A human embryonic limb cell atlas resolved in space and time

Abstract

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 outlined the fundamental processes underlying vertebrate limb development, but an in-depth characterisation of this process in humans has yet to be performed. Here we detail the development of the human embryonic limb across space and time, using both single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells, progressing from a restricted number of multipotent progenitors to myriad mature cell states, and identify several novel cell populations, including perineural fibroblasts and multiple distinct mesenchymal states. We uncover two waves of human muscle development, each characterised by different cell states regulated by separate gene expression programmes. We identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity and validate this by performing MSC knock down in human embryonic myoblasts, which results in significant upregulation of late myogenic genes. Spatially mapping the cell types of the limb across a range of gestational ages demonstrates a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncovers two transcriptionally and spatially distinct populations of the progress zone, which we term "outer" and "transitional" layers. The latter exhibits a transcriptomic profile similar to that of the chondrocyte lineage, but lacking the key chondrogenic transcription factors SOX5,6 and 9. Finally, we perform scRNA-seq on murine embryonic limbs to facilitate cross-species developmental comparison at single-cell resolution, finding substantial homology between the two species.

A single-cell transcriptome atlas of human early embryogenesis

“…3D). Importantly, 17 out of 20 patterning genes (e.g., HAND1 , ZIC3 , IRX3 ) independently selected by Zhang and colleagues in spatial transcriptome of human limb bud at similar stage 50 display consistent expression pattern on our domain map at CS15-16 (Supplementary Note 1), indicating the high fidelity of our spatial reconstruction.…”

A method for differential expression analysis and pseudo-temporal locating and ordering of genes in single-cell transcriptomic data

“…All above makes DEAPLOG a reliable and attractive approach in scRNA data analysis. Indeed, we have successfully applied it in large-scale human embryonic limb development data analysis and several other applications 37-39 .…”

“…All above makes DEAPLOG a reliable and attractive approach in scRNA data analysis. Indeed, we have successfully applied it in large-scale human embryonic limb development data analysis and several other applications [37][38][39] . Despite using a completely different framework to identify DEGs, DEAPLOG still returns inflated p-values like many existing methods.…”

A human prenatal skin cell atlas reveals immune cell regulation of skin morphogenesis

“…Single-cell T-cell receptor alpha and beta sequencing (abTCR-seq) data was also generated to accurately resolve T-cell subsets. Spatial validation was carried out using multiplex RNA in situ hybridisation (RNAScope), newly generated spatial transcriptomic (Visium) data from embryonic facial and abdominal skin, and published Visium data from embryonic limb from which only skin areas were analysed 16 (Fig. 1a).…”

“…To locate cells identified from scRNA-seq in situ, we performed Cell2location 19 analysis using spatial transcriptomic data from facial and abdominal skin (10 PCW) and embryonic lower limb skin (6-8 PCW) 16 . We assessed if specific cell types were more likely to be co-located within microanatomical tissue niches (microenvironments) using non-negative matrix factorisation and correlation analyses (see methods).…”