaine, France; N° PFS19-012) and INSERM Ethics Committee (IRB00003888).Written, informed consent was given for tissue collection by all patients.Embryonic age (post conception weeks, PCW) was estimated using the independent measurement of the crown rump length (CRL), using the formula PCW (days) = 0.9022 × CRL (mm) + 27.372.
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Life sciences study design
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Data exclusions We excluded cells based on the QC thresholds summarized in Methods section.We also removed doublets.

Replication
We used 1-2 biological and 1-2 technical replicates for human scRNA-seq and Visum; We used 1-6 biological and 1-2 technical replicates for mouse scRNA-seq.We used 2-4 biological and 1-3 technical replicates for experimental validations.All attempts at replication were successful.
Randomization Intentional randomization was not performed.Samples were allocated based on their ages.

Blinding
All human specimens were de-identified before analyses.However, selected attributes such as (developmental stage and dissected region) were available to all investigators.Blinding was not performed during tissue sample collection, analysis of scRNA-seq and Visium, as well as experimental validations, although our initial computational processing used unbiased approaches for all the sequencing samples.A majority of the downstream analyses did not adopt blinding as key sample attributes were needed for accurate cell cluster annotation and downstream analyses to create the atlas.

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